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anxiety affects quality of life in those living with parkinson's disease (pd) more so than overall cognitive status, motor deficits, apathy, and depression [13]. although anxiety and depression are often related and coexist in pd patients, recent research suggests that anxiety rather than depression is the most prominent and prevalent mood disorder in pd [5, 6]. yet, our current understanding of anxiety and its impact on cognition in pd, as well as its neural basis and best treatment practices, remains meager and lags far behind that of depression. overall, neuropsychiatric symptoms in pd have been shown to be negatively associated with cognitive performance. for example, higher depression scores have been correlated with lower scores on the mini-mental state exam (mmse) [8, 9] as well as tests of memory and executive functions (e.g., attention) [1014]. likewise, apathy and anhedonia in pd patients have been associated with executive dysfunction [10, 1523]. however, few studies have specifically investigated the relationship between anxiety and cognition in pd. one study showed a strong negative relationship between anxiety (both state and trait) and overall cognitive performance (measured by the total of the repeatable battery for the assessment of neuropsychological status index) within a sample of 27 pd patients. furthermore, trait anxiety was negatively associated with each of the cognitive domains assessed by the rbans (i.e., immediate memory, visuospatial construction, language, attention, and delayed memory). two further studies have examined whether anxiety differentially affects cognition in patients with left-sided dominant pd (lpd) versus right-sided dominant pd (rpd); however, their findings were inconsistent. the first study found that working memory performance was worse in lpd patients with anxiety compared to rpd patients with anxiety, whereas the second study reported that, in lpd, apathy but not anxiety was associated with performance on nonverbally mediated executive functions and visuospatial tasks (e.g., tmt-b, wms-iii spatial span), while in rpd, anxiety but not apathy significantly correlated with performance on verbally mediated tasks (e.g., clock reading test and boston naming test). furthermore, anxiety was significantly correlated with neuropsychological measures of attention and executive and visuospatial functions. taken together, it is evident that there are limited and inconsistent findings describing the relationship between anxiety and cognition in pd and more specifically how anxiety might influence particular domains of cognition such as attention and memory and executive functioning. it is also striking that, to date, no study has examined the influence of anxiety on cognition in pd by directly comparing groups of pd patients with and without anxiety while excluding depression. given that research on healthy young adults suggests that anxiety reduces processing capacity and impairs processing efficiency, especially in the central executive and attentional systems of working memory [26, 27], we hypothesized that pd patients with anxiety would show impairments in attentional set-shifting and working memory compared to pd patients without anxiety. furthermore, since previous work, albeit limited, has focused on the influence of symptom laterality on anxiety and cognition, we also explored this relationship. seventeen pd patients with anxiety and thirty-three pd patients without anxiety were included in this study (see table 1). the cross-sectional data from these participants was taken from a patient database that has been compiled over the past 8 years (since 2008) at the parkinson's disease research clinic at the brain and mind centre, university of sydney. inclusion criteria involved a diagnosis of idiopathic pd according to the united kingdom parkinson's disease society brain bank criteria and were confirmed by a neurologist (sjgl). patients also had to have an adequate proficiency in english and have completed a full neuropsychological assessment. ten patients in this study (5 pd with anxiety; 5 pd without anxiety) were taking psychotropic drugs (i.e., benzodiazepine or selective serotonin reuptake inhibitor). patients were also excluded if they had other neurological disorders, psychiatric disorders other than affective disorders (such as anxiety), or if they reported a score greater than six on the depression subscale of the hospital anxiety and depression scale (hads). thus, all participants who scored within a depressed (hads-d>6) range were excluded from this study, in attempt to examine a refined sample of pd patients with and without anxiety in order to determine the independent effect of anxiety on cognition. this research was approved by the human research ethics committee of the university of sydney, and written informed consent was obtained from all participants. self-reported hads was used to assess anxiety in pd and has been previously shown to be a useful measure of clinical anxiety in pd. a cut-off score of>8 on the anxiety subscale of the hads (hads-a) was used to identify pd cases with anxiety (pda+), while a cut-off score of<6 on the hads-a was used to identify pd cases without anxiety (pda). this criterion was more stringent than usual (> 7 cut-off score), in effort to create distinct patient groups. the neurological evaluation rated participants according to hoehn and yahr (h&y) stages and assessed their motor symptoms using part iii of the revised mds task force unified parkinson's disease rating scale (updrs). in a similar way this was determined by calculating a total left and right score from rigidity items 3035, voluntary movement items 3643, and tremor items 5057 from the mds-updrs part iii (see table 1). processing speed was assessed using the trail making test, part a (tmt-a, z-score). attentional set-shifting was measured using the trail making test, part b (tmt-b, z-score). working memory was assessed using the digit span forward and backward subtest of the wechsler memory scale-iii (raw scores). language was assessed with semantic and phonemic verbal fluency via the controlled oral word associated test (cowat animals and letters, z-score). the ability to retain learned verbal memory was assessed using the logical memory subtest from the wechsler memory scale-iii (lm-i z-score, lm-ii z-score,% lm retention z-score). the mini-mental state examination (mmse) demographic, clinical, and neuropsychological variables were compared between the two groups with the independent t-test or mann whitney u test, depending on whether the variable met parametric assumptions. chi-square tests were used to examine gender and symptom laterality differences between groups. all analyses employed an alpha level of p<0.05 and were two-tailed. spearman correlations were performed separately in each group to examine associations between anxiety and/or depression ratings and cognitive functions. as expected, the pda+ group reported significant greater levels of anxiety on the hads-a (u=0, p<0.001) and higher total score on the hads (u=1, p<0.001) compared to the pda group (table 1). groups were matched in age (t(48)=1.31, p=0.20), disease duration (u=259, p=0.66), updrs-iii score (u=250.5, p=0.65), h&y (u=245, p=0.43), ledd (u=159.5, p=0.80), and depression (hads-d) (u=190.5, p=0.06). additionally, all groups were matched in the distribution of gender (= 0.098, p=0.75) and side-affected (= 0.765, p=0.38). there were no group differences for tmt-a performance (u=256, p=0.62) (table 2); however, the pda+ group had worse performance on the trail making test part b (t(46)=2.03, p=0.048) compared to the pda group (figure 1). the pda+ group also demonstrated significantly worse performance on the digit span forward subtest (t(48)=2.22, p=0.031) and backward subtest (u=190.5, p=0.016) compared to the pda group (figures 2(a) and 2(b)). neither semantic verbal fluency (t(47)=0.70, p=0.49) nor phonemic verbal fluency (t(47)=0.39, p=0.70) differed between groups. logical memory i immediate recall test (u=176, p=0.059) showed a trend that the pda+ group had worse new verbal learning and immediate recall abilities than the pda group. however, logical memory ii test performance (u=219, p=0.204) and logical memory% retention (u=242.5, p=0.434) did not differ between groups. there were also no differences between groups in global cognition (mmse) (u=222.5, p=0.23). participants were split into lpd and rpd, and then further group differences were examined between pda+ and pda. importantly, the groups remained matched in age, disease duration, updrs-iii, dde, h&y stage, and depression but remained significantly different on self-reported anxiety. lpda+ demonstrated worse performance on the digit span forward test (t(19)=2.29, p=0.033) compared to lpda, whereas rpda+ demonstrated worse performance on the digit span backward test (u=36.5, p=0.006), lm-i immediate recall (u=37.5, p=0.008), and lm-ii (u=45.0, p=0.021) but not lm% retention (u=75.5, p=0.39) compared to rpda. this study is the first to directly compare cognition between pd patients with and without anxiety. the findings confirmed our hypothesis that anxiety negatively influences attentional set-shifting and working memory in pd. more specifically, we found that pd patients with anxiety were more impaired on the trail making test part b which assessed attentional set-shifting, on both digit span tests which assessed working memory and attention, and to a lesser extent on the logical memory test which assessed memory and new verbal learning compared to pd patients without anxiety. taken together, these findings suggest that anxiety in pd may reduce processing capacity and impair processing efficiency, especially in the central executive and attentional systems of working memory in a similar way as seen in young healthy adults [26, 27]. although the neurobiology of anxiety in pd remains unknown, many researchers have postulated that anxiety disorders are related to neurochemical changes that occur during the early, premotor stages of pd-related degeneration [37, 38] such as nigrostriatal dopamine depletion, as well as cell loss within serotonergic and noradrenergic brainstem nuclei (i.e., raphe nuclei and locus coeruleus, resp., which provide massive inputs to corticolimbic regions). over time, chronic dysregulation of adrenocortical and catecholamine functions can lead to hippocampal damage as well as dysfunctional prefrontal neural circuitries [39, 40], which play a key role in memory and attention. recent functional neuroimaging work has suggested that enhanced hippocampal activation during executive functioning and working memory tasks may represent compensatory processes for impaired frontostriatal functions in pd patients compared to controls. therefore, chronic stress from anxiety, for example, may disrupt compensatory processes in pd patients and explain the cognitive impairments specifically in working memory and attention seen in pd patients with anxiety. it has also been suggested that hyperactivation within the putamen may reflect a compensatory striatal mechanism to maintain normal working memory performance in pd patients; however, losing this compensatory activation has been shown to contribute to poor working memory performance. anxiety in mild pd has been linked to reduced putamen dopamine uptake which becomes more extensive as the disease progresses. this further supports the notion that anxiety may disrupt compensatory striatal mechanisms as well, providing another possible explanation for the cognitive impairments observed in pd patients with anxiety in this study. noradrenergic and serotonergic systems should also be considered when trying to explain the mechanisms by which anxiety may influence cognition in pd. although these neurotransmitter systems are relatively understudied in pd cognition, treating the noradrenergic and serotonergic systems has shown beneficial effects on cognition in pd. selective serotonin reuptake inhibitor, citalopram, was shown to improve response inhibition deficits in pd, while noradrenaline reuptake blocker, atomoxetine, has been recently reported to have promising effects on cognition in pd [45, 46]. overall, very few neuroimaging studies have been conducted in pd in order to understand the neural correlates of pd anxiety and its underlying neural pathology. future research should focus on relating anatomical changes and neurochemical changes to neural activation in order to gain a clearer understanding on how these pathologies affect anxiety in pd. to further understand how anxiety and cognitive dysfunction are related, future research should focus on using advanced structural and function imaging techniques to explain both cognitive and neural breakdowns that are associated with anxiety in pd patients. research has indicated that those with amnestic mild cognitive impairment who have more neuropsychiatric symptoms have a greater risk of developing dementia compared to those with fewer neuropsychiatric symptoms. future studies should also examine whether treating neuropsychiatric symptoms might impact the progression of cognitive decline and improve cognitive impairments in pd patients. previous studies have used pd symptom laterality as a window to infer asymmetrical dysfunction of neural circuits. for example, lpd patients have greater inferred right hemisphere pathology, whereas rpd patients have greater inferred left hemisphere pathology. thus, cognitive domains predominantly subserved by the left hemisphere (e.g., verbally mediated tasks of executive function and verbal memory) might be hypothesized to be more affected in rpd than lpd; however, this remains controversial. it has also been suggested that since anxiety is a common feature of left hemisphere involvement [48, 49], cognitive domains subserved by the left hemisphere may also be more strongly related to anxiety. results from this study showed selective verbal memory deficits in rpd patients with anxiety compared to rpd without anxiety, whereas lpd patients with anxiety had greater attentional/working memory deficits compared to lpd without anxiety. although these results align with previous research, interpretations of these findings should be made with caution due to the small sample size in the lpd comparison specifically. recent work has suggested that the hads questionnaire may underestimate the burden of anxiety related symptomology and therefore be a less sensitive measure of anxiety in pd [30, 50]. in addition, our small sample size also limited the statistical power for detecting significant findings. based on these limitations, our findings are likely conservative and underrepresent the true impact anxiety has on cognition in pd. additionally, the current study employed a very brief neuropsychological assessment including one or two tests for each cognitive domain. future studies are encouraged to collect a more complex and comprehensive battery from a larger sample of pd participants in order to better understand the role anxiety plays on cognition in pd. another limitation of this study was the absence of diagnostic interviews to characterize participants ' psychiatric symptoms and specify the type of anxiety disorders included in this study. future studies should perform diagnostic interviews with participants (e.g., using dsm-v criteria) rather than relying on self-reported measures to group participants, in order to better understand whether the type of anxiety disorder (e.g., social anxiety, phobias, panic disorders, and generalized anxiety) influences cognitive performance differently in pd. one advantage the hads questionnaire provided over other anxiety scales was that it assessed both anxiety and depression simultaneously and allowed us to control for coexisting depression. although there was a trend that the pda+ group self-reported higher levels of depression than the pda group, all participants included in the study scored<6 on the depression subscale of the hads. controlling for depression while assessing anxiety has been identified as a key shortcoming in the majority of recent work. considering many previous studies have investigated the influence of depression on cognition in pd without accounting for the presence of anxiety and the inconsistent findings reported to date, we recommend that future research should try to disentangle the influence of anxiety versus depression on cognitive impairments in pd. considering the growing number of clinical trials for treating depression, there are few if any for the treatment of anxiety in pd. anxiety is a key contributor to decreased quality of life in pd and greatly requires better treatment options. moreover, anxiety has been suggested to play a key role in freezing of gait (fog), which is also related to attentional set-shifting [52, 53]. future research should examine the link between anxiety, set-shifting, and fog, in order to determine whether treating anxiety might be a potential therapy for improving fog.

research on the implications of anxiety in parkinson's disease (pd) has been neglected despite its prevalence in nearly 50% of patients and its negative impact on quality of life. previous reports have noted that neuropsychiatric symptoms impair cognitive performance in pd patients; however, to date, no study has directly compared pd patients with and without anxiety to examine the impact of anxiety on cognitive impairments in pd. this study compared cognitive performance across 50 pd participants with and without anxiety (17 pda+; 33 pda), who underwent neurological and neuropsychological assessment. group performance was compared across the following cognitive domains: simple attention/visuomotor processing speed, executive function (e.g., set-shifting), working memory, language, and memory/new verbal learning. results showed that pda+ performed significantly worse on the digit span forward and backward test and part b of the trail making task (tmt-b) compared to the pda group. there were no group differences in verbal fluency, logical memory, or tmt-a performance. in conclusion, anxiety in pd has a measurable impact on working memory and attentional set-shifting.

PMC5075302

pubmed-2

small non-coding rnas are transcribed into mrna but remain untranslated in eukaryotic cells. they include sirna (small interfering rna), mirna (microrna), pirna (piwi-interacting rna) and snorna (small nucleolar rna). mirnas are a class of multifunctional singled-stranded small rna which are ~20 nt in length and regulate the stability or translational efficiency of targeted messenger rna depending on the base-pairing complementarity between the mirna and its target mrna [1, 2]. although over 1,000 mirna sequences have been identified from the tissues or cells of human origin and other species, as many as 1,000 to 10,000 mirnas per genome have been predicted [3, 4]. mirnas regulate a broad range of biological processes including timing of development, cell cycle progression, stem cell self-renewal, differentiation, cancer initiation, cancer cell proliferation, metastasis and apoptosis [511]. cancer is caused by multiple processes including uncontrolled cellular proliferation and inappropriate survival of apoptotic cells. many regulatory factors switch on or off genes that govern cell division and direct cellular proliferation. mirnas regulate gene expression and play important roles in the onset and progression of tumorigenesis. emerging evidence demonstrates the involvement of mirna in mammary gland tumorigenesis, functioning either as tumor suppressors or oncogenes. although the current treatment of radiation therapy, chemotherapy and hormone therapy slow mammary gland tumor growth, prolong survival and improve the quality of patients life, metastatic breast cancer still remains incurable due to our limited understanding of the molecular mechanisms through which tumorigenesis and metastasis occur. as small non-coding rnas regulate gene expression and tumorigenesis, they may represent a novel cancer therapy. unlike mrna, mirnas are transcribed but never translated. some mirnas are transcribed from non-coding regions between genes, deriving from independent transcription unit. other mirnas are transcribed together with coding mrnas from the coding region of the genome, deriving from the introns of gene transcripts [13, 14]. mirna gene copy number gain/loss and mirna gene mutation have been observed in breast cancer resulting in the aberrant expression of mirna. the first study about the altered expression of mirnas in human breast cancer patients and human breast cancer cell lines was reported in 2005 by lorio et al., in which 29 mirnas were identified with aberrant expression based on microarray and northern blot analysis of 76 breast tumor samples and 14 human breast cell lines. zhang and colleagues analyzed 283 human mirna genes on 55 human breast primary tumors and 18 human breast cancer cell lines using array-based comparative genomic hybridization. the results demonstrated a high frequency (~72.8%) of gene copy number abnormality in mirna-containing regions in human breast cancer. wang et al. collected 68 patients with newly diagnosed breast cancer and examined the expression of selected mirnas in tumor and adjacent non-tumor tissues. mir-21, mir-106a and mir-155 were significantly over-expressed in the tumor specimens compared with normal controls, whereas mir-126, mir-199a and mir-335 were significantly decreased in expression in the tumor samples. our studies of the mir-17-92 cluster demonstrated decreased expression of mir-17/20 in human breast cancer specimens compared with matching normal breast tissue from the same patient. subsequent analysis identified reduced mir-17/20 expression in node-positive compared with node-negative breast cancers and demonstrated that mir-17/20 inhibited breast cancer cell migration and invasion via a heterotypic signaling. although the tendency for a global decrease of mirna expression in human cancers originally suggested a general tumor suppressor function of mirnas, subsequent studies showing the aberrant expression of specific mirnas in breast cancer suggest mirna-specific roles in breast cancer onset and progression. many distinct mirnas have been shown to regulate breast cancer cell proliferation, apoptosis, cancer stem cell expansion, and tumorigenesis. mirna may function as either tumor suppressors or oncogenes depending on the cell type, culture conditions, target genes and pathway. the involvement of mirna in mammary gland tumorigenesis has been reviewed recently [21, 22]. le et al. described the expression pattern and regulatory network of key mirnas in breast cancer, including let-7, mir-34, mir-125, mir-200 family, mir-205, mir-21, mir-10 and the mir-17-92 cluster. adams et al. reviewed the mirna regulation of estrogen signaling pathway and erbb2/her signaling pathway in breast cancer. the understanding of how mirnas are involved in breast cancer through regulating the cell cycle remains rudimentary. herein we summarize the recent literature and research progress on the mechanism by which mirnas regulate the breast cancer cell cycle and cellular proliferation (fig. 1mirna regulation of mammary gland tumorigenesis in control of the cell cycle. through targeting different genes and different cyclin/cdk complexes, mir-17/20 and let-7 regulate the g1-s transition; mir-21 and mir-27a regulate the g2-m checkpoint mirna regulation of mammary gland tumorigenesis in control of the cell cycle. through targeting different genes and different cyclin/cdk complexes, mir-17/20 and let-7 regulate the g1-s transition; mir-21 and mir-27a regulate the g2-m checkpoint cyclin d1 is either overexpressed or amplified in ~50% of breast cancer. the abundance of cyclin d1 is rate-limiting in breast cancer cellular proliferation and g1-s phase transition [23, 24]. in addition, cyclin d1 is a critical downstream target of erbb2-, ras- and -catenin- induced breast cancers, and is sufficient for the induction of mammary tumors when targeted to the mammary gland of mice. antisense inhibition of cyclin d1 expression in vivo suppressed the growth of neut-transformed mammary adenocarcinoma cells in nude mice. conserved sequences of the cyclin d1 3utr contain potential binding sites for multiple mirnas including mir-17/20/106, mir-15/16, mir-23 and let-7. mir-17/20 binds the cyclin d1 3utr, inhibiting the expression of cyclin d1, resulting in cell cycle arrest at the g1 phase and suppression of mcf-7 cell proliferation [18, 26]. the regulation of cyclin d1 expression by mir-17-92, as well as mir-15/16, was confirmed by deshpande et al.. the let-7 family functions as a tumor suppressor in a variety of cancers including lung, colon, ovarian and breast cancer. schultz et al. demonstrated the downregulation of cyclin d1 by mirna let-7 in control of cancer cell growth. the regulation of cyclin d1 by mirna is likely of broad importance as cyclin d1 encodes the regulatory subunit of a kinase that phosphorylates and inactivates the prb family proteins to inhibit dna synthesis, and phosphorylates nuclear respiratory factor 1 (nrf-1) to inhibit mitochondria biogenesis [32, 33]. furthermore, cyclin d1 promotes breast epithelial cell angiogenesis and migration, and promotes chromosomal instability which in turn contributes to tumorigenesis. the mir-221/222 cluster regulates the cell cycle, cell growth and epithelial-to-mesenchymal transition (emt) in breast cancer. mir-221/222 inhibited p27 and p57 abundance, facilitating g1-s phase transition, thereby promoting cancer cell proliferation [36, 37]. moreover, mir-221/222 may contribute to the aggressive clinical behavior of basal-like breast cancers. the breast cancer basal-like subtype-specific mirnas, mir-221 and mir-222, promote emt in breast cancer by targeting trps1 (trichorhinophalangeal syndrome type 1) which inhibits emt by repressing zeb2 expression. mir-221 and/or mir-222 expression in mcf-7 and t47d breast cancer cells decreased er expression associated with tamoxifen resistance. the onco-mirna mir-21 is overexpressed in a wide variety of cancers including breast cancer [40, 41]. mir-21 induced cellular proliferation, migration, invasion, emt, cancer stem cell characteristics and chemotherapy resistance in human breast cancer [42, 43]. high mir-21 level is associated with poor prognosis, advanced stage, positive lymph node status and reduced survival time in breast cancer. mir-21 promotes mcf-7 cellular proliferation in part through inhibiting the expression of a tumor suppressor gene programmed cell death 4 (pdcd4). in colon cancer, mir-21 participates in a dna damage-induced g2-m checkpoint through suppressing the cell cycle regulator cdc25a. a recent report demonstrated the mir-21 regulates the cell cycle through targeting cdc25a in mcf-7 breast cancer cells. with a potential anti-cancer chemical 3,3-diindolylmethane treatment, mir-27a expression is upregulated in human breast cancer cell lines. in mda-mb-231 cells, mir-27a negatively regulated the zinc finger zbtb10 gene and myt-1, thereby promoting breast cancer cell proliferation. mir-27a suppressed myt-1, increased cdc2/cyclin b activity and promoted the g2-m checkpoint in mda-mb-231 cells. thus, distinct mirnas affect key genetic targets that govern distinct cell-cycle checkpoints including cyclins, cdks, cdk inhibitors and the g2-m regulation apparatus. in addition to cell-cycle control, breast tumor onset and progression and breast tumor stem cells are also regulated by distinct mirnas. cancer stem cells (cscs) are characterized by their self-renewal capacity, an ability to differentiate into non-tumorigenic cell progeny, and their ability to seed tumors when transplanted into animal hosts. cell surface markers such as cd44, cd24, cd133, epithelial-specific antigen and aldehyde dehydrogenase-1 are frequently used to isolate and enrich cscs. the involvement of mirnas in regulating tumor formation by cscs or tumor-initiating cells (t-ic) has been widely investigated. let-7 expression is very low to undetectable level in embryonic stem cells (es cells) and increases with differentiation. a comparison of mirna expression between breast t-ic and non-t-ic demonstrated reduced let-7 expression in t-ic and increased abundance with differentiation. transduction of breast cscs with let-7 reduced the proportion of undifferentiated cells, inhibited cell proliferation, mammosphere formation, and tumor formation in vivo. clarke and colleagues identified 37 mirnas which were differentially expressed between human breast cscs (cd44cd24lineage) and lineage nontumorigenic breast cancer cells. the mir-200 family members were downregulated in human breast cscs and normal mammary stem/progenitor cells. expression of mir-200 inhibited breast cancer stem cell expansion in vitro, and suppressed the tumor formation ability of human breast cancer stem cell in vivo. ectopic mir-34c expression reduced breast t-ics self-renewal, inhibited emt and suppressed tumor cell invasiveness via silencing notch4. zhu et al. found the reduced mir-128 expression in human breast t-ic was accompanied by bmi-1 and abcc5 overexpression, and associated with chemotherapeutic resistance and poor survival. enforced mir-128 expression increased the sensitivity of breast cancer cells to doxorubicin-induced apoptosis and dna damage. emerging evidence has demonstrated the importance of cscs in cancer initiation, cancer metastasis and drug resistance. cscs are believed to be one of the most promising targets for cure of cancer. the discovery that non-coding rnas regulate cscs widens our understanding of cscs, and may provide potential novel strategies for breast cancer therapy. deletion of chromosome 6q, including region 6q14-q16, is frequently observed in breast cancer. the small non-coding snorna u50 is a candidate tumor suppressor gene in the 6q14-16 region, playing a role in the development and/or progression of breast cancer. genomic deletion and transcriptional downregulation of snorna u50 was detected in breast cancer cell lines. re-expression of snorna u50 inhibited colony formation of the human breast cancer cells hs578 t and mda-mb-231. pirnas are small non-coding rna that form rna-protein complexes through interactions with piwi proteins. pirna was initially discovered in germ line cells, and considered as germ line-specific small rnas. emerging evidence indicates that pirna expression occurs in somatic cells [57, 58] and piwil2 expression has been identified in human breast cancer cells. high-throughput deep sequencing identified a group of small rnas matching pirna sequences in human breast cancer tissues and breast cancer cell lines. the study of these non-coding small rnas in human cancer is just starting. the identification of the expression signature of these non-coding small rnas in breast cancer subtypes, and an understanding of their functional significance to oncogene expression, tumor initiation and tumor cell metastasis may shed important new perspectives on the role of these non-coding small rnas in breast cancer. small non-coding rna-based diagnostic and therapeutic applications for human cancer are expected in the near future. although tumor-targeted delivery and local administration are still major challenges to the practical application of gene therapy for cancer, mirna-based cancer therapeutic approaches are being established and tested in animal models. synthetic mirna mimics or mirna expression vectors have been successfully applied to restore or overexpress mirna in vitro. chemically modified lna anti-sense mirna inhibitor and other approaches have been used to block mirna function in cells. kim et al. recently reported significant anti-tumor effect of virus-mediated delivery of mir-145 combined with 5-fu to treat breast cancer. intranasal delivery of let-7 and intravenous delivery of mir-34a mimics for non-small-cell lung cancer treatment and a virus-mediated delivery of mir-26a for liver cancer treatment in mouse model demonstrate the promise of mirna for treatment of cancer. dysregulated expression of mirnas has implicated components of the non-coding genome as either oncogenes or tumor suppressors of breast cancer. experimental evidence has shown specific mirnas regulating the initiation, progression, metastasis and drug resistance of breast cancer via control of the cell cycle, altering cellular proliferation, altering cellular apoptosis and/or controlling the population of tumor stem cells. dysregulated mirna expression has also been observed in cancer associated fibroblasts (caf) and in the systemic circulation [65, 66]. the circulating mirnas have the potential to serve as novel diagnostic and prognostic biomarkers for breast cancer. a specific subset of dysregulated mirnas in breast cancer cells may serve as targets for gene therapy either alone or as an adjuvant treatment to current clinical protocols for breast cancer patients.

small non-coding rnas include sirna, mirna, pirna and snorna. the involvement of mirnas in the regulation of mammary gland tumorigenesis has been widely studied while the role for other small non-coding rnas remains unclear. here we summarize the involvement of mirna in breast cancer onset and progression through regulating the cell cycle and cellular proliferation. the regulation of breast cancer stem cells and tumor regeneration by mirna is reviewed. in addition, the emerging evidence demonstrating the involvement of pirna and snorna in breast cancer is briefly described.

PMC3309138

pubmed-3

ohss is a serious complication of ovulation induction, occurring in 1-10% of in vitro fertilization patients (rizk and aboulghar, 1991; brinsden et al., 1995). this iatrogenic condition has a spectrum of clinical and laboratory manifestations ranging from mild to severe, even life-threatening conditions (rizk et al., 1990). among the serious manifestations of ohss are ascites and pleural effusion (rizk and smitz, 1992). vascular endothelial growth factor has emerged as one of the factors most likely involved in the pathophysiology of ohss (geva and jaffe, 2000; rizk and aboulghar, 2010). vascular endothelial growth factor is an angiogenic cytokine that is a potent stimulator of the vascular endothelium and appears to play an integral role in follicular growth, corpus luteum function and ovarian angiogenesis (rizk et al., 1997). vascular endothelial growth factor causes increased capillary permeability with its sequelae of ascites and possible pleural effusion (rizk et al., 1997; gomez et al., 2002). human chorionic gonadotrophin is thought to play a crucial role in the development of ohss (rizk et al. two distinct forms of severe ohss have been described: early and late forms (papanikolaou et al., 2005; mathur et al., 2000). early ohss is caused by the administration of exogenous human chorionic gonadotrophin (hcg) and it starts before the 10th day after oocyte retrieval, while the late form is the result of endogenous human chorionic gonadotrophin release in the event of pregnancy, and it starts after the 10th day following oocyte aspiration (papanikolaou et al., 2005; such strategies included the use of low dose gonadotropins and frequent monitoring during controlled ovarian stimulation, cycle cancellation, coasting, freeze all embryos and the use of gonadotropin releasing hormone (gnrh) agonist as an oocyte trigger in gnrh antagonist cycles (rizk et al. the use of antagonist in the luteal phase with total freeze of embryos to prevent severe ohss has also been suggested (lainas et al., 2012). such strategies should be used especially in patients at high risk for developing severe ohss, including those who are young, have low body weight, have polycystic ovary syndrome, those who experience high estradiol levels, rapidly increasing estradiol levels, high number of follicles during controlled ovarian stimulation and those with high number of oocytes retrieved (rizk and aboulghar, 1999; delvigne, 2009). dopamine agonists have been proposed for the prevention of ohss in women at high risk. however, the study by lvarez et al. (2007) suggested that it was only effective in reducing the incidence of moderate ohss, but not in its severe form (alvarez et al., 2007). low-dose aspirin therapy and doxycycline have also been suggested to inhibit angiogenesis (fainaru et al., 2009). (2011) believes that the concept of an ohss-free clinic has become a reality. the authors suggest that this approach should include pituitary down-regulation using a gnrh antagonist, ovulation triggering with a gnrh agonist and vitrification of oocytes or embryos (devroey et al., 2011). severe and critical forms of ohss are associated with significant ascites (golan et al., 1989; rizk et al., ascites results in an increased abdominal pressure compromising venous return, cardiac output and renal perfusion (navot et al. paracentesis is frequently needed, as prompt drainage of fluid produces a significant clinical and biochemical improvement, including spontaneous diuresis and hastening the resolution process (rizk et al., multiple paracenteses may be required to relieve symptoms and avoid serious sequelae of hemoconcentration, hypotension, decreased renal perfusion and severe respiratory compromise. instead of multiple interventions, the placement of a catheter temporarily for a few days will permit complete drainage through one procedure (rizk and aboulghar, 1999). pigtail catheters have been used for drainage in various clinical situations. in a previous study we described the use of percutaneous placement of a pigtail catheter for drainage of ascites caused by severe/critical ohss (abuzeid et al., 2003). in this study we established the efficacy and safety of pigtail catheter drainage in the management of severe/critical ohss in patients who underwent in vitro fertilization and embryo transfer at our centre between 1999 and 2001 on both inpatient and outpatient basis. the aim of the current study was to evaluate the role of the pigtail catheter drainage in outpatient management of patients with early onset severe ohss. this is a retrospective study that included all patients who underwent in vitro fertilization and developed severe or critical ohss by the golan classification (golan et al., 1989) in the period between 2004 and 2009. all patients underwent an ultrasound evaluation which documented the presence of severe ascites and bilateral ovarian enlargement. all patients presented with shortness of breath, marked abdominal distension, nausea, weight gain and lower abdominal pain. thirteen patients complained of vomiting, and 3 patients reported decreased urine output (less than 720 cc per 24 hours) and had haemoglobin 15 g/dl. all 33 patients were offered treatment using pigtail catheter insertion as an alternative to trans-vaginal paracentesis. blood studies including a complete blood count, creatinine, electrolytes, serum albumin and total protein, and coagulation panel were ordered prior to the procedure. all 33 patients were rehydrated with intravenous crystalloid solutions (0.9% normal saline). care was taken to give the patient the same amount of fluid as the amount that was drained from the peritoneal cavity. all pigtail catheter placements were performed in the operating room at our surgical centre. the catheter (6-0 french, 2.0 mm, boston scientific, quincy, ma, usa) was introduced utilizing a non-disposable metal gamete intra-fallopian treatment (gift) trocar and cannula (embryon gift catheter, ivf online, gilford, ct, usa) which was introduced into the largest accessible ascitic fluid pocket in the upper outer quadrant of the abdominal wall under local anaesthesia and trans-abdominal ultrasound guidance. the trocar was then removed and a glide wire (boston scientific corp., watertown, ma, usa) was threaded through the gift canula. the canula was removed with care so as to keep the glide wire in the peritoneal cavity. the pigtail catheter was guided into the peritoneal cavity by the wire after removal of the stylet of the pigtail catheter. the catheter was anchored to the skin using 2-0 silk suture and it was supported by gauze, which was covered by a tegaderm. the catheter was connected to a three-way stop cock which was connected to a drainage bag. initial amount of drained fluid was calculated and subsequently the patients were trained to drain 1000 cc per day (500 cc in the morning and 500 cc in the evening). one liter normal saline and 500 cc of 6% hydroxyethyle starch solution (hespan) was administered to the patient on the same day of pigtail catheter placement. any patient with critical ohss according to the golan classification, or those in whom the presenting symptoms and signs did not completely subside after drainage of ascitic fluid and iv fluid replacement, were admitted to the hospital for observation and further management. all other patients were sent home. once there was minimal or no drainage from the catheter, the patient returned to the office for removal of the catheter. the patients were encouraged to drink plenty of fluids and to increase their protein intake. all patients kept a diary for body weight, abdominal girth, and urine output before and after the procedure. patients were seen in the office as needed for evaluation and hydration with normal saline. demographic data including mean age (years), duration of infertility (years), bmi (kg/m2) and aetiology of infertility are illustrated in table i. ovarian stimulation data including number of treatment days, total dose of recombinant follicle stimulating hormone, hormone serum levels, endometrial thickness, number of follicles and number of patients receiving only 5000 iu of human chorionic gonadotrophin is illustrated in table ii. our data include the number of patients requiring early versus late placement of pigtail catheter, mean amount of ascitic fluid drained immediately after pigtail placement, number of days of pigtail catheter drainage and the number of days between oocyte retrieval and the onset of severe ohss symptoms requiring pigtail placement. table iii also illustrates the number of patients who underwent coasting, number of patients who received prophylactic hespan on day of egg retrieval. values are expressed as mean+standard deviation values are expressed as mean+standard deviation: rfsh=recombinant fsh; hmg =human menopausal gonadotropins; hcg=human chorionic gonadotropin; e2=estradiol values are expressed as mean+standard deviation three patients (9%) required an early placement of a pigtail catheter while 30 patients (91%) had late placement of the catheter (table iii). twenty-nine patients (88%) were managed on an outpatient basis without any complications. four patients (12%) required hospital admission for 1-7 days (3+2.7), three for associated severe pleural effusion requiring thoracentesis. these three patients were admitted to the hospital immediately after insertion of the pigtail catheter for management of severe pleural effusion and careful observation. one patient was admitted 48 hours after insertion of the pigtail catheter for work up for chest pain, all tests were negative. navot et al., 1992; rizk and aboulghar, 1999), but perhaps the most accepted classification of ohss is the one described by golan et al as mild, moderate and severe (golan et al., 1989). however, clinicians should be aware that ohss is a dynamic condition and moderate ohss may progress to severe ohss within hours (rizk and aboulghar, 1999; rizk, 2009). therefore, the practice committee of the american society for reproductive medicine (asrm) proposed a classification for ohss of mild, worsening and serious (the practice committee of the american society for reproductive medicine, 2008). recognizing the patients at high-risk, prevention and early recognition are the most important steps in the management of ohss (rizk, 2006). however, severe forms of ohss, especially late onset type that occurs when patients are pregnant, most likely will continue to happen, albeit at much lower rate, unless one reverts to a total freeze of all embryos after the use of gnrh agonist as a trigger shot in a gnrh antagonist cycle (donoso and devroey, 2013; devroey et al., 2011). traditional treatment consists mainly of supportive management until spontaneous resolution occurs (rizk, 1994). although moderate ohss does not usually require intervention, outpatient monitoring is essential to identify progression to a severe form (rizk, 2006). patients with moderate ohss can be managed on an outpatient basis by adequate hydration and close daily monitoring of weight gain and abdominal girth to detect progression to severe ohss, and to provide intervention, if required (rizk, 2010). patients presenting with severe forms of ohss are usually managed on inpatient basis and most patients with critical ohss are admitted to intensive care units for aggressive supportive measures to avoid maternal morbidity and mortality (the practice committee of the american society for reproductive medicine, 2008). correction of hypovolemia, electrolyte imbalance and hypoalbuminemia are the basic principles during the treatment of severe ohss (rizk, 1994, 2006; myrianthefs et al., 2000). (1992) suggested that paracentesis is the single most important treatment modality in life-threatening ohss not controlled by medical therapy. paracentesis and removal of ascites result in relieving the pressure on the inferior vena cava and improvement in venous return, cardiac output and renal perfusion (padilla et al., 1990). therefore, prompt drainage of fluid produces significant clinical and biochemical improvement, including spontaneous diuresis and hastening the resolution process (abuzeid et al., 2003; the practice committee of the american society for reproductive medicine, 2008). it is also possible that removal of ascites decreases the amount of vegf and its effects on vascular permeability. therefore ultrasound guided drainage of the ascitic fluid should be performed in severe cases of ohss with respiratory distress, oliguria or severe abdominal pain (padilla et al., 1990). this can be done either trans-vaginally (aboulghar et al., 1990) or trans-abdominally (padilla et al., 1990). however, recurrence of ascites is common in severe cases of ohss. placement of a pigtail catheter has been proposed by some investigators to allow drainage of ascitic fluid whenever it accumulates without the need for further surgical procedures (abuzeid et al., 2003). in a previous study we reported the use of percutaneous pigtail catheter for drainage of ascites in severe ohss as an alternative to repeated paracentesis (abuzeid et al., 2003). this procedure is relatively simple and requires a short period of time as compared to the time needed to gradually aspirate any fluid accumulation transvaginally. immediate relief of symptoms and signs of severe ohss after pigtail catheter insertion was an important finding. in addition, the ease by which any accumulated ascitic fluid was drained resulted in a faster resolution of ohss (abuzeid et al., no infection was reported (abuzeid et al., 2003). in that study the first 13 patients they were discharged home with the pigtail catheter in place and managed on an outpatient basis until ohss subsided; thereafter the catheter was removed. the last 13 patients were managed on an outpatient basis without any problem (abuzeid mi, et al., 2003). our previous study confirmed the safety and effectiveness of the pigtail catheter for the management of severe ohss. therefore, we embarked on the current study to evaluate the role of pigtail catheter drainage in outpatient management of patients with severe ohss syndrome. in the current study we demonstrated that patients who were diagnosed very early with severe ohss can be managed safely and effectively on outpatient basis using pigtail catheter drainage, iv fluid replacement and careful monitoring. although all 33 patients presented with shortness of breath, marked abdominal distension, nausea, weight gain and lower abdominal pain, and 13 of them complained of vomiting, (all are criteria for hospital admission according to the practice committee of the american society for reproductive medicine on ohss), 30 patients were sent home as their presenting symptoms were relieved. early diagnosis and intervention resulted in rapid elimination of the symptoms of severe ohss and normalization of vital signs, which allow us to manage the patients on outpatient basis. the pigtail catheter placements were performed without complications and patients were discharged home after a few hours of observation. the patients who needed hospital admission were those with associated severe pleural effusion requiring thoracentesis. another patient was admitted to the hospital for 1 day for work up for chest pain and possible diagnosis of pulmonary embolism. once pulmonary embolism was ruled out, she was discharged home with a pigtail catheter in place and she was managed on outpatient basis. outpatient management of ohss has no place in those who fail to have complete resolutions of their symptoms, or those with severe hyperproteinuria or electrolyte imbalance. certainly it has no place in patients with critical ohss such as those with severe pleural effusion, diminished urine output, severe hemoconcentration and those at increased risk of thromboembolic risks (rizk, 2010). it is imperative to stress that such a protocol can only be used in patients who are motivated, reliable and have the necessary support at home to allow them to return to the clinic or go to the emergency room if the need arises. it also requires dedicated in vitro fertilization nurses to educate and follow up the patient on a daily basis. contrary to other reports that state that access to intravenous albumin is a critical part of outpatient management for severe ohss, we did not use any albumin replacement (lincoln et al., 2002). the reason behind the use of intravenous albumin is to increase intravascular oncotic pressure, thereby curtailing the exodus of free water from the intravascular space. in our study we used hespan instead of albumin, as some investigators have shown that it is beneficial in reducing the incidence of severe ohss (youssef et al., 2011). by increasing the intravascular oncotic pressure in addition, the majority of our patients had late onset ohss as a result of pregnancy. hespan should be used with caution in patients with late onset ohss only when benefits outweigh risks.it is a fact that outpatient management of early severe ohss is a common practice among infertility specialists after trans-vaginal aspiration of ascitic fluid and iv fluid hydration. outpatient management by repeated abdominal or trans-vaginal paracentesis has been reported to be safe, effective and cost effective (lincoln et al., 2002; shrivastav et al., 1994; fluker et al., 2000 (1994) were the first to suggest that day care management in patients with severe ohss with abdominal paracentesis and iv hydration was simple, safe and effective compared with traditional treatment with in-patient hospitalization and supportive measures. (2002) concluded that the need for hospitalization for hyperstimulated patients can be minimized by outpatient management of severe ohss with trans-vaginal culdocentesis and rehydration with crystalloids and albumin. (2000) advocated in a pilot study an active management strategy for moderate ohss, which involved early paracentesis designed to minimize the progression from moderate to severe ohss, instead of late paracentesis aimed at speeding up recovery in severely ill patients. (2010) concluded that early outpatient paracentesis for moderate to severe ohss is the most cost effective management plan when compared with traditional conservative inpatient therapy. (2009) reported a large series of patients with severe ohss in whom the vast majority were successfully managed as outpatients with use of aggressive trans-vaginal paracentesis (smith et al., 2009). despite the findings in our study it is important to emphasize that ohss remains a serious disorder with the potential for rapid deterioration, requiring hospitalization and intensive treatment of a critically ill patient. the life-threatening complications of ohss are secondary to thromboembolism or compromised pulmonary or cardiovascular function and multi-organ failure. there have been isolated reports of women dying from the complications associated with ohss. the mortality rate related to ohss is very low and was estimated by brinsden et al. more recently, reports regarding maternal mortality rates due to ohss in the netherlands and united kingdom demonstrate an incidence of approximately 3 deaths per 100,000 in ivf cycles (braat et al., 2006; lewis, 2007). eleven lethal complications of assisted reproductive technology have been documented and summarized by braat et al. several investigators reported sporadic fatality as a result of ohss due to adult respiratory distress syndrome, massive pulmonary oedema, myocardial infarction, and carotid artery occlusion resulting in cerebral infarction. in this study we describe a modified technique of what interventional radiologists usually use for pigtail catheter placement. we observed that in the obese patients the metal introducer provided in the pigtail catheter kit is difficult to use; instead we used the non disposable gamete intra-fallopian treatment trocar and cannula to facilitate the introduction of the catheter through the abdominal wall of such patients. this technique helps to manage the patients in the office without the need for sending her to the hospital for an interventional radiologist to place the catheter. however, the patients in this study were identified from our in vitro fertilization database; the study included every patient who developed severe/critical ohss. in addition, the lack of a control group that was managed without pigtail catheter placement limits the conclusions that can be drawn from our study. in summary, drainage of ascitic fluid in patients with severe/critical ohss is an important step in the management of this condition (rizk, 2010). the placement of a pigtail catheter resulted in a safe and effective outpatient management of the majority of patients with severe ohss. however, outpatient management of this potentially fatal condition requires clear understanding that rapid deterioration may occur, which may necessitate hospitalization and intensive treatment of a critically ill patient (rizk, 2006, 2010). patients with critical ohss and those with persistence of symptoms of severe ohss despite insertion of a pigtail catheter and drainage of ascitic fluid should be admitted to the hospital for inpatient management.

objective: to evaluate the efficacy and safety of outpatient management of severe ovarian hyperstimulation syndrome (ohss) requiring placement of a pigtail catheter. methods: retrospective analysis of thirty-three consecutive patients who underwent in-vitro fertilization (2003-2009) and developed severe/critical ohss requiring placement of a pigtail catheter. patients who were managed on outpatient basis were monitored by frequent office visits, daily phone calls, and received iv normal saline for hydration when required. results: in 3 patients (9.1%) ohss started early, requiring placement of a pigtail catheter 4.3+0.6 days after retrieval. in 30 patients (90.9%) ohss started late (14 4 days after retrieval). the mean amount of ascitic fluid drained immediately after placement of the catheter was 2085 1018 cc. the pigtail catheter was removed after 7.8 5.3 days. of the 31 patients who had embryo transfer (two had total freeze), 84% conceived. twenty-nine patients (88%) were managed on outpatient basis without any complications. four patients required hospital admission for 1-7 days (3.0 2.7). one patient with severe ohss was admitted for work up for chest pain. three patients with critical ohss with severe pleural effusion requiring thoracentesis were admitted for supportive measures. conclusion: the placement of a pigtail catheter resulted in safe and effective outpatient management for the majority of patients with severe ohss.

PMC4086000

pubmed-4

the family is the cornerstone of human social support network and its presence is essential in everyone s life. changes inevitably occur in families with illness and hospitalization of a family member. in other words, among the sources of stress for families are accidents leading to hospitalization particularly intensive care unit (icu). statistics show that 8% of hospital beds in the united states are occupied by the intensive care units. stress in the family while the patient is in the icu can disrupt the harmony power of the family members and finally, it may causes disturbances in the support of the patient. in addition to the various sources of stress in intensive care units such as the patient s fear of death, financial problems, lack of awareness about the environment and etc., their satisfaction level is another important source of stress for the patient s family. today, the family needs of hospitalized patients in the icu are summarized in five sections. these factors may include receiving assurance, staying close to the patient, receiving information, feeling comfortable and receiving support. however, in many cases, the patient s family needs and their expectations in the icu will not be fulfilled which will cause dissatisfaction. since, how families deal with mental stress is the most important components of comprehensive care, it should be known that attention to the family needs of the patients hospitalized in intensive care units could increase their satisfaction and thus a reduction in stress disorders. studies showed that the nurses in particular sections focus primarily on the patient and the disease. therefore, attention to the families should be considered as an important part in patient management. effective interventions targeted at the needs and expectations of family members should help to reduce the stress and improve their satisfaction. fox et al. stated that the needs of the family members were not often overlooked by the nurses. however, the nurses spend a lot of time with the patients and their families and are in a good position to assess their needs and plan for meeting these needs with appropriate interventions. the reason of investigating the satisfaction of the families of the patients in the icu is that the patients are facing life-threatening illness experiences and complex treatments, different technologies and different equipment are used for them. these cases could potentially lead to the dissatisfaction of their families. in the meantime, the families of trauma and neurosurgical patients are more vulnerable than the other groups of patients. they need supports that are more emotional and should be accepted by the icu staff. today, there are many theories expressed that in intensive care units, the patient and his family should be considered as a single unit. by providing care to this unit, the aim of this study was to analyze the satisfaction of the families of icu patients. the tool of society of critical care medicine s family needs assessment (sccmfna) (which has been validated in 1998 by johnson) was used in that study. the results showed that most of the family satisfaction was related to the communication and patient care and the lowest level of satisfaction was about the ability of staff to patients family comfort. bailey et al. have conducted a study in 2009 with the purpose of investigating the relationship between informational support to families of special care patients and their anxiety and satisfaction. they showed that there is a direct link between the informational support and their satisfaction. the study of fumis et al. in 2008 also showed that the increase in the availability of physicians for giving the information to the patient s family and as well as nurses efforts to provide understandable explanations about the patient s condition to them will increase the patient s family satisfaction. this clinical trial was performed with respect to the existing gap for a study carried out for investigating the interventions for the satisfaction of the patients in the special care units and their families in iran. the purpose of the study was to determine the effectiveness of nursing interventions based on family needs on family satisfaction level of hospitalized patients in the neurosurgery intensive care unit of al-zahra hospital in 2010. the statistical research community was the families of hospitalized patients in neurosurgery intensive care unit of al-zahra (sa) hospital, isfahan, iran from may to september 2010. with respect to inclusion criteria, the families of 64 patients were selected with simple sampling. after obtaining the informed consents from the hospitalized patients, by using the envelopes, which were prepaid by using a table of random numbers, they were divided into two groups (intervention and control). the inclusion criteria was included as the following cases: to be a first-degree member of the family, to be older than 18 years, willing to participate in the study, having the power to speak and write in farsi, to be hospitalized one day before the study, lack of physical or mental problem in the family member, no responsibility for the care of another patient, introduced as the first person responsible for the patient care. obtaining the written informed consent, completion of the questionnaires about demographic information and the families satisfaction were performed in the second day of hospitalization of the patients in icu in both groups. johnson questionnaire for the satisfaction of the family of icu patients was used to measure the family satisfaction. the rating of the questionnaire was performed by using the likert scale with four options of 0 to 4 as follows: almost all the time (3 scores), often (2 scores), only occasionally (1 score) and never (zero score). johnson s questionnaire was the modified type of multer patient s family needs, which was evaluated in various scientific researches and there are extensive evidences for its validity. the interventions were performed in the second and the third day of hospitalization of the patient in the neurosurgery icu by the researcher in the morning shift for the intervention group. thus, the researcher provided understandable explanations about the disease to the patient s family and answered honestly to the questions about the patient and the disease. the researcher ensured them for providing the best care in the section and spoke about the prognosis of the disease. the researcher took the family member to the patient s room and gave the necessary information about the room space, equipment, personnel departments and the actions that were done for the patient. the patient family member was contacted if there was a change in the patient condition. they let the family help the patient in some cases of the public health (such as hand and foot massage and giving food). locations of rest, nutrition, prayer room, bathroom and buffet of the hospital were introduced to them. the telephone numbers of hospitals and wards (and if necessary, the social worker and supervisor) were given. various departments and personnel of the hospital were taught to accept the families and communicate with them. the hospital social worker was introduced and if necessary, the patient s family was allowed to express their feelings. these interventions were performed in the evening shift of the second and the third day of the patient reception. the actions were carried out by the fellow researcher. while, for the control group, only the routine work was done. on the fourth day, the satisfaction survey was completed by the selected family member in both of the intervention and control groups. statistical methods used in this study were 2 test, man-whitney, independent student s t-test and paired t-tests. the findings did not show statistically significant difference in the demographic characteristics of the intervention and control groups, such as: age (p<0.99), gender (p<0.79), marital status (p<0.41), educational level (p<0.12) and relation with patient (p<0.54). as shown in table 1, there was no significant difference in the mean satisfaction score before the intervention in the intervention and control groups (p>0.05). the mean satisfaction score after the intervention in the intervention group was significantly more than the control group (p<0.001). the mean satisfaction score in the intervention group after the intervention was significantly higher than before the intervention (p<0.001). there was no significant difference in the mean satisfaction score in the control group before and after the intervention (p>0.05) (table 2). comparison of mean satisfaction score (100 *) of participants in the intervention and control groups the mean of satisfaction score changes of the studied subjects in the intervention and control groups after intervention the results of the present study showed that the nursing interventions based on the family needs increased the patient s family satisfaction in the neurosurgery intensive care unit of al-zahra hospital. due to the differences in the study design, study population, the number of samples and methods of intervention, it would be difficult to compare the results. in chien et al. study, the results showed that performing the training based on the patients family needs in the icu, decreased the anxiety and increased their satisfaction. in the study of myhren et al. indicated that effective communication of the staff with the families of icu patients would increase their satisfaction. in the study by rosher and robinson, it showed that involving the families in patient care led to increase their satisfaction than before the intervention. the mentioned results were consistent with the results of this study, because all of these interventions were some parts of the interventions performed in this study. however, the study of steele et al. indicated that clinical training had no impact on the family satisfaction of the patient hospitalized in the icu. therefore, there was no statistically significant difference in the satisfaction level in the control and intervention group. in this study, it was shown that the use of nursing interventions based on family needs (confidence, support, information, proximity and convenience) had significant impact on the family satisfaction of the patient hospitalized in intensive care unit. they should be based on the family investigation and recognition and their priority needs should be determined in order to cause the greatest satisfaction. the icu nurses must also accept their role in the care of patient s family members. the findings of this study could be a basis for performing further studies about family needs of the patients in the icu especially with the different forms of culture and economy. consequently, the increase in family satisfaction of patients can reduce the stress disorders and improve their mental state and ultimately better support of the patient by the family. this study only examined the effect of nursing interventions in the first days of hospitalization on the satisfaction of the families but the effects on the patient s entire hospitalization period has not been assessed. by performing other researches in this field, it would be possible to compare the differences in the effects of nursing interventions based on the needs of families in the early days and the whole period of the patient s hospitalization.

background: since the family is a social system, the impairment in each of its component members may disrupt the entire family system. one of the stress sources for families is accidents leading to hospitalization particularly in the intensive care unit (icu). in many cases, the families needs in patient care are not met that cause dissatisfaction. since the nurses spend a lot of time with patients and their families, they are in a good position to assess their needs and perform appropriate interventions. therefore, this study was conducted to determine the effectiveness of nursing interventions based on family needs on family satisfaction level of hospitalized patients in the neurosurgery icu. materials and methods: this clinical trial was conducted in the neurosurgery icu of al-zahra hospital, isfahan, iran in 2010. sixty four families were selected by simple sampling method and were randomly placed in two groups (test and control) using envelopes. in the test group, some interventions were performed to meet their needs. in the control group, the routine actions were only carried out. the satisfaction questionnaire was completed by both groups two days after admission and again on the fourth day. findings:both of the intervention and control groups were compared in terms of the mean satisfaction scores before and after intervention. there was no significant difference in mean satisfaction scores between test and control groups before the intervention. the mean satisfaction score significantly increased after the intervention compared to the control group. conclusions:nursing interventions based on family needs of hospitalized patients in the icu increase their satisfaction. attention to family nursing should be planned especially in the icus.

PMC3702150

pubmed-5

rising levels of obesity are becoming a worldwide phenomenon and are increasingly identified as a health problem across the globe [14]. higher weight has been associated with adverse health indicators and outcomes, including cardiovascular disease [512], stroke [5, 13], cognitive and functional decline [1418], metabolic syndrome [19, 20], inflammation [21, 22], and mortality [20, 2325]. obesity among aging populations is relatively recent and aging among people who have been obese for much of their lives is also a new phenomenon. from 1980 to 2004, the prevalence of obesity in the us has continued to rise from about 17% to 25% for men aged 5059. while obesity in england has also increased during this period, from approximately 9% in 1980 to 15% in 2004 for men aged 5564, the level of obesity remains much lower in england. additionally, the difference in obesity between the us and england is more pronounced for women. the level of obesity in us women was about 24% in 1980 and rose to 37% in 2004 (age 5059); in england, levels for women were 9% in 1980 and 14% in 2004 (age 5564). the aim of this paper is to investigate differences in how obesity relates to indicators of physiological dysregulation in men and women of diverse populations. this comparison will lead to an improved understanding of how obesity might be differentially related to health and mortality across cultures and lifestyles. obesity was relatively rare in most populations until the second part of the last century, but it has now become common in many countries. the us population is recognized as among the most obese in the world, although many other countries are now approaching the us level and most countries are experiencing increasing obesity. this worldwide obesity epidemic began with the epidemiological revolution and the virtual elimination of infectious disease; the decline in manual labor needed to provide sustenance with the industrial revolution; and the increasing availability and decreasing cost of food [2729]. obesity reflects some combination of calorie intake, diet content, and amount of physical activity. in some cultures, lack of physical activity can be a more important determinant of obesity; in other cultures, overeating or food composition may be the more important determinant of obesity. it is also true that within countries, individuals could differ in the causes of obesity. for instance, changes in activity might be more characteristic of women or men resulting in different reasons for obesity by gender. these differences may affect how obesity is related to other risk factors for poor health, and it may determine the overall health risk associated with obesity. if physical activity is maintained, the overall effect of obesity may be less than if the activity is not. one indication of the cause of obesity may be the relationship between waist size and weight [30, 31]. in societies where obese people are more physically active, waist size of the obese waist circumference has also been linked to late life mortality, where high waist circumference has been associated with increased mortality among men and women in the netherlands, while high bmi was not associated with mortality. this paper builds upon current obesity research by using both bmi and waist circumference to quantify obesity in order to determine how a combined indicator of weight and adiposity is related to physiological dysregulation in populations with different cultures, diets, behaviors, and epidemiological histories. obesity has been related to many indicators of physiological dysregulation including cardiovascular risk factors such as hypertension and metabolic dysregulation in lipid levels or insulin resistance. most studies that investigate the differences in biological risk associated with excess weight have examined western populations [33, 36]. comparative studies on the health risks associated with obesity that examined the us and england reported that obese americans had an increased risk of diabetes and a higher waist circumference [37, 38]. these studies suggested that differences in physical activity, diet, and social environments may explain these national differences. while these differences have been observed between the us and england, these two western countries have roughly similar life expectancy, levels of living, history, and culture even while the us has poorer health by a number of indicators of disease prevalence and biological risk [36, 39]. comparative studies of the links between obesity and health outcomes and risk factors for obesity comparing western and non-western countries indicate important differences in the causes and consequences of obesity. a comparison of the association of disease with overweight and obesity in japan and the us indicated that the associations were stronger in the us than in japanese women and that there was no association in japanese men. links between social, demographic, and behavioral risk factors for obesity also differ markedly in japan, korea, and the us. the availability of biomarker data from taiwan a middle-income country undergoing rapid economic growth, increasing obesity, and with life expectancy recently increasing to levels similar to that of the us and england allows for investigation of the biological risk associated with obesity in a population characterized by very different cultural, behavioral, socioeconomic, and dietary parameters. we examine how elevated weight and obesity (using an indicator that considers both bmi and waist circumference) relate to having levels defined as clinical risk for cardiovascular, metabolic, and inflammatory markers in three aging societies that are now relatively similar in life expectancy but that differ in the timing of the epidemiological transition and obesity epidemic, history of economic development, socioeconomic levels, general lifestyle habits, health behaviors, and health care systems: the us, england, and taiwan. finding differences in the relationship between obesity and indicators of physiological dysregulation in these three aging societies will clarify whether increases in weight gain are equally problematic across all countries. we use data from three nationally representative samples: the us national health and nutrition examination survey (nhanes, 20032006; n=3855), the english longitudinal study of ageing (elsa, 2004-2005; n=9139), and the social environment and biomarkers of aging study (sebas) in taiwan (2000; n=1023). these surveys collect information on demographics, as well as anthropometric, physical, and biological measures. every year, approximately 5,000 individuals undergo detailed interviews and medical examinations, which include collection of several physiological measures. nhanes utilizes a complex sampling design, and when weights are applied, the sample is representative of the noninstitutionalized american population. we use the 20032006 data since nhanes data is released in two-year intervals, and this sample is centered on 2004-2005, which matches the period in which elsa was collected. for nhanes, we use individual-level data based on a sample of 1,513 fasting individuals aged 54 and older. elsa includes participants drawn from households responding to the health survey for england (hse) in 1998, 1999, and 2001 and is representative of the english population aged 50 and older. the core elsa sample (wave 1: 2002-2003) includes people living in an hse responding household who were born prior to march 1, 1952, and their partners who could be under age 50. wave 4 of elsa (2008-2009), which includes a nurse visit, includes wave 1 core members, if they are still alive and do not refuse further contact after the first interview at wave 1. it also includes a refresher sample to maintain the age structure of the sample (in waves 3 and 4), and their partners. for elsa, we use individual-level data based on a sample of 7,384 individuals aged 54 and older. sebas is drawn from a follow-up survey of the survey of health and living status of the near elderly and elderly in taiwan (also known as the taiwan longitudinal study of aging (tlsa)), a nationally representative survey of taiwanese adults (including institutionalized individuals) collected in 1989, 1993, 1996, 1999, and 2000. in 2000, a subsample of individuals was randomly selected for inclusion in sebas. sebas consists of adults aged 54 and older in 2000, with in-home interviews and medical exams taken in a hospital. for sebas, the sample averages 66.8 years of age in england, and the us and taiwan mean age is about the same (table 1). there are more men in taiwan (56%) and england (53%) and fewer in the us (44%). we examine the following indicators of physiological dysregulation often associated with obesity and also associated with increased risk for multiple adverse health outcomes and obesity [43, 44]: (1) cardiovascular markers: high systolic (sbp) and diastolic blood pressure (dbp); (2) metabolic markers: high levels of blood lipids (total and low-density lipoprotein (ldl) cholesterol, and fasting triglycerides), low levels of high-density lipoprotein (hdl) cholesterol, and high fasting glucose and glycated hemoglobin; (3) high levels of inflammatory markers c-reactive protein (crp; available in nhanes and elsa) and interleukin-6 (il-6; available in sebas), as crp and il-6 have been positively associated with bmi. for each indicator we use clinical cutpoints or widely used research-based cutpoints to indicate high levels of risk which are shown in table 1 [39, 43, 45]. there has been debate as to the best indicator of obesity: body mass index (bmi) or waist circumference. waist circumference is thought to be a better measure of abdominal adiposity than bmi and a better indicator of risk of poor health outcomes, including all-cause and cardiovascular mortality [46, 47]. for this reason we investigate the association between bmi and biomarkers across categories of bmi (underweight<18.5 kg/m, normal and overweight 18.529.9 kg/m, obese 3034.9 kg/m, and very obese 35 kg/m) and waist circumference categorized as normal or high waist (high waist: men 120 cm, women 88 cm). we create a composite measure of obesity and adiposity by categorizing individuals into five groups: (1) underweight and normal waist (all underweight individuals had a normal waist circumference), (2) normal/overweight bmi (termed normal bmi) and normal waist circumference (reference group), (3) normal/overweight bmi (termed normal bmi) and high waist circumference (termed high waist), (4) obese and high waist (all obese individuals had a high waist circumference), and (5) very obese. we also evaluate an alternate definition for obesity in taiwan based on bmi 27, as it has been suggested by some that obesity levels should be differentially defined for asians [48, 49]. a similar composite measure of obesity and adiposity was calculated using this alternate definition of bmi in taiwan. because these risk factors are all assumed to be associated with obesity and because dysregulation in multiple physiological systems has been shown to predict many of the poor health risk outcomes associated with aging, we also create two summary measures of risk based on the total number of at-risk levels of biomarkers, either 9 or 8. because crp values for sebas are not available, this measure is not included in the 9-item summary measure for taiwan, but a summary measure (range 09) was calculated for taiwan using il-6, another indicator of inflammation, instead of the crp values included for the us and england. a second alternate summary measure of biological risk, excluding the inflammatory marker (range 08), biological risk summary scores were computed for individuals who had missing values on no more than 3 biological markers. we examine multiple covariates in our investigation of the relationship between obesity and biological risk. self-reported use of antihypertensives was determined in all three countries, and use of lipid-lowering statins was only asked in the us sample. dichotomous variables were created to indicate whether the respondent reported being a current smoker and participating in at least moderate physical activity for exercise (e.g., brisk walking, running, or swimming) in the past 30 days (for the us and england) or generally exercising once a week (for taiwan). we use logistic regression models to determine the odds of having at-risk levels of a specific biomarker for obese men and women among the three populations. for all countries, the comparison group for bmi and waist circumference is the normal bmi and normal waist group. the regressions included indicators of age, use of antihypertensives, current smoking status, and having exercised in the past 30 days. ordinary least squares (ols) regression models were used to determine the relationship between the summary measures of biological risk and the composite measure of obesity and adiposity. we begin by examining national differences in the high risk levels of individual biomarkers (table 1). low levels or high risk levels of hdl cholesterol are also more common in taiwan. high total and ldl cholesterol is more common among the english; lower levels of plasma glucose, crp, and glycated hemoglobin are also characteristic of the english. few adults in england have elevated levels of fasting glucose (2.2%), while this is observed in 17.3% and 13.2% of american and taiwanese adults. most people in this age range are in the normal to overweight category (64.7%, 67.2%, and 89.4% in the us, england, and taiwan, respectively) (table 2(a)). americans are more likely to be obese (33.7%) compared to the english (32.1%) and taiwanese (7.2%). among the obese, americans are much more likely to be very obese: 13.4% of the total us sample, 10.1% in england, and about 1% in taiwan. both among the obese and very obese, the average bmi is higher in the us and england compared to taiwan. few are underweight in any country (1.7%, 0.8%, and 3.4% in the us, england, and taiwan, resp.). when we examine waist circumference, the us has the highest average waist circumference, with 65.5% of americans, 55.9% of english, and 15.8% of taiwanese having a high waist size (table 2(a)). this means that high waist characterizes a substantial number of those who would be categorized as normal weight in the us and england. among those in the normal and overweight group about half (49.5%) of americans and a third (36.4%) of the english almost all obese individuals have a high waist in the us and england (98.3% in the us and 96.5% in england), but only 78.4% of the obese in taiwan also have a high waist. when we use the alternate obese cutpoint of 27 kg/m in taiwan, less than half of the obese individuals have a high waist (not shown here). americans exhibit the highest proportion of the older population taking antihypertensive medication (47.1%) (table 1). the percentage who reports taking antihypertensives is lower in england (32.0%) and taiwan (28.6%). americans are more likely to be current smokers (24.5%) than persons in taiwan (22.5%) and england (13.9%). more than half of the population in all countries report having exercised in the past 30 days, with more english exercising (82.2%) compared to taiwan (61.4%) and the us (58.5%). men with normal bmi and high waist have a greater likelihood than men with normal bmi and normal waist size of having high-risk levels of triglycerides in all three countries. in the us and england, men with high waist are more likely to have high levels of glycosylated hemoglobin and higher crp; fasting glucose is also elevated among this group in the us (table 3(a)). men who are obese in the us have fewer elevated risk factors than those with high waist who are not obese; in the us, obese men are only more likely than normal weight men without high waist to have elevated glycated hemoglobin, fasting glucose and crp. english men who are obese have more elevated risk: both blood pressure indicators, hdl cholesterol, triglycerides, glycated hemoglobin, and crp. very obese men in england have the same elevated risk factors with the exception of dbp. very obese men in the us are more likely to have elevated fasting glucose in addition to crp and glycated hemoglobin. english and taiwanese women with normal weight and high waist are more likely to have elevated sbp, dbp, and glycosylated hemoglobin; only british women with higher waist have significantly elevated triglycerides and only the taiwanese women had more hdl risk. high risk crp is more common among both american and english women with normal bmi and high waist, and this risk of elevated crp is also higher in the obese and very obese (table 3(b)). obese women in britain had elevations in the same markers as normal bmi and high waist english women, while obese women in the us only have elevated fasting glucose, glycated hemoglobin, and crp; obese women in taiwan only had high dbp. with the exception of taiwan, levels of the inflammatory markers (crp in the us and england; il-6 in taiwan) are more likely to be elevated among persons with a high waist and normal bmi, obese or very obese, compared to their normal bmi and normal waist counterparts. among men, an increase in the biological risk summary score (range 09) is associated with having a high waist relative to being of normal bmi and normal waist in all three countries (table 4(a)). being obese or very obese is also related to a higher biological risk summary score (09) for men in the us and england. these equations explain 6 to 11 percent of the variance in the summary indicator of biological risk. these relationships are similar for women (table 4(b)), with one exception: obese taiwanese women do not have a significantly increased biological risk compared to their normal weight and normal waist counterparts. when we consider the alternate summary score that excludes our indicators of inflammation (range 08), being obese or very obese is no longer associated with a higher biological risk summary score in us men compared to men with a normal bmi and normal waist when controls for health behaviors and medication use are included (table 5(a)). the size of the effects of the obesity categories is reduced on the 8-indicator summary measure in both england and the us, indicating the strong link between crp and obesity. the r is also reduced in these equations for england and the us. for taiwan, women in england and taiwan, but not women in the us, with a higher waist but who are not obese have significantly higher physiological dysregulation compared to their normal bmi and normal waist counterparts. obese women in all three countries have elevated risk and the very obese have even higher risk (table 5(b)). the alternate biological risk summary measure (08) yields different relationships between weight and physiological dysregulation in the us and taiwan. in the us, only very obese women have a higher alternate biological risk summary score (08). in taiwan, obese and normal bmi and high waist women exhibit higher alternate biological risk summary scores compared to their normal bmi and normal waist counterparts, except when smoking status, physical activity, and use of hypertensives are included. figures 1(a) and 1(b) illustrate the predicted alternate biological risk score (08) for each weight category for men and women (respectively) aged 65 who are nonsmokers, do not engage in physical activity, and are currently taking antihypertensive medication. this figure allows for country comparisons of individuals with these characteristics within each weight category and across weight categories. the predicted values indicate that the us has the highest biological risk score within each respective weight category among men and women of the same age and lifestyle behaviors. with the exception of women in the normal bmi and high waist group, england has the second highest biological risk score within each weight category, followed by taiwan. among 65-year-old women with the noted lifestyle behaviors and with a normal bmi and high waist when we consider the alternate bmi cutpoint for obesity in taiwan (bmi 27 kg/m), our findings for the individual biomarkers and summary measures of biological risk are similar to using the bmi 30 kg/m cutoff for taiwan except that the category normal weight with high waist no longer differs from the omitted category (results not shown). this study observes three general findings about how biological risk is associated with obesity in three countries that differ in lifestyle and culture. first, obesity is associated with physiological dysregulation in all countries with differences in the links between specific indicators of biological risk and obesity. generally, obesity in england is associated with hypertension, dyslipidemia, and elevated glycated hemoglobin; americans who are obese are not more likely to have hypertension. in taiwan, obese women are more likely to have elevated dbp and obese men have an increased risk of elevated triglycerides and glycated hemoglobin compared to their nonobese, normal waist counterparts. our biological risk summary scores indicate that at all levels of weight physiological dysregulation was highest in the us, followed by england (with one exception), with taiwanese exhibiting the lowest biological risk in all groups among the three countries. second, these relationships remain after controlling for demographic factors, participation in physical activity, and other behavioral factors. third, similar to obese older adults, high waist individuals with normal bmi also exhibit greater physiological dysregulation in all countries compared to their normal bmi and normal waist counterparts. our finding of a higher physiological dysregulation, as shown by the alternate biological risk summary score, in taiwan compared to the us and england could be due to a couple of potential explanations. the prevalence of obesity in the us and england is much higher than in taiwan, indicating an earlier initial rise in obesity relative to taiwan. from 1978 to 2002, the proportion of obese americans and britons exhibited stark increases (1332% and 623% for men and women, resp.). the estimates for obesity prevalence in taiwan indicate a recent increase for men but not women. from 19931996 to 2000-2001, the age-adjusted prevalence of obesity rose from 10.5% to 15.9% for men and declined from 13.2% to 10.7% in women. it may be that the lower levels of risk among older adults who have lived longer years with obesity could be a reflection of better pharmacologic control of physiological dysregulation (e.g., through statin use), which may in turn confer less biological risk in these populations compared to populations of currently obese taiwanese adults who may have more recently begun living with obesity. a second reason for the observed country differences in obesity may be due to differences in dietary habits and lifestyle. the us and england are two modern, western populations whose diets have been influenced by increased industrialization and have over time come to be characterized by high glycemic loads and high fatty acid composition. taiwan, on the other hand, represents a country that has experienced the effects of the industrial and scientific revolutions later than that of the us and england but is currently rapidly undergoing economic development and demographic change. the recent economic changes in taiwan may indicate that obese older adults in taiwan have more recently begun to consume high-fat diets, which could result in greater initial physiological dysregulation associated with access to western-influenced dietary habits. despite controlling for lifestyle behaviors thought to be linked with health, the country differences in obesity and physiological regulation remain. moreover, the consideration of antihypertensives does not alter our substantive conclusions on these associations. this suggests that despite the greater use of medications to treat hypertension in the us, obesity among americans is associated with greater overall biological risk than the other two countries. this is supported by findings from the general population of americans relative to england, which report that the us is faced with greater health disadvantages than england in adulthood and across the life span. we also note differences in biological profiles of obese individuals between the two westernized countries: the us and england. the excess risk of hypertension associated with obesity in england was not found in the us. these differences may be due to the higher use of medications among americans compared to the english, with about 16% more men and 18% more women in the us aged 65+taking antihypertensive medication compared to their british counterparts. the greater use of hypertensive medications in the us is also noted when compared to japan and countries across europe. two notable differences in country patterns of the relationship between obesity and physiological dysregulation by sex are found. among men in england and taiwan, the order of magnitude of physiological dysregulation increases with higher weight categories; however, this is not observed for us men. this difference may be due to our inability to consider statin use in england and taiwan, which may be particularly important in the relationship between obesity and physiological dysregulation for men. conversely, the importance of considering statin use may be less vital to understanding the country differences in the association between obesity and physiological dysregulation among women, given that the relationship for women is more consistent across countries, namely in the us and england. women, underweight corresponds with higher biological risk (though nonsignificant) compared to women with normal bmi and normal waist. underweight among men in taiwan is significantly associated with much lower biological risk than their normal bmi and normal weight counterparts. further studies will be required to explore possible explanations for these differences in physiological dysregulation. the higher biological risk observed among normal bmi and high waist individuals relative to normal bmi and normal waist older adults builds upon previous studies that report on alternate indicators of body shape, which vary across countries. the importance of waist circumference is underscored by our current study, as well as a growing body of literature on the predictive value of waist circumference on indicators of health. higher rates of diabetes among older americans compared to britons have been accounted for by high waist circumference as opposed to bmi differences. additionally, increasing waist circumference is more predictive of greater risk of incident diabetes than bmi in middle-aged british men (and the european prospective investigation into cancer and nutrition (epic)-potsdam study). waist circumference, as an indicator of central fat mass, is thought to be more strongly associated with disease risk, and in our case with physiological dysregulation, compared to bmi, which is considered a cruder index of adiposity. banks and colleagues cite differences in physical activity, diet and greater psychosocial environmental challenges in america compared to england as potential mechanisms linking central adiposity and type 2 diabetes. our study considers some of these possible mechanisms (e.g., physical activity and antihypertensive use) but finds that they explain little of the relationship between biological risk and adiposity among the three countries. together, these results highlight the importance of considering waist circumference in investigating the links between indicators of health and adiposity. our finding of the biological risks associated with obesity among older taiwanese adults underscores the growing concern for risks associated with obesity in countries rapidly undergoing modernization. in comparing the biological risk of obese individuals among the three countries, we are able to use these international comparisons to our advantage to examine how differences in modernization influence the health of older adults in different populations this may have potential health policy implications that underscore the importance of addressing and controlling the rising obesity epidemic that has become most widespread in countries, like the us and england, that have long experienced high economic growth and in countries currently undergoing rapid economic development. the increasing use of biological information to inform our understanding of health represents an innovative method in biodemography that will further contribute to the testing of current comparative theory and the potential creation of new paradigms surrounding the influence of modernization on health. first is the use of a broad range of biological markers across three large-scale population surveys. the inclusion of biological information as objective precursors of health allows, to some extent, a fairly comparable comparison of indicators of health across the different populations. an exception to this uniform comparison of biomarkers across the three surveys is our use of inflammatory marker crp in the us and england and our inclusion of a different marker of inflammation (il-6) in taiwan. of note, crp seems to be more strongly associated with obesity than il-6. a growing body of literature has made distinctions between bmi and waist circumference, namely, suggesting that waist circumference is a better indicator of abdominal obesity, which in turn has been associated with obesity-related health risks. our findings generally report a similar association between increased biological risk and (1) normal bmi and high waist and (2) obese and high waist. using the us nhanes, reported that when both waist circumference and bmi were included in their analyses, only waist circumference was a significant predictor of comorbidity. although this and other studies have suggested that waist circumference may be a better indicator of obesity and risk for adverse health outcomes, our study finds the two indicators to be similarly associated with biological risk across the three countries. first, we examine population-based data from three countries at a single time point. future studies of longitudinal data will allow for further investigations of the potential role of obesity on biological risk observed in the current associations. second, we do not have measures of some lifestyle and medical behaviors for some of the datasets (e.g., statin use), which likely influence the relationship between obesity and biological risk. as such, we are unable to include such factors in our analyses of all three countries. it is possible that these lifestyle behaviors are key explanatory factors to the noted cross-country differences in obesity-related biological risk. the cross-country differences in the relationship between increased biological risk for individuals who are obese and have a high waist underscore potential differences in health and lifestyle behaviors. these behaviors may be a result of country differences in economic development that we are not able to observe in this study. the country differences in the links between obesity and physiological dysregulation are particularly marked when comparing obesity among taiwanese older adults relative to westernized populations, such as the us and england. further examination of these relationships over time and across other countries will contribute to our understanding of the potential factors responsible for these country-specific variations in biological risk, as obesity becomes increasingly more prevalent and older adults in various countries live more years with obesity and increased adiposity.

excess weight has generally been associated with adverse health outcomes; however, the link between overweight and health outcomes may vary with socioeconomic, cultural, and epidemiological conditions. we examine associations of weight with indicators of biological risk in three nationally representative populations: the us national health and nutrition examination survey, the english longitudinal study of ageing, and the social environment and biomarkers of aging study in taiwan. indicators of biological risk were compared for obese (defined using body mass index (bmi) and waist circumference) and normal weight individuals aged 54 +. generally, obesity in england was associated with elevated risk for more markers examined; obese americans also had elevated risks except that they did not have elevated blood pressure (bp). including waist circumference in our consideration of bmi indicated different links between obesity and waist size across countries; we found higher physiological dysregulation among those with high waist but normal bmi compared to those with normal waist and normal bmi. americans had the highest levels of biological risk in all weight/waist groups. cross-country variation in biological risk associated with obesity may reflect differences in health behaviors, lifestyle, medication use, and culture.

PMC3679767

pubmed-6

many studies have not been reported in literature for lumbar discectomy by destandau endospine system. we report a series of 300 patients operated for lumbar dissectomy by destandau endospine system. a total of 300 patients suffering from lumbar disc herniations were operated between january 2002 and december 2008. technique comprised localization of symptomatic level followed by insertion of an endospine system devise through a 15 mm skin and fascial incision. endoscopic discectomy is then carried out by conventional micro disc surgery instruments by minimal invasive route. the results were evaluated by macnab's criteria after a minimum followup of 12 months and maximum up to 24 months. based on modified macnab's criteria, 90% patients had excellent to good, 8% had fair, and 2% had poor results. the complications observed were discitis and dural tear in five patients each and nerve root injury in two patients. 90% patients were able to return to light and sedentary work with an average delay of 3 weeks and normal physical activities after 2 months. the advantages of use of minimal invasive spinal surgical techniques in treatment of lumbar disc herniation is small incision, limited tissue disruption, enhanced visualization due to better magnification and illumination, shorter hospital stay, and faster recovery time.13 among many posterior spinal endoscopic systems used for disc surgery, destandau endospine system and foley and smith's metrx system are seen as viable alternatives to open disc surgery.46 the aim of this study was to present results in 300 patients operated by edoscopic discectomy and to discuss technical points to shorten the learning curve. a total of 475 patients suffering from different type and level of lumbar disc herniation with radiculopathy and degenerative lumbar canal stenosis were operated between january 2002 and december 2008. the inclusion criteria were patients having lumbar disc prolapse with unilateral radiculopathy, on clinical evaluation, positive straight leg raise or femoral stretch test, and identification of a single nerve root lesion on mri. patients with bilateral symptoms, double root involvement, cauda equina syndrome and whose clinical symptoms did not match mri picture were excluded from present study. all these patients had fair trial of conservative treatment in the form of rest, medication (nsaid), activity modification, and physiotherapy (minimum 6 weeks) before they were advised to undergo surgery. however, in present study, none of the patients opted for surgery at 6 weeks after completion of conservative treatment. there were 206 males and 94 females aged between 18 to 72 years (mean, 38.4 years). levels operated upon included l1-l2 (n=3), l2-l3 (n=2), l3-l4 (n=6), l4-l5 (n=205), and l5-s1 (n=84). there were 235 extruded, 20 contained, 15 foraminal, and 30 sequestrated herniations. results were evaluated as poor, fair, and good or excellent using modified macnab's criteria. modified macnab's grading was as follows: excellent-no pain/restriction of activity and being able to do all activities; good-occasional pain with relief of presenting symptoms and returning to work with some modification; fair-some improved functional capacity but still handicapped or unemployed; and poor results-having objective symptoms of root involvement or repeat surgery at the index level. the clinical material included preoperative history, physical examination, plain x-rays and mri studies of lumbosacral spine, laboratory tests, and intraoperative video documentation. postoperative follow-up was carried out on third day, 2 weeks, 6 weeks, 3, 6, 12, and 24 months. it consists of endospine tube, trocar, and working insert [figure 1a and b]. one port for 0 degree endoscope, second for suction cannula, third port (biggest) for working instrument, and fourth port for dural and nerve root retractor. the procedure of discectomy can be carried out under general, spinal, epidural, or local anesthesia. the operative technique consists of knee chest positioning after administration of anesthesia followed by level localization by localization devise [figure 1c]. at marked point, 15 mm skin incision is made aponeurosis is incised using mayo's scissors; 1.5 cm wide periosteal elevator is used to elevate paravertebral muscles subperiostealy, thus exposing the interlaminar window and part of the affected side facet. (a) clinical photograph showing patient positioning and level marking (b) destandau endospine system (c) iitv picture of marked level (d) position of endospine tube (e) endoscopic view of decompressed nerve root the endospine tube with trocar is pushed through the incision in the direction of posterior arch over interlaminar window followed by withdrawal of trocar. the working insert any soft tissue bulging in the mouth of tube is removed till boundaries of interlaminar window such as superior and inferior lamina, facet joint are clearly visualized [figure 1e]. this follows part resection of inferior margin of the superior lamina followed by excision of ligamentum flavum leading to exposure of the dural sac and nerve root under endoscopic vision. once the nerve root has been accurately identified, it is retracted using a nerve root retractor.. it also helps to keep the field dry. depending on local findings, discectomy involving the extraction of the nucleus pulposus once satisfactory nerve root decompression is achieved, endospine tube along with working insert is withdrawn. aponeurosis is sutured using vicryl fine suture followed by closure of the skin in a subcuticular fashion. a water-impermeable dressing (a) preoperative sagittal t2wi mri of prolapsed l5-s1 disc (b) postoperative sagittal t2wi mri of l5-s1 disc after endoscopic discectomy at 2 years follow up (c) axial t2wi mri section at l5-s1 disc after endoscopic discectomy at 2 years follow up shows no compression these patients were followed up on third day, 2 weeks, 6 weeks, 3, 6, 12, and 24 months. patients were followed up for minimum of one year and maximum of 2-year duration. on second visit on third day, wound was inspected for any drainage or evidence of infection. complains about fever, backache, and leg discomfort at final follow-up, 90% patients were relieved of sciatica and were satisfied with procedure. 285 patients were operated as day care cases and were mobilized and discharged same evening from day care facility. based on modified macnab's criteria 90% patients had excellent to good, 8% had fair, and 2% had poor results. five patients who had interaoperative minor dural tears were hospitalized and were observed for any dural leak. causative factor for dural tears in present study were as follows: three patients had dural rent due to forceful retraction of dura and nerve root by dural and nerve root retractor. this was observed in patients in whom there was significant posterolateral herniation resulting in tenting of dura and nerve root at recess. in this situation, authors have found gentle mobilization of nerve root and dura by nerve root hook or approaching and debulking the offending disc through axilla before proceeding with retraction of nerve root and discectomy. this happened when kerrison rongeur was used to open the tight recess resulting in dural tear and nerve root injury. these dural tears were managed by water tight closure of muscle, fascia, and skin and bed rest for duration of one week. superficial delayed wound healing was observed in 20 patients, which healed in 21 days by regular dressings rest and administration of antibiotics. the diagnosis of postsurgical discitis was based on mainly clinical grounds and laboratory evidence of raised counts, esr, and c-reactive proteins. clinical criteria included recurrence of severe and unrelenting back pain within first week of surgery, keeping patient awake at night after initial recovery. no biopsy of disc was resorted to; however, mri of lumbosacral spine was ordered in all these patients which did not contribute much to the diagnosis. lizolid 600 mg/bd) for first week followed by oral antibiotics for 5 weeks. all these patients responded well to antibiotics and no further intervention of any kind was carried out. after initial back pain for 6 weeks, these patients had occasional residual backache which was treated by analgesics, activity modification, lumbar support, and rest during subsequent follow-up visits. nerve root injuries (n=2) were encountered while trying to do a medial facetectomy to open the recess by a kerrison rongeur causing severe laceration of nerve root. however, nerve root was in continuity., patties were used for gentle retraction and procedure of discectomy was successfully completed endoscopically without resorting to open discectomy. 276 patients were able to return to sedentary work with an average delay of 2 weeks, except 24 patients who had complains of backache, occasional leg discomfort, discitis, and nerve root injuries. medial facetectomy was resorted to in 59 patients to open the recess to decompress the nerve root in addition to discectomy. mixter and barr7 discovered the pathophysiology of discogenic sciatica and suggested laminectomy and discectomy as operative treatment. the overall results of standard discectomy range from 68 to 95% in different series.8 the operative microscope and microsurgical techniques were developed in mid-1960 's by yasargil and krayenbuhl910 and these techniques revolutionized spine surgery leading to smaller incisions, less blood loss, increased visualization of site of pathology, decreased hospitalization, shorter postoperative recovery, and earlier return to activities compared with previous operative interventional techniques. the results of microdiscectomy also range from 85 to 98%.1113 katayama et al.14 compared the results of open vs gold standard microdiscectomy and observed no difference between the surgical outcomes in both the groups but microdiscectomy gave better lighting, magnification, and therefore decreased the length of incision and tissue invasion. microdiscectomy also allowed the patients to return to early work with lesser use of narcotic medication. microendoscopic dissectomy (med) combines standard microsurgical technique with an endoscope, enabling the surgeons to address all types of disc herniations including decompression of nerve root and lateral recess. chemonucleolysis was reported by smith.15 nonetheless, based upon various randomized clinical trials, the efficacy of chemonucleolysis compared with more traditional and open procedures for the operative treatment of lumbar disc herniations remained speculative.16 the use of percutaneous nucleotomy, laser discectomy, and intradiscal electrothermal annuloplasty (idet) compared with microdiscectomy remains unclear, and it is attributed to the lack of high-quality studies.17 conclusions about the efficacy of some of the aforementioned minimally invasive procedures (e.g., chemonucleolysis, apd, idet) were questionable with regard to disc-related pathology.18 therefore, lumbar microdiscectomy remained the gold standard for addressing a herniated or sequestrated intervertebral disc; however, a movement toward more minimally invasive approaches that would yield superior outcomes, while minimizing excessive soft and bony tissue removal and minimizing soft tissue trauma, were sought. as such, an evolution in procedures toward smaller incisions, less tissue trauma, and quicker return to daily activities took center stage in spine surgery. the use of muscular retractor system was reported initially by faubert and caspar.1920 perez-curet and fessler,21 described for the first time a myriad of spine pathologies that could be addressed using tubeology. though from our initial experience, endospine technique is minimal invasive, but limitation of study has been lack of comparison with gold standard microscopic discectomy technique. however, in a study by shin et al.,22 15 cases each were compared of med and microscopic group (md). the mean cpk-mm levels were lower for the med group than for the md group at both 3 (576.1286.3 iu/l compared with 968.1377.8 iu/l) and 5 days (348.1231.0 iu/l compared with 721.7463.2) postoperatively (p<0.05). the mean vas scores for postoperative back pain were lower in the med group than in the md group, both at 1 (3.32.3 compared with 5.81.5) and 5 days (1.91.1 compared with 3.61.1) postoperatively (p<0.01). aforementioned authors concluded that the med procedure is less invasive than md, and causes less muscle damage and backache. the 90% excellent results in present study is comparable with other surgical procedures for herniated lumbar discs such as those of destandau, perez-cruet et al., and their average surgical time was 66 minutes, average blood loss was 22 ml, average hospital stay was 7.7 hours, complication rate was 5%, reoperation rate was 4%, and average return to work was 17 days with excellent result in 94% patients. average operative time was 50 minutes average blood loss was 45 ml (range, 30-70 ml). return to work (21 days) and overall results (90%) which are comparable. in another prospective and randomized evaluation of surgical treatment for lumbar disc herniation by hermantin et al.,24 satisfactory results of 97% in endoscopic group (n=30) and 93% in open laminectomy group (n=30) were reported. however, in endoscopic group, these authors had excluded large central herniations and extra ligamentous herniations between l5 and first sacral vertebra. however, present endoscopic technique could be used for all levels and all type of herniations. in our current series, there was 5% discitis and 5% incidence of dural injury. caspar and ebling,2526 authors have reported reoperation rate of 5.5, 5.7, and 3%, respectively. another measure of success is reflected by the patient's ability to return to previous employment. our patients returned to previous employment on an average at 15 days with restriction to avoid heavy manual work for 2 months. discectomy (med) by endospine system has claimed even lesser tissue invasion than microdiscectomy with even smaller skin incision, lesser use of analgesics, and early return to work. least tissue invasion is established by many reports comparing the postoperative mri signal of paraspinal muscles,27 intraoperative electromyographic findings establishing less invasion to nerve roots,28 and by measuring serum levels of biochemical parameters reflective of a postoperative inflammatory reaction and damage to the paravertebral muscles.29 our personal opinion is similar, though this was not the parameter studied in our series. minimal invasive microendoscopic decompression technique has been used not only for paracentral disc herniations, but also for all types including far lateral, cephalad, caudal migrated, and central and recurrent disc herniations.3032 one of the driving forces behind the minimal invasive spine surgery is economics, shorter hospital stay, reduced postoperative morbidity, and quicker recovery times. in our series, 90% patients were operated as day care cases. posterior paraspinous process endoscopic access to lumbar disc herniation requires creation of working space where no or little space existed before. internal view of operating site is magnified and well illuminated. with advent of this system, discectomy can be done as day care procedure ensuring reduced postoperative morbidity, minimal or no hospitalization, less pain, and faster recovery. with proper patient selection, discectomy and adequate nerve root decompression by doing foraminotomy or opening a lateral recess stenosis by minimally invasive technique can be achieved with this system. however, the endospine system has been excellent modality to address discogenic radiculopathy and to decompress lumbar canal stenosis. many surgeons are convinced of advantages of the system and have included this system as part of their inventry. however, due to difficulty in orientation with scope and two-dimensional vision, availability of less space, frustrating and steep learning curve, and inability to master hand eye coordination, majority of surgeons are not able to continue with the technique. the patience and persvrance to work through narrow confines and work closely with a surgeon who has mastered the technique is the key to learn. second step would be to become comfortable with 2 dimensional vision of endoscopic camera and to master orientation, triangulation. depth perception in these techniques comes from experience rather than observation; hence, surgeon keen to learn these techniques must combine these procedures during early phase of learning with standard procedures he is doing in his clinical practice. gradually, as surgeons master the learning curve, he will be able to use this as treatment method for his patients. there is also a need to establish cadaveric labs and dummy models on line of arthroscopic learning centers where surgeons can practice hands-on cadavers and models to improve triangulation, depth perception, and hand eye coordination.

background: posterior endoscopic discectomy is an established method for treatment of lumbar disc herniation. many studies have not been reported in literature for lumbar discectomy by destandau endospine system. we report a series of 300 patients operated for lumbar dissectomy by destandau endospine system. materials and methods: a total of 300 patients suffering from lumbar disc herniations were operated between january 2002 and december 2008. all patients were operated as day care procedure. technique comprised localization of symptomatic level followed by insertion of an endospine system devise through a 15 mm skin and fascial incision. endoscopic discectomy is then carried out by conventional micro disc surgery instruments by minimal invasive route. the results were evaluated by macnab's criteria after a minimum followup of 12 months and maximum up to 24 months. results:based on modified macnab's criteria, 90% patients had excellent to good, 8% had fair, and 2% had poor results. the complications observed were discitis and dural tear in five patients each and nerve root injury in two patients. 90% patients were able to return to light and sedentary work with an average delay of 3 weeks and normal physical activities after 2 months. conclusion:edoscopic discectomy provides a safe and minimal access corridor for lumbar discectomy. the technique also allows early postoperative mobilization and faster return to work.

PMC3270611

pubmed-7

squamous cell carcinoma of the head and neck (hnscc) is a heterogeneous disease that includes tumors arising from the mucosal epithelial surface of the oral cavity, oropharynx, hypopharynx, and larynx. although these tumors originate within different anatomic sites within the upper aerodigestive tract, they are histologically identical (95% of hnscc are squamous cell carcinomas), share common etiologic risk factors and overlapping metastatic target site profiles (reviewed in [13]). recent genetic analysis of human head and neck tumors has revealed common molecular alterations including p53 mutation, p14arf, and p16 methylation, as well as cyclin d and egfr amplification [36]. despite these similarities, the distinct anatomic subsites are associated with differing rates of regional metastasis for example, vocal cord lesions tend to metastasize less frequently than oropharyngeal or hypopharyngeal lesions. this variation may be attributed to differing densities of lymph draining vessels within each of the relevant subsites. patients who exhibit metastases into the regional nodal basin exhibit a 50% decrease in survival irrespective of treatment [715]. currently, it is the 5th leading cause of cancer by incidence and the 6th leading cause of cancer mortality in the world [16, 17]. recurrent and/or metastatic hnscc patients have a poor prognosis, with a median survival of less than 1-2 years [18, 19]. several lines of evidence indicate that cancer is a disease resulting from dynamic changes in the genome that promote the progressive transformation of normal human cells into highly malignant derivatives [20, 21]. during this process, cancer cells acquire several unique capabilities including self-sufficiency in response to growth signals, insensitivity to antigrowth signals, evasion of programmed death (apoptosis), limitless replicative potential, sustained angiogenesis as well as invasion and metastasis, reprogramming of energy metabolism, and avoiding immune destruction [21, 22]. detailed global genomic analyses of several human tumors has revealed that certain classes of signaling proteins appear to be targeted more frequently by oncogenic mutations. receptor tyrosine kinases (rtks) are a good example. of the 59 transmembrane rtks identified to date, dysregulation of ~30 rtks are associated with neoplastic transformation and cancer progression [2325]. interestingly, ninety percent of primary head and neck squamous cell cancers, irrespective of subsite, have alterations in members of the epidermal growth factor (egf) family of receptor tyrosine kinases (erbbs), in particular erbb1/egfr. ten to fifteen percent of tumors will also have an alteration in another egfr family member, the erbb2/her2/neu receptor [27, 28]. these findings suggest a strong etiologic role for rtk dysregulation in this type of tumors. given this association, patients with head and neck squamous cell cancers are well positioned to benefit from existing and future molecular targeted agents directed against oncogenic rtks such as egfr (reviewed in). rtks are a family of transmembrane proteins that mediate many important physiological processes in both normal and cancerous cells. ligand binding to the extracellular domain of rtks induces receptor dimerization and activation of rtk activity. subsequent autophosphorylation of the receptor at specific tyrosine residues within the cytoplasmic domain generates binding sites for proteins that relay downstream biological signals to regulate protein function, protein-protein interactions, and gene expression. under physiological conditions, rtk dysregulation can occur through several mechanisms including gene amplification or rtk overexpression, chromosomal translocation to produce constitutively active rtks, gain of function mutations or deletions that promote ligand-independent rtk activity, escape from negative regulatory mechanisms or local environmental changes, all of which lead to potent oncogenic signaling and hence neoplastic growth. these complex signaling networks use multiple factors to drive the outcome of rtk signaling. although often depicted as linear pathways, they actually represent an integrated network with various modes of cross-talk, overlapping and distinct functions. known signaling pathways involved in head and neck tumorigenesis include the phosphatidylinositol-3-kinase (pi3k)-akt-mammalian target of rapamycin (mtor), signal transducer and activator of transcription (stats) and raf kinase-mitogen-activated protein kinase kinase (mek)-p42/p44 mitogen activated protein kinase (mapk) signaling pathways [1, 30]. this review highlights three rtk signaling pathways involved in head and neck squamous cell carcinoma; egfr, the type 1 insulin-like growth factor receptor (igf-1r) and the hepatocyte growth factor (hgf) receptor (met). this short review will explore the relative contribution of each signaling axis to disease progression, potential modes of cross-talk, and targeted clinical approaches under investigation for disease management. the egfr family of rtks is comprised of four different receptors known as erbb1 (also referred to as egfr), erbb2 (her2/neu in rodents), erbb3 (her3), and erbb4 (her4) (reviewed in [3133]). each receptor, with the exception of erbb3, contain an intracellular tyrosine kinase domain that is activated by binding to extracellular egf-like ligands, which result in receptor dimerization and hence activation of downstream signaling cascades including mapk, pi3k/akt and stat signaling. eleven egf-like ligands have been identified to date that can be categorized into four groups those that bind egfr only (egf, transforming growth factor alpha (tgf), and amphiregulin), those that bind to egfr and her4 (heparin binding-egf, betacellulin and epiregulin), those binding directly to either her3 and her4 (neuregulin 1 and neuregulin 2) and her4 binding only (neuregulin 3 and neuregulin 4) (reviewed in). epigen, the most recently discovered member of the egf-like ligand family appears to be a low affinity and broad specificity ligand that effectively activates egfr. erbb2 is considered a ligand-less coreceptor as it does not have any known ligands that bind directly with high affinity, despite its established role as a potent oncogene in several cancer types including breast, colorectal, nonsmall cell lung carcinoma (nsclc) and hnscc [36, 37]. aberrant egfr activity has been strongly linked to the etiology of 5890% of hnscc [26, 38]. these rates can vary due to the inclusion of cancers from different subsites within the head and neck, methods used to assess gene amplification and tumor scoring methods. in contrast to lung adenocarcinomas in which activating egfr mutations result in ligand-independent signaling [3943], such activating egfr mutations are infrequent in hnscc [44, 45]. egfr gene amplification resulting in upwards of 12 copies per cell has been reported in hnscc patients compared to copy numbers detected in normal mucosa from noncancer patients. this and other pathways of ligand-independent receptor activation that do not require egfr overexpression have been characterized as the likely drivers of egfr activity in hnscc. egfr gene amplification remains a strong indicator for poor patient survival, radioresistance, and locoregional failure [4749]. egfr overexpression is detected in healthy mucosa in cancer patients (field cancerization) that will increase in proportion to observed histological abnormalities such as hyperplasia, carcinoma in situ and invasive carcinoma, indicating that it is an early event in hnscc. accordingly, significant effort has focused on egfr signaling as a therapeutic target for treating hnscc patients. cetuximab, matuzumab and nimotuzumab represent humanized antiegfr antibodies, whereas gefitinib and erlotinib are small tyrosine kinase inhibitors (tkis) (figure 1). cetuximab (erbitux) competitively inhibits endogenous ligand-binding to egfr and thereby inhibits subsequent receptor activation [5053]. cetuximab is a valuable treatment option in head and neck patients as it synergizes with current treatment modalities. cetuximab enhances the effects of many standard cytotoxic agents, including cisplatin (the conventional platinum-fluorouracil chemotherapeutic), and in combination with chemotherapy it can elicit antitumor responses in tumors that previously failed to respond to that chemotherapy. notably, cetuximab did not dramatically exacerbate the common toxic effects associated with radiotherapy of the head and neck, including mucositis, xerostomia, dysphagia, pain, weight loss, and performance status deterioration. cetuximab has been approved for use in combination with radiation for treating patients with locally advanced hnscc and as monotherapy for patients with recurrent hnscc. matuzumab (formerly emd 72000) binds to egfr with high specificity and affinity to block receptor signaling, and also modulates antibody-dependent cellular cytotoxicity (adcc) when combined with cetuximab [5860]. phase i clinical trials report excellent antitumor activity of matuzumab against several human tumor types including head and neck cancers. a randomized phase iib, four-arm, open-label study recently assessed the safety and efficacy of nimotuzumab in combination with radiation therapy (rt) or chemoradiation therapy (crt) in patients with advanced (stage iii or iva) hnscc. the addition of nimotuzumab to both the radiation and chemoradiation regimens was reported to improve the overall response rate, survival rate at 30 months, median progression-free survival and median overall survival. a combined group analysis of the nimotuzumab arms versus the non-nimotuzumab arms demonstrated a significant difference in overall survival favoring nimotuzumab. this study is compelling as patient response rates compare favorably with studies combining cetuximab with radiotherapy, but with fewer side effects. gefitinib (iressa) is a small molecule tki-targeted to the intracellular active site for phosphorylation that has been tested in clinical trials involving hnscc patients, as a single agent or in combination with radiation treatment. unfortunately, gefitinib has shown limited clinical efficacy with response rates of 1015% [63, 64]. erlotinib is a selective inhibitor of the egfr that also shows antitumor activity in hnscc comparable to standard combination chemotherapy. another promising rtk under preclinical and clinical evaluation for head and neck cancers includes the igf-1r (reviewed in [66, 67]). two ligands, insulin-like growth factor 1 (igf1) and igf2 bind to igf-1r. ligand binding to the igf-1r stimulates its intrinsic tyrosine kinase activity, activating downstream signaling networks including ras-raf, mapk and erk, and pi3k (figure 1) to drive cellular functions such as cell growth, survival and differentiation. it is widely accepted that the igf-axis activates antiapoptotic signaling, which in turn upregulates the pi3k-akt and mapk pathways in cancer cells. additionally, igf-ir also regulates vascular endothelial growth factor (vegf) production, suggesting a role in tumor angiogenesis. several studies indicate that igf-1r is overexpressed and functional in 94% of hnscc patient samples [70, 71]. consistent with this, igf-ir signaling significantly enhances the proliferation, motility and tumorigenicity of human head and neck cancer cell lines. igf-1r down regulation in a hnscc cell line using antisense oligonucleotides resulted in a dose-dependent decrease in cellular proliferation, induction of apoptosis, caspase activation and reduced expression of proangiogenic cytokines such as vegf. interest in targeting the igf-1r in hnscc was bolstered by the observation that treatment of head and neck cancer cells with either igf or egf resulted in igf-ir and egfr heterodimerization [71, 72]. however, only igf resulted in the phosphorylation of both receptors. using a mouse xenograft model for hnscc, treatment with antibodies against igf-1r, egfr or it remains to be determined whether cellular cross-talk between igf-1r and egfr has an important role in determining the biological aggressiveness of hnscc or resistance to egfr-targeted therapies. several monoclonal antibodies and tkis for igf-1r have been tested in preclinical studies and early phase clinical studies. however, the efficacy of igf-1r-targeted therapy for treating patients with hnscc, particularly cross-talk with egfr, warrants further investigation. to date, the effect of blocking oncogenic igf-1r and egfr signaling have been studied more extensively in breast cancer cell lines [7375]. treatment with gefitinib and ag1024, a tki for igf-1r reduced cell proliferation when used as single agents and showed an additive effect when used in combination [76, 77]. targeting igf-1r and egfr signaling is currently under evaluation in hormone-sensitive metastatic breast cancer using the igf-1r inhibitor osi-906 and the egfr tki erlotinib, although results are not yet available (http://www.clinicaltrials.gov/, identifier nct01205685). similarly, an exploratory study to assess the modulation of biomarkers in hnscc patients treated preoperatively with cetuximab and/or imc-a12, a humanized antiigf-1r monoclonal antibody is currently underway (http://www.clinicaltrials.gov/, identifier nct00617734). these studies will be critical for evaluating whether the use of anti-igf-1r and egfr-targeted treatments will be more effective than single-agent modalities for treating patients with hnscc. the met receptor is a single pass transmembrane protein that upon binding its ligand hgf also known as scatter factor-promotes increased cell proliferation, survival and motility (reviewed in [78, 79]). hgf is the only physiological ligand for met and is secreted as an inactive precursor polypeptide chain by mesenchymal cells. hgf is proteolytically cleaved to form an active/heterodimer by a number of serine proteases including urokinase plasminogen activator (upa), tissue-type plasminogen activator (tpa), coagulation factors x. xi and xii. met is a disulphide-linked/heterodimer derived from the proteolytic cleavage of a 170 kda precursor. the chain and n-terminal region of the -chain form sema domain, a seven -propeller structure in which blades 2 and 3 bind to hgf. the sema domain is flanked by a cysteine-rich region followed by four immunoglobulin repeats. it is proposed that the cysteine-rich region and immunoglobulin repeat domains undergo a conformational change following hgf binding allowing for met dimerization [80, 81]. binding of hgf to met results in receptor autophosphorylation at key catalytic residues and subsequent recruitment of several cytosolic signaling molecules that are shared with the egfr and igf-1r signaling pathways, including the grb2/sos complex, the p85 regulatory subunit of pi3k, gab1 and jak/stat3 (figure 1). subsequent activation of the mapk and jun-n-terminal kinase (jnk) pathways is responsible for the mitogenic and motogenic properties of met/hgf signaling resulting in invasive growth, depending on the physiological setting. increased met signaling in human cancers can be the result of enhanced ligand-binding (autocrine and paracrine), met overexpression or missense mutations that often induce constitutive kinase activity, failure of met down regulation and interactions with other cell surface receptors such as egfr (reviewed in [8284]). met is overexpressed in 84% of hnscc patient samples. interestingly, amplification of the met gene (> 10 copies per cell) is present only in 3 of 23 (13%) tumor tissues. hgf overexpression is detected in 45% of hnsccs, suggesting that hgf functions predominantly in a paracrine manner to drive met signaling in these cancers. moreover, high levels of hgf are detected in hnscc patient plasma samples supporting the idea that ligand availability is not a limiting factor for met activation. mutations in the met ligand-binding domain (t230m/e168d), transmembrane or jm domain (r988c, t1010i) and the tyrosine kinase domain (t1275i, v14333i) have also been identified in hnscc tumor samples, although their relative contribution to hnscc progression remains to be determined. two somatic met mutations have been detected in hnscc that result in constitutively active receptor signaling that confers an invasive phenotype when ectopically expressed in cell lines. the y1230c mutation confers anchorage-independent growth and an invasive phenotype in transfected cells, whereas the y1235d met mutation stimulates epithelial cells to invade reconstituted basement membrane in the absence of hgf. in the case of the mety1235d mutation, genomic analyses of hnscc patient samples detected the presence of this mutant allele in 50% of metastatic tumors versus 26% in primary tumors, raising the possibility that this could be a critical genetic lesion for the acquisition of a metastatic phenotype. alternatively, increased met signaling could afford hnscc a selective advantage for growth and/or survival in metastatic sites, such as the lymph node and lung. indeed several studies indicate that met overexpression correlates highly with lymph node metastasis, pathologic stage, and disease reoccurrence [8891]. moreover, patient survival was significantly reduced in biopsy samples with positive met expression relative to negative met expression, suggesting the association of met with hnscc disease progression. consistent with these findings, treatment with the tki pf-2341066 caused a significant reduction in tumor growth, a high level of apoptosis and cellular debris within the tumor using a xenograft animal model for hnscc. selective inhibitors of met/hgf signaling include humanized monoclonal antibodies for hgf and met, and small-molecule tyrosine kinase inhibitors directed against met (figure 1). although their efficacy for treating a variety of solid tumors is increasingly recognized, we await results of preclinical and clinical trials for head and neck cancer that are ongoing. the humanized antibody amg 102 shows high potency towards the mature and processed form of hgf with no detected effects on proteolytic activation of prohgf. amg 102 interferes with met signaling, by competing with hgf for binding to the chain of the met receptor. in phase i clinical studies in patients with advanced solid tumors, 70% of patients had a best response in terms of achieving stable disease [93, 94]. importantly, no antiamg 102 antibodies were detected and circulating hgf levels were dose dependent. another promising clinical therapeutic is the one-armed 5d5 humanized antibody (oa5d5/metmab) directed against met. metmab binds met with high affinity, preventing hgf binding, met phosphorylation, receptor internalization and downstream signaling events and has been shown to inhibit tumor growth in animal models by more than 95% [95, 96]. metmab is currently in phase i/ii human clinical trials in comparison with erlotinib in patients with nsclc (http://www.clinicaltrials.gov/, identifier nct00854308). future clinical trials will be required to determine the suitability of amg102 and metmab as either single agents or combinatorial therapeutics for treating hnscc patients. foretinib (formerly xl880) is a tki whose primary targets include met and vegf, and to a lesser extent the platelet-derived growth factor (pdgf) receptor, ron, kit and tie2 rtks. foretinib recently completed phase ii clinical trials in head and neck patients (http://www.clinicaltrials.gov/, identifier nct00725764). interim results suggest that after 12 months, 12 of 18 patients had stable disease. a phase i dose-escalation study of the safety and pharmacokinetics of xl184 administered orally to patients with advanced malignancies (showed that, on average, patients survived for more than 3 months with several up to 6 months while on treatment) (reviewed in). due to encouraging data from this study, a randomized phase iii trial of xl184 in hnscc patients was initiated to investigate xl184 as a first-line treatment (compared with placebo) for survival benefit to patients with hnscc (http://www.clinicaltrials.gov/, identifier nct00704730). arq197 (arqule) is a nonatp-site competitive, selective small molecule inhibitor of the met intracellular region. although the mechanism of arq197 is presently unknown, the results of phase i trials suggest potential antiinvasive activity for this compound. overall, met, and hgf-targeted therapies have been well tolerated in clinical trials with negligible toxicities. however, it remains to be determined whether met is a better therapeutic target than hgf. clearly, in patients where met is activated by autocrine hgf secretion, both hgf and met targeted therapies may prove to be more efficacious treatment options. acquired resistance is likely the result of several mechanisms including (1) egfr mutations initially present as well as those acquired during therapy, (2) receptor independent activation of downstream signaling cascades, (3) cross-talk with other rtks and converging signaling pathways and (4) environmental factors including inflammatory agents and viral infection. resistance to cetuximab has been associated with the coexpression of the truncated egfr mutant, egfrviii with wild-type egfr. egfrviii is the result of an in frame deletion of exons 27 spanning the extracellular ligand-binding domain. the deletion results in a truncated egfr receptor that signals in a ligand-independent manner. egfrviii expression has been detected in 42% of hnscc patient samples, and closely correlates with increased hnscc cell proliferation in vitro and increased tumor growth using in vivo xenograft models. egfrviii preferentially activates the pi3k pathway instead of the ras/raf/mek pathway, which is activated by wild-type egfr. of particular interest to the therapeutic treatment of hnscc, egfrviii expression decreases the proliferative response of egfr expressing tumor cells to cetuximab treatment relative to vector control cells. in a recent study, egfrviii cells were shown to be resistant to the antiinvasive effects of cetuximab due to an increase in phosphorylation of stat3 rather than increased pi3k signaling. egf-induced expression of the stat3 target gene hif1 was abolished by cetuximab in hnscc cells expressing wild-type egfr under hypoxic conditions, but not in egfrviii-expressing hnscc cells [102, 103]. these data suggest a role for egfrviii in mediating hnscc resistance to cetuximab. despite egfrs critical role in the development of hnscc, clinical data indicate modest clinical benefits for locoregional control and survival of head and neck cancer patients treated with egfr-targeted therapies. hnscc patients resistant to cetuximab, often succumb to local tumor recurrence as well as regional and distant metastasis. the addition of cetuximab to radiation therapy was reported to show improved locoregional disease control, progression-free survival, and overall survival in patients with locally advanced hnscc. however the data revealed a disproportionate benefit of cetuximab with radiotherapy to oropharyngeal cancer patients when compared to patients treated with hyperfractionated radiotherapy. accumulating evidence suggests that human papilloma virus (hpv) 16 status (hpv+) is an important prognostic factor associated with a favorable outcome in a subset of head and neck cancers, including oropharyngeal and tonsilar cancers. hpv+ tumors tend to have unique genetic aberrations including decreased egfr expression, whereas increased igf-1r levels characteristic of hnscc appear to be independent of hpv status. clinically, hpv+ tumors are characterized by more favorable patient prognosis regarding disease-free survival as well as overall survival [104, 105], possibly as a result of increased genomic stability associated with global gene hypermethylation in hpv+ tumors. thus it will be interesting to determine whether hpv+ status explains some of the benefits derived from the addition of cetuximab to radiotherapy in this subset of hnscc patients. at present, there are few clinical indicators of which hnscc patients will most likely respond to egfr-targeted therapies. accordingly, strategies to optimize egfr-targeted therapy remain an active area of research. additional mechanisms that result in egfr activation include activating mutations in downstream signaling components or cross-talk between different rtk pathways. activating mutations in the pi3ka oncogene occurs in 10% of hnscc tumors whereas elevated levels of phosphorylated stat3 correlates with lymph node metastasis and poor patient prognosis [108110]. conversely, h-ras mutations are infrequent in hnscc cases (less than 5%), although a higher incidence has been detected in asian populations and correlates with areca nut chewing [111, 112]. met signaling has been shown to contribute to resistance in cell lines derived from multiple tumor types including breast, gastric and lung. in one key study, nsclc with activating mutations in the egfr acquire resistance to the tki gefitinib and erlotinib, by amplification of the met gene to maintain akt and her3 signaling. these studies underscore the role of cross-talk between rtks to preferentially signal through the pi3k-akt survival pathway as a mechanism for acquired drug resistance. the relevance of met as a mechanism for escape from egfr-targeted therapy in head and neck cancers remains to be determined. hypoxia results in the transcriptional upregulation of met gene expression via hif1 in a number of tumors including head and neck, often downstream of egfr signaling. in normoxia, hydroxylation of 2 prolines in hif1 enables its binding to the von hippel-lindau tumor suppressor protein (pvhl) linking hif1 to a ubiquitin ligase complex. during hypoxia, minimal or no hydroxylation occurs enabling hif1 to avoid proteasomal degradation and dimerize to other hif family members such as hif1 and coactivators, to form an active transcriptional hif complex on the hypoxia response element (hre) of target genes such as met. the ubiquitin ligase catalyzes polyubiquitination of hif1 targeting it for proteasomal degradation. under hypoxic conditions, increased met signaling directs the invasive growth program, enabling cells to invade more oxygenated tissues. since met has been reported to promote invasive and angiogenic effects in the tumor microenvironment, the use of hgf/met inhibitors may afford a means of impairing tissue colonization as well as tumor vascularization in head and neck cancer patients. studies on other solid tumor types, most notably glioblastoma, indicate a role for igf-1r upregulation in resistance to egfr-targeted therapies. igf-1r mediates resistance to anti-egfr therapy in primary glioblastoma through the continued activation of the pi3k/akt survival pathway. the apparent cooperation between igf-1r and egfr in promoting hnscc pathogenesis as well as resistance to egfr-targeted therapy, suggests an advantage to cotargeting these signaling axes for the treatment of head and neck cancers. to date, the effect of blocking oncogenic igf-1r and egfr signaling have been studied more extensively in breast cancer lines. treatment with gefitinib and ag1024, a tki for igf-1r reduced cell proliferation when used as single agents and showed an additive effect when used in combination [76, 77]. targeting igf-1r and egfr signaling is currently under evaluation in hormone-sensitive metastatic breast cancer using the igf-1r inhibitor osi-906 and the egfr tki erlotinib, although results are not yet available (http://www.clinicaltrials.gov/, identifier nct01205685). similarly, an exploratory study to assess the modulation of biomarkers in hnscc patients treated preoperatively with cetuximab and/or imc-a12, a humanized antiigf-1r monoclonal antibody is currently underway (http://www.clinicaltrials.gov/, identifier nct00617734). these studies will be critical for evaluating whether the use of antiigf-1r and egfr-targeted treatments will be more effective than single-agent modalities for treating patients with hnscc. targeted therapies that block egfr, met, and igf-1r signaling in head and neck cancers continue to show promising results in preclinical studies and clinical trials. however, it is difficult to predict which patients are most likely to benefit from these therapeutics and potential side effects during long-term in vivo use. given the interplay between these rtk signaling pathways and the mediocre results obtained with monotherapy regimens thus far, clinical trials will be required to determine how egfr-, met-, and igf-1r-targeted therapies can be used in combination in order to definitively abrogate their common downstream oncogenic signaling networks. although gaps in our knowledge concerning the role of met and igf-1r in head and neck tumorigenesis, as well as acquired resistance to antiegfr therapies remain to be addressed, efforts to translate current information towards clinical applications continue to be impressive.

molecular therapeutics for treating epidermal growth factor receptor-(egfr-) expressing cancers are a specific method for treating cancers compared to general cell loss with standard cytotoxic therapeutics. however, the finding that resistance to such therapy is common in clinical trials now dampens the initial enthusiasm over this targeted treatment. yet an improved molecular understanding of other receptor tyrosine kinases known to be active in cancer has revealed a rich network of cross-talk between receptor pathways with a key finding of common downstream signaling pathways. such cross talk may represent a key mechanism for resistance to egfr-directed therapy. here we review the interplay between egfr and met and the type 1 insulin-like growth factor receptor (igf-1r) tyrosine kinases, as well as their contribution to anti-egfr therapeutic resistance in the context of squamous cell cancer of the head and neck, a tumor known to be primarily driven by egfr-related oncogenic signals.

PMC3135278

pubmed-8

global and regional left ventricular (lv) functions are well-known indicators of cardiac disease. quantitative values of ventricular volumes and of myocardial mass are independent predictors of morbidity and mortality in patients with coronary artery disease. classically, echo has been used to evaluate lv volume and function because it is relatively inexpensive and noninvasive. however, a component of operator dependence and poor contrast between blood and myocardium are considerable limitations of this technique. cardiac magnetic resonance imaging (mri) is considered the clinical gold standard for lv function assessment, but it is expensive, of limited availability, and can not be performed in patients with implanted pacemakers or defibrillators. in recent years, multidetector ct (mdct) has gained acceptance as a promising imaging method for coronary arteries. mdct acquired in a single breath-hold with retrospective electrocardiogram (ecg) gating can cover the entire heart with 1-mm slice thickness with a temporal resolution of 125-250 ms. when performed for coronary imaging, this method provides excellent opportunity to create, image reformation in any desired plane, including anatomically optimized long axis, short axis, or four-chamber views. diastolic and systolic images can easily be produced from the same data set with a retrospective ecg-gating technique, thus obtaining lv end-diastolic and end-systolic volumes (edvs and esvs). mdct has a potential of being utilized as tool for the combined assessment of the coronary anatomy and lv function. in addition, ventricular wall motion can be assessed visually by the use of cine loop displays of multiple cardiac phases. recently we observed increasing tendency for utilizing low radiation dose, prospective gating for coronary angiography, thus limiting possibility of volumetric assessment of ventricular function. however, a small number of patients may need a retrospective gating, thus providing possibility of reconstructions in various phases of cardiac activity. according to published reports, measurements for various lv functional parameters with mdct were well-correlated and agree with measurements obtained with mri, two-dimensional transthoracic echocardiography (2d-tte), and ecg-gated single photon emission ct (spect). experience with 64-slice mdct for cardiac function assessment remains limited by small patient numbers and the inclusion of homogeneous patient populations. the purpose of this study was to assess lv ejection fraction (lvef) using 64-slice as a byproduct of mdct coronary examination and to compare efficacy of technique with 2d-tte in a heterogeneous patient population. also, review the role of mdct lv function with a relation to evolution in the technology of coronary mdct imaging. study included 113 patients referred for 64-slice mdct coronary angiography for evaluation of coronary artery disease. all patients were scanned on 64 slice ge-helical ct (ge high speed advantage) scanner and had an echo done within 1 week of the ct scan. this prospective study was approved by the institutional review board and written informed consent was obtained from all patients. patients with absolute contraindication to contrast or radiation were excluded from the study. in patients with relative contraindications such as atopy, asthma, and renal failure scan was performed if the benefit of examination outweighed the risk in such patients. patients with arrhythmias and ectopic heart beats were excluded as stable heart rate is required for ct coronary angiogram. apart from the routine contraindications, patients with pacemaker and ventricular septal defect were excluded from the study as successful segmentation of the lv blood pool is not possible in these patients due to artifacts and incorrect segmentation by software. all patients undergoing ct coronary angiogram, who had heart rate of more than 60 bpm, were premedicated with 50-200 mg oral b adrenergic blocking agent: metoprolol, 1-h prior to the study. a 60-120 mg calcium channel blocker: diltiazem, cta was performed with contrast volume of 1.2 ml/kg body weight of iohexol 350. the intravenous contrast agent was followed by 30 ml of saline chaser bolus at the same injection rate. scan parameters were 0.35 s rotation time, 120 kv tube voltage, 600-800 effective ma, 0.6 mm collimation, and a helical pitch of 0.22:1. the image acquisition was caudocranial for post-coronary artery bypass grafting (cabg) patients and craniocaudal for the rest of the patients. no complications encountered in any of the patients. retrospectively, ecg-correlated image reconstruction was performed. the reconstruction was performed with the reconstruction window starting at 10% of r-r interval and up to 90% r-r interval with increment of 10%. this included data sets reconstructed in systole, if diastolic data sets showed motion artifact. diastolic and systolic axial image sets were then transferred to the scanner's workstation-ge advanced workstation advantage windows 4.4 p. image data were evaluated with a prototype version of a commercially available program (auto ejection fraction, circulation; ge medical solutions) that performs a fully automatic segmentation of the blood volume in the lv by defining the mitral valve plane and the lv. the software uses this mitral valve plane as an upper boundary for the segmentation of the lv. the software identifies the hinges of the mitral and aortic valve leaflets closest to the ventricle wall and selects these as defining points for the plane [figure 1a]. all ct scans were analyzed according to this method, which allowed for optimal segmentation of the lv. papillary muscles were automatically excluded from the blood pool, which allows for precise determination of blood volume in the lv. multiplanar reformats are then performed by the software in long and short axes of left ventricle. the long axis image is obtained parallel to the interventricular septum connecting the lv apex and the middle level of mitral valve. the short axis images are obtained parallel to the plane of mitral valve [figure 1b]. lv=left ventricular (b) line perpendicular to long axis of left ventricle (parallel to mitral valve) plane for short axis images once the region of interest is finalized, the edv and esv are measured by this software using simpson's method by summing the endocardial area of all lv ed and es short-axis slices multiplied by the slice thickness [figure 2a and b]. the stroke volume (sv) and ef were automatically calculated from these values and displayed by the software [figure 3a d]. (a) lv short axis image in diastole (b) short axis image in systole 3d display of volumetric data in different patients. (a) patient 1: left ventricular ejection fraction by ct 58.4% (echo 58%). (b) patient 2: left ventricular ejection fraction by ct 55.7% (echo 53%). (c) patient 3: left ventricular ejection fraction by ct 60.0% (echo 60%). (d) patient 4: left ventricular ejection fraction by ct 38.3% (echo 35%). 3d=three-dimensional, echo=echocardiography, ct=computed tomography two-dimensional echo examination was performed either 1 week before or after coronary cta in either of two ultrasound units, an acuson sequoia (siemens medical systems usa, mountain view, ca) or a ge vivid 3 (ge healthcare, milwaukee, wi). images were obtained using a 3.5 mhz transducer and images were acquired in standard apical and parasternal two- and four-chamber views. the chamber and wall dimensions were measured using standard recommendations for chamber quantification in consensus. results on continuous measurements are presented on mean sd (min max) and results on categorical measurements are presented in number (%). the mean standard deviation (sd) lvef calculated by clinical echo similarly mean sd of lvef calculated by fully automated software based on cta data was obtained [table 2]. agreement for lvef was determined by the use of pearson's regression analysis [figure 4] and calculating correlation coefficient (r). bland-altman analysis [figure 5] was used to compare the lvef measured with mdct and that with 2d-tte. mountain plot [figure 6] was used to see the relationship between two groups. echo-ejection fraction (ef) mean standard deviation (sd): 58.67 4.53, echo=echocardiography ct-ejection fraction (ef) mean standard deviation (sd): 58.93 5.43, ct=computed tomography pearson regression analysis between echo-ef and ct-ef. r=pearson correlation coefficient, p=p-value, ef=ejection fraction bland and altman showing the correlation of echo-ef and ct-ef sd=standard deviation mountain plot showing the correlation of echo-ef and ct-ef. in our study, 86 (76.1%) were males and 27 (23.9%) were females. the mean age of the patients was 51.19 10.10 years and majority of subjects belonged to age group between 51 and 60 years. 28.3% were between 41 and 50 years, 15.9% subjects were between 61 and 70 years, and 12.4% subjects between 31 and 40 years. most of the patients had at least one symptom, the commonest being chest pain in 88 (77.9%) cases. the most common coronary risk factor association was hypertension, accounting for 77.9% of the cases. the mean heart rate of the patients at the time of scan was 61.5 8.6 bpm with maximum patients having a heart rate range of 61-70 bpm. the mean lvef calculated by clinical echo was 58.67 4.53% with maximum number of patients having an ef range of 56-60%. the mean lvef calculated by fully automated software based on cta data was 58.93 5.43% with maximum number of patients having an ef range of 61-65%. in our study using the fully automated software, the pearson's regression analysis showed a good interstudy correlation, with a correlation coefficient of 0.503 (p<0.001). bland-altman analysis showed a trend towards mdct resulting in slightly higher values for lvef when compared with echo; however, this observation was not statistically significant. the mountain plot analysis reinforced that ef measured by ct correlated well with that measured by echo. mdct coronary angiography has emerged as a valuable technique for the evaluation of coronary artery disease in patients with low to intermediate pretest probability of ischemic heart disease. utilizing analytic software, gated volumetric ct data can be processed to provide quantitative functional analysis of the lv in patients with coronary artery disease. we were able to obtain satisfactory artifact free datasets from 113 consecutive subjects who underwent coronary cta. all of our patients were either known cases of coronary artery disease or had suggestive clinical symptoms. patients with pacemaker, ventricular septal defect were excluded from the study as successful segmentation of the lv blood pool was not possible. achieving a stable heart rate for the examination was variable component of the examination. however, no examination had to be postponed or cancelled due to this limitation. our study using automated software showed a good interstudy correlation, with a correlation coefficient of 0.503 (p<0.001). the bland-altman plot revealed a slight mean difference between ef measurements on ct and echo with most differences falling within two sds of the mean. hence, we found that software is user-friendly and capable of providing good reproducibility for ef measurements in comparison with echo. in a previous study by krishnam et al., similar results were recorded, though the number of subjects was small. in a study by cury et al., a trend of mdct slightly underestimating lvef compared with tte was observed. we observed a trend towards mdct resulting in slightly higher values for lvef when compared with echo, contrary to expected mild reduction in beta blocked patients. trend however was not statistically significant, could be due to recognized limitation of evaluation technique leading to over or underestimation. mean difference in ef measurements between cta and echo is small; although standard deviation of the mean difference is quite high, leading to wide limits of agreement. possibly observation results from the fact that the ef measurements from echo were obtained in a clinical setting, on visual estimation and calculation of ef using simpson's method based on geometrical assumptions. our observations are in accordance with the previous studies of 64-slice coronary ct, confirming that lvef estimation is feasible with the mdct data and may be regarded as a useful clinical index, correlating with results of echo. there are studies with semiautomated software for quantitative functional analysis of lv with user defined mitral valve plane and an arbitrary point within the lv, with the option to expand or reduce the area of segmentation. many earlier studies have compared the use of 4-, 8-, and 16-slice ct scanners for evaluation of lv volumes. larger detector configuration in 64-slice ct scanner, has the advantage of being faster, capable of smaller slice thickness and higher temporal resolution. relatively higher radiation dose results from the protocol optimized for thin slice high-resolution imaging of the coronary arteries. the ecg-dependent tube current modulation is currently the most effective tool for dose reduction and may reduce patient dose by up to 50%. it is important to note that two points of the cardiac cycle (end-systole and end-diastole) with modulation of tube current were not used in our study because ecg-gated dose modulation was only applicable to 50-90% of the rr interval on ecg. if the aim is to evaluate coronary arteries only, it is recommended to use an ecg-dependent dose modulation technique or newer prospective gated techniques. new developments in mdct technology is allowing examination of patients with higher heart rates and reducing the dose of beta-blockers. presently, mdct examination is possible which contains all the phases with a considerably lower dose of the order of 2-3.3 msv. the software identifies the lv blood pool based on hounsfield unit values and continuity of adjacent voxels. in patients with a ventricular septal defect, there is contrast opacification in both ventricles and a bridge of contrast through the septal defect. therefore, the software identifies the right and lv blood pools as a single chamber, resulting in incorrect segmentation and thus inaccurate assessment of lv functional parameters. in patient with pacemakers, software identifies a pacemaker wire as high-density contrast and segments the pacing wire as part of the ventricle, resulting in failed segmentation. the version of the software used for this study it was not able to segment the myocardium in order to quantify the lv myocardial mass, which may be important in certain cardiac diseases such as hypertrophic cardiomyopathy. it is important to note that cardiac mri is considered as a gold standard for lv function assessment. cardiac mri (cmri) provides excellent temporal and spatial resolution, image acquisition in any desired plane, and a high degree of accuracy and reproducibility. concerning quantitative measurements cine mri technique is potentially the most comprehensive cardiac imaging modality available because of its excellent contrast between blood-filled ventricles and the surrounding myocardium. yamamuro et al., have shown high linear correlation between ef measurements on ct and on mri. the temporal resolution of many available mdct is still considerably lower than that of echo and cine mri. this lower temporal resolution can make evaluation of isovolumetric ed and es phases of the cardiac cycle, and thus ef, less precise. there is considerable improvement in temporal resolution of mdct with improvement in gantry rotation, multiple and partial segment processing techniques. tr of 80-250 ms has been achieved in state of the art units. though it is short of fluoroscopic resolution, currently available options are more than sufficient for motion free systolic and diastolic cardiac imaging with heart rate below 100/min. additionally, the use of a b-blocker to reduce the heart rate to less than 65 bpm can influence the functional parameters that are to be measured. beta blocker do influence the lvef, lead to underestimation. overestimation or underestimation of the lv volume has been reported because of the different criteria for selecting the endocardial boundary or the inclusion/exclusion of papillary muscle. ideally estimation of the real-ef from all 20 phases is more precise; however, this significantly increases effort and processing time. effect of beta-blocker has to be factored in interpretation of ef by mdct. introduction of new ct imaging methods, including dual source ct in clinical practice will overcome these two problems significantly owing to its two-fold increase in temporal resolution. also, newer options in mdct technology may partially obviate need for use of beta blockers. functional parameters derived from 2d-tte are compared with ct derived 3d volumetric data, which are not strictly comparable. our study design did not allow realistic comparison of mdct and echo lv volume data. it would be interesting to compare the same for assessment of accuracy of respective data. with evolution of speckle-tracking 3d echography and new low radiation, high tr scanning such studies are possible. the delay time between ct and echo and premedication with -blockers could have changed myocardial contraction and lv volumes as measured with the two methods. in the present set up, the number of patients referred to coronary angiography who will have volumetric data will be significantly smaller, limiting the application of this utility to a smaller offset of patients. the radiation issue will certainly be an important consideration to use this technique in the larger group of patients. emerging new applications of echography in the form of 2d and 3d speckle-tracking echo certainly will have a greater role to play in the future noninvasive assessment of the myocardial function. in conclusion our study confirms useful complementary functional information in coronary cta datasets, using fully automated analysis software for rapid assessment of lvef. it is irrational to utilize mdct alone to assess lv function in clinical patients, given the radiation exposure involved. however, additional clinically useful information from a clinically indicated coronary ct examination with a lowest possible radiation dose is invaluable in patients known or suspected of ischemic heart disease. validating consistency of results with mri will further lend support to the use of mdct derived results. going forward with changing trends in ccta imaging, it is conceivable that number of patients undergoing cta with retrospective gating will substantially be reduced, thus limiting the functionality to a small group of patients.

background: coronary computed tomography angiography (ccta) is a frequently performed examination for coronary artery disease. when performed with retrospective gating, there is an opportunity to derive functional parameters of left ventricle utilizing automated software. complementary information, if validated with established standards, will enhance the total value of study. objective:study evaluates the usefulness of fully automated software for the assessment of left ventricular ejection fraction (lvef) using 64-slice ccta data and to correlate ct results with echocardiography (echo). role of ct derived lv function is reviewed in the light of emerging technologies and recent developments in multidetector ct (mdct). materials and methods: a total of 113 patients referred for mdct ccta for evaluation of coronary artery disease. all patients were scanned on 64 slice ge-helical ct scanner and had an echo done within 1 week of the ct scan. retrospectively electrocardiogram (ecg)-correlated image reconstruction was performed with the reconstruction at 10% r-r interval increment. axial image sets were analyzed with advanced workstation using a program-auto ejection fraction, circulation: ge medical solutions. results:the mean lvef calculated by clinical echo was 58.6 4.5% and by fully automated software based on cta data was 58.9 5.4%. the pearson's regression analysis showed a large correlation, with a correlation coefficient of 0.503 (p<0.001). bland-altman analysis showed a trend towards mdct resulting in slightly higher values for lvef when compared with echo. conclusion:the fully automated software is simple, reliable, and user-friendly, and can provide rapid assessment of lv functional parameters with good reproducibility. despite of good correlation, fewer patients are likely to benefit, in future, from this function due to smaller number of patients undergoing ccta with retrospective gating.

PMC5353405

pubmed-9

moraxella catarrhalis is a gram-negative, aerobic diplococcus human mucosal pathogen which causes middle ear infections in infants and children [13], and it is one of the three major causes of otitis media along with streptococcus pneumonia and haemophilus influenzae. although moraxella catarrhalis is frequently found as a commensal of the upper respiratory tract, recently it has emerged as a genuine pathogen and is now considered an important cause of upper respiratory tract infections in healthy children and elderly people, lower respiratory tract infections in adults with chronic obstructive pulmonary disease [1, 5], and hospital-acquired pneumonia. amikacin, cefixime, fosfomycin, cefuroxime, cotrimoxazole, doxycycline, and erythromycin resistant strains of moraxella catarrhalis were isolated and the widespread production of a -1actamase enzyme renders the bacterium resistant to the penicillin [79]. this has led to the search for new and effective therapeutic alternatives among natural compounds. plants remain an important source of diverse chemical entities which have been used as drugs or provide scaffolds from which new drugs have been derived. the selection of a suitable candidate species for investigations can be done on the basis of long-term use by humans. this approach is based on an assumption that the active compounds isolated from such plants are likely to be safer than those derived from plant species with no history of human use. aristolochia is an important genus in the family of aristolochiaceae and is widespread across tropical asia, africa, and south america. it is used in traditional medicine as a gastric stimulant and in the treatment of cancer, lung inflammation, dysentery, and snakebites. it is also used in the treatment of tumors and malaria and for fevers, but its usage as an antimalarial is not recommended in its crude form. aristolochia bracteolata showed a definite positive effect on wound healing, with significant increase in the level of powerful antioxidant enzymes. the whole plant was used as a purgative, antipyretic, and anti-inflammatory. organic solvent extracts of the plant showed antibacterial activities while the water extract showed antifungal activity. although aristolochia has been used for thousands of years in many cultures for many indications due to its various pharmacological activities, it was later discovered that consuming these plants can certainly be dangerous. the genus of aristolochia contains a naturally carcinogenic compound aa which has been shown to be the cause of so-called chinese herb nephropathy or aa nephropathy [19, 20], and mutations in the cells of people who consume it, causes more mutations than two of the best-known environmental carcinogens: tobacco smoke and uv light [21, 22]. there are many cases of nephropathy reported in the literature caused by the systemic and long term application of chinese snakeroot (aristolochia fangchi); this highlighted the risk of using preparations which contain aristolochic acids. although aristolochia is being known in many countries that is containing a toxic compound aa, but this has not stopped it from being a popular herbal remedy for thousands of years. it is still extensively used in india and in traditional chinese medicine for slimming, menstrual symptoms, and rheumatism. it is also widespread used in sudan and other african countries as one of the most effective herbal remedies for infectious diseases. therefore, it was our objective to assess the potential antimicrobial activity of aristolochia bracteolata using a bioassay-guided fractionation, in order to produce pure compound that can act as the lead compound in developing new, safe, and effective drug to replace the use of the harmful crude plant material. sephadex lh-20 (pharmacia fine chemical co. ltd) was used for column chromatography. precoated silica gel plates (merck, kieselgel 60 f254, 0.25 mm) and precoated rp-18 f254s plates (merck) were used for thin-layer chromatography (tlc) analysis. high resolution fab-ms and esi-ms were recorded on jeol jms700n and jms-100td, respectively. h- and c-nmr, h-h cosy, noesy, hsqc, and hmbc spectra were recorded with a unity plus 500 spectrometer (varian inc., u.s.a.) operating at 500 mhz for h and 125 mhz for c, respectively. h-nmr chemicals shifts are expressed in values referring to the solvent peak h 2.49 for dmso and coupling constants are expressed in hz. c-nmr chemical shifts are expressed in values referring to the solvent peak c 39.5 for dmso. piperonylic acid was purchased from commercial sources (tci) and used without further purification. the plant material (whole) was collected in the period from (october to december 2012) from khartoum state in sudan. standard strains: moraxella catarrhalis (gtc 01544), klebsiella pneumoniae (atcc 13883), escherichia coli (k12), salmonella typhimurium (atcc 14028), streptococcus pyogenes (atcc 19615), streptococcus agalactiae (atcc 13813), staphylococcus epidermidis (atcc 12228), neisseria lactamicus (atcc 23970), enterobacter cloacae, (atcc 23355), bacillus subtilis (atcc 6633), staphylococcus aureus (209p), and pseudomonas aeruginosa (ifo 3445).clinical strains: moraxella catarrhalis, bacillus cereus, aeromonas hydrophila, salmonella typhi, vibrio cholerae, and yersinia enterocolitica. standard strains: moraxella catarrhalis (gtc 01544), klebsiella pneumoniae (atcc 13883), escherichia coli (k12), salmonella typhimurium (atcc 14028), streptococcus pyogenes (atcc 19615), streptococcus agalactiae (atcc 13813), staphylococcus epidermidis (atcc 12228), neisseria lactamicus (atcc 23970), enterobacter cloacae, (atcc 23355), bacillus subtilis (atcc 6633), staphylococcus aureus (209p), and pseudomonas aeruginosa (ifo 3445). clinical strains: moraxella catarrhalis, bacillus cereus, aeromonas hydrophila, salmonella typhi, vibrio cholerae, and yersinia enterocolitica. in addition to a sea urchin (anthocidaris crassispina) derived bacillus sp. which obtained from the laboratory in medical plants garden, nagasaki university.(c)fungal strains: the fungal strains used were aspergillus niger (nbrc 33023), penicillium crustosum (nbrc 33015), schizophyllum commune (nbrc 30749), trichophyton concentricum (nbrc 31068), and candida albicans (nbrc 10108). fungal strains: the fungal strains used were aspergillus niger (nbrc 33023), penicillium crustosum (nbrc 33015), schizophyllum commune (nbrc 30749), trichophyton concentricum (nbrc 31068), and candida albicans (nbrc 10108). the air-dried powdered whole plant (200 g) was exhaustively extracted with cold maceration method with sufficient quantity of 70% methanol for 7 days at room temperature. the methanolic extract was passed through whatman number 1 filter paper (whatman england) and the concentrated extract (40 g) was digested with 100 ml distilled water and successively partitioned with n-hexane (4 400 ml), chloroform (3 400 ml), ethyl acetate (5 400 ml), and n-butanol (2 400 ml). each fraction was concentrated under reduced pressure to a constant weight to give the corresponding n-hexane fraction (0.4 g), chloroform fraction (2 g), ethyl acetate fraction (0.7 g), n-butanol fraction (6 g), and aqueous fraction (30 g). the most active fraction against bacillus sp. and m. catarrhalis (chloroform fraction) was subjected to sephadex lh20 column chromatography to give three subfractions (a-c). fraction (b) was resubjected again to sephadex lh20 to afford very active and pure compound aa-1 (150 mg). to the solution of aa-1 (50 mg; 0.23 mmol) in dimethylformamide (dmf), 1 ml potassium carbonate was added (95 mg; 0.69 mmol). to the resulting suspension iodomethane was added (72 l; 1.15 mmol) and stirred for 8 hours. the reaction mixture was poured onto water (10 ml) and ethyl acetate (20 ml). the organic layer was washed with 1 m hcl (10 ml) three times and then with brine (10 ml) once. after removal of solvent under reduced pressure, the residue was purified by silica-gel chromatography (chloroform-methanol) to afford the titled ester (88%). h-nmr (400 mhz, cdcl3, tms, r.t.) (ppm): 3.88 (3h, s), 4.06 (3hs), 6.38 (2h, s), 7.11 (1h, d, j=7.8 hz), 7.72 (1h, dd, j=7.8 hz, 8.0 hz), 7.77 (1h, s), 8.70 (1h, d, j=8.0 hz), and 8.83 (1h, s). suspension of the tested bacteria (100 l of 10 cfu/ml) was spread onto solid media plates. the sterile paper discs (6 mm in diameter) which were impregnated with the plant extract (14 mg) and pure compound (10100 g) were placed aseptically over the bacterial culture on nutrient agar plates. after incubation at 37c for 24 hours, the zone of inhibition around the discs was measured by millimeter scale. sterile, blank paper discs impregnated with only sterile solvents served as negative control each time. the sterile paper discs (6 mm in diameter) which were impregnated with individual extract were placed on the inoculated plates. these plates were incubated for 2472 h at 2528c and the growth was evaluated visually by comparing a particular plate with the negative control plates (having only test fungi). the antifungal activity was evaluated by measuring the inhibition zone diameter (in millimeter) observed. the mic and mbc values were determined by broth dilution method in accordance with clsi methodology. bacterial strains were cultured for 24 h at 37c on nutrient broth and then suspended in sterile distilled water to give a final inoculum concentration of 1.5 1.0 10 cfu/ml. dilutions ranging from 1.56 to 400 g/ml of the compound were prepared in tubes including broth and dmso 10% (v/v), in addition to one negative control (broth+dmso 10% v/v+test microorganism) to ensure that the final concentration of dmso in the assays did not interfere with the bacterial growth and one sterility control (broth+dmso 10% v/v+test compound). a 100 l suspension of test microorganism was added to individual tubes and incubated at 37c for 24 h. the mic of the compound was defined as the lowest concentration that inhibited the visible bacterial growth and the mbc was defined as the lowest concentration that prevented the growth of the organism after subculture onto antibiotic-free plates. initial steps in new drug discovery involve identification of new chemical entities, which can be either sourced through chemical synthesis or can be isolated from natural products through biological activity guided fractionation. bioassay-guided fractionation of the identified plant may lead to standardized extract or isolated bioactive lead compounds as the new drug. the whole plant of aristolochia bracteolata was extracted successively with meoh and subjected to liquid-liquid fractionation with n-hexane, chloroform, ethyl acetate, and n-butanol. the resulting fractions were tested for antibacterial and antifungal activities. the crude extract and chloroform fraction were significantly active against sea urchin-derived bacillus sp. and both standard strain and clinical isolates of moraxella catarrhalis and were moderately active against s. aureus, b. subtilis, and ps. the n-hexane fraction had moderate activity against s. aureus and b. subtilis while ethyl acetate fraction showed moderate activity against ps. the crude extract failed to inhibit the growth of all test fungi in addition to the following bacterial strains: klebsiella pneumoniae, escherichia coli, salmonella typhimurium, streptococcus pyogenes, streptococcus agalactiae, staphylococcus epidermidis, neisseria lactamicus, enterobacter cloacae, bacillus cereus, aeromonas hydrophila, salmonella typhi, vibrio cholerae, and yersinia enterocolitica. the chloroform soluble fraction was therefore selected for further chromatographic separations and resulted in the isolation of known compound aa-1 (figure 1). aa-1 showed strong activity against moraxella catarrhalis (standard strain and clinical isolates) and sea urchin-derived bacillus sp. (table 2), with equal mic and mbc values of 25 and 50 g/ml. both the piperonylic acid moiety of aa-1 (figure 1) and aa-1-methyl ester showed no activity against bacteria (table 2), which suggests that the coexistence of phenanthrene ring and free carboxylic acid is essential for aa-1 antibacterial activity. bioguided fractionation of methanolic extract of aristolochia bracteolata led to isolation of aa-1 and its structure was elucidated by interpretation of its nmr and ms data and by comparison with those reported in the literature. electrospray ionization mass spectrometry (esims) showed pseudomolecular ion signal at m/z 364.03 [m+na] and high resolution fast atom bombardment mass spectrometry (hr-fabms) afforded m+1 ion signal at m/z 342.0620 which was corresponding to the molecular formula c17h12no7 (calculated for 342.06138). h nmr and c-nmr spectra were matched with those of aa-1 which were previously reported. two weak signals which did not show any correlation with proton in hsqc and hmbc spectroscopy were considered as quaternary carbons at positions 5 (124.3 ppm) and 6 (143.5 ppm), respectively. laboratories of the world have found literally thousands of phytochemicals which have inhibitory effects on all types of microorganisms in vitro. more of these compounds should be subjected to animal and human studies to determine their effectiveness in whole-organism systems, including in particular toxicity studies and an examination of their effects on beneficial normal microbiota. in spite of the fact that herbal remedy is a mixture of many chemicals in unknown doses and might result in unpleasant side effects, many people believe that treatments that are natural are somehow magically safe and effective. aristolochia is used in traditional medicine for the treatment of various diseases [13, 15], including those associated with bacteria. this study showed clearly that the excellent effect of aristolochia in treating such diseases is due to the toxic compound aa-1. although aa-1 is highly effective in killing m. catarrhalis, it is ineffective against the other microorganisms tested. this highlights the importance of m. catarrhalis in discovering the cellular target of aa-1 and the mechanism of aa-1 toxicity. the widespread use of aristolochia is not sufficient to ensure that it is effective or even that it is safe. therefore, hit-to-lead exploration is necessary to identify related compounds with low toxicity, low cost, and improved potency that can replace the use of the harmful crude plant material. it is impossible to ban the use of these remedies, especially in the rural areas in sudan and other african countries; therefore, we strongly recommend educating the public of the risks versus the benefits of aristolochia and gradually replacing them with either economical new drugs or standardized extracts and homogenous batches of other plant material with known levels of safe active constituents. using bioassay-guided fractionation technique, the present study directly linked the antibacterial activity of aristolochia bracteolata to the aa-1. although aa-1 had strong activity against m. catarrhalis, it had a narrow spectrum of activity than expected based on the activity of the crude extract from which it was isolated or from its traditional usage. this may be the result of synergism between different compounds in the complex extracts or may be due to placebo effect.

a bioassay-guided fractionation of methanol extract of aristolochia bracteolata whole plant was carried out in order to evaluate its antimicrobial activity and to identify the active compounds in this extract. antibacterial and antifungal activities of methanol extract against gram-positive, gram-negative, and fungal strains were investigated by the agar disk diffusion method. among the strains tested, moraxella catarrhalis and sea urchin-derived bacillus sp. showed the highest sensitivity towards the methanol extract and hence they are used as test organisms for the bioassay-guided fractionation. from this extract, aristolochic acid 1 (aa-1) has been isolated and has showed the greatest antibacterial activity against both standard strain and clinical isolates of moraxella catarrhalis with equal minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) values of 25 and 50 g/ml. modification of the aa-1 to aa-1 methyl ester completely abolished the antibacterial activity of the compound and the piperonylic acid moiety of aa-1 which suggested that the coexistence of phenanthrene ring and free carboxylic acid is essential for aa-1 antibacterial activity.

PMC4745564

pubmed-10

anti-neutrophil cytoplasmic antibodies (ancas) correlate well with a wide spectrum of vasculitis manifestations, including wegener's granulomatosis, microscopic polyangiitis, and churg-strauss syndrome, all of which are commonly referred to as anca-associated vasculitis (1). several infections, particularly infective endocarditis, have been reported to show positive findings on anca tests; furthermore, their clinical features have been acknowledged to mimic anca-associated vasculitis, which may lead to a misdiagnosis and inappropriate treatment (2,3). however, to our knowledge, infected aortic aneurysms have not been reported to show positive findings on anca tests. we herein report a patient with an infected thoracic aortic aneurysm mimicking anti-proteinase 3-antineutrophil cytoplasmic antibody (pr3-anca)-associated vasculitis. vegetations in the descending aorta, which were detected using transesophageal echocardiography, contributed to the diagnosis in this case. a woman in her 60s was admitted to the nagoya city university hospital complaining of a fever, which had persisted for three weeks, along with a loss of appetite, proteinuria, hematuria, and renal dysfunction. her renal function test values had been in the normal range three months prior to the admission. she also had a history of orbital mucosa-associated lymphoid tissue lymphoma, which was in remission after treatment with only radiation therapy. a physical examination at the time of admission revealed a body temperature of 38.1, pulse rate 109 beats/min, and blood pressure 104/53 mmhg. the laboratory tests revealed a white blood cell count of 18,900/mm, hemoglobin 6.6 g/dl, blood urea nitrogen 30.8 mg/dl, serum creatinine 2.24 mg/dl, and c-reactive protein (crp) 9.44 mg/dl (table). the pr3-anca titer was 47.4 iu/ml (normal range<10 iu/ml), although her myeloperoxidase-antineutrophil cytoplasmic antibody (mpo-anca) titer was not elevated. a urinalysis gave results of 2+for protein and 3+for occult blood in a dipstick examination, and a microscopic examination detected more than 100 red blood cells per high-power field. alt: alanine aminotransferase, ana: antinuclear antibodies, ast: aspartate transaminase, c3: compliment 3, c4: compliment 4, ch50: 50% hemolytic unit of complement, crp: c-reactive protein, dna: deoxyribonucleic acid, ldh: lactate dehydrogenase, mpo-anca: myeloperoxidase-antineutrophil cytoplasmic antibody, rf: rheumatic factor, pr3-anca: anti-proteinase 3-antineutrophil cytoplasmic antibody she was initially diagnosed with pr3-anca-associated vasculitis and was thereafter treated with methylprednisolone pulse therapy (500 mg/day) for three days (fig. however, streptococcus sanguis was serially detected in blood cultures; therefore, the administration of steroids was stopped. transesophageal echocardiography also revealed no vegetation in the heart; however, it showed many oscillating masses attached to the intima in the descending aorta (fig. 2). contrast-enhanced computed tomography (ct) revealed a new descending aortic aneurysm (fig. effects of antibiotics on fever and c-reactive protein during hospitalization. the patient finally became afebrile and has c-reactive protein levels within the normal range after receiving antibiotics. crp: c-reactive protein, dap: daptomycin, pcg: penicillin g, taz/pipc: tazobactam/piperacillin, tx: therapy transesophageal echocardiographic images of the descending aorta, with sectioning planes at 90 (a) and 0 (b). many oscillating masses attached to the intima were seen in the lumen of the descending aorta. contrast-enhanced computed tomographic images in the axial (a) and frontal (b) sections. these images show a descending aortic pseudoaneurysm and a contrast defect (arrow) beside the aneurysm that is compatible with echo-documented vegetation. based on the results of microbiological testing, antibiotic treatment with intravenous penicillin g (24,000,000 unit/day) was started, after which she became afebrile. we added intravenous daptomycin (300 mg every 24 hours) and tazobactam/piperacillin (2.25 g every 6 hours) to the penicillin g because recurrent fever was observed two weeks later. this additional antibiotic treatment made her afebrile again, and her renal function was recovered (fig., she developed a janeway lesion, which was confirmed by the pathological finding of a skin biopsy specimen (fig. 4). transesophageal echocardiography finally demonstrated that the vegetation had disappeared after the antibiotic treatment (fig. 5), but contrast-enhanced ct revealed that the aneurysm remained unchanged in form. a nontender hemorrhagic macular on the sole of the foot (a). photomicrograph of the macular lesion shows microembolization with fibrin and infiltration of neutrophilic cells hematoxylin and eosin staining (b, c). a transesophageal echocardiographic image of the descending aorta after the antibiotic treatment, with the sectioning plane at 90. many oscillating masses have disappeared. on the 54th hospital day, she underwent endovascular aortic repair (evar) of the aneurysm (fig. 6) after confirmation that her crp level was in the normal range and once blood cultures were consistently negative. one year after the procedure, she continues to take oral antibiotics; no complications related to stent-graft deployment or recurrent infections have been encountered. her pr3-anca titer has normalized. a contrast-enhanced computed tomographic image after endovascular stent graft repair. in this case, an infected aortic aneurysm exhibited elevated the patient's pr3-anca level, and its clinical course mimicked pr3-anca-associated vasculitis. the detection of ancas is highly specific for a diagnosis of anca-associated vasculitis (4); however, a number of infections can result in a positive anca test. there are several reports of infective endocarditis with the presence of ancas that mimic the clinical manifestations of an anca-associated vasculitis such as glomerulonephritis (2,3). in the present case, based on the finding of positive blood cultures, we suspected that the patient was experiencing not anca-associated vasculitis but infective endocarditis. however, no vegetations were detected in the cardiac chambers using transthoracic echocardiography or transesophageal echocardiography. instead, contrast-enhanced ct revealed a pseudoaneurysm in the descending aorta, which suggested that the patient had an infected aneurysm. her renal dysfunction and elevated pr3-anca levels normalized with clinical resolution of the infected aneurysm after the administration of appropriate antibiotic therapy and evar. the patient has remained free of any evidence of systemic vasculitis during follow-up. the presence of pr3-anca may have been falsely positive in the present patient. to our knowledge, this is the first report of an infected aortic aneurysm with a positive pr3-anca test and clinical features mimicking anca-associated vasculitis. the presence of vegetations in the heart is one of the most characteristic findings of infective endocarditis. these vegetations are composed of bacteria, platelets, and inflammatory cells interspersed with fibrin mesh, either on damaged endocardium, including native cardiac valves, or prosthetic valves (5). in the present case, because these masses disappeared after antibiotic treatment, they were considered to be vegetations. a pathological examination of the macular hemorrhages on the patient's sole indicated not a cholesterol embolization but rather a janeway lesion, which was the consequence of septic embolic events. we therefore suspected that these masses were not mobile plaques but bacterial vegetations. to our knowledge, few cases of infected native aortic aneurysm manifesting vegetations in the aneurysm have been reported (6-8). several mechanisms have been proposed regarding how ancas are formed during the course of infection, such as autoantigen complementarity, molecular mimicry, epigenetics, neutrophil extracellular traps, and microbial-sensing proteins called toll-like receptors (9). there may be a great degree of overlap in the clinical and laboratory manifestations between anca-associated vasculitis and anca-positive infected aortic aneurysm, potentially leading to a misdiagnosis. it is important to correctly diagnose these cases in order to avoid administering inappropriate therapy, such as immunosuppressive treatment, which might have life-threatening consequences in a patient with an infected aortic aneurysm. positive blood cultures and imaging findings, such as a pseudoaneurysm and/or vegetations in the aorta, suggest the presence of an infected aortic aneurysm. a combination of conventional surgical treatment with resection of the aneurysm, extensive local debridement, and revascularization by in situ reconstruction or extra-anatomic bypass may be the gold standard, but such a treatment strategy is accompanied by a high mortality and morbidity. in the present case, it was recently reported that evar of an infected aortic aneurysm is feasible and, for most patients, a durable treatment option (10). given that the continuous administration of antibiotics for the infected aneurysm should prevent a recurrence of local infection, we continued using antibiotics until the infection was controlled and carefully monitored the shape of the pseudoaneurysm on contrast-enhanced ct. we were ready to perform an emergency surgical correction at any point during this patient's treatment course. in conclusion, we herein reported an unusual case of infected descending aortic pseudoaneurysm with luminal pathognomonic oscillating vegetation and with serological findings and clinical features mimicking pr3-anca-associated vasculitis.

we herein report an unusual case of an infected descending aortic pseudoaneurysm with luminal pathognomonic oscillating vegetation with serological findings and clinical features mimicking anti-proteinase 3-antineutrophil cytoplasmic antibody-associated vasculitis. the positive blood cultures and imaging findings, including a pseudoaneurysm and vegetations in the aorta, suggested the presence of an infected aortic aneurysm. the patient was successfully treated with antibiotics and endovascular aortic repair. a precise diagnosis is crucial in order to avoid inappropriate therapy such as immunosuppressive treatment, which could result in life-threatening consequences in a patient with an infected aortic aneurysm.

PMC5216144

pubmed-11

neonatal diabetes mellitus (ndm) presenting within the first 6 months of life includes permanent neonatal diabetes mellitus (pndm), which require lifelong therapy, and transient neonatal diabetes mellitus (tndm) where the condition shows remission during infancy but relapses in adolescence. almost all cases of neonatal diabetes have monogenic etiology in contrast to the autoimmune diabetes presenting in children beyond 6 months of age. pndm is associated with defects in pancreatic beta cell development and function. activating mutations in the kcnj11 gene, encoding the subunit kir6.2, and abcc8 gene, encoding the sulfonylurea receptor 1 (sur1) of atp-sensitive potassium (katp) channel, which has a key role in insulin secretion in glucose metabolism, are the most common causes and account for approximately 40% of all cases of pndm12). mutations in the glucokinase (gck) and insulin (ins) genes have also been reported in patients with pndm. sulfonylureas close the katp channel by an atp-independent route, leading to increase insulin secretion4). therefore, in many patients with pndm with kir6.2 and sur1 mutations, insulin therapy can be replaced by oral sulfonylureas which offer more improvement in glycemic control and better quality of life35). we report a case of pndm caused by a novel p.h186d heterozygous mutation in the kcnj11 gene whose treatment was successfully transitioned from insulin to oral sulfonylurea. her parents were unrelated, and her family was healthy and had no history of diabetes. laboratory results at diagnosis were: blood glucose, 1,041 mg/dl; ph, 7.025; hco3, 5.1 mmol/l; pco2, 19.8 mmhg; sodium, 147 mmol/l; potassium, 5.6 mmol/l; chloride, 113 mmol/l; blood urea nitrogen, 31 mg/dl; creatinine, 1.2 mg/dl; urine ketone,++ +; glycosylated hemoglobin (hba1c), 7.7%; and c-peptide, 0.2 ng/ml. after recovery from diabetic ketoacidosis, she had injections of neutral protamine hagedorn (nph) insulin two times a day and regular insulin (ri) four times a day. her maximal daily insulin dose was at 1.5 u/kg. her insulin requirement was gradually decreased to 0.44 u/kg/day by 4 months of age. although there were several hypoglycemic events, insulin injection for glucose control after the infancy period could not be stopped. genetic study for neonatal diabetes using genomic dna extracted from peripheral lymphocyte was done at 4.5 years of age, and a novel heterozygous mutation of the kcnj11 gene located on chromosome 11p15.1 and encoding kir6.2, c.556c>g (p.his186asp), was found (fig. transition of treatment from insulin to sulfonylurea was attempted at the age of 4.8. before the trial, laboratory findings were as follows: hba1c, 6.9%; fasting glucose, 205 mg/dl; c-peptide, 0.84 ng/ml; 24-hour urine c-peptide, 2.2 g; negative for islet cell antibody; anti-insulin antibody; and anti-gad antibody. she was 103.2 cm in height (25th-50th percentile), 17 kg in weight (50th-75th percentile) and did not have any specific neurological deficit. oral sulfonylurea (glibenclamide) was initiated at a dose of 0.1 mg/kg/day divided into two equal doses. the glibenclamide dose was gradually increased to 0.4 mg/kg/day over 1 week and the insulin was stopped. oral glucose tolerance test (glucose, 1.75 g/kg) was done at 6 months and 1 year after completion of the sulfonylurea transition. insulinogenic index [insulin (30 min-0 min)/glucose (30 min-0 min)], a marker of beta cell function, became increased from 0.10 to 0.18, and acute c-peptide response [c-peptide (30 min-0 min)1,000/glucose (30 min-0 min)] became increased from 6.48 to 8.29 (table 1). the patient is now 7.5 years of age with a height of 121.4 cm (50th percentile) and weight of 23.4 kg (50th percentile). blood glucose was well controlled without episodes of hypoglycemia and the hba1c has been lower than during insulin injection (fig. ndm is a monogenic form of diabetes that presents within the first 6 months of life. heterozygous activating mutations in the kcnj11 and abcc8 gene encoding the subunits kir6.2 and sur1 of katp channel in pancreatic beta cells are the most common and account for nearly half of all cases of pndm12). mutations in the glucokinase (gck), insulin (ins) genes have also been reported in patients with pndm. the causes of rare syndromic pndm includes recessive mutations in several genes, such as pdx1, eif2ak3, gck, ptf1a, foxp3, neurog3, neurod1, rfx6, ier3ip1, hnf1b, glis3, pax6, slc19a2, slc2a2, and wfs167). in tndm, imprinted locus at chromosome 6q24 seen in more than half of cases8), and kcnj11 and abcc8 gene mutations are also found in some cases. glucose increases intracellular atp level and it induces the closure of katp channels that lead to insulin secretion by pancreatic beta cells. activating mutations in the kcnj11 or abcc8 gene of katp channel lead to katp channels remaining open despite the presence of glucose, thus, insulin secretion can not be increased1). on the contrary, loss of function mutations cause congenital hyperinsulinemia due to the closure of the katp channel and lead to increased insulin secretion. sulfonylureas (su) can close the katp channel and increase insulin secretion4). treatment response is not expected in patients with mutations in other genes, such as glucokinase gene, foxp3, and ipf1. therefore, molecular diagnosis in neonatal diabetes may help identify patients that are likely to respond to oral sulfonylureas. pearson et al.3) reported the responses to sulfonylurea according to mutation types in diabetic patients caused by kcnj11 mutation. there were no differences in blood insulin, c-peptide level, and insulin injection dose between the successful group and unsuccessful group. v59 m, r201c, f35v, h46y, r50q, g53r, r201l, e322k, y330s, f333i mutations also belong to the successful group. switching to su was unsuccessful in patients with q52r, i296l, and l164p mutations. in addition, failure of switching to su was more related with the presence of neurologic features (14% in successful group and 80% in unsuccessful group) and older age at initiation of su. median age was 6 years old (intraquartile range, 3-12 years) in the successful group and 18 years old (intraquartile range, 6-35 years) in the unsuccessful group3). the report about a mother and daughter carrying the same kcnj11 mutation showed that the daughter could be switched from insulin to su at age 8.5 years, but her mother had an imcomplete response9). the trial of su switching in a poorly controlled diabetic 19 years old patient with r201h mutation, a mutation type expected to respond successfully, failed10). these observations suggest that factors that can affect the success of switching to su include; mutation type, severity of mutation, and duration of the diabetes. a long-standing diabetes may lead to islet beta cell exhaustion resulting in nonresponse to su9). ndm caused by kcnj11 gene mutations is inherited in autosomal dominant pattern, but most of these cases result from new mutations without family history. the patient in this case has a novel mutation (p.h186d) in the kcnj11 gene and presented with a mild form of pndm. switching to su with an initial dose 0.4 mg/kg/day was successful at 4.8 years of age. oral glucose tolerance test in index patient showed improvement in insulin secretion during the treatment at 6 months and 12 months after the use of su. although she had hypoglycemia every 2 or 3 weeks and experienced a few episodes of hypoglycemia with mental change during insulin treatment, there was no more hypoglycemia after changing to su treatment. oral sulfonylurea therapy is both safe and better than insulin for metabolic control. reported side effects of oral su are transitory diarrhea and tooth discoloration in a few patients311). mean hba1c was 8.1% in patients with insulin injection and 6.4% in patients with su therapy312). chronic su treatment seems to develop no beta cell desensitisation in pndm patients with kcnj11 mutation13). early su therapy at disease onset can permit insulin hypersensitivity and maintained basal insulin secretion, then provide long-term remission in animal subset14). allowing for the potential beneficial effect on neurodevelopmental outcome and glycemic control, empiric tiral of su before genetic testing in neonatal diabetes patients can be considered15). in conclusion, as the mutation type, severity of mutation, and duration of the diabetes are the major factors affecting the success of switching to su, early genetic analysis and trial of sulfonylurea is important in the management of neonatal diabetes. further studies looking at the result of su treatment and long term follow in pndm patients are needed.

permanent neonatal diabetes mellitus refers to diabetes that occurs before the age of 6 months and persists through life. it is a rare disorder affecting one in 0.2-0.5 million live births. mutations in the gene kcnj11, encoding the subunit kir6.2, and abcc8, encoding sur1 of the atp-sensitive potassium (katp) channel, are the most common causes of permanent neonatal diabetes mellitus. sulfonylureas close the katp channel and increase insulin secretion. kcnj11 and abcc8 mutations have important therapeutic implications because sulfonylurea therapy can be effective in treating patients with mutations in the potassium channel subunits. the mutation type, the presence of neurological features, and the duration of diabetes are known to be the major factors affecting the treatment outcome after switching to sulfonylurea therapy. more than 30 mutations in the kcnj11 gene have been identified. here, we present our experience with a patient carrying a novel p.h186d heterozygous mutation in the kcnj11 gene who was successfully treated with oral sulfonylurea.

PMC4573445

pubmed-12

radiocontrast-induced nephropathy (rin) can lead to acute renal failure (arf), which may require dialysis therapy. arf increases treatment cost due to sepsis, hemorrhage, respiratory failure, and a long hospitalization.[13] rin is an important cause of hospital-acquired arf and is responsible for 12% of cases. renal medullary hypoxia and the direct toxic effects of iodinated contrast agents on renal tubules are possible mechanisms responsible for the pathophysiology of rin. identified specific risk factors for rin are current renal insufficiency, diabetes mellitus, and high contrast volume, dehydration, advanced age (> 70 years), congestive heart failure (chf), and nephrotoxic drug use (angiotensin converting enzyme inhibitor and nonsteroidal antiinflammatory drugs). patients at risk for radiocontrast nephropathy are recommended to use nonionic iso-osmolar or nonionic low osmolar contrast agents. increases in serum creatinine levels are useful for detecting rin. in the majority of patients, plasma creatinine levels rise within the first 2448 h after administering the radiocontrast agent, reach a peak within 35 days, and return to normal after 13 weeks. recent studies have reported that urine levels of il-18 (a pro-inflammatory cytokine), kidney injury molecule-1, and neutrophil gelatinase-associated lipocalin (ngal) levels are important for early detection of rin. some reports have shown that il-18 levels start to increase within 46 h and peak at 12 h in patients with acute renal injury. in this study, we aimed to compare the plasma creatinine levels with spot urine il-18 levels following radiocontrast administration. twenty patients (11 males and 9 females) underwent diagnostic and therapeutic contrast-enhanced examinations at the department of internal medicine from january 2009 to march 2009. the study was approved by the institute ethics committee and written consent was obtained from the selected patients based on a low mehran risk score (5). patient demographic characteristics a precontrast-enhanced examination of serum blood urea nitrogen (bun), creatinine, na, k, cl, ca, p, creatinine clearance was analyzed and they were repeated at 24, 48, and 72 h following contrast administration. spot urine il-18 levels were measured before and 6 and 24 h after radiocontrast administration with a human il-18 elisa kit (biosource invitrogen human il-18, california). intravenous iopromid (623 mg/ml, 1.5 ml/kg; ultravist 300), a three-way oral and rectal contrast material for abdominal ct scans, and 650 mg/ml meglumine diatrizoate (urovist, 100 ml) were used for every patient. glomerular filtration rate was calculated using the cockcroft 1 h before the procedure, 8.4% nahco3 plus 5% dextrose (3 ml/kg/h) with 1200 mg/day n-acetylcysteine was given to all patients prophylactically. after radiocontrast agent administration, the same prophylactic treatment was continued (1 ml/kg/h) for 6 h. during this time, hydration and urine output were followed by monitoring the intake and release of fluids. patients with no history of kidney disease, plasma creatinine values<1.2 mg/dl, gfr60 ml/min, nondiabetic, no urinary infection, and no decompensated heart failure were included. urinary il-18 levels were measured with a human il-18 elisa kit (biosource invitrogen human il-18, carlsbad, ca, usa). the statistical analysis was performed with the ncss pass 2007 and 2008 statistical software (kaysville, ut, usa). serum creatinine levels increased after radiocontrast administration compared with precontrast levels, although the result was not statistically significant [table 2]. a slight increase in creatinine levels occurred at 48 h after radiocontrast administration but they fell to precontrast values at 72 h. a slight increase in plasma creatinine levels at 24 and 48 h following radiocontrast administration was observed compared with precontrast values, but it was not statistically significant (p=0.052 and p=0.285, respectively) [table 2]. plasma creatinine levels in patients before and after radiocontrast agent administration compared with precontrast urine spot il-18 levels, postcontrast 6 and 24 h urinary levels of il-18 increased significantly (p=0.048 and p=0.028, respectively; table 3). a tendency for postcontrast 24-h urinary il-18 levels to increase was observed compared with 6 h, but the increase was not statistically significant (p=0.808; table 3). spot urine il-18 levels in patients before and after radiocontrast agent administration plasma creatinine levels and spot urine il-18 were weakly but positively correlated with those during the precontrast period, although this finding was not statistically significant (r=0.246, p=0.126). similarly, postcontrast 24-h plasma creatinine levels and spot urine il-18 levels were weakly but positively correlated, although this result was also not statistically significant (r=0.254, p=0.276). there was no difference between pre- and postcontrast values of serum blood urea nitrogen (bun), creatinine, na, k, cl, ca, p, and creatinine clearance. the most common definition of rin is plasma creatinine levels of 0.5 mg/dl or higher 72 h after contrast administration or 25% higher than the basal plasma creatinine level. plasma creatinine levels began to rise within 24 h in 80% of the patients with rin, peaking at 4872 h, and returning to baseline after 2 weeks. the first 24 h remains unclear in patients with acute renal injury, but il-18 levels start to increase within 46 h, peaking at 12 h. additionally, plasma creatinine is affected by age, body weight, total body volume, gender, race, drug use, muscle mass, and protein intake so researchers are looking for a diagnostic marker for rin that can be measured easily and is not affected by nonrenal factors. most studies related to these parameters include serum and urine cystatin c, serum and urine ngal, and urine il-18 in the analysis. as proinflammatory cytokine il-18 levels increase in urine, tubular inflammation, such as ischemia, reperfusion injury, allograft rejection, cisplatin toxicity, and endotoxemia occur. parikh et al., found that 72 patients with acute tubular necrosis and delayed graft reaction have significantly higher il-18 levels than other kidney diseases (urinary tract infection, chronic renal failure, nephrotic syndrome, or prerenal azotemia). reported that il-18 generally showed a low sensitivity but high specificity, respectively, for assessing an acute kidney injury diagnosis and risk classification. in our study, we examined spot urinary il-18 levels in comparison with plasma creatinine levels. a weak positive correlation was found between precontrast creatinine and urine il-18 levels, although it was not statistically significant. furthermore, we also found a weak positive correlation between postcontrast 24 h creatinine and urine il-18 levels, although this was not statistically significant either. a slight increase in plasma creatinine levels at 24 h and 48 h following radiocontrast administration was observed compared with precontrast values, but it was not statistically significant which was regressed to precontrast values at 72 h. a statistically significant increase in the level of spot urinary il-18 levels at 6 and 24 h postcontrast was observed, compared with precontrast spot urine il-18 levels and difference between 6 and 24 hour levels were not statistically significant. one of the limitations of this study is that there are other early biomarkers of acute kidney injury such as ngal and kim-1. another limitation is that urine il-18 measurement was indexed to serum creatinine instead of urine creatinine since serum creatinine is considered to be a better and commonly used marker in the diagnosis of acute kidney injury. in conclusion, spot urine il-18 levels at sixth hour following radiocontrast administration suggesting that it may be an earlier parameter for identifying kidney injury.

radiocontrast administration is an important cause of acute renal failure. in this study, compared the plasma creatinine levels with spot urine il-18 levels following radiocontrast administration. twenty patients (11 males, 9 females) underwent radiocontrast diagnostic and therapeutic-enhanced examinations. the rin mehran risk score was low (5). the radiocontrast agents used were 623 mg/ml iopromid (1.5 ml/kg), and 100 ml of 650 mg/ml meglumine diatrizoate as three-way oral and rectal contrast material for abdominal computed tomography (ct) scans. serum blood urea nitrogen, creatinine, na, k, cl, ca, p, creatinine clearance, and spot urine il-18 levels were analyzed before and repeated at 24, 48, and 72 h after radiocontrast administration. six and 24-h urinary il-18 levels were measured with a human il-18 elisa kit following radiocontrast administration. an increase in plasma creatinine 24 and 48 h following radiocontrast administration was observed compared with precontrast values, but it was not statistically significant (p=0.052 and p=0.285, respectively). a statistically significant increase in il-18 levels was observed at 6 and 24 h, compared with precontrast values (p=0.048 and p=0.028, respectively). a tendency for postcontrast 24-h urinary il-18 levels to increase was observed compared with 6 h, but the increase was not statistically significant (p=0.808). our results show that plasma creatinine starts to increase at 24th hour; however, spot urine il-18 levels go up at 6th hour following radiocontrast administration implying urine il-18 to be an earlier parameter for kidney injury.

PMC3459524

pubmed-13

according to the u.s. national institutes of health, up to 80% of human bacterial infections involve biofilm-associated microorganisms 1. among these, implant-related infections do still have a tremendous impact in orthopaedics and trauma 2, with high social and economic costs 3, 4, posing challenging diagnostic and therapeutic dilemmas 5. in fact, peri-prosthetic joint infection (pji) remains one of the most feared complications in orthopaedic surgery and among the first reasons for implant failure 6. moreover, given the increasing number of hip and knee arthroplasties performed, the prevalence of this complication is rising, with increasing costs for national health systems and increasing biological costs for the patients, such as loss or reduced joint function and deterioration in their physical and psychological health 7. according to the widely accepted model of the ' race for the surface ' for pji development, host and bacterial cells compete for surface colonization, with a low probability of bacterial attachment if host cells adhere to implant first, and vice versa. in the event of bacterial adhesion to an implant, in addition, the matrix protects the biofilm cells from various microbicidal agents and stresses, including dehydration, toxins, ultraviolet light, chemical disinfectants, temperature and osmotic shock, and lead them to increased resistance against antimicrobials 9, 10. to address the limited efficacy of existing antibiotics in the treatment of established bacterial biofilms, novel approaches are required to prevent bacterial adhesion and biofilm formation 11. adhesion is a necessary first step in microbial colonization and pathogenesis and provides a good theoretical target for new preventive and treatment strategies 12. bacterial adhesion to surfaces can be divided into a first, reversible phase and a second, irreversible phase. once an implant is inserted into the body, it is covered by a conditional protein layer composed of host proteins, such as albumin and complement, that act as a reservoir of several receptors for bacterial adhesive ligands, mediating adhesion of free-floating bacteria to the surface of the biomaterials 14, 15; these first adhesions are, however mechanically and biologically unstable. few minutes after this first, reversible phase, bacterial clusters attached to the surface starts to express biofilm related genes, produce glycocalyx and form mature biofilm, thus transforming the adhesion from reversible to irreversible 16. full-formed biofilm can be found few hours after the first bacterial adhesion 17. antimicrobial surface coatings can be based on an anti-adhesive principle that prevents bacteria to adhere and form biofilms 18. in fact, some polymer coatings, like the hydrophilic polymethacrylic acid, polyethylene oxide or protein-resistant polyethylene glycol can be applied to the surface of titanium implants and result in significant inhibition of bacterial adhesion 19-22. hydrophobic and superhydrophobic surface treatment technologies have also shown a great repellent antibacterial effect in preclinical studies 23-25. however, clinical application of completely novel coating technologies and compounds, not otherwise previously tested in humans, appears particularly challenging 26. bacterial colonization can also be blocked by an inhibitor interfering with ligand-receptor interaction for bacterial attachment. one of these inhibitors could be hyaluronic acid (ha), a glycosaminoglycan made up of glucuronic acid and n-acetylglucosamine disaccharide units. it is a uniform, linear and unbranched molecule, with highly variable length and molecular weight (up to 106 da). it is abundant in skin (up to 56%) and in connective tissues, with a turnover ranging from several hours to a few days depending on tissues. hyaluronic acid constitutes one of the main components of extracellular matrices. because of its biological properties, ha has several clinical applications (aesthetic surgery, dermatology, dentistry, orthopedics and opthalmology) 27. extensive studies on the chemical and physicochemical properties of ha and its physiological role in humans, together with its versatile properties, such as its biocompatibility, non-immunogenicity, biodegradability, and viscoelasticity, have proved that it is an ideal biomaterial for medical and pharmaceutical applications 28, 29. among its various properties, several studies have recently shown the ability of ha to protect against various infectious agents 30, depending on ha concentration and molecular weight 31, 32, while more recently ha interference on bacterial adhesion and biofilm formation has been extensively investigated 33. given its high biocompatibility and well known safety profile and the anti-adhesive capabilities, ha and its composites represent an attractive, non-antibiotic, option to mitigate the impact of biofilm-related infections in various clinical settings including implant-related infections. aim of this review is to provide an update of the current evidence concerning ha ability to reduce/prevent bacterial adhesion and biofilm formation. nearly two decades ago, pavesio et al. 34 were probably the first to describe the ability of ha to resist bacterial adhesion, with particular reference to staphylococcus epidermidis, and its non-fouling properties 35, proposing coated polymeric medical devices (e.g., intraocular lenses, stents and catheters) to reduce implant-related infections. in particular, a hydrophilic ha overlayer, linked to the surface of polymethylmethacrylate intraocular lenses (iols), was shown to be able to prevent fibroblasts adhesion and to greatly reduce staphyloccous epidermidis adhesion to the implant surface 36. the impact of slime dispersants and anti-adhesives on in vitro biofilm formation on iols was further investigated by kadry and co-workers 37, using a staphyloccous epidermidis wild strain, isolated from a patient with endophtalmitis; the authors reported the ability of hyaluronan to reduce bacterial adhesion to iols to 30%, compared with untreated control cells. the authors suggested the use of adjuvant therapy such as dispersants or anti-adhesives, in addition to the antibiotics in irrigating solutions for bacterial ocular infections. more recently, the in vitro antiadhesive and antibiofilm activity of hyaluronic acid towards bacterial species commonly isolated from respiratory infections was investigated by drago et al. 33. in this study, the interference exerted on bacterial adhesion was evaluated by using hep-2 cells, while the antibiofilm activity was assessed by means of spectrophotometry after incubation of biofilm with hyaluronic acid and staining with crystal violet. the experimental findings clearly demonstrated how hyaluronic acid is able to interfere with bacterial adhesion to a cellular substrate in a concentration-dependent manner. moreover, staphylococcus aureus biofilm was found to be more sensitive to the action of ha, compared to that produced by haemophilus influenzae and moraxella catarrhalis. concerning more specifically the antimicrobial activity, ha has also been shown to exert varied bacteriostatic, but not bactericidal, dose-dependent effects on different microorganisms in the planktonic phase 31, 38. in this regard, radaeva et al. reported the inhibiting activity of ha with respect to some pseudomonas species 39, while ardizzoni and co-workers 30 investigated the effects of ha on 15 atcc bacterial strains, representative of clinically relevant bacterial and fungal species. their results showed that different microbial species and, sometimes, different strains belonging to the same species, are differently affected by ha. in particular, staphylococci, enterococci, streptococcus mutans, two escherichia coli strains, pseudomonas aeruginosa, candida glabrata and c. parapsilosis displayed a ha dose-dependent growth inhibition, while no ha effects were detected in e. coli atcc 13768 and c. albicans and s. sanguinis was favoured by the highest ha dose. comparing the potential bacteriostatic effect of some of the most commonly used biomatrix materials (collagen type i, hyaluronic acid, hydroxyapatite, polylactic acid and polyglycolic acid) on the growth over the first 12h of exposure of some of the most common orthopaedic bacterial pathogens (staphylococcus aureus, staphylococcus epidermidis, -hemolytic streptococcus, pseudomonas aeruginosa), carlson and co-workers 38 found that ha had the most significant bacteriostatic properties on the studied organisms. 31 investigated the potential bacteriostatic effect of hyaluronic acid in different concentrations and molecular weight on oral and non-oral microorganisms (staphylococcus aureus, propionibacterium acnes, actinobacillus actinomycetemcomitans, pavotella oris and porphyromonas gingivalis) with potential application in dentistry surgery; the results showed that different hyaluronan solutions exerted varied bacteriostatic effects on all the bacterial strains. the authors concluded that the clinical application of hyaluronan in form of membranes, gels, or sponges during surgical therapy may reduce bacterial contamination of the surgical wound, thereby lessening the risk of postsurgical infection and promoting more predictable regeneration. concerning possible orthopaedic applications, in 2004 harris and richards 40 showed the visualization and quantification of s. aureus adhering to a variety of different treated/coated titanium surfaces. in their study, coating titanium with sodium hyaluronate significantly decreased the density of s. aureus adhering to the surfaces and its potential use in osteosynthesis, orthopaedics or dental applications was suggested out. in a very recent review on polysaccharide-based coatings, that have been proposed over the last ten years to impede biofilm formation on material surfaces exposed to bacterial contamination, hyaluronic acid was discussed as one of the most studied, with demonstrated non-fouling properties on glass surfaces 41; displaying hydrophilic characteristics (contact angle of 22), this coating was in fact reported to reduce adhesion of s. epidermidis and e. coli by several orders of magnitude compared to the unmodified glass slide. similarly, adhesion of s. aureus on ti foils functionalized with hyaluronic acid-catechol was lower than on pristine substrates. based on ha antiadhesive properties, a novel ha-based hydrogel has been recently proposed, in order to protect implanted biomaterials in orthopaedics, trauma and dental surgery from bacterial colonization 42; this fast-resorbable hydrogel coating, composed of covalently linked hyaluronan and poly-d, l-lactide (defensive antibacterial coating, dac, novagenit srl, mezzolombardo, italy), has been found to have a synergistic antibiofilm activity with various antibacterials and able to be effectively manually spread onto the surface of various biomaterials commonly used in orthopaedics, trauma and dental surgery 43 (fig. 1). the ability to completely cover even sand-blasted titanium surface and resist scraping has in fact been confirmed by scanning electron microscopy (sem) analysis (cf. this is an important requirement in order to reduce the exposed surface of a biomaterial, thus creating a uniform coating of the surface and leaving no pores or cracks that could eventually be colonized by planktonic bacteria. in unpublished experiments (novagenit srl, data on file), in order to evaluate dac ability to prevent bacterial adhesion, 200 mg of hydrogel were homogenously spread on the surface of sterile titanium discs. hydrogel-coated substrates and uncoated substrates (controls) were then placed into sterile 6-wells polystyrene plates and overlaid with a standardized inoculum (10 cfu/ml) of bacterial cells for 15, 30, 60 and 120 minutes. the remaining adhered cells were detached by adding a solution of 0.1% w/v dithiothreitol (dtt) (sigma-aldrich, milan, italy) to each well and stirring for 15 minutes at room temperature. then, 100 l of each sample were plated onto tryptic soy agar (tsa; merck, darmstadt, germany) and incubated at 37c for 24 hours for cfu counts. the results showed that the adhesion density of s. aureus on titanium discs pre-treated with dac, was significantly lower than adhesion on untreated controls at each time point (fig. 2). in particular, reductions of adhered bacteria equal to 86.8%, 80.4%, 74.6% and 66.7% vs untreated discs were observed after 15, 30, 60 and 120 minutes of incubation, respectively, while an increase of adhesion density during time was observed for both control and pre-treated discs (fig. further analyses were conducted to show the ability to dislodge previously adhered bacteria; to this aim, titanium discs were placed into sterile 6-wells polystyrene plates and overlaid with a standardized inoculum (10 cfu/ml) of bacterial cells in order to allow the adhesion of bacterial cells. afterwards, 200 mg of hydrogel were spread on the surface of contaminated titanium discs in order to remove previously adhered bacteria. non-adherent bacteria were removed by rinsing with sterile saline, while the remaining adhered cells were detached by adding 0.1% dtt as previously described. then, 100 l of each sample were plated onto tsa and incubated at 37c for 24 hours for cfu counts. the results showed that dac hydrogel treatment of discs reduced the amount of adhered bacteria in respect to control discs after 15, 30, 60 and 120 minutes of 84.0%, 72.8%, 72.3% and 64.3%, respectively (fig.4). once again, an increase of adhesion density during time was observed for both control and treated discs (fig. dac hydrogel showed similar or superior in vitro activity, compared to various antibacterials and a synergistic activity when used in combination (fig. were grown on chrome-cobalt devices in 6-wells polystyrene plates containing tsb for 24 hours at 37c. then, growth medium was removed together with non-adherent bacteria and new broth added. the plates were incubated at 37c in ambient air, until a visible biofilm was obtained. gentamycin and vancomycin were tested at a final concentration of 20 mg/ml. similarly, when mixed with the hydrogel, 60 mg of gel powder were reconstituted with 1 ml of water for injections containing gentamicin or vancomycin at 20 mg/ml concentration. amount of biofilm at each time was determined before hydrogel and antibiotic agents addition and after 0.5, 1, 2, 4, 6, 24 and 48 hours of incubation by a spectrophotometric assay. in particular, at each time, broth was removed and biofilm stained with crystal violet. after elution of the stain from implants with absolute ethanol, the amount of biofilm was quantified by reading optical density (o.d.) at a wavelength of 595 nm against blank (consisting of ethanol). amount of biofilm at each time was compared with that formed on the same type of implant before treatment. each assay was performed in duplicate and repeated for three times. at each time point, both for gentamycin and vancomycin showed only a partial inhibition of biofilm formation (ca. 40-50% for vancomycin), with minor difference between the two studied microorganisms. on the other side, 50% in comparison to the untreated controls, while a combination of the hydrogel with either antibacterial resulted in a larger reduction of biofilm formation (approximately 75 to 80% in comparison with untreated controls). both these experimental studies show the ability of the dac hydrogel to significantly reduce bacterial adhesion and biofilm formation of common bacterial pathogens, thus potentially providing an effective protection of the implant; however, these data also point out how, in the clinical setting, in the absence of an adequate immune response from the host and/or of sufficient local levels of antibiotics, a passive antiadhesive coating 18 like ha can be overcome by the remaining bacteria in a time-dependent manner. for this reason, any passive antiadhesive coating of implants 44 should probably better be seen as a tool to reduce and delay bacterial adhesion and biofilm formation to a variable degree, also depending on the local environment, the contaminating bacterial species and initial bacterial load; this may still provide an additional advantage to the host's cells to first colonize the implanted biomaterial and win the competition with the microorganisms that may eventually be present, thus contributing to reduce the occurrence of implant-related infections. several clinical local applications of ha to reduce the impact of biofilm-related infections have been reported, in different clinical settings, with favourable results and no adverse events. 45 recently described topical administration of hyaluronic acid in children with recurrent or chronic middle ear inflammations and chronic adenoiditis. in this prospective, single-blind, randomised controlled study, otoscopy, tympanometry and pure-tone audiometry in children which received the daily topical administration of normal 0.9% sodium chloride saline solution (control group) or 9 mg of sodium hyaluronate in 3 ml of a 0.9% sodium saline solution was performed. the final analysis was based on 116 children (49.1% boys; mean age, 62.9 17.9 months): 58 in the control group and 58 in the study group. at the end of follow-up, the prevalence of patients with impaired otoscopy was significantly lower in the study group (p value=0.024) compared to baseline but not in the control group. in comparison with baseline, the prevalence of patients with impaired tympanometry at the end of the follow-up period was significantly lower in the study group (p value=0.047) but not in the control group. the reduction in the prevalence of patients with conductive hearing loss (chl) (p value=0.008) and those with moderate chl (p value=0.048) was significant in the study group, but not in the control group. the mean auditory threshold had also significantly improved by the end of treatment in the study group (p value=0.004) but not in the control group. several studies have also reported the beneficial effect of topical ha in chronic urinary tract infections (uti). in contrast to traditional antibiotic therapy, which aims at eradicating pathogens, treatment with ha targets bacterial adherence to the bladder mucosa with the presumption that a damaged glycosaminoglycan mucous layer facilitates bacterial adherence and therefore recurrent uti 46. among others 47, 48, lipovac and colleagues evaluated the efficacy of nine ha bladder instillations over 6 months in 20 women with a history of recurrent uti. their status was assessed prospectively but compared with a retrospective review of patients ' charts. the number of infections per year per patient was significantly reduced (from 4.990.92 to 0.560.82, p>0.001) and the mean time to recurrence (from 76.724.6 to 178.325.5 days, p>0.001) was prolonged significantly. nevertheless 65% of treated patients were free of recurrences until the end of study (47.6 weeks) 49. were able to provide a higher level of evidence by reporting a prospective, randomized, double-blind, placebo-controlled study, in which a significant reduction of 77% (p<0.0002) in the uti rate per patient per year versus placebo was observed at the end of the study. moreover, mean time to uti recurrence was significantly prolonged (185.278.7 versus 52.733.4 days, p<0.001) after treatment compared with placebo. overall urinary symptoms and quality of life measured by questionnaires significantly improved compared with placebo 50. very recently a multicentre european study confirmed the efficacy of intravesical administration of combined hyaluronic acid and chondroitin sulphate (cs) for the treatment of female recurrent urinary tract infections 51. a total of 276 adult women received intravesical administration of ha+cs or standard of care (antimicrobial/ immunoactive prophylaxis/ probiotics/cranberry). at follow-up, 181 patients treated with ha+cs and 95 patients treated with standard of care from 7 centres were available. the crude and adjusted ors (95% ci) for bacteriologically confirmed recurrence within 12 months were 0.77 (0.46 to 1.28) and 0.51 (0.27 to 0.96), respectively. studies were also undertaken to determine the effect on clinical variables, sub-gingival bacteria and local immune response brought about by application of hyaluronan-containing gels in early wound healing after scaling and root planing (srp) in dentistry 52, 53. in the study reported from eick et al. the exclusion criteria were: antibiotics intake in the 6 months before the study, periodontal treatment received during the previous year, pregnancy, nursing, smoking, chronic diseases such as diabetes mellitus or rheumatoid arthritis, and allergy to ingredients in the drug. in the test group (n=17), a 0.8% hyaluronan-containing gels (ha) was introduced into all periodontal pockets during srp and a 0.2% ha gel was applied by the patients onto the gingival margin twice daily during the following 2 weeks while the control group (n=17) was treated with srp only; no placebo was used. probing depth (pd) and clinical attachment level (cal) were recorded at baseline and after 3 and 6 months, and subgingival plaque and sulcus fluid samples were taken for microbiologic and biochemical analysis. in both groups, pd and cal the changes in pd and the reduction of the number of pockets with pd5 mm were significantly higher in the test group after 3 (p=0.014 and 0.021) and 6 (p=0.046 and 0.045) months. six months after srp, the counts of treponema denticola were significantly reduced in both groups (both p=0.043), as were those of campylobacter rectus in the test group only (p=0.028). although to date no surface modification has been reported to be able to fully prevent bacterial adhesion and biofilm formation 55, available data show that hyaluronic acid has a proven in vitro antiadhesive/antibiofilm effect against some of the most common pathogens and it has been used safely, alone or in combination with other polymers, with satisfactory results in different conditions associated with biofilm-related chronic infections. clinical data in various applications, including dentistry, urology, wound management, dermatology and orthopedics, allow to consider the potential use of ha as a protective coating barrier of implants particularly safe and feasible on a large scale basis. while antibacterial coatings to mitigate the occurrence of implant- and biofilm-related infections are regarded as one of the most needed technology, currently only few and insufficient options are available for clinical use in orthopedics and trauma surgery 18. considering the pathogenesis of implant-related infections, any protection offered by a fully biocompatible antiadhesive barrier, like ha and some of its derivatives, could be extremely useful to reduce the tremendous burden of implant-related infections. on the other hand, it should be noted that hyaluronic acid as a passive protective barrier has some limits. among others, the antiadhesive/antibiofilm effect is limited and may vary, depending on the type of the microorganism, the bacterial load, the local environment, etc.; moreover, ha protection may be neutralized by the possible ability of some bacteria to produce hyaluronidase, an enzyme that catalyzes the degradation of hyaluronic acid 56, while collagen and hyaluronan may even become possible ligands for microbial attachment in particular situations 57, 58. to overcome at least some of these limits, possible loading of hyaluronic-based hydrogels with antibiotics is technically feasible and has been proposed by different authors 59-62, being a possible option for future developments and large scale clinical applications, provided that regulatory requirements can be met.

living in biofilms is probably the most common condition for bacteria and fungi and biofilm-related infections account for the majority of bacterial infectious diseases worldwide.among others biofilm-related infections, those associated with implanted biomaterials have an enormous and still largely underestimated impact in orthopaedics and trauma, cardio-surgery and several other surgical disciplines.given the limited efficacy of existing antibiotics in the prevention and treatment of bacterial biofilms, new strategies are needed to protect implants and host tissues, overcoming the striking ability of the microorganisms to adhere on different surfaces and to immediately protect themselves by forming the biofilm matrix.adhesion is a necessary first step in microbial colonization and pathogenesis and provides a potential target for new preventive and treatment approach.among various polymers, tested as antibacterial coatings, hyaluronic acid and some of its composites do offer a well-established long-term safety profile and a proven ability to reduce bacterial adhesion and biofilm formation.aim of the present review is to summarize the available evidence concerning the antiadhesion/antibiofilm activity of hyaluronic acid and some of its derivatives to reduce/prevent bacterial adhesion and biofilm formation in various experimental and clinical settings.

PMC5423572

pubmed-14

in 2011, who declared combat drug resistance: no action today, no cure tomorrow.. in recent years presently, antimicrobial resistance (amr) poses a major threat to patient's treatment as it leads to increased morbidity and mortality, increased hospital stay, and severe economic loss to the patient and nation [3, 4]. the clinical isolates such as pseudomonas aeruginosa, methicillin resistant staphylococcus aureus (mrsa), enterococci especially vancomycin resistant enterococci (vre), and members of family enterobacteriaceae, for example, klebsiella pneumoniae, e. coli, and proteus sp. in the last two decades, there were so much increase of infectious diseases that the standard of public health in many parts of the world is equivalent to preantibiotic era. as per standardized international terminology created by european centre for disease control (ecdc) and centre for disease control&prevention (cdc), atlanta, the multidrug-resistant (mdr), extensively drug-resistant (xdr), and pandrug-resistant (pdr) bacteria have been well defined. multidrug resistant (mdr) was defined as acquired nonsusceptibility to at least one agent in three or more antimicrobial categories. extensively drug resistant (xdr) was defined as nonsusceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e., bacterial isolates remain susceptible to only one or two antimicrobial categories). pandrug resistant (pdr) was defined as nonsusceptibility to all agents in all antimicrobial categories. hence, this short term study was undertaken to detect the incidence of mdr, xdr, and pdr bacterial isolates in a tertiary care hospital of central india. this short term cross-sectional study was conducted in the department of microbiology from 15th of april to 15th of july, 2014. the bacterial strains were isolated from different clinical samples and were identified by conventional methods. the clinical specimens from indoor patient departments (ipd) only were included in the study. antibiotic susceptibility test of bacterial strains was done by kirby bauer disc diffusion method as per clinical laboratory standard institute (clsi) guidelines. antibiotics used for gram positive cocci (gpc) were penicillin, erythromycin, ciprofloxacin, tetracycline, amikacin, vancomycin, and linezolid and for gram negative bacilli (gnb) were amikacin, ceftazidime, ceftazidime-clavulanic acid, ciprofloxacin, imipenem, and colistin, respectively. linezolid and colistin were used as supplemental drugs. for urine sample, instead of ciprofloxacin and tetracycline, for routine quality control of antibiotic susceptibility test, s. aureus atcc 25923, e. coli atcc 25922, and pseudomonas aeruginosa atcc 27853 were used. mdr, xdr, and pdr strains were detected as per criteria described by ecdc and cdc. methicillin resistant staphylococcus aureus (mrsa) strains were detected by meca-mediated oxacillin resistance using cefoxitin disk (30 g) on mueller hinton (mh) agar plate inoculated with test strains as per standard disk diffusion recommendations and incubated at 3335c for 1618 hours. inhibition zone 21 mm with cefoxitin disk was interpreted as meca positive according to clsi guidelines. extended spectrum -lactamases (esbl) producing strains were detected by combined disk method using ceftazidime (30 g) and ceftazidime plus clavulanic acid (30 g plus 10 g). an increase in diameter of 5 mm with ceftazidime plus clavulanic acid as compared to ceftazidime disk alone was considered positive for esbl detection. 138 clinical samples were received from intensive care unit (icu) and 742 clinical samples were received from wards of different clinical specialities. 698 clinical samples had single bacterial growth and 182 had mixed bacterial growth. out of these 182 clinical samples, 172 samples had 2 bacterial isolates, 4 samples had 3 bacterial isolates, and 6 samples had 1 bacterial isolate along with candida albicans. figure 1 shows that 314 (29.6%) bacterial strains were gram positive cocci (gpc) and 746 (70.4%) were gram negative bacilli (gnb). out of 314 gpc, 252 (80.3%) were coagulase positive staphylococci. amongst 746 gnb, 261 (35%) were e. coli, followed by pseudomonas aeruginosa 212 (28.4%). during the study period, figure 2 shows the incidence of mdr and xdr strains isolated. out of total 1060 bacterial strains studied, 393 (37.1%) bacterial strains were mdr and 146 (13.8%) strains were xdr. amongst 314 gpc strains isolated, 143 (45.5%) and 56 (17.8%) were mdr and xdr, respectively. out of 746 gnb isolates, 250 (33.5%) strains were mdr and 90 (12.1%) were xdr. out of total 9304 patients admitted, 393 (4.2%) and 146 (1.6%) were positive for mdr and xdr strains, respectively. figure 3 shows the incidence of mdr and xdr gram positive cocci isolated. out of total 252 coagulase positive staphylococci isolated, 125 (49.6%) were mdr and 38 (15.1%) were xdr. 10 coagulase negative staphylococci were isolated and 5 (50%) were mdr, whereas 2 (20%) were xdr. 79 (31.3%) coagulase positive staphylococci strains were mrsa and 2 (20%) coagulase negative staphylococci were mrcons. out of total 45 enterococci isolated, 13 (28.9%) were mdr and 16 (35.6%) were xdr. no vancomycin intermediate staphylococcus aureus (visa), vancomycin resistant staphylococcus aureus (vrsa), or vancomycin resistant enterococci (vre) were isolated. all streptococcus species including group a, nongroup a, and pneumococcus were sensitive to penicillin. no mdr or xdr strain was isolated from streptococcus sp. all (100%) gram positive cocci were sensitive to vancomycin and linezolid. figure 4 shows incidence of mdr and xdr strains isolated from each species of gram negative bacilli. in the present study, e. coli was the commonest isolate 261 (35%), followed by pseudomonas aeruginosa 212 (28.4%). 79 (30.3%) and 22 (8.4%) e. coli strains were mdr and xdr, respectively. out of 200 klebsiella pneumoniae strains isolated, 75 (37.5%) and 25 (12.5%) were detected as mdr and xdr, respectively. out of 42 acinetobacter and other nonfermenter species isolated, 19 (45.2%) and 8 (19%) were mdr and xdr strains, respectively. amongst 250 gnb-mdr strains isolated, the commonest mdr strains were detected from e. coli 79/250 (31.6%), followed by klebsiella pneumoniae 75/250 (30%). similarly, out of 90 gnb-xdr strains isolated, the commonest xdr strains were detected from pseudomonas aeruginosa 29/90 (32.2%), followed by klebsiella pneumoniae 25/90 (27.8%). in the present study, 137 (18.4%) others include wards like dermatology, pulmonary medicine, orthopedics, and cardiovascular and thoracic surgery (cvts). the different icus include neonatal icu (nicu), medicine icu (micu), operation theatre icu (ot icu), and paediatric icu (picu). out of total 393 mdr strains detected, 127 (32.3%) (the highest number) mdr strains were isolated from surgery wards followed by 72 (18.3%) mdr strains from different icus. amongst total 146 xdr strains isolated, 41 (28.1%) (the highest number) were isolated from surgery wards also. out of 72 mdr strains detected from different icus, 29 (40.3%) (the highest number) mdr strains were isolated from nicu, followed by 20 (27.8%) and 18 (25%) from ot icu, and only 5 (6.9%) from picu. even in the total 26 xdr strains isolated from different icus, 10 (38.5%) (the highest number) the percentage of mdr and xdr strains isolated from different icus was 72/138 (52.2%) and 26/138 (18.8%), respectively, which were again much more than mdr and xdr strains isolated from wards 321/742 (43.3%) and 120/742 (16.2%), respectively. 275 patients were admitted to nicu, of whom 29 (10.5%) were positive for mdr strains and 10 (3.6%) were positive for xdr strains. out of total 1907 patients admitted to surgery ward, 127 (6.7%) were positive for mdr strains whereas 41 (2.1%) were positive for xdr strains. in micu, 545 patients were admitted and 20 (3.7%) were positive for mdr strains and 8 (1.5%) were positive for xdr strains. the clinical and financial burden to patients and health care providers for mdros is really challenging. barbara soule, joint commission resources practice leader, infection prevention and control services, has told, patients who are infected with mdros often have an increased risk of prolonged illness and mortality. the cost of care for these patients can be more than double as compared to those without mdro infection. since the year 2000, only 4 new classes of antibiotics have been approved by food and drug administration (fda), us, for example, linezolid, streptogramins, daptomycin, and tigecycline. the problem is that the bacteria are developing resistance at a much faster pace than the new drug development. regarding public health attention, mdros are described as superbugs having very limited treatment options. for some mdros, only 1 or 2 antibiotics can be effective with toxic side effects. in 2009, boucher et al. have reported eskape organisms as bad bugs, where e stands for enterococcus faecium, s for staphylococcus aureus, k for klebsiella pneumoniae, a for acinetobacter baumannii, p for pseudomonas aeruginosa, and e for enterobacter species. in the year 2009 only, peterson has reported the escape group of organism, which was the same as the above list but k was replaced by c, that is, clostridium difficile, and the last e stands for enterobacteriaceae. in the present study, amongst 250 gnb-mdr strains isolated, the commonest mdr strains were detected from e. coli 79/250 (31.6%), followed by klebsiella pneumoniae 75/250 (30%). similarly, out of 90 gnb-xdr strains isolated, the commonest xdr strains were detected from pseudomonas aeruginosa 29/90 (32.2%), followed by klebsiella pneumoniae 25/90 (27.8%). aly and balkhy reported that most prevalent mdro in their study was e. coli followed by klebsiella pneumoniae. in another study, carried out in a tertiary care hospital in riyadh, it has been reported that most frequent mdr pathogens were pseudomonas aeruginosa followed by e. coli. the percentage of mdr e. coli strains was more than klebsiella pneumoniae and even pseudomonas aeruginosa in our study probably because a total number of e. coli strains isolated (261) were also higher. the slightly increased incidence of drug resistant strains observed in our study may be because our hospital is a tertiary care center in a rural setup and patients from adjoining districts and even villages are admitted for treatment. before attending the hospital, most of the patients get different antibiotics from general practitioners or due to over-the-counter sell of antibiotics often in improper dose. the limitation of this study is that this is a single center study for only three-month period in a tertiary care hospital in central india. to reflect the trend of infections caused by mdr and xdr strains of bacteria in the region, a multicenter study involving all types of healthcare setups for a minimum period of one year there is paucity of data regarding mdros in health care setup not only in india but also worldwide. unless and until multidrug resistant organisms are detected and their incidence is known, the strategies for their control can not be adopted properly in healthcare setup. hence, detection, prevention of transmission of mdros by following infection control practices, antimicrobial surveillance, and stewardship are need of the hour. misuse and overuse of antibiotics, over-the-counter selling of antibiotics without prescription to common people, must be stopped by strict implementations of rules and regulations. we hereby conclude that early detection and close monitoring of mdr, xdr, or even pdr bacterial strains must be started by all clinical microbiology laboratories to reduce the menace of antimicrobial resistance which is now a global problem.

background and objective. antimicrobial resistance is now a major challenge to clinicians for treating patients. hence, this short term study was undertaken to detect the incidence of multidrug-resistant (mdr), extensively drug-resistant (xdr), and pandrug-resistant (pdr) bacterial isolates in a tertiary care hospital. material and methods. the clinical samples were cultured and bacterial strains were identified in the department of microbiology. the antibiotic susceptibility profile of different bacterial isolates was studied to detect mdr, xdr, and pdr bacteria. results. the antibiotic susceptibility profile of 1060 bacterial strains was studied. 393 (37.1%) bacterial strains were mdr, 146 (13.8%) strains were xdr, and no pdr was isolated. all (100%) gram negative bacterial strains were sensitive to colistin whereas all (100%) gram positive bacterial strains were sensitive to vancomycin. conclusion. close monitoring of mdr, xdr, or even pdr must be done by all clinical microbiology laboratories to implement effective measures to reduce the menace of antimicrobial resistance.

PMC4749793

pubmed-15

the majority of women with menorrhagia, postcoital bleeding, intermenstrual bleeding, or postmenopausal bleeding ultimately undergo diagnostic hysteroscopy with endometrial sampling as part of their assessment, particularly if symptoms persist or pelvic imaging suggests a uterine abnormality. dilatation and curettage (d&c) has been widely considered to be the method of choice for obtaining endometrial samples for histopathological evaluation. however, the needs for admission and general anesthesia and their associated costs have made this option less favorable. in the outpatient setting, endometrial sampling is an effective and acceptable method for obtaining endometrial samples for histopathological assessment [3, 4]. inadequate sampling is more problematic in postmenopausal women, for whom up to 68% of endometrial samples are reported to be inadequate. in our institution, the only sampling tool available to perform the outpatient sampling procedure is the uterine explora model i-mx120 (http://www.coopersurgical.com/) (figure 1). in addition, the device is sterile and disposable (one-time use). the advantages of using explora rather than d&c as a sampling device include a reduction in hospitalization costs, extra convenience for the patient and physician, and the minimal complications of the procedure. the purpose of this study is to compare the effectiveness of the explora model i tool with the conventional d&c technique for obtaining adequate endometrial samples that are capable of providing specific and informative histopathologic diagnoses. after obtaining the approval of our institutional review board, all endometrial samples received at the histopathology department in king khalid university hospital (kkuh, riyadh, ksa) between january 2007 and december 2010 were included in this study. a total of 1270 endometrial samples were included (table 1). two hundred seventy-four samples (21.6%) were obtained by conventional d&c in the surgical theater, while the remaining 996 samples (78.4%) were obtained by senior obstetrics and gynecology residents who used a standardized biopsy technique in the outpatient procedure rooms. during the usage of the explora model i, the syringe provided with the instrument was used to create a negative pressure, and the explora was rotated as it was withdrawn. after withdrawal, the tip was cut off, and the tissue was placed in 10% buffered formalin saline fixative and was sent for pathological examination. the pathologists who interpreted the endometrial samples were blinded to the instrument or method used to obtain the samples. an inadequate sample was defined as consisting of only blood, cervical mucus, endocervical epithelium, or blood with fragments of endometrial glands or stroma insufficient for histopathological assessment and diagnosis. the age, gravidity, parity, menstrual history, uterine size, hysteroscopy findings (when available), and the presence or absence of any cervical abnormality were recorded on the request forms, which were reviewed by the investigators. for each of the two methods used (explora model i and d&c), the numbers and percentages of inadequate samples and age group clustering were calculated and statistically analyzed. of the 1270 endometrial samples obtained, 253 samples (19.9%) were scored as inadequate. of these samples, the explora sampler was used to collect 224 samples (88.5%), whereas 29 samples (11.5%) were obtained by d&c (figure 2). thus, the insufficient tissue percentage was higher with the explora (17.6%) than with d&c (2.2%), which was a statistically significant difference (p<0.0001). age group clustering (i.e., numbers of premenopausal and postmenopausal women) of inadequate sample results was also calculated (figure 3). of the 253 inadequate samples, 82.6% were from women 45 years of age and older (i.e., postmenopausal) compared to 17.4% in premenopausal women; the age difference was significant (p<0.0001). the detection rates of endometrial hyperplasia and carcinoma using both methods were assessed and calculated. of the 73 samples with a diagnosis of endometrial hyperplasia, 50 (68.5%) were diagnosed by d&c, and 23 (31.5%) were diagnosed using the explora sampler. however, of the 18 samples with a diagnosis of endometrial cancer, the rates of detection were similar between the two methods. endometrial sampling for the evaluation of dysfunctional uterine bleeding and the diagnosis of endometrial hyperplasia and carcinoma and other indications remains one of the most commonly performed gynecological procedures [14]. in recent years, less hazardous and more inexpensive and convenient outpatient sampling methods have replaced the traditional, in-hospital, endometrial curettage. the advantages of outpatient endometrial biopsy include reduced cost and less risk for the patient, as no anesthesia is required. furthermore, the discomfort and pain produced by sampling have been reported to be minimal. however, it is essential to ensure that outpatient endometrial sampling is quantitatively adequate and comparably accurate to conventional dilatation and curettage. a sample is judged as adequate if a specific diagnosis can be given from the histological examination of the endometrial fragments obtained. adequacy can be measured by comparison of either outpatient biopsy with curettage histological evaluation or outpatient biopsy with the results of pathological examination of hysterectomy specimens [3, 4]. many techniques for obtaining an endometrial sample without the need for curettage have been described in the literature. chicago, il, usa) and the novak biopsy curette with a 10 ml syringe functioning as an aspiratory device, which have been shown to be equally effective compared to d&c in detecting an endometrial pathology [69]. however, the vabra aspirator and novak biopsy curette, although widely available and relatively inexpensive, have several disadvantages, including the need for an electric vacuum pump to perform the aspiration in the former technique and the pain caused by both methods. as a result of these drawbacks, smaller inexpensive and self-contained instruments have been developed and the prototype of this class of endometrial samplers is the pipelle. the pipelle has been shown to have a diagnostic accuracy comparable to that of vabra aspiration and the novak curettage while causing less pain [911]. all of these instruments (i.e., the vabra aspirator, the novak biopsy curette, and the pipelle) have low rates of false-negative and insufficient tissue results for the detection of endometrial abnormalities, as determined by comparison to hysterectomy specimens [1113]. it was found that pipelle biopsy had a sensitivity of 99.2% in pinpointing high grade cancer and a sensitivity of 93% in detecting low grade malignancies; the sensitivities defined for d&c were 100% and 97%, respectively. while excellent agreement was generally noted between preoperative histology and grade and the final pathology, pre-operative endometrial sampling more commonly provided underestimates of final grade (low grade versus high grade) than overestimates. the explora is somewhat similar in its design to the pipelle, but clinical studies on its effectiveness are scarce, with the effectiveness ranging between 14.6 and 15% according to various studies [6, 15]. our own findings revealed that the rate of obtaining inadequate samples using the explora was much higher (17.6%) than the rates reported in the literature. however, most of these cases (82.6%) were obtained from postmenopausal women with atrophic endometrial status. this finding is in keeping with the rates reported by other investigators [58, 16]. this retrospective study suggests that traditional d&c produces better endometrial sample adequacy than the explora technique. this finding indicates that clinicians performing endometrial sampling would benefit from more experience and training using the explora technique. additional studies comparing the adequacy of samples obtained with different endometrial sampling techniques and devices are warranted. furthermore, we recommend using the d&c procedure when the explora-obtained samples are inconclusive or when the use of the explora sampler is accompanied by ultrasound findings that are suspicious of endometrial hyperplasia or carcinoma.

aims. our aim is to compare the adequacy and diagnostic yield of samples obtained by the endometrial explora sampler i-mx120 with endometrial specimens obtained by conventional dilatation and curettage (d&c). methods. a total of 1270 endometrial samples were received in the histopathology laboratories at the king khalid university hospital, riyadh, saudi arabia, between 2007 and 2010. in the outpatient clinic, the uterine explora model i was used to obtain 996 samples. the remaining 274 samples were obtained by conventional d&c. sample adequacy and the clustering of inadequate specimens according to age groups by the two different techniques were compared and statistically analyzed. results. out of 1270 endometrial samples, 253 (19.9%) were inadequate. the uterine explora was used in 88.5% of these inadequate samples (253 samples), and the remaining 11.5% were obtained by d&c. the insufficient tissue incidence was higher with the explora (17.6%) than with the d&c (2.2%) and the difference was statistically significant (p<0.0001). the ages of the patients, as well as the clinical indications for the procedures, were recorded. conclusion. this retrospective study demonstrated better specimen adequacy when d&c was used compared to the higher rate of sample insufficiency obtained with the explora.

PMC4334055

pubmed-16

the geological and (palaeo-)biological evolution of lowland amazonia during neogene and quaternary times remains fascinating since the early days of natural sciences (for summaries of research history see e.g., loczy, 1963 and wesselingh, 2008). many, but partly highly controversial models have been introduced to explain its historical development (for recent compilations see e.g., lundberg et al., 1998 ;, 2006; wesselingh and salo, 2006; rossetti and mann de toledo, 2007; haffer, 2008; hoorn and wesselingh, 2010; hoorn et al., 2010; generally, it is widely accepted that around the onset of the miocene (23 ma) a mega-wetland (pebas system, also called lake pebas) developed in western amazonia due to the subsiding subandean foreland (e.g., hoorn, 1994, 2006; wesselingh et al., 2002; wesselingh and salo, 2006; shephard et al., 2010). short-lived marine incursions or even a transcontinental seaway from the caribbean sea through the venezuelan/columbian llanos basin southwards to the argentinean paran basin are proposed (e.g., rsnen et al., 1995; 2009) but heavily disputed (e.g., campbell et al., 2006; cozzuol, 2006; westaway, 2006; latrubesse et al., 2007, 2010 ;, 2003; hoorn and vonhof, 2006; wesselingh, 2006). in the late miocene this mega-wetland disintegrated due to enhanced uplift of the northern/central andes. the drainage pattern of northern south america started to reverse completely to today s easterly course and the modern amazon system became established during the early pliocene (hoorn, 2006; figueiredo et al., 2009; hoorn et al., 2010; latrubesse et al., 2010). beside the vast size of amazonia and the still fragmentary regional coverage with field surveys, there are considerable inconsistencies in palaeoenvironmental reconstructions and, in particular, in the chronology and correlation of scattered outcrops. wesselingh (2008, p. 5) stated: the lack of geological data has led to the emergence of many grand theories about the origin of present-day amazon system and its highly diversity, often based on dubious interpretations of the little data available. the present paper aims to contribute basic sedimentological data from a barely studied region (eirunep, 2.000 km sw manaus; fig. 1a), which is supposed to be placed at the south-eastern margin of the pebas system (wesselingh and ramos, 2010). we demonstrate that there is no evidence for a long-lived lake (sensu gorthner, 1994) or any marine influx. conversely, we document a well-structured, aggrading fluvial system of late miocene age, which is in agreement with the sedimentation model and chronology proposed by latrubesse et al. the studied sections are located along the juru and tarauac river, ne respectively se of the city eirunep (state of amazonia, western brazil; 245 km s of benjamin constant; fig. delineations and subdivisions of basins in western amazonia diverge notably and several authors attribute the region of eirunep to the solimes basin (eirunep subbasin; e.g., caputo, 1991; roddaz et al., 2005; ramos, 2006; wesselingh and salo, 2006; wesselingh et al., however, based on subsurface information, obtained by extensive hydrocarbon and coal exploration campaigns during the 1970 s, this region is situated west of a basement high (miura, 1972; iquitos arch; corresponds to the carauari arch of caputo, 1991) on the eastern margin of the acre basin (e.g., del arco et al., 1977; maia et al., 1977; latrubesse et al., 2010; fig. beside quaternary deposits (terraces, alluvium), the scattered outcrops along river banks expose sediments of the upper parts of the solimes formation (del arco et al., 1977; maia et al., 1977; paz et al., in press). the solimes fm. comprises more than 1000 m thick, largely pelitic sandy alternations with lignitic intercalations and covers a huge part of western amazonia (fig.. uncertainties in its definition, its stratigraphical and geographical extent as well as its depositional environments basically reflect the ongoing debate about amazonia s history through neogene times. (1977), hoorn (1993, 1994), latrubesse et al. the sections were vertically logged by visual inspection of the lithofacies (including colour, grain size, bedding planes, sedimentary structures, macrofossil content). for lithofacies coding the scheme of miall (1996) was used: capital letters=dominant grain size (g, gravel; s, sand; f, fine sand clay) followed by a lowercase letter=sedimentary structures and/or biogenic features (c, clast-supported; m, massive/faint lamination; h, horizontal bedding/lamination; t and p, trough and planar cross bedding; r, ripple bedding; s, scour fill; l, lamination; r, rooted; c, coal; p, pedogenic overprint). additionally the outcrops were mapped laterally as far as possible. due to poor outcrop conditions an application of the architectural element concept (miall, 1996) was only loosely possible. for micropalaeontological investigations bulk samples (12 kg) were taken from all outcrops. 500 g of dried sediment (40 c, 24 h) were washed by using diluted hydrogen superoxide for disintegration through standard sieves (h2o2: h2o=1: 5; 63/125/250/500 m). wet sieve residuals were washed with ethanol prior to drying (40 c, 24 h). an in-depth taxonomic examination of the obtained microfossils (including the sieve fractions<250 m) will be subject of further studies. for preliminary stable isotope analyses (o, c; 18 samples) two or three ostracod valves (4060 g; cyprideis spp.) from earlier sampling campaigns were used (collected by m.i.r). for analyses a thermo-finnigan kiel ii automated reaction system and a thermo-finnigan delta plus isotope-ratio mass spectrometer were used (university of graz; standard deviation=0.1 relative to nbs-19; results in per mille relative to vpdb). more detailed investigations are in preparation (m. fonseca/belm, m. caporaletti/graz). location: 12.6 km ne eirunep (s 063355.5/w 0694611.7, altitude: 108 m), left cutbank of the juru river (fig. 2). description: the basal 1.8 m (layers 19) of the section consists of partly ripple bedded sands and laminated silts; some cm-thick clay intercalations were observed. above follows a 0.65 m thick alternation of clay with incorporated calcareous nodules and laminated silty sand or ripple bedded sand (1017; fig. the top is rich in vertebrate remains (crocodiles, turtles; 17; fig. massive to finely laminated, occasionally bivalves-bearing (silty) clay with coaly and sandy layers forms the next 1.14 m (1823; fig. beds 2425 (1.85 m thick) comprise massive, horizontally or trough cross bedded sand and finely laminated or massive (sandy silty) clay/fine sand (fig. grey mottled, rooted and massive clay (paleosol) with a calcareous horizon on its top (26). up-section, a 4.2 m (2739) thick alternation of massive or laminated clay, silt and partly ripple bedded fine sand is developed (a slight coarsening-upward trend is anticipated). bed 32 displays a slightly erosive base with intraclasts in the lower part (mud pebbles). layers 3139 are cut by a channel (40) with a multiphase fill. large scale cross bedded sands (-cross stratification) with sporadic intraclasts (mud pebbles) on reactivation surfaces and trough cross bedded and ripple bedded fine microfossil contents: samples from the basal part of the section (pd2) yielded only scarce valves of cyprideis spp. and rare fish elements. interpretation: the lithofacies of the basal part of the outcrop (117) refers to a deposition in a fluvial overbank environment. the diffuse layers of calcareous nodules (17) are supposed to be largely a diagenetic feature (groundwater caliche). however, it may also hint to soil-forming processes at seasonally dry floodplain areas (retallack, 2001). up-section the sediments become finer and contain several (sandy silty) coal-rich layers (1823). these beds are assigned to the formation of a floodplain pond, which was inhabited by opportunistic ostracods, freshwater bivalves and fishes. however, crevasse splays influenced episodically that shallow lacustrine environment (sandy and organic matter-rich layers). later (layers 2425), the influx of crevasse splays into the pond, which is still populated by ostracod faunas and fishes increased (more prominent sandy beds, partly scour-fills). pedogenic processes are indicated by incipient soil formation in layer 26 (mottled, roots). floodplain pond conditions ended above (beds 2739) due to enhanced clastic input from an approaching channel. finally (bed 40), the overbank fines were intersected by a channel of at least 23 m in depth, which was filled up by large scale cross bedded sand. more or less perpendicular to the main accretion surfaces oriented ripple bedding indicates deposition by lateral accretion of a point bar. location: 17.9 km ne eirunep (s 063251.1/w 0694239.4, altitude: 107 m), left cutbank of the juru river (fig. 4). description: the lower 5 m of the outcrop are formed by (section 1): (1) greenish grey, mottled clay with ferran cutans and cm-sized carbonate nodules (fig. 5a); (2) dark grey, massive or indistinctly laminated clay with cm-sized carbonate concretions (fig. 5b), thin, pyrite-rich coaly layers, plant fragments, root traces and vertebrate remains (crocodiles, turtles); (3) greenish yellowish, mottled clay with ferran cutans (passes gradually into bed 4); (4) violet-red green-grey, mottled clay/paleosol (fig. 5c); and (5) green grey, mottled clay with vertebrate remains (reptiles) on top. a trough cross bedded sandy layer with coaly interlayers in the se part of the site mentioned by paz et al. (in press) was not exposed during our observation (section 4). in the nw part (section 1), above layer 5, ripple bedded fine sand with intraclasts (mud pebbles) at its base is recorded, which contains bivalves and coalified wood remains (6). laminated sand alternations (7) and massive sandy clay, which is rich in molluscs in its lower and upper part (8). up-section (9), an alternation of clay and coal layers, rich in molluscs (i.e., mycetopoda sp.), as well as a thinly bedded succession of laminated sand and thin clay interlayers (10) follow. towards the se (section 24) an up to 2 m thick, large scale cross bedded sandy unit is developed (fig. occasionally, intraclasts and vertebrate fragments are found at the base of this element or on accretion surfaces, which dip around ne. the dip of ripple foresets is approximately perpendicular oriented to these surfaces (sw). this unit is overlain by a layer of calcareous nodules (11; section 3) and laminated clay (silt)coal alternations with bivalve and vertebrate remains (13 and 14=equivalent to 9). bed 12 (plastic, strongly weathered clay) is an equivalent of layer 10. microfossil contents: samples mn1 and mn2 lack microfossils, in mn8a and mn8b only scattered valves of cypria sp. and heterocypris? mn9 and mn 14 yielded many fish, gastropod and bivalve (sphaeriids) remains as well as abundant ostracod valves (cyprideis spp.). earlier reports on fossil material: celestino and ramos (2007; see also wesselingh and ramos, 2010) documented the following ostracod taxa: cyprideis graciosa, cyprideis lacrimata, cyprideis longispina, cyprideis machadoi, cyprideis pebasae, cyprideis olivencai, cyprideis sp. 1 and 2. further findings (plants, fishes, reptiles) are mentioned in del arco et al. the later authors also reported the occurrence of cyathecidites annulatus, echitricolporites spinosus and grimsdalea magnaclavata pollen. greenish to yellowish and dark grey colouration might indicate more or less water-logged conditions, whereas reddish colours may hint to rather well-drained, oxidizing environments. however, diagenetic alteration can hamper such an interpretation significantly (retallack, 2001). layer 2 is less affected by pedogenic processes (faint lamination, coaly layers, plant and reptile fragments) and perhaps represents floodplain lake/backswamp deposits. the paleosols (overbank fines; 15) are overlain by a sandy channel fill (point bar) with lag deposits at its base and repeatedly occurring intraclasts at its lateral accretion surfaces (channel depth>2 m based on the preserved height of this unit). in the nw part of morada nova layers 68 represent an abandoned channel fill, which could be the fill of a subordinate channel or the fill of a chute channel. up-section, the influx of the active channel decreased and a floodplain lake developed (9, 13, 14), which enabled the establishment of plentiful ostracod and aquatic molluscs faunas. the pond itself or its surroundings were richly vegetated (coaly layers) and settled by semi-aquatic reptiles. finally, enhanced input of sandy matter points to an increasing influence of the channel, probably due to crevasse splays. (s 063140.8/w 0693952.0; altitude: 106 m), left cutbank of the juru river (fig. description: section 1 (w part) starts with a 1.6 m thick succession of laminated, ripple and cross bedded silty sand with coaly intercalations (layers 16/1). up-section (714/1), a 2.5 m thick sequence of ripple bedded silty fine sand layers with commonly erosive basal boundaries (incl. convolute bedding and m-thick concretions are frequent in its upper part (1114/1). silty clay; 1520/1) and comprise coal and mollusc-rich pelitic (16/1, 19/1) and coaly layers (18/1) as well as carbonate-cemented clay (20/1). layer 21/1 (0.75 m thick) is composed of greenish violet, mottled or poorly laminated clay with scattered bivalve shells. intercalated are several layers (10 cm-thick), which consist of 12 mm large, rounded clay clasts (fig. it is overlain by 1 m of massive clay with abundant mollusc (e.g., sheppardiconcha septencincta; fig. leaves; 22/1) and a>0.5 m thick (weathered) coaly layer (23/1) with mollusc fragments (e.g., anodontites? section 2 (e part; 120 m downstream of section 2) starts with>0.35 m thick, laminated fine sand with coaly interlayers (layer 1/2). it is cut by a 4 m wide and 0.8 m deep channel fill (25/2; fining-upward, from base to top: laminated, coal-rich fine sand with rare mollusc and plant remains; ripple bedded fine sand, rich in plant fragments; massive clay, rich in bivalves (sphaeriids), several wood remains; fig. this channel fill is topped by a 0.5 m thick, massive poorly laminated clay with thin fine sand interlayers (6/2) and 0.2 m of massive, partly indurated (cemented) clay (7/2). coalified wood remains are rare; bivalves (sphaeriids) are abundant (especially upper part of 6/2). layer 6/2 and 7/2 taper off towards the w. up-section (1 m thickness), follow massive, silty sandy clays (89/2) and ripple bedded silty fine sand with silt interlayers (1014/2). especially layer 8/2 is rich in molluscs (sphaeriids), 1314/2 contain coaly fragments; clay intraclasts are observed at the base of 13/2. these beds are topped by 2.7 m of mottled silty fine sand with coaly fragments in the lower 0.3 m (15/2).. layer 16/2 (> 1.2 m thick) comprises coal coaly fine sand finely laminated clay alternations (fig. 7f) and is rich in bivalves (sphaeriids) in the lowermost 57 cm. whereas samples aq5/1 and aq6/1 contain only rare ostracod remains, aq16/1 yields, beside fish fragments, some valves of cytheridella sp. and darwinulids, accompanied by rare specimens of cypria sp., cyprideis spp. and, darwinulids, cypria sp., ilyocypridids) and fish bones. several (aq22a/1) respectively rare (aq22b/1) cyprideis spp. valves are recorded in the samples of section 1 above, together with fish remains and gastropods (aq22b/1). sample aq3/2 of section 2 delivered only rarely cyprideis spp. and some fish remains. in the samples aq 5-6-8-13/2 plentiful ostracod faunas (cytheridella sp., ilyocyprinids, darwinulids, cyprideis spp. are documented along with fish remains and sphaeriid bivalve shells (aq8/2 and aq13/2). aq15/2 and aq16/2 are almost barren of ostracods but contain fish and bivalve (sphaeriids) fossils. earlier reports on fossil material: celestino and ramos (2007; see also wesselingh and ramos, 2010) recorded the following ostracod taxa: alicenula (darwinula) fragilis, cypria aqualica, c. longispina, c. pebasae, cyprideis sp. 3, cytheridella purperae, heterocypris? (2006b) re-examined mollusc material studied by roxo (1937) from aquidab and described: ampullariidae sp. sheppardiconcha septemcincta. additional findings (including vertebrates and plants) are mentioned by del arco et al. the basal, sandy silty layers (16/1=1/2) possibly represent crevasse splay deposits, which are followed by a series of crevasse splay and/or crevasse channel sediments (714/1 respectively 25/2). alternatively, these layers could represent channel deposits of an avulsive river arm. in section 2 a subordinate abandoned channel fill (floodplain pond; 67/2) is developed. afterwards (1523/1 and 816/2) the influx of crevassing respectively the active channel successively decreased and led to the formation of a floodplain lake, which was only influenced by flash floods in more proximal settings (section 2; e.g., 12/2). aquatic faunas (bivalves, ostracods, fishes) and semi-aquatic crocodilians inhabited that lake, which was surrounded by a densely vegetated backswamp. these beds display a mottled appearance and contain (21/1) layers of crumb peds (clast-supported layers of rounded but not interlocked tiny mud balls), which indicate bioturbation due to invertebrate activity (retallack, 2001). finally, the lake became replaced by a swampy environment (23/1 and 16/2). location: 27.4 km ne eirunep (s 063122.0/w 0693542.8; altitude: 105 m), left cutbank of the juru river (fig. description: at the base>2 m of slightly southwards inclined (< 10) rippled bedded silty fine sand (layer 1) are followed by 2.7 m thick violet-brown (2) and yellowish (3) mottled paleosols with calcareous nodules (between 2 and 3 a pedogenically overprinted clay plug is intercalated). layers 13 are cut by a channel, whose fill starts (4) with>1.5 m thick, massive poorly laminated silty clay with gastropods, rare bivalves and plant (leaves) remains. bed 4 grades into a 0.8 m thick, brownish-grey, blocky structured silty clay (5) with a calcareous crust on its top. it is overlain by 2.4 m of violet-brown, mottled clay (6) with intercalated layers of calcareous nodules. strata 13 as well as the channel fill (46) are topped by a 0.4 m thick alternation of laminated silty clay and laminated or rippled bedded fine medium sand with a high amount of plant detritus (also wood remains) and abundant vertebrates (turtles; 7). the badly exposed, 7.2 m thick beds up-section (8) consist of cross and ripple bedded fine sand with intercalations of clayey lenses (2 m lateral extension, 20 cm thickness), which are formed by clay intraclasts (mud pebbles, 1 cm diameter). repeatedly coaly layers are intercalated and rarely vertebrate (crocodiles) remains were found in the intraclast conglomerate. layer 9 represents a 1.2 m thick alternation of horizontally or ripple bedded fine the topmost 1.2 m thick layer 10 consists of ripple bedded silty fine sand and silty clay alternations, which form cm-thick, fining-upward couplets. microfossil contents: sample re4 yielded only a few valves of cyprideis spp. as well as some fish and gastropod remains. interpretation: layer 1 could represent a crevasse splay or levee deposit, which is overlain by pedogenically altered floodplain fines (23) with intercalated minor channel fills. these overbank deposits are cut by a channel (? 20 m wide and 5 m deep), which became filled by pelitic, afterwards pedogenically overprinted sediments. the rare (a matter of preservation ?) mollusc and ostracod record indicates at least at the beginning (4) aquatic life in the abandoned channel. above, a crevasse splay (7) points to rising proximity of the active channel. large wood remains and vertebrate fragments were accumulated by this splay. the sandy succession up-section (8) can only be attributed to a sandy bedform sensu miall (1996) due to insufficient outcrop conditions. intercalated shallow scours, filled up with mud balls indicate deposition of reworked material and variations of the flow regime. the more or less rhythmically stratified, sandy-pelitic alternations at the top of remanso (910) mark a shift towards overbank environment and might represent a bar-top assemblage. location: 17.5 km se eirunep (s 064923/w 0694704, altitude: 117 m), left cutbank of the tarauac river (fig. description: the basal 1.7 m (layers 13) consist of massive or laminated pelite- and fine sand with rare bivalve remains. they are topped by 0.2 m of reddish-brown, massive clay (4). the sandy silty layers 59 display a general coarsening-upward trend and contain only rarely bivalve fragments. up-section (10), a 0.25 m thick intraclast conglomerate (mudstone pebbles) with abundant mollusc (e.g., ampullariidae? this stratum is overlain by thin sand and clay layers (1117) with abundant mollusc remains (12, 14, 16, lower part of 17). layer 14 is rich in coal fragments (wood, leaves) at its top. the 2.57 m thick succession above (1823) starts with thin, ripple bedded fine sand beds (1819), followed by massive silty/sandy clay with coaly fragments (20) and cm-thick alternations of fine sandy silt and silty fine sand (21; forming couplets of small-scale fining-upward cycles). beds 22 and 23 comprise alternations of massive, fine sandy silt and ripple bedded, silty fine sand. 24 consists of 10 cm-thick alternations of indistinctly ripple bedded silty fine sand and massive or poorly laminated pelite. the upper part (1.5 m) is mottled and primary sedimentary structures are obscured due to pedogenic processes. these samples contain several fish remains and in to12 rare characean gyrogonites were found additionally. the samples to20 and to 23 delivered only rare ostracod (cypria sp. earlier reports of fossil material and facies: ramos (2006) mentioned the following ostracod taxa: c. aqualica, c. graciosa, c. lacrimata, c. longispina, c. pebasae, c. purperae, cytheridella sp., darwinula fragilis as well as additional vertebrate and crab findings. interpretation: the lowermost sandy-pelitic layers (14) might be deposited in a floodplain pond/lake, which was influenced by crevasse splays. the coarsening-upward trend of layers 59 hints to a progressive influx of a crevasse channel (layer 10). it remains unclear if the ostracods of this layer are allochthonous or if they are incorporated in that layer due to subaquatic inflow of the crevasse splay (crevasse delta) into a floodplain pond/lake. up-section (1124), plentiful ostracod and mollusc faunas (especially in the lower part: 1117; compare ramos, 2006) indicate the return to an aquatic environment and the abandonment of the crevasse channel. small-scale fining-upward alternations of ripple bedded sand and pelite document individual flooding events or surges. location: 22.1 km sse eirunep (s 065218.3/w 0694705.1; altitude: 120 m), left cutbank of the tarauac river (fig. description: layers 16 represent a 3.2 m thick succession of greenish (1), reddish (2), yellowish (3, 5, 6) or violet grey (4) coloured, mottled, frequently calcareous nodules-bearing paleosols. in layer 6 vertebrate remains (crocodile teeth) and gastropods were found. above follows a 1.1 m thick, indistinctly bedded clay to fine sand (7), which contains in its lower half coaly plant fragments and becomes towards the top and laterally progressively mottled (paleosol). the 0.65 m thick sediment column up-section (811) is composed of massive clay (8), ripple bedded fine sand (9), laminated silty fine sand to silt (10) and fine sand (11). each of these layers displays a slightly erosive base and an internal fining-upward trend. the beds 8, 9 and 11 each start with a basal layer of concretionary mud clasts in which (layer 8 and 11) abundant vertebrate remains were found. layer 12 is formed by 1.2 m thick, ripple bedded fine sand with cm-thick, laminated silty clay interlayers. up-section a 0.7 m thick, orange-grey, mottled paleosol with mud clasts and rare vertebrate remains at its base (13) as well as a>1.5 m thick, violet-purple coloured, mottled paleosol are developed (14). microfossil contents: the samples ba7a and ba7b contained several ostracod valves (mainly cyprideis spp., some specimens of cypria sp. interpretation: the basal layers (16) represent a succession of paleosols, which could be seriously altered floodplain deposits. due to the occurrence of ostracods, probably bed 7 was deposited in a floodplain lake environment. afterwards, a series of crevasse splay (maybe partly also crevasse channel) deposits follows (813), which is topped by pedogenically overprinted floodplain fines (14). the sedimentary record of the investigated outcrops documents vertically as well as laterally highly variable fine-grained clastic successions (predominantly clay fine sand, along with fine medium sand). based on the lithofacies and, as far as possible, lateral observations, these sediments represent fluvial deposition within active channels as well as in overbank environments. macroforms, generated within the active channel include lateral accretion deposits (sandy point bars; figs. 2 and 4) and undifferentiated sandy bedforms (stacked dune fields ?; fig. 8), which are probably partly intersected by chute channels or are overlain by overbank fines. these comprise pelite sand-alternations of levee, crevasse splay and crevasse channel deposits. frequently, a lag of mud pebbles and vertebrate fragments is found at the base of crevasse channels/splays. in part, some splays may have entered floodplain ponds/lakes via crevasse deltas (e.g., figs. 6 and 9). abandoned, fine-grained channel fills (mud plugs) were observed (fig. decimetre-thick, clay silt beds with rich ostracod and mollusc faunas indicate the presence of short-lived, shallow ponds or lakes within the floodplain (floodbasin; e.g., fig. 6) point to partly swampy, poorly-drained conditions and a densely vegetated floodplain, which is, however, influenced by high clastic input. intensively mottled paleosols with root casts and calcareous nodules are ubiquitous (e.g., fig. differences in colour and occasional calcic horizons may reflect diverging water-logging due to e.g., local topography and/or seasonal fluctuations in discharge or climate (kaandorp et al. nevertheless, diagenesis may have altered significantly primary features and more specific investigations are required (retallack, 2001). we are aware that restricted outcrop conditions and the limited areal and stratigraphical extent (30 m stratigraphical thickness) of the current investigation hamper conclusions about the nature of fluvial style. reconstructions of fluvial geomorphology, based solely on lithofacies assemblages and fragmentary known fluvial elements, remain to some degree tentative (miall, 1996; bridge, 2003). however, it seems obvious that we are dealing with a suspended load river system due to the high amount of overbank fines, probably within a tropical to subtropical wet dry climate (kaandorp et al., 2005; latrubesse et al. we observed: (a) a large proportion of overbank deposits, (b) laterally extensive, heterogeneous, fine-grained avulsion deposits (including crevasse splays), (c) predominantly sandy channel deposits, which change laterally and vertically rapidly into overbank deposits as well as (d) lacustrine and coaly deposits. some sandy point bars (of secondary channels ?) indicate subordinate lateral sediment accretion. these features are not exclusive to anastomosing river deposits, however, they largely coincide with the standard model of an anastomosing river sensu miall (1996) as well as with characteristics of such a system as reviewed by makaske (2001; compare also smith, 1983). the general setting (subsiding foreland basin, extensive sediment input from the andes, possibly incompletely framed by tectonically active basement highs towards the east) as well as a wet dry seasonal climate (seasonal floods, channel banks stabilized by vegetation) would provide the conditions for anastomosing river systems (smith, 1983; nanson and knighton, 1996; makaske, 2001; latrubesse et al. (late miocene) in the state of acre (sw brazil) led to similar assumptions (latrubesse et al. these authors proposed a megafan system, which originates in the andes and encompass a complex mosaic of avulsive rivers and associated wetlands (including abandoned channels, floodplain lakes, floodbasin deltas, backswamps and well-drained floodplains; see also wilkinson et al. this system is suggested to reach to the east at least as far as 67 w (iquitos arch sensu del arco et al., 1977) and probably also beyond (purus arch; latrubesse et al., 1997, 2010; fig according to that, the sections around eirunep were located in a more distal position, which could be the reason for a more pronounced anastomosing-anabranching pattern due to a lower gradient (compare also interpretations of the late miocene tariquia formation, southern bolivia; uba et al. (2007, 2010) compared the depositional environment of the late miocene solimes fm. in acre with i.e., the quaternary megafans of the chaco plain and the pantanal wetland (iriondo, 1993; horton and decelles, 2001 the mollusc fauna, recorded from floodplain pond/lake deposits, is dominated by sphaeriids and the pachychilid sheppardiconcha (fig. 7b) and indicates freshwater conditions within a fluvio-lacustrine environment (wesselingh et al., 2006b; wesselingh and ramos, 2010). typical freshwater ostracods (darwinulids, ilyocyprinids, cypria, cytheridella) are associated with diverse species of cyprideis. this genus is a holoeuryhaline taxon, able to cope with fluctuating salinities, aberrant water chemistries, variable temperatures and oxygenation (e.g., meisch, 2000; keyser, 2005). as it could adapt to freshwater conditions (e.g., lake tanganyika; wouters and martens, 2001) and co-occurs here with exclusively freshwater taxa, there is no constraint to conclude a brackish water environment (ramos, 2006). preliminary stable isotope data (o, c) measured on cyprideis valves from aquidab, morada nova and torre da lua, yielded very negative values (o: 5.7 to 9.7, c: 10.3 to 12.5), which exclude any marine influx. there is still intensive debate on definition and timing of palynological biozonation concepts (e.g., hoorn, 1993, 1994; latrubesse et al., 2007, 2010; jaramillo et al., 2010; silva-caminha et al., however, palynostratigraphy remains a cornerstone for correlation across amazonia up to now and provides at least a rough stratigraphical framework. (2006a) are highly linked to the concept established by hoorn (1993). based on the presence of some index taxa (c. annulatus, e. spinosus, g. magnaclavata) a late miocene age (probably asteraceae zone sensu lorente, 1986) is proposed by paz et al. (in press) for the outcrops morada nova and torre da lua recently. the vertebrate fauna from torre da lua (ramos, 2006) is related to the so-called acre fauna, which is one of the best documented faunas of northern south america (cozzuol, 2006; latrubesse et al. the acre vertebrate assemblage displays significant affinities with the mesopotamian faunas of argentina and uruguay as well as with the urumaco assemblages (venezuela) and is dated to the late miocene (huayquerian south american land mammal age; cozzuol, 2006; latrubesse et al. is still poor and has to be extended by later works (wesselingh and ramos, 2010; see roxo, 1937; wesselingh et al., 2006b; wesselingh, 2008, and outcrop descriptions herein). from a stratigraphical point of view the mollusc faunas of aquidab and torre da lua (2006b) to be of late miocene origin and post-date the pebas fauna (compare celestino and ramos, 2007; wesselingh, 2008; wesselingh and ramos, 2010, which suggested a late middle miocene age based on the ostracod record). a late miocene age seems to be in agreement with the lack of typical endemic pebasian molluscs (wesselingh and ramos, 2010). the recorded cyprideis-species from aquidab, morada nova and torre da lua (ramos, 2006; celestino and ramos, 2007; wesselingh and ramos, 2010) have generally long ranges according to muoz-torres et al. (1998, 2006; see also whatley et al., 1998). the occurrence of c. lacrimata and c. pebasae might be indicative for an age not younger than the (early) late miocene. nonetheless, due to some contradictions (e.g., c. lacrimata is supposed to appear first after the last appearance of c. longispina but both are co-occurring at morada nova and torre da lua) and the lack of key taxa, the current ostracod zonation needs some adjustment and is not further stressed here. however, to date, a late miocene age seems most plausible for all outcrops described herein by considering the palaeontological record. moreover, this fits well to the palaeogeographic context of this region as proposed by latrubesse et al. sedimentological observations derived from outcrops around eirunep (south-western part of the state of amazonia) document various subenvironments of a fluvial system (upper part of the solimes formation, late miocene). beside sandy channel deposits, the main part comprises overbank sediments of levees, crevasse splays/channels/deltas, abandoned channels, backswamps and floodplain paleosols. based on the lithofacies and the general geological setting, this system can be possibly related to an anastomosing river style. there is not any indication for a long-lived lake (lake pebas) or any marine influx in this region during the late miocene. (1997, 2007, 2010; compare also cozzuol, 2006; westaway, 2006).

in miocene times a vast wetland existed in western amazonia. whereas the general development of this amazing ecosystem is well established, many questions remain open on sedimentary environments, stratigraphical correlations as well as its palaeogeographical configuration. several outcrops located in a barely studied region around eirunep (sw amazonas state, brazil) were investigated to obtain basic sedimentological data. the observed deposits belong to the upper part of the solimes formation and are biostratigraphically dated to the late miocene. vertically as well as laterally highly variable fine-grained clastic successions were recorded. based on the lithofacies assemblages, these sediments represent fluvial deposits, possibly of an anastomosing river system. sand bodies formed within active channels and dominant overbank fines are described (levees, crevasse splays/channels/deltas, abandoned channels, backswamps, floodplain paleosols). lacustrine environments are restricted to local floodplain ponds/lakes. the mollusc and ostracod content as well as very light 18o and 13c values, measured on ostracod valves, refer to exclusively freshwater conditions. based on palaeontological and geological results the existence of a long-lived lake (lake pebas) or any influx of marine waters can be excluded for that region during the late miocene.

PMC4599590

pubmed-17

with increasing availability, widened indications, and technical refinements of computed tomography (ct) and magnetic resonance imaging (mri), the number of incidentally discovered adrenal lesions, such as adrenal incidentalomas and incidental adrenal enlargement, is increasing. in a recent study by tang et al., among a total of 564 eligible ct studies (patients undergoing ct without prior known malignancy, trauma, or endocrine disease), adrenal hyperplasia was detected in 64 cases, giving a prevalence of 11.3%. this indicated that incidental adrenal enlargement had a significant prevalence and has become a common clinical problem. it should be emphasized that the term incidental adrenal enlargement represents the way the lesion was detected (incidentally), rather than the etiology or diagnosis. it is a common term for a variety of adrenal disorders, but its cause must be properly assessed so that patients needing treatment, such as those with hormone hypersecretion or malignant disease, can receive appropriate care. however, there is a lack of literature on functional status and its follow-up to provide comprehensive insight to these findings. patients with incidental adrenal enlargement were evaluated in a tertiary referral hospital with endocrinological departments in china. this study aimed to determine the primary clinical presentation that most frequently leads to the discovery of adrenal enlargement, to evaluate clinical characteristics and functional status of these patients, and to analyze risk factors for functional lesions. this retrospective study included 578 patients with adrenal imaging features showing adrenal enlargement who were hospitalized at the department of endocrinology in pla general hospital (beijing, china) between january 1993 and july 2013. data retrieved included patient demographics, final functional diagnosis, adrenal imaging features, and concomitant diseases. patients were classified as having incidental adrenal enlargement when abdominal imaging was performed for indications unrelated to adrenal disease. patients with diseases known to cause adrenal enlargement, such as known endocrine disorders which could affect adrenal size, trauma, and underlying malignancy, were excluded. among all enrolled patients, 78 presented with incidental adrenal enlargement. the ct imaging technique used was not standardized due to the various clinical indications. however, to be included in their entirety on a maximum of 5 mm section thickness, the upper limit of normal was set as 10 mm for the body of the gland and 5 mm for each limb as documented by vincent et al.. the type of enlargement, based on subjective evaluation of the adrenal glands, was recorded as either smooth or nodular. nodular enlargement was diagnosed if the adrenal gland had an irregular contour, contained nodules, and had normal adrenal tissue interspersed between the nodules. smooth enlargement was defined as enlargement of the gland with a smooth contour and no measureable or diffuse nodules. after obtaining patient history and physical examination, all patients underwent biochemical evaluation to assess their functional status. patients with elevated 24-hour urine-free cortisol level (2 times) or those in whom plasma cortisol levels did not decrease after an overnight low-dose dexamethasone test (1 mg dst; cutoff 50 patients with an aldosterone-rennin ratio (arr)>20 underwent any 1 of 3 confirmatory tests (saline infusion, captopril challenge, or postural stimulation) to confirm or exclude definitively primary hyperaldosteronism (pa). categorical data such as gender and clinical/radiologic features were compared using -test or fisher's exact test. variables that resulted in a p<0.05 in the univariate analyses were entered into logistic regression analysis to assess the risk factors of functional lesions. the hospital ethics committee approved this study, and written informed consent was obtained from all patients or their parents. of 578 patients with adrenal enlargement, 78 cases (13.49%) were detected incidentally. every 2 years, the numbers of total cases were 17, 11, 14, 24, 33, 31, 54, 55, 114, and 225, respectively. the numbers of incidental adrenal enlargement cases were 0, 0, 0, 1, 1, 2, 5, 4, 16, and 49, respectively. in addition, the proportion of incidental adrenal enlargement gradually increased (0, 0, 0, 4.17%, 3.03%, 7.32%, 9.26%, 7.27%, 14.04%, and 21.33%). 39 cases had unilateral enlargement on the left side and 3 on the right side, and the remaining 36 were bilateral enlargement. as shown in table 1, routine medical checkup was found to have the greatest chance (43.59%) of revealing clinical onsets leading to the discovery of adrenal enlargement. predominant complaints included low back pain (10.26%) and abdominal pain (3.85%). in addition, there were 30 (38.46%) cases in which the lesions were incidentally detected during hospitalization for underlying diseases, such as diabetes mellitus, hypertension, and coronary heart disease, among others. biochemical and functional evaluation revealed that 54 (69.23%) cases were nonfunctional and 12 (15.38%) were subclinical cushing syndrome (scs); among these patients, 10 cases were diagnosed as aimah, the other 2 were diagnosed as adenomas, and they were all confirmed by pathology results. primary hyperaldosteronism 6 (7.69%), metastatic 1 (1.28%), the primary cancer was gastric cancer. there were 5 patients (6.41%) whose functional status remained unclear because of failure to finish the functional evaluation (figure 2). nodular adrenal enlargement (or 7.306; 95% ci, 1.72728.667; p=0.006) was the risk factor for functional lesions. as outlined above, incidental adrenal enlargement is detected with increasing frequency, most likely due to widespread increase in cross-sectional imaging, and is gradually emerging as a common clinical problem. our study shows that the proportion of incidental adrenal enlargement has gradually increased by year. mean age at diagnosis was 50.32 years, which is in line with other incidentally detected adrenal findings, namely, adrenal incidentaloma. the increasing age of the general population and a research trend towards more advanced investigations in the elderly population may be contributing to the high detection rate in this age group. our results indicate that, for the elderly patients, it is essential to place emphasis on these incidental findings. 's study indicated that, of the total 64 patients, of which 40 (63%) were men and 24 (37%) were women, 43 (67%) cases were bilateral enlargement and 21 (33%) cases were unilateral. in addition, smooth enlargement was more common, in 53 (83%) cases, and together these statistics reflect the likelihood that adrenal enlargement will be bilateral, smooth, and found in men. however, our study did not show this tendency, likely because the research goals and thus, study populations, differed between the 2 studies. 's study aimed to explore prevalence, while the present study aimed to evaluate functional status. in addition, patients enrolled in our study were hospitalized at the department of endocrinology. it should be noted that admitting was more or less selective, especially in tertiary referral hospitals, and that economic considerations in parts of china were still a problem. clinically, upon discovery of incidental adrenal enlargement, 2 issues arise: functionality and malignancy. in the relevant literature [48], adrenal enlargement can result from endocrine disorders, such as adrenocorticotropic hormone- (acth-) dependent or independent cushing syndrome, pa, multiple endocrine neoplasia type 1 (men-1), and congenital adrenal hyperplasia. other potential causes include nonfunctional lesions, defined as a radiographic adrenal enlargement without clinical or biochemical manifestations, inflammation, neoplastic processes, obesity, or depression. 12 patients were found to have subclinical cushing syndrome (originated from the adrenal gland) and 6 patients were diagnosed with pa. however, the variety of disease spectrum in the study was only moderate, perhaps due, in part, to the limited number of included cases. it is important to note that even though reported prevalence was up to 11.3%, patient referrals to endocrinologists are relatively rare. this is likely related to the poor radiological awareness of this issue and its potential clinical significance. the clinical significance of lesion location and patient gender is smaller. in the present study, acth-independent macronodular hyperplasia (aimah) and primary pigmented nodular adrenal hyperplasia often manifest as adrenal hyperplasia. the clinical features of aimah tended to be atypical. nodules usually distorted and completely obscured the normal adrenal glands and were characteristically ginger-like. as for pa, adrenal glands affected by idiopathic hyperaldosteronism (iha) may be normal on the ct scan or show nodular changes, and small aldosterone producing adenoma (apas) may be interpreted incorrectly by the radiologists as iha on the basis of ct findings of bilateral nodularity or normal-appearing adrenal glands [1013]. thus, it is a simple matter to explain why nodular enlargement could be a predictive factor of functional lesions. meanwhile, this also suggests that if incidentally detected lesions were nodular enlargements, evaluating its functional status should be a priority. in addition, the present study suggests that lesions on the left side were likely to be nonfunctional. in addition, there was only one patient with malignant lesion in the present study, and thus we were unable to analyze incidence of malignancy. incidental adrenal enlargement is a frequent radiographic finding and it is accompanied by diverse clinical factors that require proper diagnostic evaluation and management. in functional evaluation ,

aim. to investigate incidental adrenal enlargement clinical characteristics and functional status and analyze functional lesion risk factors. materials and methods. this retrospective study included 578 patients with adrenal imaging features showing enlargement. incidental adrenal enlargement cases (78) were considered eligible. demographics, functional diagnosis, adrenal imaging features, and concomitant diseases were analyzed. results. the number of adrenal enlargements and proportion of incidental adrenal enlargement increased each year. mean patient age was 50.32 years. thirty-nine cases had unilateral enlargement on the left side and 3 on the right side; 36 had bilateral enlargement. routine medical checkup was found to have the greatest chance (43.59%) of revealing clinical onsets leading to discovery. biochemical and functional evaluation revealed 54 (69.23%) cases of nonfunctional lesions, 12 (15.38%) of subclinical cushing syndrome, 6 (7.69%) of primary hyperaldosteronism, 1 (1.28%) of metastasis, and 5 (6.41%) of unknown functional status. nodular adrenal enlargement (or, 7.306; 95% ci, 1.72728.667; p=0.006) was a risk factor for functional lesions. age and lesion location were not significant factors. conclusion. incidental adrenal enlargement is a frequent radiographic finding and is accompanied by diverse clinical factors that require proper evaluation and management. nodular adrenal enlargement was a risk factor.

PMC4369954

pubmed-18

pursuing the goal of improved health literacy requires more alliances between health and education sectors to improve literacy levels in the population. it is important for health educators to know about knowledge transfer to meet their needs in transferring their knowledge to public. knowledge transfer, which means the synthesis, exchange and ethically application of knowledge within a complex system of relationship among researchers and users, has become one of the recent priorities in research centers. the word the method of knowledge transfer, apart from its characteristics, requires active interactions between researchers and users. caplan proposes the two- communities theory indicating a gap between researchers and policymakers, whereas these days another gap has been formed between researchers and other users. in addition to illiteracy in developing countries that hampers effective health education, there are multitudes of non-medical specialists who do not have enough information about daily health affairs, and this lack is more remarkable in countries that welcome immigrants. immigrants often have significant language and health literacy difficulties, which are further exacerbated by cultural barriers. alongside with sophisticated methods like computer and internet, using simple methods help us bring to achieve our health goals[68]. also, the lack of health literacy needs more attention when it is manifested in caregivers. it is believed that some of the undesirable health outcomes in children are because of inadequate health knowledge among caregivers. in the usa, therefore researchers have considered different methods to come up with this deficiency by having children to cooperate in their health programs. a research group in india endeavored to transfer knowledge on leprosy in cooperation with children and informed their parents through them. jacob et al (1994) started conducting a similar research that yields promising results. rimal and flora (1998) express that parental dietary behavior is partially affected by children. the findings from studies in this field have encouraged other experts to apply similar methods to conduct their research projects, allahverdipour and bashirian, onyango-ouma and mwangagain from the influence of children in order to teach different parts of their society[1316]. despite the researcher's trends in using novel methods in this field, none of them have used childish poem as a medium for knowledge transferring. in this study, we try to raise families health knowledge about infectious diseases by a method that uses children as health agents. infectious diseases are still the leading cause of mortality in children less than 60 months in developing countries. juvenile age is accompanied by learning childish poems, which can be sung contnuously at home and potentially become as part of both children's and parents memories. we decided to transfer health knowledge to families through altering this information to childish poems and teaching children at kindergartens. in this method the outcome of transferring health knowledge through childish poems has been assessed in this study. in this study, we use the method of interventional pre and post series to conduct our research. the proposal of this non-invasive project was approved by the research committee of hamadan medical university in january 2009. it was also accepted by hamadan welfare organization, which is the responsible organization of kindergartens in hamadan. our methodology is briefly explained as follows. at first, a sub specialist in pediatric infectious diseases provides seven short texts about health and common pediatric infectious diseases prevention. then, a poet transfers these conceptions to childish poems for the first time (appendix 1&2). in the process of preparing an accurate questionnaire, at first 30 questions with three choices of, false and do not know were made, but 24 items of those are confirmed as valid questions by two experts of health education and infectious diseases. to assess the reliability of the questionnaire a pilot study on a 40 participant sample was performed and cronbach's alpha of the questionnaire was determined as 83 percent. in addition, we defined knowledge mark (km) as a parent mark in both the pre-test and/or post-tests. among 35 kindergartens in hamadan, we selected 7 kindergartens by simple random method. the parents (either father or mother, the one who is responsible to take her/his child) of all five to six year old children of the selected kindergartens are asked to participate in the survey and a verbal consent is obtained from those who accepted. we excluded the following children from our sample: those whose parents are physicians, nurses, health-care workers, and those children whose parents work at the kindergarten (exclusion criteria). we asked the parents of the sample survey to participate in a pretest by answering to a questionnaire when they are in the kindergartens. also, they were asked to come to the kindergartens personally one more time when it is required (to answer to the post test). then, we asked our kindergarten tutors to teach children seven musical poems about hydatid cyst, antibiotic misuse, botulinum toxin in home-canned foods, dysentery, the importance of sixth tooth, brucellosis and tetanus. in this step, children were not allowed to take home these texts, but they were asked to sing the poems at home and want their parents to re-write the poems on a paper and give them the papers to take to the kindergarten. the teaching stage took between three to four months. during the next stage, parents took a post-test survey with the same 24 questions. parents of 115 kids participated in the pre-test and 103 of them completed the post-test. statistical analysis is done on 103 who completed both tests by paired t-test. the sample data consists of 103 parents who provided solutions to both the pre- and post-test question survey. of the participants 77 were female and 26 male. from the level of education point of view, 19 were below high school diploma, 45 had high school diploma, 8 had associate degree, 27 had ba, 3 had ma, and 1 had phd. the results of the survey show a significant difference between the correct answers in both pre-test and post-test stages (fig. 1). the frequency of correct answers to the tests table 1 shows the mean of the correct answers in the pre-test stage is 59.22 while the same figure for the post-test step is 81 (p<0.001). in addition, we define knowledge mark (km) as a parent mark in both the pre-test and/or post-test stages. the mean km shows a significant increase (5.01) from 13.62 in the pre-test to 18.63 in the post-test (table 2). mean percent of correct answers to pre-test and post-test categories analyzed by paired t-test sd: standard deviation mean of knowledge mark in pre-test and post test analysed by paired t-test the difference of km promotion among females and males was not significant (21.6120.17 in males and 20.331.61 in females, p=0.8). the km was increased to 19.9325.06 among parents who at most had high school diploma and 23.4720.84 among parents with upper educational levels (p=0.5). health has been the concern of poets such as fiona sampson for years, however, these poems never had educational purposes. the results of this study confirm our hypothesis about the role of children in raising family's knowledge on health. the results show that neither gender nor the grade of knowledge degree (below and upper high school diploma or below and upper ba) affect answers. therefore, people with different educational status have received the information similarly implying that this method can be extended to general cases in a straightforward manner. 1 shows that the percentage of correct answers has increased in 23 out of 24 questions. the percent-age of correct answer to post-test has decreased only in one item (item 11) which compares the risk of brucellosis transmission via unpasteurized milk and yogurt. although the term yogurt has not been mentioned directly in the related poem, but there is a hint about boiling mechanism and we expect that the audience discover the answer by logical thinking about the necessitation of boiling milk for preparing yogurt. the decrease in answering is 2 percent which is not significant; however, it shows that it would be better to mention the conceptions directly in these types of poems in order to avoid any misconceptions. moreover, the percentage of correct answers to the questions of selected topics in pre-test and post-test has been compared (table 1) and shows that parents have done better in post-test about all topics except one. the difference of correct answers to the questions about brucellosis does not show a significant increase in post-test. this finding is probably due to higher basic knowledge about brucellosis among parents. in other words, parents have had acceptable information about brucellosis even without our educational program, so we observe lower contrast between pre-test and post-test about brucellosis. it should be taken into consideration that brucellosis is an endemic disease in hamadan and people have been educated in different ways about this disease in recent years. although the lowest contrast between pre-test and post-test correct answers belongs to a question about botulism, the overall assessment shows that parent's knowledge about this topic has been promoted significantly (p<0.001, table 1). the significant difference between the pre-test and post-test km, based on the results of table 2, is the sign of successful knowledge transfer through childish poems. the researcher's method is impressively successful; however, the fact that he mentions that some mothers can not distinguish the semantic difference between information and advice, needs serious attention. in addition, since daughters may misunderstand the information, it would be probable to transfer the misconceived information to their mothers and this fact threats the successfulness of study in applying. while, in our study; firstly, childish poems do not imply sententious, secondly, if the poem is sung wrong, the disturbance in rhythm will be appeared, so the information which is transferred through poems is not at risk of alteration. in evans research, parents get familiar with educated topics with the help of their children in homework assignments designed for asthma management, and are taught about asthma indirectly. the advantage of our study in comparison with evans is that in evans method knowledge transfer brings to bear via homework assignment, while in our study there is no obligation for doing home works, and parents are educated while enjoying their child's singing. on account of the fact that making communication with younger children is not as difficult as with adolescents for parents, we are sure that knowledge transfers within a family through a continuous and friendly communication will work. christensen expressed the importance of children's role in promoting the family health status. in his article, he emphasized on the activities that children can perform to enhance their health and promote their family health situation via the health efforts for themselves. we showed that children, additionally to what christensen mentioned, are able to affect their family's health status directly. they can improve their family's health as little teachers in health. it should be taken into consideration that the limitations of this study was the impact of other media on participant's knowledge, which was not preventable. one of the acceptable results of this survey was publishing the poems as a book for children which was republished and welcomed by public. this study's results suggest that health knowledge transfer to families through childish poems is an applicable method that has many advantages. by applying this joyful, cost-effective and easy-to-use method however, it should be taken into consideration that this method is applicable for families with trainable children, for teaching in larger scales more general ways should be added to this method. moreover, the simplicity of poems and expressing the topics in a less elaborative form are the key factors that affect the successfulness of this way.

objectivethe purpose of this study is to propose an innovative method of knowledge transfer that aims to improve health literacy about pediatric infectious diseases prevention in families. children have an appreciable role in this scheme. methodsthis study is a before and after trial that has been conducted in hamedan in 2009. after changing seven infectious disease topics into childish poems, we selected five kindergartens randomly and taught these poetries to the children. teaching process held after a pretest containing 24 questions that examined 103 of parents about mentioned topics. the same post-test was given after 4 months of teaching process. findingsthe mean of correct answers to the pretest was 59.22% comparable with 81.00% for post-test (p<0.00). gender and knowledge degree could not change the results significantly. assuming one's correct answers to the questions as his/her knowledge mark, the mean of this variable increased to 5.32 by this method. conclusionthis cost-effective and joyful method had successful results in promoting health knowledge. children are able to play an active role in family's health situation. learning within family atmosphere without any obligations makes our scheme a solution for paving the knowledge transferring way.

PMC3533150

pubmed-19

septic arthritis of the shoulder is uncommon in adults. it is a surgical emergency as joint destruction occurs rapidly and can lead to significant morbidity and mortality. a 43 years old male came to our outpatient department with complaints of pain and stiffness of his left shoulder. on examination, his shoulder movements were severely restricted. further evaluation with mri revealed septic arthritis of left gleno-humeral joint for which emergency arthroscopic debridement was done. septic arthritis of shoulder may not present with classical clinical features. hence, a through clinical and radiological evaluation will help us prognosticate and treat accordingly thereby preventing complications like septic shock, osteomyelitis. patients with septic arthritis usually develop moderate to severe joint pain, warmth, tenderness, effusion, restricted active and passive motion, and sometimes redness. we report an unusual presentation of shoulder septic arthritis in a 43 years old man with no other clinical signs and symptoms of classical septic arthritis. a 43-year-old man presented to the orthopaedic outpatient clinic with 2 months history of pain and limited range of motion in his left shoulder. his pain was insidious in onset, mild to moderate in intensity, aggravated by activities and associated with moderate rest and night pain. patient was diagnosed as frozen shoulder at an outside facility and had been given intraarticular depomedrol 40 mg injection for the same 40 days ago with no improvement in his symptoms. he is diabetic and has liver cirrhosis and is on treatment. on physical examination, the skin colour and temperature of the left shoulder were normal, but the shoulder was tender to touch over the anterior joint line. mri was obtained [fig 2] which showed extraosseous soft tissue enhancements around left shoulder with soft tissue abscess in inter muscular planes of supraspinatous, infraspinatous, subscapularis. blood test revealed normal white cell count with normal differential count, crp-13.4 mg/l (normal<5.0 mg the erythrocyte sedimentation rate was raised, at 88 mm/hour (normal range, 0-20 mm/hour) aspiration of shoulder joint was performed and fluid was sent for aerobic and anaerobic culture, afb staining, mycobacterium culture, and mycobacterium genetic testing. arthroscopic lavage of the joint was done and articular fluid sent for repeat aerobic and anaerobic culture. joint visualization was markedly limited because of severe inflammation and fibrinous changes of the joint. the articular cartilages of glenoid and humeral head were completely eroded down to the bone. susceptibility of the isolate was determined with the disk diffusion method and it was susceptible only to colistin. 3) fibrocollagenous and fibrovascular inflammed connective tissue containing proliferated blood vessels and perivascular mixed inflammatory cells and lymphoplasmacytoid cells. based on preoperative and intraoperative culture report, a definitive diagnosis of polymicrobial septic arthritis of the shoulder was established and patient was treated with combination antimicrobial treatment. our patient had muted inflammatory response probably due to immunocompromised state (cirrhosis and diabetes) which is not uncommon. most often it is hematogenous seeding of shoulder joint, however it can also happen after intraarticular steroid injections. septic arthritis of the shoulder is more common in immunocompromised patients and intravenous drug abusers. in this case report, the patient had atypical clinical symptoms and is immunocompromised and his primary source of infection seems to be hematogenous. our experience with this patient highlights the importance of maintaining high index of suspicion for septic arthritis in immunocompromised patients presenting with atypical shoulder pain associated with stiffness. in the setting of suspected septic arthritis, our initial pre-investigation diagnosis was atypical shoulder pain and stiffness of non specific etiology in an immunocompromised patient and infection was part of the differential diagnosis not the only diagnosis. esenwein et al in his report highlighted the importance of early intervention to prevent chondral damage, osteomyelitis and also to prevent systemic spread. various authors have highlighted the importance of early diagnosis and management of septic arthritis failing which could lead to osteomyelitis and septic shock. in our patient, the diagnosis was delayed due to atypical presentation and over reliance on clinical findings at an outside center. we strongly recommend early imaging studies in immunocompromised patients presenting with shoulder pain and practitioners should avoid loosely diagnosing as shoulder pain with associated stiffness as frozen shoulders. klinger et al. did a retrospective study on 21 patients who underwent surgical treatment for septic arthritis of the shoulder joint between 2000 and 2007, and he concluded that patients with symptoms for less than 2 weeks did well with arthroscopic approach and early infection can be managed arthroscopically. our reports show that arthroscopic washout can give good result even after 2 weeks of clinical symptoms if there is no evidence of osteomyelitis. septic arthritis of the shoulder is very often due to hematogenous spread and diagnosis is often clinical. laboratory investigations and imaging studies like mri and usg may be useful in establishing the diagnosis but confirmation is usually by joint aspiration. patients who are immunocompromised and have insidious onset of moderate to severe pain and which fails to respond to trial of conservative treatment should be subjected to either ultrasound or mri instead of x-ray because very often in these group of patients it is primarily a soft tissue pathology like impingement, rc tear, calcific tendinitis or rarely infections and malignancies. we recommend that moderate pain of more than 4 weeks duration with severe stiffness in immunocompromised patient (liver cirrhosis, renal failure, steroid treatment) should be further evaluated with mri or ultrasound in the setting of normal x-ray and should not be loosely diagnosed as frozen shoulder. goldenberg in his report emphasized the role of local as well as systemic factors that predispose patients with cirrhosis to gram-negative bacterial joint infections. malnick also reported a case of spontaneous septic arthritis in a cirhottic patient that was due to e.coli. our study as well as other studies by goldenberg and malnick highlight the importance of including broad spectrum antibiotics with gram negative cover whilst waiting for final culture sensitivity. septic arthritis of shoulder may not present with classic clinical features. a through clinical and radiological evaluation should be executed and treatment initiated promptly to prevent complications like septic shock and osteomyelitis. the case highlights the importance of establishing anatomical and pathological diagnosis using mri in patients with shoulder pain instead of loosely diagnosing them as impingement or frozen shoulder. the case also challenges the practice of routine shoulder depomedrol steroid injection, in the setting of secondary frozen shoulder, atleast in immunocompromised individuals. primary idiopathic frozen shoulder is a rare condition and secondary frozen shoulder cases are often due to underlying shoulder pathology.

introduction: septic arthritis of the shoulder is uncommon in adults. it is a surgical emergency as joint destruction occurs rapidly and can lead to significant morbidity and mortality. accurate diagnosis can be particularly challenging in patients with underlying liver disease. mri is a useful adjunct in early detection of atypical causes of shoulder pain. case report: a 43 years old male came to our outpatient department with complaints of pain and stiffness of his left shoulder. on examination, his shoulder movements were severely restricted. further evaluation with mri revealed septic arthritis of left gleno-humeral joint for which emergency arthroscopic debridement was done. conclusion:septic arthritis of shoulder may not present with classical clinical features. hence, a through clinical and radiological evaluation will help us prognosticate and treat accordingly thereby preventing complications like septic shock, osteomyelitis.

PMC5040566

pubmed-20

split-liver transplantation (slt) is an attractive alternative procedure to expand the donor pool in patients waiting for liver transplantation. the standard split-liver procedure for a child and an adult (adult/pediatric split liver ttransplantation, a/p slt), by splitting segment ii-iii for pediatric recipient and segment i-iv-v-vi-vii-viii for an adult, is an accepted surgical option with good results both for the adult and for pediatric recipient. biliary complications continue to be the major cause of morbidity after slt with a reported high incidence ranging between 10% and 32% [1, 2]. as a consequence of bile duct anatomic variations, slt requires a precise knowledge of the liver anatomy [3, 4]. the challenge of this procedure is represented by a preoperative radiological assessment of the biliary anatomy often unavailable at the donor's hospital. thus the risk of biliary duct injury during the splitting procedure is usually considered higher than during living donor procedure. in this report we describe an uncommon late biliary complication that occurred after slt and was successfully treated by a multidisciplinary approach. a 63-year-old male with hepatitis c-related cirrhosis was referred for liver transplantation to our institution. we performed a conventional a/p slt with in situ technique providing the left lateral segment for a child (segments ii-iii) and leaving the right lobe graft (segment i-iv-v-vi-vii-viii) for an adult recipient. the celiac trunk was left on the left graft while the right hepatic artery remained on the right graft. the patient was transplanted using the piggy-back technique without a veno-venous bypass. the biliary tract was reconstructed performing a duct-to-duct anastomosis using a t-tube by our standard technique previously described. a cholangiography through the t-tube was performed on postoperative day 14, and the t-tube was clamped before patient discharge. three months after a/pslt the t-tube was removed after a cholangiography with normal findings. one year after transplant the patient showed abnormal liver function tests, hyperbilirubinemia, leukocytosis, and elevated g-glutamyl transpeptidase (ggt), and mild elevation in alanine transaminase (alt). the patient underwent a doppler ultrasound that showed (a patent hepatic artery) an intrahepatic bile duct dilatation and an anastomotic biliary stricture. the anastomotic stricture was treated by stenting the main biliary duct during an endoscopic retrograde cholangiopancreatoghaphy (ercp) without any evidence of intrahepatic biliary dilatation. after this procedure the patient was submitted to a percutaneous transhepatic cholangiography (ptc) showing a complete obstruction of segments vi and vii biliary branches near the duct-to-duct biliary anastomosis (figure 1). the patient was discharged leaving the external biliary drain open allowing bile drainage and an easy access for repeated radiologic treatment and an internal stent in the common bile duct. three months later the patient underwent a surgical revision because of repeated episodes of cholangitis. during surgery an intraoperative cholangiography was performed through the biliary drain confirming a bile duct dilatation at the level of segments vi and vii. it was impossible to cross the biliary stricture by a torque catheter and by hydrophilic guide wire (figure 2). we supposed that the segmentary bile duct branch of posterior segments vi and vii draining in the left bile duct was unidentified and tied at the time of the in situ split-liver procedure during the parenchymal transection. a primary biliary reconstruction by biliary repair or biliodigestive anastomosis was considered at high risk because of the presence of postsurgical adhesions sand due to fibrotic tissue replacing the parenchymal transected area. a liver resection of the dilated segments vi and vii was considered at high risk of leaving an inadequate liver mass. we decided to perform a permanent intraoperative obliteration of the dilated intrahepatic ducts by a percutaneous embolization using a nonresorbable agent. with a fluoroscopic guidance through the transhepatic access we positioned a 5-french polyethylene catheter inside the ducts, preliminary flushed by a nonionic dextrose solution. we then injected the tissue adhesive agent n-butyl cyanoacrylate (nbca, glubran 2, gem, viareggio, italy) mixed with ionized oil (lipiodol, guebert, aulnay-sous-bois, france) for opacization in a ratio of 1:5. this solution completely filled the biliary duct, and the occlusion was totally accomplished in a few seconds (figure 3). during the first 3 days after the chemical bile duct embolization, the patient had a low fever with a slightly abnormal liver function test. a computed tomography (ct) scan performed 6 months later showed no sign of hepatic abscesses, and the bile duct dilatation was completely occupied by the nbca-lipiodol mixture. one year after the procedure patient showed normal liver function tests without no episodes of cholangitis. chemical bile duct embolization treatment could represent a valuable solution to treat uncommon biliary complications. these tissue adhesive glues are low-viscosity liquid monomers that undergo rapid polymerization and solidification when they come into contact with organic fluids such as bile. nbca is a permanent liquid embolic material that produces long-term occlusion in vessels of various size through an inflammatory tissue response resulting in vessel thrombosis or tissue atrophy. little is known about the use of cyanoacrylate compounds, and unlike european countries the use of glubran has not been approved by the food and drug administration yet. other authors have described the efficacy of biliary duct ablation by nbca in patients with persistent postsurgical bile leaks after lobectomy or cholecystectomy. vu et al. treated six patients with persistent postsurgical bile leaks as a complication after hepatic lobectomy or cholecistectomy using nbca glue for the obliteration of isolated segmental bile ducts in four cases. endoscopic treatment of biliary leakage by nbca has been described by seewald in nine patients in whom primary stent placement or nasobiliary drain was unsuccessful. described the use of a cyanoacrlylate in the treatment of a pancreatic fistula after distal pancreatectomy. the percutaneous interventional technique represents an effective valuable approach to reduce mortality and morbidity in the treatment of biliary complications after liver transplantation. the use of nbca in obliteration of a dilated bile duct seems to be a safe procedure with good results providing a less invasive option than hepatic resection above all in high-risk patients with posttransplant bile duct injuries, decreasing the morbidity associated with chronic external biliary drainage. futher studies are needed to determine whether this approach is effective and safe and whether it could reduce hospital stay and costs.

biliary complications continue to be a major cause of morbidity after split-liver transplantation (slt). in this report we describe an uncommon late biliary complication. one year after slt the patient showed an intrahepatic bile dicy dilatation with severe cholangitis episodes. the segmentary bile duct of hepatic segment vi-vii draining in the left duct was unidentified and tied at the time of the in situ split-liver procedure. we perform a permanent obliteration of the dilated intrahepatic ducts by a percutaneous embolization using an n-butyl cyanoacrylate (nabc). the management of biliary complications after slt requires a multidisciplinary approach. the use of nbca in obliteration of a dilated bile duct seems to be a safe procedure with good results providing a less invasive option than hepatic resection and decreasing the morbidity associated with chronic external biliary drainage. further studies are needed to determine whether this approach is effective and safe and whether it could reduce hospital stay and cost.

PMC2809321

pubmed-21

ribonucleotide reductase (rr) inhibitors have been studied as radiation sensitizers for over 30 years in both the lab and the clinic. the first of these, hydroxyurea, has been studied in both cervical and head and neck cancers, among others. although initially promising, many of the clinical trials produced negative results, or those that were difficult to interpret. there has recently been a significant advance in our understanding of this pathway from a number of well-done pre-clinical studies. in addition, the discovery of new rr inhibitors with increased potency and improved binding characteristics has produced a significant increase in interest in this area. this review will synthesize the data detailing the response of rr to ionizing radiation (ir) and will provide a perspective on the use of rr inhibitors as radiosensitizers in the treatment of human cancers. over the years, many groups have explored the use of rr inhibitors as chemotherapeutics in their own right, or as adjuncts to dna damaging molecules, particularly in hematologic malignancies. while this area looks promising, this subject will not be reviewed here. ribonucleotide reductase is the rate limiting enzyme in the synthesis and repair of dna and is the only enzyme responsible for the conversion of ribonucleoside diphosphates to deoxyribonucleotide diphosphates, the fundamental building blocks of dna synthesis and repair. r1 (also called rrm1 or m1) is the larger, regulatory subunit that is constitutively expressed throughout the cell cycle. it binds allosteric modulators, ribonucleoside diphosphates, and the nucleoside analogs gemcitabine and fludarabine. there are currently two known smaller subunits that bind r1 to form the active enzyme; r2 (also called rrm2 or m2) and a more recently discovered p53 inducible homolog of the r2 subunit, known as p53r2. both contain a tyrosine free radical stabilized by a non-heme iron complex that is critical in the reduction of ribonucleotides. r2 is known to be cell cycle regulated, with the highest levels during s phase, however the precise roles of the r2 and p53r2 subunit in the response to ir are an area of active debate, as outlined below (figure 1). given the pivotal role of rr in dna synthesis and repair, many studies have investigated the effect of dna damaging agents, including ir, on rr and its subunits. even so, there is currently still a great deal of controversy surrounding the exact mechanism of rr response to ir. the first is that the small subunit r2 is up-regulated and provides dntps for dna repair in addition to its usual role in dna synthesis during s phase. this is supported by a number of studies, including one by kuo et al., who characterized the response of rr in the human cervical cancer cell line, caski. they demonstrated an increase in r2 protein levels after treating cells with ir, which was correlated with an increase in rr activity. however, there was no increase in the transcription of r2, implying that the protein increase was due to post-transcriptional regulation (kuo and kinsella, 1998). this finding was reinforced by studies that demonstrated dna damage dependent stabilization of the r2 protein without any change in mrna after ir exposure in mouse balb/3t3 cells (chabes and thelander, 2000); however it was in contrast to earlier work showing transcriptional activation of the r1 and r2 promoters after exposure to ir in the same cell line (filatov et al., 1996). even though the precise mechanism of r2 response to ir damage is unclear, work has shown that human nasopharyngeal cancer cells overexpressing r2 demonstrate a significant increase in radioresistance and fewer dna double strand breaks (dsb) after exposure to ir (kuo et al., 2003), confirming the concept that r2 is important in the cellular response to ir the second theory involves the p53 inducible subunit, p53r2, first reported by tanaka et al. they showed that p53r2 was not cell cycle regulated (unlike r2), but was significantly induced after exposure of normal fibroblasts to ir (tanaka et al., 2000) and was found to translocate to the nucleus, the proposed site of most important dntp production (r2 did not). in cells that lacked wild-type (wt) p53, there was no induction, indicating that functional p53 was necessary for p53r2 up-regulation. cells transfected with p53r2 were resistant to dna damage induced cell death. in agreement with other studies, they found no increase in r2 mrna, but did not measure protein levels. additional studies have subsequently shown that p53r2 forms an active complex with r1 (guitett et al., 2001) and rr activity increased in correlation with the increase in p53r2, however the response of r2 was variable, with decrease in some lines and a moderate increase in the others (yamaguchi et al., 2001). finally, other models are possible, including one described by xue and colleagues. in their study, both subunits were found to bind p53 in human oropharyngeal carcinoma cells, and in response to ir, were released to bind r1 in the nucleus (xue et al., 2003), highlighting a third possibility in contrast to those previously presented. clearly, there is still work to be done in elucidating the response of rr to ir. many of the differences seen in the studies discussed can likely be attributed to the use of different techniques, materials, and especially cell lines. what should be clear is that rr is involved in the cellular response to ir and targeting it is both rational and likely desirable in enhancing the treatment of cancers with ir. it is likely that both r2 and p53r2 are involved to a different degree in different cancers, with cellular phenotype likely playing a key role in determining their relative significance. hydroxyurea (hu) is a hydroxylated analog of urea and was the first rr inhibitor to be extensively studied. it has directed activity at the tyrosine radical moiety of r2 and was first found to be active against cancer cells in 1963 (stearns et al., 1963). subsequent experiments in vitro showed that in addition to its direct inhibition of dna synthesis, it was also a sensitizer of cell killing by x-rays, particularly if given before or after ir (sinclair, 1968). later experiments showed that this was also the case in in vivo animal tumor models using isotransplants of spontaneous c3h/he mouse mammary carcinomas (piver et al., 1972). the total dose of ir to cure 50% of tumors was reduced when hu was combined with fractionated ir, although this effect was nt seen with single fraction ir treatments. given these encouraging pre-clinical results during the 19601970s, hu was subsequently examined in a number of clinical trials in a variety of human cancers. the majority of these trials have occurred in cervical cancer, most commonly in locally advanced disease. in particular, there were a number of prospective randomized controlled trials in the 1970s and 1980s that examined the effect of hu plus radiotherapy vs. radiotherapy alone. and the gynecologic oncology group (gog) enrolled 190 women with figo stage iiib or iva cervical carcinoma. hu was administered orally at 80 mg/kg starting on the first day of irradiation and every 3 days thereafter for 12 weeks. patients received at least 50 gy minimum tumor dose to the whole pelvis followed by a single brachytherapy treatment of 20 gy to point a. in spite of the large number of patients enrolled, only 90 were eligible for assessment of response. this was due to ineligibility (wrong stage, wrong cell type, etc.) and those that were inevaluable (refused treatment, periaortic node irradiation, improper field, etc.). the data were impressive, with a complete response (cr) of 68.1% in the hu group vs. 48.8% in the placebo (p=< 0.05), and a median progression free survival (pfs) of 13.6 vs. 7.6 months (hreshchyshyn et al., 1979). however, myelosuppression was more common in the hu group, with seven grade iii or iv myelotoxicities. they recruited 148 women with figo iib or iiib cervical carcinoma and again compared hu to placebo in the setting of conventional radiotherapy, with hu given every 3 days for 12 weeks. of the stage iib patients, 74% receiving hu had no evidence of disease at the completion of therapy compared with 43.5% of the placebo (p=< 0.01). of the stage iiib patients, the cr rates were 52.5 vs. 33% (p=0.22; piver et al., 1977). again, 78% of patients in the hu group developed leucopenia vs. 11% in the placebo group indicating a significant toxicity in addition to the improved clinical effect. at the time these studies were published, it was felt that hu added significant benefit to the treatment of locally advanced cervical carcinoma, and hu plus radiotherapy became the standard of care for the gog. however, over time, much of the data in these studies has been challenged, particularly in a systematic review by symonds et al. they found a number of methodological problems with the studies such as small sample size, large numbers of exclusions post randomization, subgroup analyses of already small groups and questionable censoring. they concluded that hu appears to add to acute toxicity and probably increases late complications and that there is no convincing evidence of sufficient quality to suggest a therapeutic effect of this drug although the gog initially used hu as its standard of care in the treatment of locally advanced cervical cancer, it was nt long before this combination was supplanted by a new adjunct to radiation therapy. one of the most important studies prompting this paradigm shift was the gog 120 trial, first reported in 1999 (rose et al., 1999) and recently updated (rose et al., 2007). gog 120 was a randomized phase iii study comparing cisplatin alone vs. cisplatin, fluorouracil, and hu vs. hu alone, in conjunction with pelvic irradiation for patients with locally advanced cervical cancer and pathologically negative para-aortic nodes. they reported a significant improvement in pfs and overall survival (os) in both cisplatin containing arms (p<0.001), with relative risks for progression of disease or death of 0.57 and 0.51 for cisplatin alone and cisplatin, fluorouracil and hu, respectively, when compared with hu alone. in addition, toxicities were similar in all groups. further, a similar study by whitney et al. compared the efficacy of standard radiotherapy (rt) plus hu with standard rt plus fluorouracil (5-fu) and cisplatin in a randomized controlled trial. in 368 women with figo stage iib, iii, or iva cancer of the uterine cervix, there were significant improvements in pfs and os with the 5-fu and cisplatin combination (whitney et al., 1999). adverse effects included leucopenia: 4% of 5-fu/cisplatin patients and 24% of hu patients experienced grade iii or iv toxicity. given the findings of these studies, concurrent cisplatin and radiotherapy became the standard of care in locally advanced cervical cancer, spelling the end of hu use in the treatment of cervical cancer. in addition to its study in cervical carcinoma, hu has also been extensively investigated in other cancers, particularly head and neck. while early studies had significant flaws in methodology, a phase i study showed a 90% response rate in patients treated with hu, 5-fu, and palliative dose fractionated ir (vokes et al., 1989), prompting a series of trials by the same group and others. this included work by mantz et al., who examined the benefit of hu when added to a more extensive chemoradiation protocol including cisplatin, 5-fu, leucovorin, and interferon-2b induction chemotherapy followed by 5-fu and hu with fractionated radiotherapy in 32 laryngeal cancer patients. median follow up was 44.5 months, and, after completion of all therapy, the cr rate was 94%. median os was 44.5 months, and median pfs was 86, 78, and 78% at 1, 3, and 5 years, respectively, which compared favorably with other published data and for the first time saw patients with stage iv head and neck cancer failing distantly more often than locally (mantz et al. the increased local control, unfortunately, was associated with increased toxicity, which has also been seen in other studies. one phase i study with prolonged infusion of hu with hyperfractionated, accelerated, external radiation in patients with advanced squamous cell cancer of the head and neck showed an increase in the severity of swallowing toxicity compared with previous trials, with severe edema, and reductions in motility and mobility of pharyngeal and laryngeal structures (beitler et al., 1998). the same group later published a study examining the long term impact on swallowing that showed persistent, severe swallowing dysfunction (smith et al., 2000). interestingly, these studies examined continuous hu infusions based on pre-clinical data that suggested that hu should always be given concurrently with ir to maximize effect. even though local control was excellent, with just 4 of 26 patients experiencing recurrence, quality of life is of great importance in these patients, and the late follow up reported esophageal strictures for the first time after chemoradiotherapy, suggesting that this particular regimen may be too aggressive. the state of hu in head and neck cancer is well reviewed by argiris et al. (2003) and while the authors are optimistic about the future of chemoradiation in head and neck cancer, it remains to be seen where hu and other rr inhibitors will fit in the future treatment of this disease site. even as hu has slowly progressed in the clinic, work has continued on examining the mechanism by which it sensitizes cells to the effects of ir. in particular, work by kuo et al. has shown that the sequence with which cells are treated with hu plays an important role. they demonstrated that in the caski cervical cancer cell line, clinically relevant concentrations of hu had a significant interaction with ir, with post-ir exposure>pre-ir (kuo et al., 1997). this was associated with increased g2 delay, suggesting a decrease in the repair of damaged dna. in addition, in cells overexpressing the r2 subunit, hu is able to return ir sensitivity to baseline (kuo et al., 2003), demonstrating that r2 inhibition is the likely mechanism for hu radiosensitization in these cells. these findings are potentially informative about the failure of hu to become established as a radiosensitizer in cervical cancer. in the majority of the early trials, given that hu works best in vitro when dosed immediately after ir exposure, one could conclude that these trials were not optimized for best effect. in addition, hu has recently been shown to have a significant effect on the mechanism of dna dsb repair employed by cells after exposure to ir. burkhalter et al. showed that cells pre-incubated with hu were unable to use homologous recombination (hr) to repair dsb, and instead relied on non-homologous end joining (nhej). in addition, cells that were nhej deficient had significantly more dsb after hu treatment (burkhalter et al., 2009). given that nhej is thought to be the dominant dsb repair mechanism in cells treated with hu, rr inhibitors are likely to have enhanced activity in tumors that are nhej deficient. even with new studies on its mechanism of action, hu will likely remain consigned to history due to the many inadequacies it has as a drug molecule. while its oral absorption is almost complete and it is completely distributed in the water compartments of the body, hu has a short half-life (between 1.6 and 4.45 h; gwilt and tracewell, 1998) and its effectiveness is limited by relatively low affinity for rr and by the development of resistance. one area where it could potentially find use in the future is in cns neoplasms, as it does cross the blood recent studies have examined its use in progressive meningioma in combination with 3d-conformal radiotherapy and adjuvant chemotherapy. in one trial, pfs at 1 and 2 years was 84 and 77%, which is similar to other adjuvant studies (hahn et al. in spite of the mixed clinical data for hu, there is sufficient proof of concept to suggest that a rr inhibitor can be efficacious as a radiosensitizer in human cancers. thus, there has been a concerted effort to discover more potent molecules with more favorable pharmacokinetics and pharmacodynamics for this purpose. one of the more promising of these is triapine, a thiosemicarbazone that destroys the tyrosyl radical in r2/p53r2 by forming a redox active complex with iron, producing reactive oxygen species. in studies comparing it with hu in vitro, triapine was shown to have significantly higher potency in both enzyme and cell assays. in addition, it was fully active against hu and gemcitabine resistant cells and was equally potent against r2 and p53r2, whereas hu was approximately threefold less potent at binding p53r2 (zhu et al., 2009). in addition, in in vivo models, triapine was active against hu resistant l1210 and kb cell lines and caused significant inhibition of solid tumor growth in mouse xenograft models (finch et al. further studies have examined the radiosensitizing properties of triapine in a number of human cell lines. used a panel of three human tumor cell lines, including glioma, pancreatic, and prostate cancer cells, with triapine enhancing radiosensitivity when delivered 16 h before or immediately after ir by 1.5- to 2-fold. ir interaction was associated with a reduction in the repair of dna dsb as evidenced by a persistence of h2ax foci at 24 h (barker et al., 2006). a similar effect was seen in mouse tumor xenografts, again, with greater effect if triapine was dosed just after ir. the most effective temporal relationship between triapine dosing and ir is similar to that seen with hu in earlier pre-clinical studies. interestingly, normal human fibroblasts were only sensitized when triapine was given before, not after ir, suggesting a potential for an improved therapeutic index for ir triapine sequencing that may be incorporated into future clinical studies. are ic50 (concentration of compounds producing 50% inhibition of recombinant rr activity) values for both compounds in an in vitro rr activity assay with r2 or p53r2 bound to r1 (zhu et al., 2009). also shown are elimination half-lives (t1/2) for both compounds (gwilt and tracewell, 1998; kunos et al., 2010b). of note, many cancers have virally or mutationally silenced p53 that allows rr activity to continue unchecked. in these cancers, it is potentially of increased importance to inhibit the r2 and p53r2 subunits that have lost p53 regulatory control. this is the case in cervical cancer, where the vast majority have dysfunctional p53 due to hpv infection. in one study, three cervical cancer cell lines with mutated or dysfunctional p53 were irradiated 6 h after triapine exposure. in all cases, the cell lines were sensitized to ir and sustained dna damage as measured by persistence of h2ax foci. in addition, by measuring dctp levels, the investigators were able to show reduced rr activity in cells with and without functional p53, demonstrating that the inhibition of rr is p53 independent (kunos et al., 2009). these findings were reinforced by further work of the same group that also showed a synergistic effect when cisplatin was added in addition to triapine and ir (kunos et al., 2010a). these promising pre-clinical data have prompted the initiation of a number of clinical trials. indeed, triapine has so far been studied in 27 clinical trials in the usa at various stages of recruitment (www.clinicaltrials.gov), including those in both solid and hematologic malignancies. in particular there are three trials investigating the radiosensitizing potential of triapine, with data from one phase i study being published by kunos et al. the purpose of their study was to assess the safety/tolerability, pharmacokinetics, and clinical activity of triapine three times weekly in concert with once weekly cisplatin and pelvic radiation in 11 patients with gynecologic malignancies. triapine was dosed in 2 h infusions and the half-life was found to be 2 h. all 10 patients with advanced stage ib to ivb cervical cancer achieved complete clinical response, with a median 18 month follow up showing no disease progression in any of the patients. five of the 10 patients had pet/ct evident pelvic or para-aortic lymphadenopathy before treatment, with complete resolution on follow up imaging after treatment (kunos et al. in addition to the clinical data, the authors also examined objective markers of disease response. late responders had a significantly higher rr activity on day 10 vs. day 1 and there was no temporal change in rr activity in the early responders, indicating that the expected spike in rr activity after ir was suppressed by triapine. the fact that the late responders experienced durable responses in spite of elevated rr activity at day 10 suggests that there may be other mechanisms of triapine activity that require further study. the promising results from this phase i trial prompted a phase ii trial which is now underway. over the last 10 years, there have been a number of major leaps forward in the field of radiation oncology that allow us to deliver higher doses of radiation in ever more specific ways to our patients. still, in the vast majority of cases, we are constrained by dose limiting toxicities that must be accounted for when designing treatment plans and balanced against the therapeutic benefit realized. therefore, the field of radio modulation and specifically radiosensitization will continue to grow in importance in the coming years as we search for ways to maximize the therapeutic index of our treatments. while there are many different radiosensitizers currently being studied, the rr inhibitors are among the oldest of targets, and are getting a new lease of life in recent times with the development of more modern drug molecules. as outlined in the review above, the story began with the study of hu in combination with ir in a number of pre-clinical trials in the 1960s, leading to a large amount of interest in the clinic, culminating with the gog adopting hu and ir as its standard of care in locally advanced cervical cancer. while its success in this arena was short lived, many parallel studies investigating the mechanism of action of hu were carried out, resulting in a far clearer understanding of the biological interactions between ir and rr inhibitors. even as hu was fading from view, investigators were working on successors including the intriguing molecule, triapine. as shown above, this drug works in a similar fashion to hu, but has significantly improved potency, pharmacokinetics, and pharmacodynamics. the phase i study in cervical cancers that was recently published includes some very encouraging data, and the field waits in anticipation for the publication of further trials. it remains to be seen what the best dosing regimen for triapine will be, however the weight of pre-clinical data, including that with hu, suggests that daily dosing shortly after radiation therapy will be most effective and provide the greatest therapeutic window. these authors would encourage studies incorporating this type of dosing schedule in the clinic, although this must obviously be weighed carefully against the potential for increased toxicity. in addition to triapine, there are other new rr inhibitors being tested at various stages, including trimidox and motexafin gadolinium. (1994), in a paper that reported 100-fold more potency at rr than hu in a cell based assay. subsequent in vitro studies have demonstrated that while it acts as a radiosensitizer in panc-1 human pancreatic cancer cells (ahmed and hassan, 2000), it is less potent than hu and further work is ongoing to fully assess its potential. motexafin gadolinium is a texaphyrin molecule that targets thioredoxin reductase in addition to rr. in in vitro experiments, it was shown to inhibit rr with an ic50 of 26 m (hashemy et al., 2006), although given that it has a number of other cellular effects, it is unclear how much of its potency as a radiosensitizer is due to rr inhibition alone. it is likely that the coming years will continue to see the emergence of new rr inhibitors with unique properties. the recent advances in the understanding of rr biology and the development of new inhibitors places us at an important crossroads in the story of rr inhibitors. there are sufficient data to provide proof of concept for the target from a biological standpoint, however clinical trials to this point have not been wholly convincing. it will be interesting to follow the development of the field in the next 510 years, particularly with the clinical trials of triapine in cervical cancer, and hopefully at least one of the many rr inhibitors being studied will eventually bring additional therapeutic benefit to patients in the near future. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

ribonucleotide reductase (rr), the rate limiting enzyme in the synthesis and repair of dna, has been studied as a target for inhibition in the treatment of cancer for many years. while some researchers have focused on rr inhibitors as chemotherapeutic agents, particularly in hematologic malignancies, some of the most promising data has been generated in the field of radiosensitization. early pre-clinical studies demonstrated that the addition of the first of these drugs, hydroxyurea, to ionizing radiation (ir) produced a synergistic effect in vitro, leading to a large number of clinical studies in the 19701980s. these studies, mainly in cervical cancer, initially produced a great deal of interest, leading to the incorporation of hydroxyurea in the treatment protocols of many institutions. however, over time, the conclusions from these studies have been called into question and hydroxyurea has been replaced in the standard of care of cervical cancer. over the last 10 years, a number of well-done pre-clinical studies have greatly advanced our understanding of rr as a target. those advances include the elucidation of the role of p53r2 and our understanding of the temporal relationship between the delivery of ir and the response of rr. at the same time, new inhibitors with increased potency and improved binding characteristics have been discovered, and pre-clinical and early clinical data look promising. here we present a comprehensive review of the pre-clinical and clinical data in the field to date and provide some discussion of future areas of research.

PMC3356024

pubmed-22

the value of the ketogenic diet (kd) has been recognized in the treatment of epilepsy, although the exact mechanisms by which it exerts its effect remain an enigma. they seem to be different from those of regular antiepileptic medications (aeds), and discovering what they are may lead to its use in clinical situations other than epilepsy as well. the kd is comprised of four elements, the changes of any of them can potentially lead to losing its anticonvulsant effect: (1) increased amount of fat, usually in a ratio of 3 to 4 grams of fat for each gram of protein and carbohydrates, (2) as low a consumption of glucose as possible, (3) caloric restriction, and (4) fluid restriction. although there is some debate about the last component, clinical practice has shown that stopping fluid restriction can lead to seizure recurrence much in the same way as when stopping glucose restriction. the most important result of observing the diet is the increased blood level of free fatty acids (ffas). the ffas are transferred into the mitochondria, a process which requires the presence of an appropriate amount of carnitine, where they are degraded into ketone bodies through oxidation. these ketone bodies include hydroxybutyrate, acetoacetate, and acetone (figure 1). the effect of the increased amount of ketone bodies seems to be the most prominent of all other suggested antiepileptic mechanisms of the kd. the degradation of the ketone bodies delivers acetyl-coa directly to the tricarboxylic acid cycle, thus increasing its turnover while bypassing the need to depend on acetyl-coa coming from glycolysis to produce atp. unlike glucose, which requires a transporter to cross the blood brain barrier, the ketone bodies penetrate it easily. when this transporter is deficient, as in glut 1 deficiency, the kd is the preferred way of antiepileptic treatment, since it allows for bypassing the need for glucose. children in whom the conversion of pyruvate to acetyl-coa is blocked, for instance, in pyruvate dehydrogenase (pdh) deficiency, will also benefit from bypassing this route. hydroxybutyrate is the predominant ketone body measured in the blood, and it is used to monitor the degree of ketosis during therapy. the breath of a patient who is ketotic while on the diet will often even smell of acetone. acetone is one of the ketone bodies that have an anticonvulsant effect in several types of mouse seizure models. the mechanism of this effect is unknown, although an effect on k2p channels has been proposed. by means of the tca cycle, acetyl-coa increases the level of the neurotransmitters glutamate and -aminobutyric acid (gaba) and the major excitatory and inhibitory neurotransmitters in brain, respectively. another product of an elevated level of free fatty acids is polyunsaturated fatty acids (pufas). the potential ability of pufas to block seizure activity in the brain is speculated to be associated with some rather more complicated mechanisms, including (1) directly inhibiting voltage-gated sodium and calcium channels, (2) activating a lipid-sensitive potassium channel, (3) enhancing the activity of the sodium pump to limit neuronal excitability, (4) activating peroxisome proliferator-activated receptor- (ppar), and (5) inducing the expression and activity of brain-specific uncoupling proteins in the mitochondria, thereby inducing a neuroprotective effect. neuroprotection can contribute to the anticonvulsant effect, but it may have other effects as well, which can lead to other clinical uses of the kd. appleton and de vivo reported that the kd increased the total quantity of bioenergetic substrates (adenosine triphosphate (atp)) and elevated the energy charge in rat brain. acetoacetate, a product of hydroxybutyrate dehydrogenation, is transferred into acetyl-coa which enters the tricarboxylic acid (tca) cycle. the increased turnover of the tca cycle generates protons and electrons that are channeled to the electron transport chain. this, in turn, drives the formation of atp from adenosine phosphate (adp) by atp synthase. enhanced atp can either be converted to phosphocreatine for energy storage or broken into adenosine. enhanced atp levels provide energy reserves for a neuron to continue functioning under stress. increased extracellular adenosine offers a neuroprotective buffer against insults, reduces excitation, and averts excessive atp demands, thus providing local seizure control and neuroprotection. it was also suggested that the kd influences toward an upregulation of transcripts encoding energy metabolism enzymes and increase in the density of mitochondria in neuronal process, leading to heightened energy reserves. an improved energetic status can support seizure prevention, for instance, by supporting gabaergic inhibition. it is suggested that adaptive processes to the metabolic changes induced by the diet lead to changes in gene expression which in turn result in some of the above-noted changes. other path of neuroprotection is modulated through decreased generation of ros which is considered to be related to pufas effect on uncoupling proteins. the fact that the kd is considered a proven therapy with relatively few adverse affects and wide clinical experience, particularly in children, led to recent studies investigating new potential uses for other neurological disorders. one of the most intriguing and active fields of research is the effect of a high-fat caloric-restricted diet on the survival of brain tumors cells. it is hypothesized that mitochondrial abnormalities impair the ability of brain tumors to generate energy from ketone bodies. unlike normal cells, malignant tumor cells have impaired genetic adaptability due to their genetic abnormalities and, therefore, increased susceptibility to environmental stress, such as fasting or caloric restriction. the same genomic defects that are involved in the creation of brain tumors can be exploited for their destruction [3, 10, 11]. in 1995, nebeling et al. reported two young girls with unresectable advanced stage brain tumors who had poor response to radiation and chemotherapy. they were treated with a kd and their response was remarkable, both clinically and according to positron emission tomography follow-up scans. described a patient with glioblastoma multiforme whose tumor, which is very malignant, improved on the kd. surprisingly, despite the appealing efficacy of this treatment, no further human studies or clinical trials on the kd as a therapy for brain tumors have been conducted. several laboratory studies in mouse and rat models have recently confirmed that inhibition of brain tumor growth is directly related with reduced levels of glucose and elevated levels of ketone bodies. moreover, the kd was shown to reduce reactive oxygen species (ros) in the brain. cancer cells need high levels of ros for the induction of angiogenesis and the production of tumor growth factors, thus, through this mechanism the kd can be protective. the clinical manifestations were mainly intractable seizures that necessitated repeated admissions to the intensive care unit, as well as severe cognitive and alertness decline. after the oncologists decided that antitumor treatment would be ineffective, she was started on the kd. after four weeks of trial, it did not have any effect on the tumor progression. however, it did have a notable improving effect on cognition, alertness, and mood of the girl, in spite of her devastating condition. the beneficial effect of the kd on cognition, alertness, and mood is well recognized, and clinical experience shows that in many times it is not less important than the anticonvulsant effect. the potential neuroprotective effect of the kd was what motivated investigations into its potential as a treatment option in other neurologic disorders. there are increasing numbers of reports that ketosis achieved by starvation or administration of a kd has a consistent neuroprotective effect after various brain injuries in animal models. one human pilot study and several animal model studies have shown improvement in autistic behavior parameters with kd treatment. it remains to be further clarified whether this improvement is related to reduced epileptic activity found in up to 30% of these patients or to a primary effect of the kd. a factor that can be crucial for the application of the kd in medical conditions other than intractable epilepsy is the inherent difficulties in its use. dietary restriction can pose a significant problem in a child with a progressive tumor that undergoes massive chemotherapy, who may already be cachectic. the kd may not be an option in a grown-up hyperactive autistic child. thus, the neurologist must evaluate the appropriate clinical, familial, and environmental situations very cautiously prior to the recommendation of the kd. in conclusion, the major metabolic effect of the kd is in supplying the brain with an increased amount of free fatty acids. their degradation into ketone bodies, together with the load of pufas, leads to major changes in the metabolic, bioenergetic, mitochondrial, and even genetic constellation. whether it may also be effective in other pathologies, especially in treating malignancies, awaits future research.

although the ketogenic diet (kd) has been widely accepted as a legitimate and successful therapy for epilepsy and other neurological disorders, its mechanism of action remains an enigma. the use of the kd causes major metabolic changes. the most significant of them seems to be the situation of chronic ketosis, but there are others as well, for instance, high level of polyunsaturated fatty acids (pufas). these primary influences lead to secondary, in part adaptive, effects, for instance changes in mitochondrial density and gene expression. clinically, the influences of the diet are considered as anticonvulsive and neuroprotective, although neuroprotection can also lead to prevention of seizures. potential clinical implications of these mechanisms are discussed.

PMC3420518

pubmed-23

the endotracheal tube (ett) should be placed at the optimal level to avoid inadvertent complication. if the ett is too deep, it increases the risk of unintended single lung ventilation. on the other hand, if the ett is too shallow, it may cause vocal cord injury by the ett balloon or accidental extubation. there are many methods for determining the appropriate depth of ett in adults; fixed insertion depth according to sex (23 and 21 cm from the upper incisors in adult males and females, respectively), the use of depth marks on the ett, suprasternal palpation of the ett tip or cuff, and bilateral auscultation. although chest radiography and bronchoscopy are considered an accurate method, they are not always feasible and the costs are considerable. considering the individual variation in the length of the trachea, using fixed depths or marks on the ett may result in inadequate placement of the ett. to reduce the risk of single lung ventilation or vocal cord injury, the segment between the proximal edge of the cuff and the ett tip should be placed at the mid-trachea level. if the length of the trachea can be predicted at bedside before intubation, the ett can be placed at a safe depth for each patient. the purpose of this study was to determine whether surface anatomical landmarks can be used to predict the mid-tracheal level in adult patients. neck computed tomography (ct) images of adults obtained between 2009 and 2014 were reviewed by a single reviewer after obtaining approval form seoul national university hospital institutional review board (july 17, 2015/no. patients with laryngeal, tracheal, or thoracic abnormalities, tracheostomy or significant tracheal deviation caused by mass lesions were excluded. in addition, poor ct image quality, absent sagittal ct images, ct images that did not cover the entire trachea, or ct images taken in neck hyperextension (boidin angle>130 on scout view) or hyperflexion (boidin angle<110 on scout view) position were also excluded. levels of subglottic and tracheal airway segments that include the vocal cords (vc), the cricoid cartilage (cc), and the carina were identified using the method described by sirisopana et al. the vocal cords were identified as the most cranial level of the upper airway, with a teardrop shape below the laryngeal ventricle. anatomic level of suprasternal notch (ssn) the level of manubriosternal junction (msj) was defined as the middle level between the manubrium and the body of the sternum where the second rib attaches to the sternum. the level of carina was defined as the bifurcation of right and left main bronchus and identified by the figure 8 shaped lumen. the external locations of the cc, ssn, and msj were defined on the skin surface. the distances between the external points of the cc and the ssn (extcc-ssn), and those of the ssn and the msj (extssn-msj) were measured. the distances between the subglottic structures at the levels of the vc, the cc, ssn, and carina were measured. tracheal length was calculated as the summation of the distances measured from the vc to the carina. the mid-trachea level was calculated from tracheal length and the ideal position of ett is shown in fig. 2. identification of subglottic segments on axial view (right), and defining the distances between subglottic segments on sagittal view (left) in a 45-year-old male patient. cc=cricoid cartilage, extcc-ssn=the surface distances from the cricoid cartilage to the suprasternal notch, extssn-msj=the surface distances from the suprasternal notch to the manubriosternal junction, msj=manubriosternal juction, ssn=suprasternal notch, vc=vocal cord, tracheal length=a+b+c+d. the ideal position of endotracheal tube (ett) to minimize the risk of endobronchial intubation and vocal cord injury. the mid-point between the proximal edge of the cuff and the ett tip was matched to the level of the middle of the trachea (mtl), which was calculated from the tracheal length. cc=cricoid cartilage, ett=endotracheal tube, msj=manubriosternal juction, mtl=the level of the middle of the trachea, ssn=suprasternal notch, vc=vocal cord. the correlations between age, height, weight, and tracheal length were analyzed using linear regression. the relationships between surface measurements and the mid-tracheal level were analyzed with bland the distance between the cc and the ssn was longer in females (p<0.001), whereas distances between the vc and the cc, the ssn and the carina, and the tracheal length were longer in males (p<0.001). the extcc-ssn was longer in females (p<0.001), whereas extssn-msj was longer in males (p<0.001). demographic data and measured distances between subglottic structures of trachea, external landmarks, and calculated tracheal length. the calculated tracheal length was 131.9 (10.3) mm and ranged from 106.0 to 169.8 mm. tracheal length showed weak correlation with height (r=0.2188) and weaker correlation with age (r=0.0004) and weight (r=0.0466) (fig. the calculated mid-tracheal level was 66.0 5.1 mm from the vc. altman plots showing the difference between the mid-tracheal level and the surface measurements are shown in fig. the bias was calculated by subtracting the mid-tracheal level from the surface measurements. the difference between the extcc-ssn and the mid-tracheal level was 6.6 (12.5) mm, and the difference between the extssn-msj and the mid-tracheal level was 19.2 (6.1) mm. altman plots showing the agreement between the mid-tracheal level (mtl) and the surface measurements of surface anatomical landmarks. (left) the bias (subtraction of the mid-tracheal level from the surface distance from the cricoid cartilage to the suprasternal notch) and 95% limit of agreement as the mean difference were 6.6 [31.1 to 17.9] (mm). (right) the bias (subtraction of the mid-tracheal level from the surface distance from the suprasternal notch to the manubriosternal junction) and 95% limit of agreement as the mean difference were 19.2 [31.1 to 7.2] (mm). extcc-ssn=the surface distances from the cricoid cartilage to the suprasternal notch, extssn-msj=the surface distances from the suprasternal notch to the manubriosternal junction, mtl=the level of the middle of the trachea. altman plots of the difference between the extcc-ssn and the mid-tracheal level in female and male patients. the calculated mid-tracheal level was 64.5 (4.6) mm in females and 67.9 (5.0) mm in males. the difference between the extcc-ssn and the mid-tracheal level was 1.7 (11.7) mm in females and 12.8 (10.7) mm in males (p<0.001). altman plots of female (left) and male (right) patients showing the agreement between the mid-tracheal level (mtl) and the surface the surface distance from the cricoid cartilage from the suprasternal notch. (left) the bias (subtraction of the mid-tracheal level from the surface distance from the cricoid cartilage to the suprasternal notch) and 95% limit of agreement as the mean difference were 1.7 [24.6 to 21.2] (mm) in females. (right) the bias (subtraction of the mid-tracheal level from the surface distance from the cricoid cartilage to the suprasternal notch) and 95% limit of agreement as the mean difference were 12.8 [33.8 to 8.2] (mm) in males. extcc-ssn=the surface distances from the cricoid cartilage to the suprasternal notch, mtl=the level of the middle of the trachea. this study showed that mid-tracheal level could be estimated by the surface distance between the cc and the ssn (extcc-ssn). compared with males, extcc-ssn more accurately predicts the mid-tracheal level in females. in clinical practice, physicians can use extcc-ssn to determine the depth of ett so that the segment between the proximal edge of the cuff and the ett tip lies at the mid-tracheal level. there are several studies that used the distance between the surface anatomical landmarks to estimate the airway length and the optimal insertion depth of endotracheal tube. lee et al showed that the distance between the upper incisor and the manubriosternal angle in the neck extension position correlates with the distance between the upper incisor and the carina in the neutral neck position. evron et al used the sum of the 2 distances from the mouth angle to the jaw angle and from the jaw angle to the sternal manubrium to determine the depth of ett. however, these methods are complicated than single measurement of extcc-ssn due to the difficulty in locating the sternal manubrium, more than 1 measurement, and the need for full neck extension. the physicians can easily apply our method with a simple measurement of the extcc-ssn to estimate the mid-tracheal level, and apply this depth during endotracheal intubation. however, according to previous studies in adults, flexion and extension of the neck from neutral position are associated with the inward and outward movements of the ett. conrardy et al showed that the orally intubated ett can move 1.5 (range 0.52.0) cm toward the carina during flexion and 2.4 (range 1.34.3) cm away from the carina during extension. kim et al showed that the ett can migrate 1.3 (sd 0.6; range 0.52.5) cm toward the carina during flexion and 1.7 (sd 0.8; range 0.43.1) cm during extension. therefore, the position of the neck should be considered when determining the optimal depth of the ett. first, this study was a retrospective analysis of ct images and the distances were measured in the neutral position. second, since the vc is used as a baseline of the depth of intubation in our method, it can be difficult to apply our method to patients with high cormack grades. lastly, the efficacy of this method is yet to be demonstrated in clinical practice. in conclusion, the mid-tracheal level, which is considered the optimal depth of ett, can be predicted by the surface distance between the cc and ssn in adults, especially in females.

abstractendotracheal tube (ett) should be placed at the optimal level to avoid single lung ventilation or accidental extubation. this study was performed to estimate the mid-tracheal level by using surface anatomical landmarks in adult patients.neck computed tomography images of 329 adult patients between the ages of 16 and 79 years were reviewed. in the midline sagittal plane, the levels corresponding to the vocal cords, cricoid cartilage, suprasternal notch, manubriosternal junction, and carina were identified. the surface distances from the cricoid cartilage to the suprasternal notch (extcc-ssn) and that from the suprasternal notch to the manubriosternal junction (extssn-msj) were measured. the relationship between mid-tracheal level and the surface distances was analyzed using bland altman plot.the difference between the extcc-ssn and the mid-tracheal level was 6.6 (12.5) mm, and the difference between the extssn-msj and the mid-tracheal level was 19.2 (6.1) mm. the difference between the extcc-ssn and the mid-tracheal level was smaller in females compared with males [1.7 (11.7) mm vs 12.8 (10.7) mm; p<0.001].the mid-tracheal level, which is helpful in planning the insertion depth of an ett, can be predicted by the surface distance between the cricoid cartilage and suprasternal notch in adults, especially in females.

PMC5371447

pubmed-24

thyroid cancer is the most common endocrine neoplasia which accounts for about 1% of human cancers. mtc originates from calcitonin-producing cells (c-cells) of the thyroid gland and accounts for 35% of thyroid cancers. mtc is relatively slow-growing tumor but, if metastasized or relapsed, it becomes very aggressive causing more than 13% of all thyroid cancer-related mortality. in the united states, mtc has an incidence in children of 0.03 per 100 000 population per year with a fairly equal female to male ratio [4, 5]. mtc occurs either sporadically or in an inherited autosomal dominant manner. in adults, sporadic mtc accounts for 6575% of mtc, but in children, sporadic mtc is very rare; the vast majority of mtc diagnosed in the childhood is hereditary. hereditary mtc occurs as a familial mtc (fmtc) or as a part of multiple endocrine neoplasia (men) type 2a and b syndromes, wherein other endocrine glands are also affected. mtc diagnosed during childhood almost always results from a dominantly inherited or de novo activating mutations in the ret proto-oncogene, which encodes the ret receptor tyrosine kinase [69]. advances in predictive genetic testing for ret mutations have enabled early diagnosis of hereditary men syndromes and prophylactic thyroidectomy in presymptomatic patients to prevent mtc. the early onset of mtc in hereditary syndromes makes it an important endocrine disease that is increasingly managed by pediatric providers [1012]. in this review, we discuss the etiology of pediatric mtc and currently available therapeutic modality for the cancer. ret encodes a receptor tyrosine-kinase which is expressed in the neural crest-derived cell types, including thyroid parafollicular cells, neuronal cells, and adrenal medullary chromaffin cells. in these cell types, ret plays a central role in regulating cell proliferation, growth, differentiation, migration and survival. in humans after alternative splicing at the 3 end, ret transcripts encode three protein isoforms with distinct c-terminal ends that contain either 9 (ret9), 51 (ret51), or 43 (ret43) amino acids. ret exon 19 is present in all transcripts and its differential splicing at the 3 end produces distinct transcripts wherein exon 19 is either unspliced, spliced to exon 20, or spliced to exon 21. all three resulting ret isoforms commonly contain a tyrosine (tyr1062) whose phosphorylation is critical for their activation. the major ret isoforms in vivo are ret9 and ret51, which consist of 1072 and 1114 amino acids, respectively, and are usually co-expressed. ret consists of an extracellular ligand binding domain, a trans-membrane domain, and an intracellular kinase domain (figure 1). the extracellular domain includes four cadherin-like repeats and a highly conserved cysteine-rich region, which is located near the cell membrane. the intracellular domain consists of two tyrosine-kinase subdomains, tk1 and tk2, which contain multiple tyrosine residues that are phosphorylated during receptor activation and are required for the activation of different downstream signaling pathways of ret [19, 20]. the ligands for ret are the glial cell line-derived neurotrophic factor (gdnf) family proteins, including gdnf, neurturin, artemin, and perseptin. activation of ret also requires the formation of a heterodimeric complex recruiting a gdnf-family receptor alpha (gfr). when unbound by a ligand, ret is monomeric, unphosphorylated, and inactive. when a ligand and the gfr co-receptor bind to the extracellular domain of ret, ret undergoes dimerization and autophosphorylation of the tyrosine residues in their kinase domains. this generates the docking sites for their downstream effectors that contain the src homology 2 domain. for example, gdnf-mediated stimulation of ret results in activation of the pathways regulated by phosphatidylinositol 3-kinase (pi3k) and different mitogen-activated protein kinases (mapks), including the extracellular regulated kinases (erks), c-jun amino-terminal protein kinases (jnks), the p38 mapk and the big map kinase (bmk1) erk5 [22, 23]. ret is one of the first receptor tyrosine-kinases (rtks) that have been found to play a role in neoplasia, being most well-known as a key etiological factor for thyroid cancer [6, 24]. activating mutations of ret abnormally enhance ret activity and can trigger tumorigenesis in certain organs although the exact underlying mechanisms are as of yet unclear. first, mutations of the six cysteine residues (cys609, 611, 618, 620, 630, and 634) in the extracellular domains can promote ret dimerization via disulfide bonds and result in constitutive ligand-independent activation of ret. second, mutations affecting the tyrosine kinase domains can also confer ligand-independent catalytic activity to monomeric ret. these ret mutants exhibit different patterns of autophosphorylation and altered substrate specificity [2628]. indeed, activation of different downstream signaling pathways is associated with different clinical features of ret mutant thyroid cancers, as observed in men2 syndromes discussed below. for example, the hirschprung disease, a congenital disorder of neural crest development is caused by a loss-of-function ret mutation. of note, the hirschprung disease is closely associated with men2a, demanding a genetic screening for men2a for children with familial hirschsprung's disease. a strict correlation exists between specific ret mutations and the onset of hereditary mtc (table 1) [31, 32]. the detailed and up-to-date information of ret sequence variations can be obtained from the men2 ret database (www.arup.utah.edu/database/men2/men2_welcome.php), which also contains links to selected men2 literature reviews, gene and protein information, and ret reference sequences. the men2a subtype, accounting for 9095% of the men type 2 cases, is a highly penetrant, autosomal dominant endocrine tumor syndrome characterized by the development of mtc in>90% of ret mutation carriers. in association with mtc in addition, rare variants of men2a are also associated with cutaneous lichen amyloidosis and hirschsprung disease [35, 36]. patients with men2a usually have mutations in the extracellular cysteine-rich region of the ret tyrosine kinase receptor, usually in exon 10 (codons 609, 611, 618 or 620) or exon 11 (codon 634) (table 1, figure 1) [31, 37]. more than 80% of men2a patients exhibit a specific substitution, i.e., cys634arg, on exon 11. mtc is generally the first manifestation of men2a syndrome and develops during early childhood, usually before age six and sometimes before age two. the men2b subtype accounts for approximately 510% of the men type 2 cases. men2b patients typically feature early coincident onsets of mtc, pheochromocytoma, and gastrointestinal mucosal ganglioneuromas. visible physical symptoms include mucosal neuromas of lips (bumpy lips) and tongue, and asthenic marfanoid body habitus [36, 40]. men2b patients usually have mutations in the tyrosine kinase domain 2 (tk2) in the intracellular region of ret, which almost always (> 95%) lead to a single substitution, i.e., met918thr, in exon 16 (figure 1) [31, 37]. de novo mutations, which usually occur on the paternal allele, are also common in men2b. men2b is characterized by the early development of an aggressive form of mtc in all affected individuals, typically during the first year of life. thus, apart from the genetic testing of ret mutations in children born to a parent with men2b, early diagnosis of men2b remains challenging. individuals with men2b are likely to develop metastatic mtc at an early age if they do not undergo prophylactic thyroidectomy before age one. without this intervention, fmtc is considered as the least aggressive clinical variant of men2a with decreased penetrance and/or delayed onset of the other endocrine pathologic manifestations [39, 43]. similarly to sporadic cases, familial mtcs are isolated and are not associated with other endocrine tumors. patients with fmtc harbor mutations similar to men2a in either the extracellular or intracellular region of the ret tyrosine kinase receptor [6, 44]. the onset of fmtc is relatively late, not appearing until the second or the third decade of life, and its penetrance is lower than the mtc caused by men2a and men2b [31, 39, 45, 46]. therefore, it is often difficult to determine fmtc based upon a family history and only careful genetic screening can distinguish between inherited and sporadic forms of mtc. mtc cells secrete the polypeptide hormone, calcitonin, and the glycoprotein carcinoembryonic antigen (cea), and these are used as diagnostic biomarkers for mtc. mtc is most commonly diagnosed by immunohistochemical staining of fine-needle aspiration of a new thyroid nodule for calcitonin, chromogranin a, or cea. serum calcitonin is the primary biochemical marker used for detection, staging, postoperative management, and prognosis for mtc patients. however, in very rare cases, certain mtc cells do not secrete calcitonin, which makes diagnosis and patient follow-up difficult. symptoms of mtc include neck pain, a palpable neck mass, and/or diarrhea resulting from hypercalcitoninemia. the clinical course of mtc in men2 patients is variable and is determined by the codon specific mutations. in hereditary form, an age-related progression of malignant disease is observed, with lymph-node and distant metastases being typically detected years after the onset of tumorigenesis. metastatic spread to cervical and regional lymph nodes (i.e., parathyroid, paratracheal, jugular chain, and upper mediastinum) or to distant sites including the liver, lungs, or bone is common and is frequently present in individuals with a palpable thyroid mass or diarrhea. positive lymph-node status and higher stage at diagnosis predict lower disease-free survival and higher mortality [5, 5456]. men2 is one of few hereditary cancer syndromes for which predictive genetic testing is recommended at childhood. genetic testing for hereditary mtc syndromes has had an enormous impact on reducing the incidence of mtc in the families affected by these hereditary syndromes [57, 58]. genetic counseling is indicated for all children diagnosed with mtc and others who either carry or are at risk of inheriting a ret mutation. children of patients with men2b should undergo ret analysis at birth, and children of patients with men2a or fmtc should undergo ret analysis before age six [59, 60]. even 610% of apparently sporadic cases of mtc demonstrate de novo germ-line ret mutations, thus making genetic testing worthwhile in all patients with mtc. thus, all children with an affected parent in this setting retain a 50% risk of mtc, and surgical decisions must rely solely on clinical testing. the standard treatment for mtc is surgical removal of all thyroid tissue including the posterior capsule [39, 62]. early thyroidectomy in all men2 patients can change the course of disease, either in a preventive or a curative fashion. the american thyroid association guidelines task force has classified mutations based upon a model that uses the genotypephenotype correlations to rank the mutations into risk levels for the development of aggressive mtc from the lowest a to the highest d (table 1). this classification may be used to predict phenotype, to recommend the timing of prophylactic thyroidectomy and the extent of surgical intervention, and to begin biochemical screening for pheochromocytoma and hyperparathyroidism. the ages at which the prophylactic thyroidectomy is recommended for the children tested positive for the ret gene mutation are as follows: ages 01 for ret mutations that carry the highest risk for aggressive metastatic mtc at young ages, i.e., classified as ata-d; before age 5 for ret mutations that carry a lower, yet still high risk of aggressive mtc at any age, i.e., classified as ata-c; after age 5 for ret mutations that carry a lower risk of aggressive mtc, i.e., classified as ata-b or ata-a, so long as the affected children have no other clinical signs of mtc development. there is ongoing debate on what age the thyroidectomy should be recommended for fmtc patients. some clinical institutes suggest the prophylactic surgery at age 1015, depending upon the exact mutation and family history, while recommending yearly test of calcitonin levels prior to deciding the surgery. indeed, the ata management guideline for mtc has been very recently revised. in the setting of a prophylactic thyroidectomy, the lymph-nodes are not routinely removed since metastases are not expected to occur at this stage. in the case of clinically apparent mtc, whether sporadic or hereditary, thyroidectomy and concomitant central and compartment-oriented lateral neck dissection should be performed to increase clinical outcomes. primary hyperthyroidism is rare during childhood; therefore, parathyroidectomy is usually avoided, particularly during a prophylactic procedure. dissection and autotransplantation of parathyroid tissue is not typically performed at the time of thyroidectomy unless there is enough biochemical evidence for hyperparathyroidism. thyroidectomy in children is usually associated with a higher rate of complications, such as recurrent laryngeal nerve injury and hypoparathyroidism, as compared to the surgery in adults. therefore, pediatric thyroidectomy must be performed by highly experienced thyroid surgeons. for individuals with a ret mutation who have not had a thyroidectomy, annual biochemical screening of calcitonin levels is recommended and, if the results are abnormal, immediate thyroidectomy is required. annual serum calcitonin screening should begin at age six months for children with men2b and at age 35 for children with men2a or fmtc. biochemical evidence of disease recurrence includes elevation of calcitonin and cea levels. all individuals who have undergone thyroidectomy need thyroid hormone replacement therapy along with annual screening for pheochromocytoma and hyperparathyroidism depending upon the ret mutation present in the patients. mtc does not respond well to radiation therapy or the standard cytotoxic chemotherapeutic agents, including doxorubicin, dacarbazine, capacitabine, and 5-fluorouracil [43, 71]. of note, the mechanism-based targeted therapies that inhibit ret and other receptor tyrosine kinases have become available for the treatment of surgically inoperable progressive mtc. these include the multi-kinase inhibitors, vandetanib (zd6474, caprelsa) and cabozantinib (xl-184, cometriq), which have been recently approved by the us food and drug administration [72, 73]. a recent phase i/ii trial of vandetanib in children with mtc reported partial responses in 47% patients. in general, the drug efficiency and the primary side effects, i.e., diarrhea, rash, headache, hypertension, and nausea, were similar between children and adults. phase iii trial of cabozantinib demonstrated a 28% response rate in adults with significant adverse effects. therefore, there is a critical need for more effective therapies for patients with advanced mtc. characterization of additional molecular pathways responsible for mtc development may allow the discovery of therapeutic targets that can be exploited to induce reduction of tumor size, disease stabilization, and symptomatic improvement [7782]. mtc and the men type 2 syndromes are rare but significant endocrine diseases that are increasingly encountered by pediatricians. our understanding of mtc has been greatly increased by the discovery of ret and the genotype genetic tests according to the established guidelines should be performed whenever diagnosis of mtc is made. due to limited adjuvant treatment options, adequate surgical treatment is critical for initial control of the disease and prophylactic thyroidectomy is recommended for children with men2a and men2b at an early age, sometimes during infancy. emerging newer treatments are expected to better treat this rare but life-threatening malignancy.

medullary thyroid carcinoma (mtc), which originates from thyroid parafollicular c cells, accounts for 3 to 5% of thyroid malignancies. mtc occurs either sporadically or in an inherited autosomal dominant manner. hereditary mtc occurs as a familial mtc or as a part of multiple endocrine neoplasia (men) type 2a and b syndromes. a strong genotype-phenotype correlation has been observed between hereditary mtc and germ-line gain of function mutations of the ret proto-oncogene. most cases of pediatric mtc are hereditary whereas sporadic mtc is rare in children and is usually diagnosed in adults. therefore, mtc in children is most often diagnosed in the course of a familial genetic investigation. the standard treatment of mtc mainly requires surgery involving total thyroidectomy and central neck node dissection before extrathyroidal extension occurs. to prevent mtc development in hereditary syndromes, prophylactic thyroidectomy is performed in presymptomatic patients. an appropriate age at which the surgery should take place is determined based upon the data from genotyping, serum calcitonin measurements, and ultrasonography. for the treatment of advanced mtc cases, the broad spectrum receptor tyrosine kinase inhibitors vandetanib and cabozantinib, which also inhibit ret, are used although they are not always effective.

PMC4803041

pubmed-25

neurosurgery is characterized by the delicate balance between surgical success and potential for devastating side effects. thanks to multiple technological improvements, the morbidity of neurosurgical interventions has substantially decreased over the last decades, allowing for the resection of previously inoperable lesions. in particular, image-guided neurosurgery (igns) devices allow the use of coregistered and fused multimodality 3d images to guide the surgeon's hand and help define preoperatively the boundaries of pathological and predefined functional structures. meanwhile, new modes of medical imaging have also improved the localization of pathological lesions and their characterization. medical imaging nowadays includes a wealth of different techniques, such as computed tomography (ct), structural and functional magnetic resonance imaging (smri and fmri), diffusion tensor imaging (dti), and positron emission tomography (pet). although the overall accuracy of igns is estimated to be 12 mm, current neuronavigation systems can not, however, adapt to changing conditions over time. skull-opening brain shift, brain retraction, cerebrospinal fluid suction, lesion resection, perfusion, and pharmacological manipulation during surgery indeed all alter the 3d morphology of the structures [25]. these changes can lead to localization errors that are one order of magnitude larger that igns accuracy [1, 2, 6] and may result in incomplete resections or unexpected damage to normal brain. such inaccuracies could be reduced if one could acquire, throughout surgery, fresh images of the same modalities and quality as the preoperative ones intraoperative images such as intraoperative mr (imr) images are with the exception of a handful surgical facilities usually acquired using low-field mri scanners that provide lower resolution and contrast than their preoperative counterparts, and, to this date, several useful imaging modalities, such as pet and possibly meg, can not be acquired intraoperatively. one solution is to bring over the high-quality preoperative multimodality images into the intraoperative configuration of the brain using a nonrigid registration technique [710]. one category of nonrigid registration techniques uses physics-based models, where landmarks are tracked in successive reduced-quality intraoperative images, and their displacement fields drive the deformation of a biomechanical model. so far, most of the mechanical conditions of the brain can not be estimated in the operating room, such as the volume of cerebrospinal fluid flowing out of the skull cavity, intercellular fluid volume changes that result from mannitol injection, or changes in blood volume and vessel permeability. the fact that an intraoperative image can provide the knowledge of the current state of the brain after some deformation partly eliminates the need for a complete evaluation of these mechanical conditions. the nonrigid registration technique replaces them with the landmark displacements evaluated from successive intraoperative images. using a nonrigid registration technique based on a biomechanical model, three types of brain deformations have been identified that require specific modeling, although they depend on common parameters, such as csf suction, perfusion, or pharmacological manipulation. the first deformation is the brain shift, which appears at the beginning of surgery with the opening of the skull and dura. the suction or leakage of csf, as well as the release of intracranial pressure caused by tumor growth, generally cause such shift of the brain (note that in this work, we name brain shift the specific shift of the brain that occurs after the opening of the skull and dura, before any other surgical act has happened). however, for these deformations, we will consider that the shift is a part of these two deformations. the second deformation is the retraction; when target tissues are located deep inside the brain, the surgeon incises brain tissues and inserts a retractor to spread out the tissues, and to create a path towards the target. the third deformation is the resection, that is, the removal, of lesion tissues. three deformations can thus be defined in terms of the two elemental actions that change the topology of the brain: the introduction of a discontinuity and the removal of some tissues. most studies of brain deformation based on biomechanical models have focused on shifts (the topology of the brain is not modified), that occurs just after the opening of the skull and dura [1125]. a good review of these different studies can be found in [24, 2628]. resection and retraction are more complex to model than (brain) shift. until recently, their modeling for the specific application of preoperative image update has received much less attention. one of the difficulty for modeling resection and retraction is that both induce a topological change of the brain because some tissue are cut. a method of mesh adaptation [2931] or remeshing [3235] must be used in conjunction with fem if an accurate representation of the location of the cut, for example, the resection cavity or retraction path, is needed to deform the model. indeed, fem can not directly handle discontinuities that go through the fes, and requires to realign the discontinuity with fe boundaries. in the field of fracture mechanics, which studies the growth and propagation of cracks in mechanical parts, some methods were developed to avoid using fem in conjunction with mesh adaptation or remeshing. the extended finite element method (xfem or x-fem) appeared in 1999 and has been the object of considerable research since then. xfem works by allowing the displacement field to be discontinuous within some fes of the mesh. the mesh does not have to conform to the discontinuities, so that these can be arbitrarily located with respect to the underlying fe mesh. because xfem allows an accurate representation of the discontinuities while avoiding mesh adaption or remeshing, and because of the similarity between cracks in mechanical parts and cuts in tissue, we proposed the use of xfem for handling cut, resection, and retraction in the updating of preoperative images. this paper presents a complete 3d framework for updating multimodal preoperative images with respect to surgical brain deformations, due to brain shift and successive resections, followed and quantified using imr images. our approach is modular, and is applied iteratively each time a new intraoperative image is acquired. we take into account successive deformations based on a linear elastic biomechanical model which is deformed using fem or xfem, depending on the type of deformation occurring between the pair of imr images under consideration, namely, brain shift or resection. while some 3d results have already been presented for brain shift, and initial 3d results for resection modelings, this paper is the first complete and detailed account of the generalization to 3d of our 2d previous work. we present the state-of-the-art of resection modeling for preoperative image update. in section 3, we describe our basic strategy for updating preoperative images based on successive intraoperative images. in section 4, we give detail about our methods and algorithms. in section 5, we consider two patient cases that illustrate our approach for handling brain shift followed by successive resections. among studies that take into account resection for preoperative image update, one should distinguish two categories. the first category of studies models brain deformation using two time-point images, the first image being acquired before surgery has started, the second image being acquired after resection. in this category, the methods that existed for a second image showing some brain shift are adapted for a second image showing some resection. the second category of studies models brain deformation using more than two time-point images, and models at least two successive resections. among the first category of studies, hagemann et al. developed a 2d method for modeling brain deformation between a preoperative mr image and a postoperative mr image, the postoperative image showing a complete resection. the 2d mesh of the biomechanical model corresponded to the underlying pixel grid of the 2d image. the biomechanical model included four different linear elastic laws for the skull/skin region, the whole-brain region, the csf region, and the image background. they computed the correspondence of the skull boundary, the whole-brain region boundary in the neighborhood of the tumor, and the posterior midline between the two images. they also computed the correspondence between the internal tumor region boundary visible in the preoperative image, and the resection cavity boundary visible in the postoperative image, both boundaries corresponding under the assumption that the resection is complete. the displacements fields of these landmarks drove the deformation of the biomechanical model. as a result, the biomechanical model presented high deformation in the tumor region, which is not physically plausible. however, the resection was complete, and, thus, they were not interested by the displacement field of the biomechanical model in the tumor region itself. clatz et al. developed a 3d method for modeling the brain deformation between a preoperative mr image and an imr image, the latter showing partial or complete resection. the biomechanical model was deformed based on a sparse volume displacement field evaluated from the two images, using a block matching algorithm. in their algorithm, blocks of voxels that presented discriminant structures were selected in the preoperative image. the blocks were then matched to blocks in the imr image using a similarity criterion, for example, a coefficient of correlation. the value of the similarity criterion was used as a value of confidence in the displacement measured by the block matching algorithm. the biomechanical model was then deformed iteratively, driven by the sparse displacement field of the matched blocks, where a block rejection step was included for measured block displacements initially selected but considered as outliers. in the imr image, a part, or the totality, of the tumor tissues the blocks were thus selected and matched in only the healthy-brain region of the two images. they tested their algorithm on six patient cases, and used for validation nine landmarks picked up manually in each image. they found a mean and maximum error on displacements of 0.75 mm and 2.5 mm, respectively. they explained this phenomenon by the fact that a substantial number of block matchings were rejected in the tumor neighborhood. the deformation of the biomechanical model in the tumor neighborhood was thus essentially governed by the linear elastic law, and the result might show the limitation of this model. archip et al. also tested the nonrigid registration method presented in on eleven patient cases, and used the 95% hausdorff distance for evaluating the alignment of the nonrigidly registered images. as a result, they obtained a mean error of 1.82 mm. among the second category of studies, miga et al. they built a linear poroelastic biomechanical model and preoperatively tagged the tetrahedron fes that were going to be removed to simulate the brain deformation due to successive resections. second, a boundary condition was applied to the new boundary of the resection cavity, in order to model the relaxation of strain energy, induced by preoperative tumor growth or surgery acts, stored in the resected tissues, and released after their removal. in this approach, the tissue discontinuity was represented as best as possible with a jagged topology defined by the fe facets defining the boundary of the resection cavity. [45, 46] also modeled the removal of tetrahedra in order to model the action of an ultrasonic aspirator in the context of real-time surgery simulation. [13, 47, 48] modeled successive resections based on several time-point imr images. between two successive images, they deformed the biomechanical model, in its current state of update, to take into account the (partial) resections(s) that took place between these two images. first, the biomechanical model, in its current state of update, was deformed in accordance with the displacement field of the healthy-brain boundary between the pair of images under consideration. second, the fes that fell into the resection cavity in the second image of the pair were removed, while the fes that laid across the resection-cavity boundary were cut. to ensure the link between the successive deformed configuration of the biomechanical model, their algorithm kept track of the topology modification between fes and nodes of the mesh before and after the removal of fes. they tested their algorithm on one patient case including five imr images (the first two imr images being used for brain shift modeling), and used for validation thirty-two landmarks picked up manually in each image. they found a mean and maximum error on the displacements of 0.9 0.7 mm (mean standard deviation) and 3.7 mm, respectively. they explained this phenomenon by the limited accuracy in the process of picking landmarks in that region, and because the retraction occurring between the second and third images was modeled as a resection, that is, a removal of tissues, even though the tissues were not removed but simply spread out. the methods described above have been all developed using an fem-based biomechanical model for intraoperative image registration. the objective of a surgical simulator is to provide an interactive manipulation with force feedback of the anatomical part to be operated using various surgical instruments. in order to model a large range surgical procedures, jebkov and kuhlen have applied nonlinear xfem for simulating cut, and have shown that xfem is successfully efficient for such purpose. the block diagram of figure 1 shows our global approach for updating preoperative images using successive imr images acquired at different critical points during surgery. although the principles of the approach are quite general, they are tailored for use based on images acquired with a 0.5 tesla intraoperative ge signa scanner, which guarantees that the full volume of brain tissues is included in the image field of view. in our present strategy, the preoperative images are updated incrementally. at the end of each update, the preoperative images should be in the best possible alignment with the last imr image acquired. prior to surgery, a patient-specific biomechanical model is built from the set of preoperative images. because the patient does not necessarily lie in the same position during the acquisition of each of the preoperative images, one may need to perform a rigid registration (involving translations, rotations, and scales) to bring these images into correspondence, assuming, in first approximation, there is no local, that is, nonuniform, brain deformation between preoperative images. once the 1st imr image has been acquired prior to the opening of the skull, the set of registered preoperative images and the biomechanical model are registered to the 1st imr image via a rigid transformation. in the present situation, it is assumed that the patient's brain imaged in the 1st imr image has the same physical shape as the brain imaged in the preoperative images (note that in the following, when an imr image is defined by a number, this number is the index of the imr image in the series for a specific patient case. the 1st imr image thus corresponds to the very first imr image of the series). as each imr image is acquired, this new image and the preceding imr image are used to estimate the deformation of the brain. the update of the preoperative images is done incrementally with each new pair of successive imr images. for each pair, we proceed as follows. a set of common anatomical landmarks the use of surface structures rather than volume structures seems more appropriate given the reduced-quality of typical intraoperative images, and would be more easily adapted to intraoperative modalities other than imr, such as ius. the landmark surface displacement fields resulting from the matching are then applied to the biomechanical model, which is deformed using fem or xfem, depending on the type of deformation occurring between the acquisition times of the imr images in the pair under consideration, namely, brain shift, or resection. the resulting displacement field of the biomechanical model is finally used to warp the set of preoperative images in their current state of updating. note that, for each deformation modeling, the biomechanical model is deformed in accordance with the landmark displacements tracked between the pair of successive imr images under consideration. because intraoperative deformation can follow a reverse direction, it is important to track the landmarks between the next-to-last and the last acquired imr images, rather than track the landmarks between the first and the last acquired imr images. for the patient cases treated in section 5, we assume that the brain undergoes relatively small deformations (small strains and small displacements), and we use a linear finite-element formulation in the biomechanical model. a consequence of using this linear formulation (linear elasticity) is that the equations of solid mechanics can be solved based on the initial configuration of the solid. actually, knowing the displacement field increment un=u u at the anatomical landmarks between configuration n and increment n+1, one can apply this constrained displacement field increment un to the initial configuration, and the finite element analysis will lead to the deformation tensor increment n between the configuration n and n+1. the final deformation tensor or the body is thus simply obtained from =k=0k. remark that rigorously, the increment of constrained displacement field at the landmark should be applied to the balanced solution of the solid reached after increment n, but as we are using a linear elasticity model, this step can be skipped owing to the superposition principle: if =c, then =k=0k=ck=0k=c, where c is the hooke tensor. as a summary, with this approach, the process of deformations is modeled as a succession of deformations k, for example, brain deformation composed of shift followed by successive resections and the current configuration of the brain biomechanical model, after a specific deformation can then be recovered by adding the computed volume displacements for all successive incremental deformations. remark that this is not a limitation of the method as we could easily extend it to nonlinear model by simply keeping in memory the previous deformed configuration n and adding the constrained displacement field increment un to compute the new deformed configuration at increment n+1, simply this would be less computationally efficient. because we use a linear formulation (and, thus, the incremental volume displacement fields can be added to recover the current configuration of the biomechanical model), we could theoretically obtain an identical deformed configuration of the biomechanical model using the two following approaches. the first one would consist of computing and adding the successive incremental deformations of the biomechanical model based on the landmarks tracked between the next-to-last and the last acquired imr images. the second approach would consist in computing directly the deformed configuration of the biomechanical model based on the landmarks tracked between the first and the last acquired imr images. however, the landmarks selected to drive the deformation of the biomechanical model vary depending on the type of deformation, namely, brain shift or resection. in addition, part of the biomechanical model is cut, using xfem, to model resection. consequently, we would not get an identical deformed configuration of the biomechanical model by these two approaches. in order to use a maximum of information from the imr images, we track, as explained for the first approach, the landmarks between the next-to-last and the last acquired imr images. the problem of updating preoperative images between more than two critical points during surgery, that is, based on more than two imr images, is addressed in only a small number of studies. in our previous work [39, 41], and in, the biomechanical model was successively deformed, and this was done using a linear formulation. the framework proposed here, where the initial biomechanical model is always used, instead of using it in its successive states of deformation, has the important advantage of using a good quality mesh for each deformation modeling rather than using a mesh whose quality progressively deteriorates with each successive deformation modeling, and which would require remeshing or mesh adaptation for getting back good fe quality. to summarize, for each deformation, the landmarks are tracked between the two successive imr images under consideration. because we use a linear formulation, the displacement fields of these landmarks are applied to the initial, rather than current, configuration of the biomechanical model. the resulting volume displacement field corresponds to the deformation that the brain undergoes between the two imr images. this volume displacement field is used to deform the preoperative images in their current state of update, that is, registered (at the previous step, if any) to the first imr image of the pair. after the deformation, the preoperative images are thus in as good as possible registration to the second imr image of the pair. in all the rest of this work, we make a simplification of the approach just presented, by using the 1st imr image as a substitute for the preoperative images. the biomechanical model is thus built based on structures visible in the 1st imr image, instead of in the preoperative images, and the structures used in the model are limited to the ones visible in the intraoperative image. except for the rigid registration between the preoperative images, the biomechanical model, and the 1st imr image, this simplified approach allows us to discuss, illustrate, and test all key aspects of the system. the above strategy allows us to focus on the main issue of this paper, that is, the estimation and handling of 3d deformations. even though the issues involved in the update of preoperative images will need to be addressed in a operational image update system, the present strategy of deforming the imr images remains useful for calibration purpose, even in the operating room. this section details the different methods that are commonly used for updating preoperative images in presence of brain shift and resection. more specifically, the block diagram of figure 2(a) shows the building of the biomechanical model from the preoperative images. specific regions from the preoperative images are segmented, meshed, and assigned appropriate constitutive laws. the block diagram of figure 2(b) shows, for any pair of successive imr images, a detailed view of the calculation of the volume displacement field of the initial biomechanical model that corresponds to the deformation that has occurred between the acquisition time of these images. all along surgery, the patient is lying inside the 0.5 tesla intraoperative ge signa scanner. although the patient's head is fixed, one can not totally rule out the possibility of slight head motion. imr images thus have to be rigidly coregistered to take into account this potential rigid motion. the rigid registration that we use is the point-based landmark transform available in vtk (http://www.vtk.org/). the segmentation of imr images into specific regions, for example, healthy-brain and tumor regions, is first performed manually using 3d slicer (http://www.slicer.org/) and then smoothed to minimize the dependance of the results on segmentation roughness. it is clear that performing a manual segmentation in the operating room is not acceptable, and that this process needs to be automated as completely as possible to test the feasibility of our framework online. however, while there exist sophisticated segmentation algorithms that could be used [5052], in particular for extracting the whole-brain region (skull and external cerebrospinal fluid masked out), the segmentation of the tumor region is still challenging. as mentioned above, the biomechanical model is built, in the present context, from the 1st imr image rather than from the preoperative images. thanks to the use of xfem instead of fem for modeling discontinuities, this biomechanical model can be built offline before the operation starts and does not need to be repeated (through remeshing) during the surgery. with respect to fem-based approaches, the execution time thus ceases to be a limiting parameter, which is a remarkable advantage of our approach. it thus requires specific techniques, and we use the meshing software tool isosurf (http://svr-www.eng.cam.ac.uk/~gmt11/software/isosurf/isosurf.html). our goal is to model the boundaries of healthy-brain and tumor regions as two connected surfaces meshes. however, isosurf can only mesh the boundaries of one or several separate regions, and, thus, does not allow one to mesh connected region boundaries with common nodes at their intersections. we thus start by building two separate surfaces meshes that we connect using our own routines based on vtk. the two connected triangle surfaces are then jointly meshed into a single volume mesh of tetrahedra that conform to the two surface meshes using gmsh (http://www.geuz.org/gmsh/). further details on the building of the biomechanical model, in particular the building of the connected surface meshes, can be found in. a linear elastic law is assigned to the biomechanical model, with young modulus e=3000 pa and poisson ratio =0.45. because displacements, rather than forces, are applied to the model using a linear formulation, the fem or xfem solution is independent of young modulus e. we choose as surface landmarks the whole-brain and internal tumor region boundaries. to evaluate the surface deformations of these region boundaries between two imr images, we use an active surface algorithm [55, 56]. because these region boundaries to match must be closed surfaces, we thus use as surface landmarks the whole-brain and healthy-brain region boundaries. the surface deformation of the internal tumor region boundary will be derived from the active surface algorithm of the healthy-brain region boundary. in our active surface algorithm coming from [13, 47, 48], the external forces f(x) are computed using a gradient descent on a distance map of the region boundary. with such external forces, the active surface algorithm is not able to take correctly into account local rigid motion due, as an example, to lateral or tangential movement depending on the head orientation. for the whole-brain region, any rigid transformation that could have occurred has already been taken into account by the rigid registration of the imr images (section 4.1). however, for the healthy-brain region, it can happen that the internal tumor region boundary moves partly in a rigid way. therefore, the active surface, initialized from the healthy-brain region boundary in the first imr image, is first locally transformed in a rigid way along the internal tumor region boundary using the iterative closest point transform available in vtk. then, this resulting surface is deformed using the active surface algorithm as explained above. further details on the local rigid registration of the healthy-brain region boundary can be found in. before applying the displacements whole-brain and internal tumor region boundaries to the biomechanical model nodes, the two surface displacement fields are smoothed based on a weighted-distance average, that is, the displacement of each node is averaged with the displacements of its n closest neighbor nodes. this smoothing will make them consistent with each other, and compatible with the volume mesh in order to avoid element flipping, in particular at the intersections between whole-brain and internal tumor region boundaries. the displacement fields of the surface landmarks are applied to the biomechanical model, which deforms according to the laws of solid mechanics. the equations of solid mechanics are solved using fem or xfem, depending upon the type of circumstances, namely, brain shift or resection. we use the fem-software tool metafor (http://metafor.ltas.ulg.ac.be/) developed in our mechanical-engineering department, to which we have added an xfem module. the initial stress state of the brain is unknown and is thus set to zero for each fem or xfem computation, as in [10, 13]. fem discretizes the solid of interest into a mesh, that is, into a set of fes interconnected by nodes, and approximates the displacement field u(x) by the fem displacement field u(x) defined as (1)ufem(x)=iii(x)ui, where i is the set of nodes, the i(x) 's are the nodal shape functions (nsfs), and the ui's are the nodal degrees of freedom (dofs). each i(x) is defined as being continuous on its compact support i, which corresponds to the union of the domains of the fes connected to node i. in our approach, we use linear nsfs. in contrast, xfem handles a discontinuity by allowing the displacement field to be discontinuous within fes [37, 5860]. the xfem displacement field generalises the fem displacement field (1) with (2)uxfem(x)=iii(x)ui+iji(x)j=1neigj(x)aji. the first term corresponds to the fem displacement field (1), where i is the set of nodes, the i(x) 's are the fem nsfs, and the ui's are the nodal fem dofs. the heart of xfem is the enrichment that adds a number, n, of dofs aji to each node i of the set j, which is the subset of nodes of i whose support is intersected by the discontinuity of interest. these dofs are multiplied by the nsfs i(x) and the discontinuous functions gj(x). the use of specific xfem enrichment functions gj(x) for a node i j depends on the type of discontinuity, for example, crack, hole, material interface, and so forth, to be modeled. suppose that our goal is to model a crack, characterized by a discontinuity in the displacement field (as opposed to a material interface for instance, characterized by a discontinuity in the derivative of the displacement field). when the crack fully intersects the support of the node, a simple choice is a piecewise-constant unit function that changes sign at the boundary across the crack, that is, the heaviside function (3)h(x)={1for (xx)en>0,1for (xx)en<0, where x is again the position of a point of the solid, x*is the position of the point on the crack that is the closest to x, and en is the outward normal to the crack at x *. in case of resection deformation, the goal is to model a discontinuity such that the part of tissues corresponding to tissue removed by the resection has no influence on the deformation of the remaining part of the tissues. one is actually interested in the deformation of the remaining part of the tissues only. in that sense, the hole function as the following equation: (4)v(x)={1for (xx)en>0,0for (xx)en<0, could be used as xfem enrichment function, instead of the heaviside function, and would be totally sufficient. the results that we would obtain on the remaining part of the tissues would be identical. however, because the heaviside function is necessary for retraction modeling, we have used the same function for the resection modeling even if it was not strictly necessary. when minimizing the total deformation energy, the resulting xfem equations remain sparse and symmetric as for fem. whereas fem requires a remeshing and the duplication of the nodes along the crack to take into account any discontinuity, xfem requires the identification of the nodes whose support is intersected by the crack and the addition of dofs: (1) any node whose support is not intersected by the discontinuity remains unaffected and thus possesses three dofs; (2) any node whose support is fully intersected by the discontinuity is enriched with three heaviside dofs and thus possesses six dofs. to qualitatively estimate the similarity between two images, we compare the edges extracted from these images using the canny edge detector available in itk (http://www.itk.org/). indeed, although potentially useful for the sake of comparing methods on a mathematical basis and defining unique correspondences, landmark-based target analysis presents several relevant limitations in the present setting. having experts picking landmarks introduces significant intra- and interobserver variability. picking landmark points, as ferrant et al. did, is rather difficult when it comes to define enough visible landmarks especially in the tumor region on the 5 different images (and not 2 images only, as majority of studies focusing on brain shift are using). rather than point targets, linear tumor contours, and limits between structures and potential eloquent structures matter most in the practical case of tumor ablation neurosurgery. these are the reason why we chose to use the canny edges in order to evaluate the registration. besides, while it is true that these edges do not necessarily physically correspond between the successive imr images, these images have been acquired with the same image protocol (mr sequence, voxel size, grayscale value range), which should limit this problem. to quantitatively estimate the similarity of the two edge maps, we compute the modified hausdorff distance between the sets of edge points, that is, voxels representing the edges, in these two images. the modified hausdorff distance (a, b) between two sets of points a and b is defined as (5)(a, b)=max(h(a, b),h(b, a)) with h(a, b)=1naaad(a, b), where the directed hausdorff distance h(a, b) is a measure of the distance of the point set a to the point set b, na is the number of points in set a, and d(a, b) is the distance of point a a to the closest point in b, that is, d(a, b)=minbb||a b||, where ||a b|| is the euclidean distance. the directed hausdorff distance h(a, b) thus computes the average distance of points of a to points of b. the averaging minimizes the effects of outlier points, for example, due to image noise. the value of the modified hausdorff distance (a, b) increases with the amount of difference between the two sets of edges points. in the following, we denote by (ia, ib) the modified hausdorff distance of the edges extracted from the whole-brain region of the images ia and ib, that is, with the skull and external cerebrospinal fluid masked out from them. in this section, we apply our methods, respectively, of brain shift and resection (imr images are acquired with the 0.5 tesla intraoperative ge signa scanner of the brigham and women's hospital, boston, usa. imr image size is 256 256 60 voxels, and voxel size is 0.9375 0.9375 2.5 mm). two patient cases, each including five imr images, are treated to illustrate our modeling and brain shift followed by successive resections. in both cases, the 1st imr image was acquired prior to the opening of the skull; the 2nd imr image was acquired after the opening of the skull and dura, and shows some brain shift; the 3rd, 4th, and 5th imr images were acquired after successive resections. the modelings of brain shift, 1st, 2nd, and 3rd resection are performed using different techniques, as detailed below. except where otherwise noted, the following discussion applies to both patient cases (the result of each deformation modeling is shown for the two patient cases at the end of section 5.2.3). to model brain shift based on the 1st and 2nd imr images, we estimate the surface displacement fields of the whole-brain region boundary and the internal tumor region boundary from the two imr images. no tissue discontinuity is involved in the brain shift deformation, so the biomechanical model is deformed using fem. this results in the volume displacement field of the biomechanical model, which is illustrated in figure 3 for the first patient case. this volume displacement field is used to warp the part of the 1st imr image corresponding to the whole-brain region. in the following sections, the three successive resections are modeled separately, because they require different types of processing. matching two region boundaries to get a displacement field makes sense only if they correspond to the same physical entity. once the resection has started, we can no longer rely on the entirety of the whole-brain region boundary, since a part of it is now missing. for modeling the successive resections, we thus evaluate the displacement field for the boundary of the healthy-brain region only. the 1st resection occurs between the times the 2nd and 3rd imr images are acquired. however, since the corresponding removal of tissues is most likely accompanied by deformation, one can not exactly determine what tissue is removed based just on the two imr images. we thus decided to model the 1st resection by still relying on the displacement fields of key surfaces, here the healthy-brain region boundary, to deform the biomechanical model. this indeed appears to be the only reliable information concerning the deformation due to resection that we can extract from the 2nd and 3rd imr images. consequently, we do not model explicitly the removal of tissue, but we model directly the deformation resulting from it, without introducing any tissue discontinuity. using the surface displacement field of the healthy-brain region boundary, we compute the deformation of the biomechanical model via fem. then, using the resulting volume displacement field, we warp the part of the 2nd imr image corresponding to the whole-brain region, in the same way as we did in the case of for brain shift. the image resulting from the 1st resection modeling is now registered to the 3rd imr image, except outside of the healthy-brain region boundary, that is, for the tumor region. finally, we alter the resulting image to reflect the effect of resection. for this, we assign the background color to the voxels corresponding to the resected tissue volume the significant feature of the 2nd resection is that some tissue has already been removed by the 1st resection, which means that this tissue can not have any physical influence on subsequent brain deformations because it does not recall that the biomechanical model has been deformed to model the brain shift and the 1st resection and is thus registered to the 3rd imr image. so, using the 3rd imr image, we can define the boundary of the 1st resection, that is, the tissue discontinuity to include in the deformed biomechanical model (figures 4(a) and 4(b)). we then enrich the nodes whose supports are intersected by the discontinuity with heaviside dofs. consequently, when the xfem-based biomechanical model deforms, the part corresponding to tissue removed by the 1st resection has no influence on the deformation of the remaining part of the brain. for the first patient case illustrated in figure 4, the tetrahedron mesh consists of 3,317 nodes, which corresponds to 9,951 fem dofs. the biomechanical model is deformed in accordance with the displacement field of the healthy-brain region boundary evaluated from the 3rd and 4th imr images. the bottom part of the mesh, representing the tissue remaining after the 1st resection, has been deformed according to the displacement field of the healthy-brain region boundary, while the top part, representing the tissue removed by the 1st resection, has been subjected to a translation, but only for visualization purposes. even though the mesh is displayed as two separate parts, it is, in fact, a single entity. indeed, a main feature of xfem is its ability to handle the effect of a discontinuity without modifying the underlying mesh, that is, without remeshing. for modeling the 2nd resection, the edges of fes straddling the discontinuity have been made discontinuous and their nodes moved apart. using the xfem volume displacement field, we warp the part of the 3rd imr image corresponding to the whole-brain region. the resulting image is then masked out with the whole-brain region segmented out from the 4th imr image. one significant feature of the procedure described for modeling the 2nd resection is that it can be applied repetitively for each subsequent resection visible on successive imr images, no matter how many there are. the modeling of the 3rd resection is thus identical to the modeling of the 2nd resection. the tissue discontinuity due to the 2nd resection is defined from the 4th imr image, and used to appropriately enrich the nodes of the biomechanical model. then, this biomechanical model is deformed using xfem, in accordance with the displacement field of the healthy-brain region boundary evaluated from the 4th and 5th imr images. for the first patient case, a simplification for the modeling of the 3rd resection can be made because, by the time the 5th imr image is acquired, the resection is complete. this means that we only need to compute the volume displacement field of the healthy-brain region. since we apply displacements exactly to the boundary of the healthy-brain region, the results obtained with fem and xfem will be identical. using the fem (for the first patient case) or xfem (for the second patient case) volume displacement field, we warp the part of the 4th imr image corresponding to the whole-brain region. the resulting image is then masked out with the whole-brain region segmented out from the 5th imr image. figures 5 and 6 show the results of warping the imr images, as well as the edges extracted from them, after brain shift and each successive resection modeling for the two patient cases. as explained in section 5.2.3, since we apply displacements exactly to the boundary of the healthy-brain region, the results obtained with fem and xfem are identical in the healthy-brain region. one can deduce that using xfem for modeling resection is interesting when the neurosurgeon needs to have an accurate displacement field of the remaining tumor tissues. in this case, it is interesting to evaluate the impact of using fem, instead of xfem, to model the resection as if no resection was performed before. using fem for modeling resection is equivalent to ignoring the presence of resection on intraoperative images. to illustrate the comparison between fem and xfem results, we choose the 3rd resection modeling of the second patient case indeed, it is the deformation with remaining tumor tissues that shows the largest magnitude, and, thus, that is likely to give a maximum difference between the two computations. the healthy-brain and tumor regions segmented out from the 4th and 5th imr images are respectively shown in figures 7(a) and 7(b). the volume displacement fields of the biomechanical model using xfem and fem are respectively shown in figures 7(c) and 7(d). the part of the 4th imr image corresponding to the whole-brain region is warped, first with the volume displacement field obtained via fem, and then with that obtained via xfem. the difference between the two warped images is shown in figure 7(e). as expected however, the difference between the two volume displacement fields is smaller than the image resolution (although the difference between the two volume displacement fields is smaller than the image resolution, the difference between the images resulting of the warping using these two volume displacement fields is nonzero. this is explained by the fact that the (gray) value of each voxel of the warped image is defined as a weighted-value of voxels of the original image. the weights are defined based on the overlapping ratio of the voxel of the warped image, with voxels (determined using the volume displacement field) of the original image). in addition, the deformed 4th imr images, using the xfem- and the fem-based deformations of the biomechanical model, show the same similarity, computed based on the modified hausdorff distance, with the 5th imr. two reasons explain that the differences between the fem and xfem results are so small. first, the brain deformation itself due to the 3rd resection is small, and, thus, it is expected to obtain small differences between the two resulting brain deformations. second, in the case the remaining tumor tissues are close to the healthy-brain region boundary, it implies that they are close to the boundary where surface displacement fields are applied to drive the deformation of the biomechanical model. although this comparison between fem and xfem should be done on more patient cases, we suggest that, in first approximation, fem could be used for modeling resection cases with small brain deformations. nevertheless, the presentation of the successive resections using xfem shows the generality of our framework, and details how xfem is implemented. note that in section 6 devoted to validation, the warped images are the ones deformed with xfem. for each deformation modeling based on a pair (ik, ik+1) of two successive imr images that are already rigidly registered, we compare the similarity between these ik and ik+1 images, as well as the similarity between the ik and ik+1 images, where ik is the result of warping ik. this gives us an estimate of how well we are able to capture, and compensate for, the local deformations between ik and ik+1. the goal of the nonrigid registration is, however, to deform the preoperative images. by warping ik for each deformation modeling, we do not take into account the fact that an error of alignment after each deformation modeling could propagate and amplify through the successive deformation modelings. to evaluate the effect of this error amplification on the results, we also perform the required succession of warpings on i1, and we denote the resulting image by i1,k. we then compare, for each deformation modeling, the similarity between i1 and ik+1, together with the similarity between i1,k and ik+1. this allows one to evaluate the propagation, that is, the amplification, of alignment error on the results. the modified hausdorff distance computed for each pair of imr images are given in tables 1 and 2. table 1 shows, for each deformation modeling based on a pair (ik, ik+1) of two successive imr images, the values of the modified hausdorff distances (ik, ik+1) and (ik, ik+1). these values are computed using the canny edges extracted from the pair of images (ik, ik+1) (figures 5 (d) and 6 (d)) and (ik, ik+1) (figures 5 (e) and 6 (e)). we observe that the values for the images nonrigidly registered are relatively constant, that is, 1 mm, for each deformation modeling. six out of eight deformation modelings give smaller modified hausdorff distances when the imr images are (rigidly and subsequently) nonrigidly registered. however, the modified hausdorff distance increases for the 3rd resection modeling of the first patient case, as well as for the brain shift modeling of the second patient case. to understand if the nonrigid registration is responsible for the increase of the misalignment of the two imr images everywhere in the whole-brain region, or if this effect is localized, we compute the modified hausdorff distance in the region and neighborhood of the tumor only (volume region that extents by 25 mm the tumor region segmented in i1 for both patient cases). the modified hausdorff distance decreases from (i4, i5)=1.70 mm to (i4, i5)=1.37 mm for the first patient case, while it decreases from (i1, i2)=1.36 mm to (i1, i2)=1.28 mm for the second patient case. this indicates that the nonrigid registration enhances the alignment of the two imr images within the tumor region and its neighborhood, which is in fact the location requiring the best modeling accuracy. this behavior could be explained by the fact that a maximum of information from the imr images is used in this region, that is, one or two (in case of brain shift modeling) surface displacement fields are applied around it. the increase of misalignment elsewhere in the brain volume could be explained by two reasons. first, the landmarks tracked from the imr images are surfaces. as a consequence, the nonrigid registration is expected to give better results near the tracked surfaces than far from them in the volume. the volume misalignment could point out the need for better parameters values and/or other constitutive laws. table 2 shows, for each deformation modeling based on a pair (ik, ik+1) of two successive imr images, the values of the modified hausdorff distances (i1, ik+1) and (i1,k, ik+1). so far, igns systems allow one to rigidly register preoperative and successive imr images. (i1, ik+1) thus represents the navigation accuracy that we can obtain with an igns system at the present time. the comparison of (i1, ik+1) with (i1,k, ik+1) gives the improvement that could be practically achieved in the alignment with our approach. as expected, table 2 shows that the igns accuracy decreases through the successive deformations. indeed, the modified hausdorff distance increases from (i1, i2)=1.24 mm to (i1, i5)=1.78 mm for the first patient case, and from (i1, i2)=1.01 mm to (i1, i5)=1.68 mm for the second patient case. six out of eight deformation modelings give smaller modified hausdorff distances when the imr images are nonrigidly registered. to understand if the modified hausdorff distance increases everywhere in the whole-brain region for the brain shift and 1st resection modeling of the second patient case, we compute the modified hausdorff distance in the neighborhood of the tumor region (in the same way as explained for table 1), and observe the improvement of the alignment within the tumor region and its neighborhood. as opposed to the values of the modified hausdorff distances in table 1, the values for the images nonrigidly registered in table 2 increase through the successive resection modeling. this amplification error is due to the fact that, after having modeled brain deformation between a pair of imr images, the deformed biomechanical model is not in perfect alignment with the second image of the pair. since, for the subsequent deformation modeling, the surface landmarks are initialized based on the deformed biomechanical model, this can thus ampliy a misregistration error. we developed a complete 3d framework for serial preoperative image update in the presence of brain shift followed by successive resections. the nonrigid registration technique used an homogeneous linear elastic biomechanical model, driven by the deformations of whole-brain and internal tumor region boundaries for brain shift modeling, and healthy-brain region boundary for resection modelings, tracked between successive imr images. the biomechanical model was deformed using fem for brain shift modeling, and fem or xfem for resection modeling, depending upon whether some brain tissues were previously resected or not. we showed that our approach was modular, and could be applied each time a new imr image is acquired. we used a linear formulation to characterize the deformation of the brains of both patients because the brains underwent relatively small deformations and displacements. while nonlinear biomechanical models have proven effective to decrease yet do not abolish the inaccuracies of fem-based modeling methods of large brain deformations, the deformations observed in our patients during surgery remained moderate (47 mm), thus reducing the theoretical benefit of using nonlinear models. this allowed us to use simpler linear models and focus on the added value of xfem to simultaneously account for surgical deformations, namely, shift and resection. using a linear formulation implied that, for each new deformation modeling, one could use the initial configuration rather than the last-deformed configuration of the biomechanical model. this had the important advantage of using a good quality mesh for each deformation modeling rather than using a mesh whose quality progressively degraded with each successive deformation modeling. this also had the advantage that we did no longer need to reconnect the deformed mesh for each new xfem calculation, which was one drawback of our previous method, presented in [39, 41], where the biomechanical model was successively deformed. we also showed how xfem could handle a discontinuity for modeling resection without any remeshing or mesh adaptation while the representation of the discontinuity remained accurate, that is, the representation of the discontinuity was not based on a jagged topology using fe facets. xfem thus also avoided making the mesh resolution richer in the neighborhood of the resection-cavity boundary for improving the accuracy of the representation of the discontinuity for that purpose only. we showed that our nonrigid registration technique improved the alignment of the successive imr images for most of the deformation modeling of both patient cases. when our nonrigid registration failed, it still improved the alignment locally, that is, within the tumor region and its neighborhood. we tested the explicit modeling of the lateral ventricles ' region with a soft, compressible law in addition to the whole-brain region law used in the homogeneous biomechanical model. in addition to the validation that is usually performed for successive deformation modelings, that is, validation between pairs of successive intraoperative images, shown in table 1 of section 6 or in the work of ferrant et al. [13, 47, 48], we also evaluated the fact that an error of alignment after each deformation modeling could propagate and amplify through the successive deformation modelings. as a result, shown in table 2 of section 6, we showed that our approach suffered from the propagation of misregistration through the successive deformation modelings. we expected that this was due, at least partly, to the algorithms used to evaluate intraoperative surface displacements fields from the whole-brain and healthy-brain region boundaries. the surface displacement fields were computed using active surface algorithms, and smoothed to make them compatible with the biomechanical model. because of these two smoothings, the deformed biomechanical model was likely to not be in a perfect alignment with the imr image to which it was registered. because the surface displacement fields evaluated for the next deformation modeling were initialized based on the deformed biomechanical model, we expected to observe an amplification of the misregistration, which was confirmed by our quantitative evaluation. at the present time though, commercial igns systems allow one to register preoperative images and successive imr images, but in a rigid way only. consequently, although the effect of error amplification exists, our technique still enhances the current capabilities of commercial igns systems. future work on modeling of brain shift followed by successive resection is required in five main areas. first, the effect of error amplification through the successive brain deformation modelings calls for further research. consequently, the segmentation, and the subsequent smoothing, as well as the evaluation of surface displacement fields, should be improved to minimize the effect of error amplification. second, further research is required to include additional structures in the biomechanical model in general, and to study the best way to include the lateral ventricles in particular. the use of a poroelastic model in order to model the cerebrospinal fluid filling the ventricles could be considered [17, 18]. indeed, these images provide volume information (rather than surface information only), are of good quality in comparison to other intraoperative modalities, and possess a field of view that includes the full volume of brain tissues (for the 0.5 tesla ge signa scanner). these images thus allow one to evaluate what, and how, new structures of the brain could be used, to enhance the modeling of brain shift. some regions, for example, the lateral ventricles ' region, could be extracted from the two imr images, and used as surface landmarks to drive the deformation of the biomechanical model [13, 62]. indeed, the workflow presented in this paper has the advantage of being easily adaptable. in case the tumor region would not be visible (enough) on the imr images, these new structures, easier to segment, could also adequately replace the tumor for driving the deformation. fourth, our global approach should no longer be based on the 1st imr image used as a substitute for preoperative images, but on the preoperative images themselves. fifth, we should implement, for the surgery cases involving large deformations of the brain, a nonlinear formulation of fem [63, 64], and, particularly, a nonlinear formulation of xfem, which is the subject of recent research [65, 66].

current neuronavigation systems can not adapt to changing intraoperative conditions over time. to overcome this limitation, we present an experimental end-to-end system capable of updating 3d preoperative images in the presence of brain shift and successive resections. the heart of our system is a nonrigid registration technique using a biomechanical model, driven by the deformations of key surfaces tracked in successive intraoperative images. the biomechanical model is deformed using fem or xfem, depending on the type of deformation under consideration, namely, brain shift or resection. we describe the operation of our system on two patient cases, each comprising five intraoperative mr images, and we demonstrate that our approach significantly improves the alignment of nonrigidly registered images.

PMC3263624

pubmed-26

acquired angioedema (aae), an acquired deficiency of c1esterase inhibitor, is a medically treatable condition which can cause severe abdominal pain mimicking an acute surgical abdomen. this disorder is strongly associated with chronic lymphocytic leukemia (cll) and other indolent lymphoplasmacytic disorders. we describe a patient with known cll who developed incapacitating, recurrent severe abdominal pains, culminating in partial bowel resection. wider appreciation of the possibility of aae, particularly in patients with lymphoproliferative disorders, could lead to preventive therapy and spare unnecessary surgery. acquired angioedema (aae) is due to acquired deficiency of c1 inhibitor (c1inh), resulting in excessive complement and bradykinin activities. blood vessel permeability is increased; thus, angioedema occurs. just as with hereditary angioedema (hereditary c1inh deficiency; hae), common clinical manifestations are skin swelling, laryngeal edema, and/or abdominal pain.1,2 aae often occurs in the context of lymphoplasmacytic disorders, such as monoclonal gammopathy of unknown significance (mgus), non-hodgkin s lymphoma, or chronic lymphocytic leukemia (cll).1,3 among 32 patients with aae, castelli found that 13 (40%) had mgus and 9 (28%) had lymphoproliferative disease.3 therefore, all cases of aae should be evaluated for the possibility of underlying lymphoplasmacytic disorder. conversely, when patients with known lymphoproliferative disease manifest compatible symptoms, aae should be expeditiously considered. this is important because aae can be effectively treated medically, but delayed diagnosis can lead to unnecessary diagnostic procedures, therapeutic interventions, or life-threatening complications, well-illustrated by our case. a 78-year-old woman with atherosclerotic vascular disease was transferred to our hospital with abdominal pain and underwent emergent laparotomy. one year earlier, she had been diagnosed with rai stage i cll which had been observed without treatment. two months earlier, she presented with severe abdominal pain, nausea, and vomiting. over the next 8 weeks, she had six emergency room and/or hospital admissions for identical symptoms. pains would begin at rest in the lower abdomen, spread to the upper abdomen, described as gas-like, non-radiating, constant. extensive evaluation included colonoscopy, endoscopic retrograde cholangiopancreatography, magnetic resonance angiography, and abdominal aortography. benign colon polyps and small gallstones were removed, mesenteric stenoses ruled out, yet pains recurred unabated. there was no dysphagia, change in bowel habits, gi bleeding, fever, or sweats. physical examination on transfer revealed blood pressure 130/88 mmhg, pulse 88 beats per minute, respiratory rate 20 cycles per minute, pulse oximetry of 95% on ambient air, and temperature 97.0f. hemoglobin was 18.1 g/dl, hematocrit 55%, platelets 146 10/l, and leukocytes elevated to 34,500 cells/l with 47% neutrophils and 48% lymphocytes. ct scan of abdomen and pelvis with iv contrast showed multiple abnormal loops of small bowel with contrast-enhanced bowel wall edema (fig.. 1a ct scan of abdomen and pelvis with intravenous contrast shows several abnormal loops of small bowel with a target appearance indicating bowel wall edema. b a section of small bowel shows massive submucosal edema. there is no infiltration of the wall by lymphocytes a ct scan of abdomen and pelvis with intravenous contrast shows several abnormal loops of small bowel with a target appearance indicating bowel wall edema. b a section of small bowel shows massive submucosal edema. there is no infiltration of the wall by lymphocytes at laparotomy, massively swollen small bowel was encountered and resected. c4 was 3 mg/dl (normal 1746), c3 66 mg/dl (85200), and c1inh activity reportedly 83% (68200%). serum protein electrophoresis revealed two faint bands immunofixing as monoclonal igm kappa and igg kappa. lymphocytosis and lymphadenopathy improved and c1inh activity increased to 110%. over 3 years of follow-up, abdominal symptoms never recurred. approximately 145 cases have been reported of aae,3 and this is one of four cases that we have diagnosed in the last decade with abdominal pains from aae with associated cll. table 1 shows characteristics of patients with cll and aae we have seen (some briefly mentioned in a prior report).4 this is our only case to undergo surgery, allowing unique and dramatic demonstration of massive bowel edema visible radiographically, on surgical inspection and on histopathology. an initial c1inh activity was reported low normal, but there can be no doubt about the diagnosis based on radiologic/surgical/histologic findings, further laboratory results, and the clinical course. characteristic are very low c4 level and low c3; diagnostic recommendations currently add c1q and anti-c1inh antibody levels. autoantibody is demonstrable in up to 70% with aae.1 our patient had monoclonal gammopathy, which frequently corresponds to the c1inh autoantibody. patients with the hereditary form (hae) usually manifest before age 20 and give a family history of symptoms. in all our patients with cll and aae, chemotherapy and androgens increased c1inh and produced durable remission. table 1characteristics of patients with aaepatientunderlying diagnosismanifestations of acquired angioedemamonths before diagnosislaboratory valuesinterventions prior to diagnosistreatmentangioeedema episodes post-treatment (years of follow-up)pretreatmentpost-treatment78 years old/fcllrecurrent abdominal pain2c1 inh activity: 83% (68200)c1 inh activity: 110% (68200)exploratory laparotomy with small bowel resectionchemotherapydanazolnone (3 years)c4: 3 mg/dl (1746)ct scanc3: 66 mg/dl (85200)colonoscopysmall monoclonal gammopathyercpmraabdominal aortography74 years old/fsll/cllrecurrent abdominal pain24c1 inh activity: 1% (68200%)c1 inh activity: 117% (68200%)ct scanchemotherapynone (6 years)episodic oropharyngeal swellingc1 inh quantitative: 6.7 mg/dl (> 11 mg/dl)c1 inh quantitative: 21 mg/dl (1025)colonoscopydanazolc4: 13 mg/dl (1647)c3: 70 mg/dl (75161)small monoclonal gammopathy61 years old/mcllrecurrent abdominal pain5c1 inh activity: 4% (68200%)not availablect scanchemotherapynone (10 years)small monoclonal gammopathycolonoscopydanazol67 years old/mcllrecurrent abdominal pain3c1 inh activity: 6%(68200)c1 inh quantitative: 38 mg/dl (2139)colonoscopychemotherapynone (3 months)oropharyngeal swellingc1 inh quantitative: 3 mg/dl (2139)laryngoscopydanazolc1q: 3.6 mg/dl (58.6)c4:<2 mg/dl (1746)small monoclonal gammopathy characteristics of patients with aae angioedema should be borne in mind among medical illnesses that can mimic acute surgical abdomen, along with such other disorders as porphyria, familial mediterranean fever and sickle cell disease (fig. 2). one may need to discern whether a true surgical emergency might supervene even when one of these disorders is present. our patient had typical symptoms of acute bowel edema, including diffuse abdominal pain, occasionally rebound tenderness and vomiting, with spontaneous resolution within 15 days.1,2 some patients with aae have cutaneous or upper respiratory edema in addition, or instead of, bowel symptoms. because of cardiovascular comorbidities, there was a high suspicion for ischemic bowel in our patient, but radiography and endoscopy did not support this. in the differential diagnosis, angiotensin-converting enzyme inhibitors rarely precipitate angioedema, but our patient had taken benazepril many years and continued it without incident after aae therapy. fig. 2algorithm for diagnosis and treatment for suspected aae algorithm for diagnosis and treatment for suspected aae in 2009, the us fda approved the c1inh concentrate berinert p and the kallikrein inhibitor ecallantide (kalbitor) for treatment of acute attacks, and the c1inh concentrate cinryze for prophylaxis in severely affected patients.5 fresh frozen plasma can be given when these are unavailable. c1inh concentrates are highly safe and effective; the standard of care for decades in other countries, but us approval was delayed by concerns of virus transmissibility.1,59 doses used in the clinical hae trials may need to be higher for aae because of increased enzyme clearance. a bradykinin b2 receptor antagonist icatibant (firazyr) is under investigation. for long-term control, commonly used for prophylaxis are anti-fibrinolytic drugs or the attenuated androgen danazol, which increases c1inh synthesis at low cost.10 in conclusion, earlier suspicion for aae in our known cll patient could have spared her the morbidities of recurrent abdominal pains, hospitalizations, morbid interventions, and bowel resection. wider appreciation of this disorder takes on added importance as our ability to effectively treat the problem has grown.

introductionacquired angioedema (aae), an acquired deficiency of c1esterase inhibitor, is a medically treatable condition which can cause severe abdominal pain mimicking an acute surgical abdomen. this disorder is strongly associated with chronic lymphocytic leukemia (cll) and other indolent lymphoplasmacytic disorders.discussionwe describe a patient with known cll who developed incapacitating, recurrent severe abdominal pains, culminating in partial bowel resection. signs, symptoms, laboratory and pathologic findings demonstrated aae. conclusionwider appreciation of the possibility of aae, particularly in patients with lymphoproliferative disorders, could lead to preventive therapy and spare unnecessary surgery. this is more important now that more effective medical therapies are available.

PMC3220812

pubmed-27

a 68-year-old woman visited the clinic with the chief complaints of pain, swelling, and a warm sensation in her left thigh for over 2 days prior to her visit. the patient also complained about a warm sensation during the nighttime but body temperature was normal when measured in the clinic. she had undergone a left bipolar hemiarthroplasty following a hip fracture 24 days prior to the current visit and was wearing compression stockings to prevent the occurrence of a deep vein thrombosis. she had been taking aspirin 100 mg daily, fexonadine 180 mg daily, and hydrea 500 mg twice a day for a diagnosis of polycythemia vera for 1 year and was phlebotomized in a hemato-oncology setting on an irregular basis. in advance of the operation, her erythrocyte sedimentation rate (esr), and c-reactive protein (crp) levels were 30 mm/hr and 4.99 mg/dl, respectively. three days after her hip surgery, her esr and crp levels were 23 mm/hr and 6.35 mg/dl, respectively. medical care was administered in close cooperation with the hemato-oncology department and the level of hemoglobin was maintained at<12 g/dl to prevent a thrombosis prior to her operation. the patient did not have a fever and her skin color seemed normal despite swelling and a slight warm sensation in the left hip and proximal femur. results of a blood test showed that her complete blood count, electrolyte levels, and liver function were all within normal ranges (white blood cell count [wbc]: 4,700/l, hemoglobin: 10.6 g/dl, hematocrit: 32.3%, platelet: 213,000/l, neutrophil count 67.2%, lymphocyte 24.1%, monocyte 6.8%) whereas the esr and crp level were 38 mm/hr and 8.25 mg/dl, respectively. when compared to tests performed previously, no further observations of additional fractures, signs of infection, or loosening of inserts after the surgery acute or sub-acute inflammation was diagnosed given the perfusion image of soft tissue in the left hip area as well as the blood pool image (fig. the patient was injected intravenously with antibiotics for 1 week while staying in the hospital, however, symptoms were not alleviated. in order to prevent a deep vein thrombosis, the patient started to move with the aid of a wheelchair and exercised her knee joints 3 days after the operation. g/dl, a color doppler ultrasonography was performed due to her history of polycythemia vera. results revealed a deep vein thrombosis in the common femoral vein as well as in the superficial femoral vein (fig. symptoms were alleviated following the administration of low-molecular-weight heparin (enoxaparin, 40 mg) and warfarin (5 mg) for 5 days. additional warfarin was prescribed (2.5 mg daily) for maintenance at discharge. during her first follow-up visit 2 weeks later, no symptoms were noted and all blood test parameters were within normal ranges. it has been reported that patients with polycythemia vera present with various complications related to thrombosis due to excessive blood viscosity1). several putative reasons have been postulated to explain such complications including hypervolemia, an increase in the volume of red blood cells, telangiectasia due to decreased blood flow velocity as well as vascular elasticity, thrombocytosis, and complex hemostatic disorders attributable to inefficient blood clotting processes2). thrombosis is the leading cause of death in those with polycythemia vera and has been associated with high morbidity as well. approximately 12-49% of patients with polycythemia vera experience thrombosis and 20-40% will die as a result2). thromboembolism is one of the major complications of hip surgery and contributes to its poor prognosis. warwick et al.3) reported that the incidence rate for a deep vein thrombosis was about 1.89% if proper medication was not provided after total hip arthroplasty. therefore, in the case of the patient described in the current case report, it was reasonable to expect that she would be susceptible to the high incidence rate of thrombosis as she underwent the hip surgery based on her history of polycythemia vera. for prophylactic purposes, aspirin was administrated (100 mg daily) and early exercise and active movements were recommended for the patient in order to lower the risk of venous thromboembolism. in the current case report, a patient with polycythemia vera exhibited clinical symptoms and signs of a postoperative infection following bipolar hemiarthroplasty due to femoral neck fractures. there was, however, a deep vein thrombosis which rendered the intravenous administration of antibiotics ineffective in the alleviation of symptoms. early symptoms of a deep vein thrombosis, including swelling, pain, oppressive pain, and a warm sensation, can mimic those of a postoperative infection. furthermore, features of systematic inflammatory responses such as increased levels of interleukin-6, interleukin-8, and crp may be observed in patients with a deep vein thrombosis. collectively, these findings suggest that appropriate clinical examinations are warranted at an early stage in order to avoid making an inaccurate diagnosis4,5). on the first visit after the operation, the patient was suspected to have had a postoperative infection due to the simultaneous increase in esr and crp levels. however, the specificity of esr and cpr levels pertaining to the diagnosis of infections is questionable, as most patients display elevated levels of esr and crp following a hip surgery6). these inflammatory responses stimulate the production of cytokines due to the fractures and operation per se, thus biochemical parameters for inflammation are known to be elevated in a non-specific manner. in cases without postoperative infections, elevated crp levels recover to the normal range within 3 weeks of the operation6,7). in the current case report, the elevated crp level that was maintained for up to 24 days after the operation led us to suspect a postoperative infection. however, the abnormally increased level of acute phase reactants (e.g., crp) may be observed in patients with chronic myeloproliferative diseases (e.g., polycythemia vera) and therefore further specific clinical examinations are required for an infectious diagnosis8). regarding the diagnosis of a postoperative infection, elevated level of serum procalcitonin may be more valuable than wbcs and crp levels7). regrettably, the level of serum procalcitonin was not measured in the current case but could have been informative in distinguishing between postoperative infection and a deep vein thrombosis. despite the presentation of similar clinical symptoms between a postoperative infection and a deep vein thrombosis, their etiological mechanisms are completely distinct. therefore, different treatments are warranted, as patients receiving inappropriate medical treatments may experience exacerbated symptoms and conditions. the potential for a deep vein thrombosis requires continuous thought and attention regarding relevant examinations for patients at high risk for thrombosis as well as for embolism (e.g., polycythemia vera).

a 68-year-old woman who suffered from polycythemia vera presented at our clinic with the chief complaints of pain, swelling, and a warm sensation in her left thigh. she had undergone a left bipolar hemiarthroplasty following a hip fracture 24 days prior to this presentation. her erythrocyte sedimentation rate and c-reactive protein (crp) levels were elevated. in addition, a postoperative infection was suspected in the 3-phase bone scan; therefore, she received intravenous antibiotic therapy. this approach proved to be ineffective and she was subsequently diagnosed with a deep vein thrombosis via color doppler ultrasonography. it is interesting to note that a deep vein thrombosis can present with symptoms similar to those of a postoperative infection. furthermore, an elevated crp level is frequently observed in patients suffering from polycythemia vera. therefore, the two conditions, which require completely different treatments, can be confused. we report on this case with a review of the relevant literature.

PMC4971116

pubmed-28

brachial plexus surgery using the da vinci surgical robot is a new procedure. to evaluate the advantages and the restrictions of the technique, a cadaveric study of supraclavicular and axillary approaches was conducted. we found that the axillary approach was useful and advantageous for lower roots, particularly for thoracic outlet syndrome (tos). this report will focus on the evaluation of axillary robotic approach as the advantages and disadvantages of supraclavicular robotic intervention have been widely discussed in the literature. a human cadaver was subjected to this experiment in paris university ecole europenne de chirurgie anatomy laboratory and da vinci robot system was used. the left arm was tucked along the side and the right arm was placed in a semiflexed position extending toward the anesthesia location near the head, supported by foam and blankets (figure 1). a 6 cm long incision was made at the right axillar line, lateral to the edge of the pectoralis major muscle (figure 2). a self-retaining chung retractor was placed into the incision to elevate the pectoralis major muscle flap. the robot was docked as a camera; right and left robotic arm were adapted in the incision area (figure 3). a 10 mm 0 downlooking scope, maryland forceps, and a curved scissors the working space was maintained with the self-retaining retractor, without co2 insufflation (figure 4). the subclavian artery was seen in front of the truncus and was positioned to the posterior of the working space. subclavian artery was dissected from the plexus and truncus of the lower plexus was exposed with blunt dissection. the plexus was exposed thoroughly from t1 to c7 levels. in this surgical setting, the operating surgeon, who has a wide experience in open brachial surgery of the brachial plexus, reported that lower brachial plexus exposure was easier from the axillary working area and a more wide range of motion was achieved to manipulate the robotic tools compared to the supraclavicular exposure for lower part of the brachial plexus. the development of robotic-assisted minimally invasive techniques began in urology, general surgery, and gynecology because of the generally large working spaces available in the abdomen for these types of surgeries [14]. since then, other surgeons have sought to use robotic devices in other areas, such as the brachial plexus [5, 6]. brachial plexus dysfunction can be the result of shoulder trauma [7, 8]. it can also occur with tos, which encompasses three separate disorders involving compression of the subclavian artery, subclavian vein, or brachial plexus in the triangular space bordered by the first rib, clavicle, and scalene muscles [9, 10]. compression of the vessel-nerve package at the thoracic inlet has been treated with soft-tissue (scalene muscle) release and/or bone (first rib) resection. surgical approaches to first rib resection may be transthoracic, transaxillary, supraclavicular, infraclavicular, or thoracoscopic [9, 10]. however, these approaches are typically associated with incomplete resection of the most medial portion of the first rib and neurovascular complications. theoretically, a minimally invasive transthoracic approach can obviate these problems, enabling complete resection of the offending portion of the first rib without neurovascular complication., respectively, reported successful results of robotic en bloc first rib resection for tos treatment via transthoracic and transaxillary approaches [9, 11]. 's techniques were only bony interventions and as being intrathoracic these need to be lung collapsed and lung complication can be waiting risk. although liverneaux et al. reported techniques and results of upper brachial plexus injury intervention via robotic surgery with a supraclavicular approach, they described the disadvantages as a narrow working space and difficulty to expose the c7 vertebra [1, 5, 12]. to our knowledge, this report is the first to objectively describe robotic axillar brachial plexus exposure. thus, we discuss the theoretical and clinical advantages and disadvantages of the axillary approach in the present report. the development of robot-assisted surgery has revealed new perspectives in peripheral nerve microsurgery. minimally invasive robot-assisted surgery could lead to modification of the classic algorithm for the treatment of traumatic brachial plexus lesions [6, 8]. to date, exploration of these lesions has not been attempted less than 3 months after the traumatic event because clinical examination can not provide an accurate diagnosis or reliable prognosis in these first weeks. early intervention may enable initial assessment of the lesion and repair of potentially graftable nerve roots. several robotics properties are particularly adapted to microsurgery, such as high-resolution three-dimensional (3d) visualization with up to 40 magnification, up to 10-fold magnification of surgical movements, elimination of physiological tremors, and the provision of ergonomic work conditions for otherwise uncomfortable surgery. robotic surgical systems allow high-definition magnified 3d visualization of the operative field, provide significant instrument maneuverability, even within a confined space, and may overcome the shortcomings of conventional approaches [2, 5]. axillary (infraclavicular) brachial plexus intervention via robotic surgery has not been described previously. axillary intervention was previously performed as an open procedure to expose the plexus or resect the first rib for the treatment of tos. martinez et al. described first rib resection via robotic surgery but not to address plexus injury without transthoracic exposure, a novel minimally invasive approach to the first rib from inside of the chest. in addition, gharagozloo et al. reported first rib resection via transthoracic robotic surgery for paget-schroetter disease. 's techniques were considered more useful for lower brachial plexus viewing and assessing according to gharagozloo et al. the experience of the whole surgical team with robotic technology is important for the procedure. during learning curve period, two staff surgeons are required to participate in all procedures to ensure the safety of the program [5, 9]. martinez et al. reported importance of the learning curve, not only for the surgeon but also for the entire surgical team and 180 minutes for the initial 10 cases. reported exclusion criteria include a history of previous incision in the same area and obesity, which present difficulties in robotic surgery initiation. other drawbacks of this new surgical approach are the increased cost of surgical equipment and longer operating time, especially during the learning curve period. however, we believe that the avoidance of a classic incision leads to significant patient satisfaction for cosmetic reasons and we believe that demand for this procedure from a select group of patients justifies the exploration of alternative ways to avoid classic brachial plexus exposure. this report presents our initial experience with robot-assisted axillary exposure of the brachial plexus region. in our opinion robotic surgery will be used routinely in the future for brachial plexus surgery and particularly for tos that is caused by bone and/or soft tissue. however, newer dedicated surgical instruments need to be developed and further studies should be conducted to evaluate in vivo application and results of this novel approach.

brachial plexus surgery using the da vinci surgical robot is a new procedure. although the supraclavicular approach is a well known described and used procedure for robotic surgery, axillary approach was unknown for brachial plexus surgery. a cadaveric study was planned to evaluate the robotic axillary approach for brachial plexus surgery. our results showed that robotic surgery is a very useful method and should be used routinely for brachial plexus surgery and particularly for thoracic outlet syndrome. however, we emphasize that new instruments should be designed and further studies are needed to evaluate in vivo results.

PMC4130329

pubmed-29

gastric cancer is the second most frequent neoplasm of the alimentary tract after the large intestine. 5,103 people in poland were affected by it in 2008. the case-to-death ratio of around 1 indicates unfavourable prognosis as to recovery from this disease [1, 2]. this poor result is determined by the fact that it is rarely (only in around 8%) detected in the form of early gastric cancer, in the mildly symptomatic or asymptomatic phase. in most patients it is diagnosed at a higher degree than the 1st degree of disease progression and its classic symptoms are weight loss, continuous and dull pain in the epigastrium, loss of appetite, nausea, vomiting and chronic bleeding [3, 4]. the existence of gastric cancer metastasis to the ureter has been described twice in the literature to date. a female patient, age 67, was diagnosed at the district hospital (22.04-30.04.2010) because of intensified symptoms of left-sided renal colic. based on the conducted usg and single-phase computed tomography tests of the abdominal cavity and the pelvis, dilation of the ureter was found because of its infiltration by a pathological focus with the dimensions of 28 mm 15 mm. another lesion was located nearby at the level of the left iliac muscle 30 mm 27 mm 20 mm, adhering to the sigmoid colon. tissue infiltration of the pelvis minor wall was found descending in the direction of the left appendages. colonoscopy was conducted and in this test the large intestine was described without pathology, while the gynaecological usg test confirmed the presence of fluid in the pelvis. during her stay the patient was treated with analgesic and diastolic medication and then referred for further treatment at the regional oncology centre. because of reported pain complaints, she was immediately admitted to the department of oncological surgery on the day of her visit to the outpatient clinic (04.05.2010). the urologist consulting the patient indicated the possibility of kidney damage due to ureteral obstruction with recommendation for an accelerated operation. therefore, pre-operative diagnostics were not extended beyond the tests received from the district hospital. the patient underwent surgery on 10.05.2010 and intraoperatively, besides the expected neoplastic tumour of the left ovary with infiltration of the ureter, numerous neoplastic foci were also found: a sigmoid colon tumour, a caecum tumour, a tumour of the body of the stomach and two single tumours in the omentum. because of the resectability of the neoplastic foci described above, the operation plan adopted earlier was changed and the following were performed in succession with palliative intention: partial gastrectomy by the rydygier method, right-sided hemicolectomy, left ovariectomy and sigmoid resection. after restoring the continuity of the alimentary tract, the tumour occluded the lumen, which is why segmental ureterectomy was performed with end-to-end anastomosis over a pigtail catheter. the patient passed the post-operative period without complications, except a two-day fever. because of the expected alimentary tract failure, parenteral nutrition was included on the 1st day after the procedure and blood deficits were supplemented with 2 units of erythrocyte mass and 7 units of plasma. on the 9th day after the operation, the patient was discharged home in a good general condition with recommendations for further treatment. the received postoperative histopathological protocol indicated that the stomach was the origin of the neoplastic process. to lauren), type i (acc. to goseki), of a stomach adenocarcinoma with a g2 malignancy degree, with occupation of the whole thickness of the stomach wall, with the following immunohistochemical characteristics: ck7(+++), ck20(++), ca125(+), mucicarmine(+). neoplastic infiltrations along nerves and neoplastic embolisms of blood vessels, as well as metastatic foci in the greater omentum, were observed in the specimen. because of the palliative partial gastrectomy type, only 3 lymph nodes were described in this specimen and all of them contained neoplastic cells. besides the above, histopathological confirmations were obtained of the metastatic character of the foci in the ovary, caecum and sigmoid colon. the cancer occupied large and small intestine walls without infiltration of the mucous membrane and the immunohistochemical characteristics were identical as for the primary lesion. according to the above protocol, excision within tissues with a healthy margin of these organs was achieved. the widening of the left ureter is visible (marked by arrow) focus of metastatic carcinoma in the ureter wall. microscopic section, he staining, magnification 40 neoplastic invasion in the ureter wall. microscopic section, he staining, magnification 100 it is interesting biologically and constitutes the basis for this paper that the ureteral tumour was described by the pathologists not as a neoplastic infiltration encroaching on the ureter, but as a metastatic focus to the ureteral wall. after the operative treatment, the patient was qualified for palliative chemotherapy and received it from 28.07.2010. the first treatment course was according to the eox regimen (oxaliplatin and capecitabine), but because of the occurrence of neutropenic fever the regimen was changed to pf (cisplatin and 5-fluorouracil). she again received only one course and the treatment was changed once more because newly formed metastatic foci in the liver were located in imaging tests. next, three chemotherapy courses were administered according to the folfiri regimen (irinotecan, leucovorin and 5-fluorouracil), but only until 11.11.2010 because due to progression of changes in the liver described in examinations the chemotherapy was discontinued and the patient was qualified for symptomatic treatment. according to our knowledge treatment of gastric cancer in the disseminated phase of the disease aims at extension of life and achieving a good palliative effect. the recommended methods, allowing the above to be achieved, are chemotherapy or combination radiochemotherapy. in an advanced stage, palliative operations on this organ are performed for life reasons and serve to eliminate complications such as bleeding, perforation or obstruction of the organ. it is indicated that the above complications forced surgical intervention in 1/4 of the patients previously disqualified from operative treatment because of the presence of metastatic foci. according to literature data, patients operated on by palliative resection in the disseminated phase of the disease achieve a survival time of between 9 and 15 months. the survival time is limited by the number of metastatic foci; when their number is higher than two foci, no statistically significant differences are observed in this scope. the value of these procedures is increasing because of the reported low perioperative mortality and the observed significant improvement in the further quality of life for these patients [513]. it seems that the operative procedure conducted in the presented patient allowed a good palliative effect to be achieved along with loss of severe colic and maintenance of kidney function. the literature often presents, as characteristic of gastric cancer, blood-borne metastasis to the ovary, termed a krukenberg tumour. this is a term generally defining metastasis to the ovary, mainly gastric cancer and next colon cancer. metastases from other organs, such as the lungs, the mammary gland and the uterus, are also possible, though much rarer. krukenberg tumours are encountered in the course of 2-4% of disseminated neoplastic processes. this is a negative prognostic factor, with varying median survival time after its diagnosis: 12-13 months in the course of gastric cancer compared to 17-29 months in the course of large intestine cancer. this undoubtedly results from biological differences in the course of these neoplasms [1423]. in summary, the available literature sources have described this only twice to date (in 1976 and 2000). however, nephroureterectomy was conducted in these quoted cases [24, 25]. in the presented patient the kidney was spared and healing of the ureter after end-to-end suture was achieved. the dissemination of a neoplasm with extremely rarely encountered symptomatology described above is an interesting experience which we wanted to share. the beginning of the symptomatic disease as a left-sided renal colic is, in itself, a previously unreported case.

the typical symptoms of advanced cancer of the stomach are well known in clinical practice. the presented case concerns a patient with symptoms of left-sided renal colic, caused by a malignant tumour involving the ureter, which was diagnosed with a ct scan. the multifocal process, involving the stomach, two parts of the colon, the left ovary and the side of the pelvis, was confirmed only during surgery.the resection or partial resection of the above-mentioned organs involved by the malignant process and reconstruction of the alimentary tract as well as the ureter were performed at time of this operation. the patient's recovery was without any complications. the histopathological findings support the diagnosis of this malignant process as disseminated stomach cancer.in the available literature only two cases of stomach cancer metastasis to the ureter have been described. in both cited examples resection of the ureter with nephrectomy was performed. the review of the literature supports the value of stomach palliative resection in prolonging life and improving quality of life.

PMC3687400

pubmed-30

eosinophilic gastritis is an extremely rare disease that is characterized by eosinophilic infiltration of the various layers of the gastrointestinal tract in the absence of any definite causes of eosinophilia. patients with eosinophilic gastritis have diverse symptoms, including abdominal pain, emesis, abdominal distension, and weight loss. these symptoms are associated with eosinophilic infiltration of the various layers of the gastrointestinal tract. the disease has to be distinguished from generalized eosinophilic disorder presenting with involvement of other organs. a 52-year-old female patient visited another hospital because of epigastric pain and tenderness. she had no history of allergic diseases such as eczema or atopy, or food or drug allergies., she had undergone esophagogastroduodenoscopy (egd), which revealed multiple focal ulcerative lesions with diffuse discoloration and edematous change of the rugae in the gastric fundus, cardia, and upper body (fig. the symptoms had been recalcitrant to treatment with proton pump inhibitors, fasting, and fluid. she had reported no nausea, vomiting, hematemesis, or melena. on admission, her blood pressure was 110/60 mm hg, pulse rate 76 beats per minute, respiratory rate 22 breaths per minute, and body temperature 36.2. physical examination revealed tenderness of the epigastric area. her white blood cell count was 22,770/mm with markedly increased eosinophils (5,009/mm, 22%). the c-reactive protein concentration was 13.95 mg/dl. her other blood chemistry test results were normal. the tests for viral markers included hepatitis a, b, and c virus and human immunodeficiency virus; those for autoimmune antibodies included anti-nuclear, anti-double stranded dna, and anti-neutrophilic cytoplasmic antibodies; and those for tumor markers included -fetoprotein, carbohydrate antigen 19-9, and carcinoembryonic antigen. the serological test for toxocara antibodies (igg) was positive, whereas those for echinococcus, paragonimus westermani, sparganumi, and trichinella antibodies were negative. the chest and abdomen radiographic examinations were normal. to investigate the cause of the epigastric pain and tenderness, abdominal computed tomography was performed, which revealed severe edematous wall thickening with focal localized low attenuation of the fundus and cardia of the stomach (fig. she underwent a repeated egd, which showed diffuse necrotic change in the fundus, cardia, and upper body (fig. a biopsy specimen was obtained during egd; a rapid urease test (clotest) revealed no evidence of helicobacter pylori. on histopathologic evaluation, the gastric surface was found to be eroded and the underlying lamina propria showed dense eosinophilic infiltration. she was treated with empirical intravenous antibiotics (cefoperazone and metronidazole) immediately after her transfer because infectious gastritis was not ruled out. although she was treated with a broad-spectrum anthelmintic (albendazole), antibiotics, proton pump inhibitor, and prokinetic agent during the next 5 days, her symptoms became worse. eosinophilic gastritis was diagnosed according to the clinical pictures, laboratory findings, and endoscopic findings. she was immediately started on methylprednisolone (62.5 mg/day; this dose was maintained for 7 days). therefore, she underwent a repeated short-term follow-up egd that showed regenerative epithelial tissue with peeling off, of the necrotic tissue (fig. she underwent a repeated egd that showed the replacement of white color scar tissue (fig. currently, she is being treated with 30 mg/day prednisone and is showing considerable clinical improvement. eosinophilic gastroenteritis is an uncommon and rarely reported disorder characterized by eosinophilic infiltration of the various tissue layers of the digestive tract in the absence of any definite causes of eosinophilia, without eosinophilic infiltration in other organs. the diagnosis of eosinophilic gastroenteritis is based on the following criteria: the presence of gastrointestinal symptoms, histological presentation of eosinophilic infiltration in tissue layers of the digestive tract, presence of high eosinophil count in ascites, and no evidence of parasitic infection or eosinophilic involvement of extraintestinal organs. eosinophilic gastroenteritis can involve any portion of the gastrointestinal tract from the esophagus to the rectum, and the stomach is the most commonly involved organ, especially the antrum. in eosinophilic gastritis, the endoscopic features are rather extensive: rugal fold thickening, erythema, friability, nodularity, gastric outlet obstruction, gastric ulcer, and even a normal mucosa. although not clearly defined, the common pathophysiological mechanism of this disease is associated with eosinophilic infiltration and degranulation in specific tissue layers of the digestive tract. this eosinophilic recruitment and activation regulated by diverse cytokines is a part of the host immune mechanism in the gastrointestinal mucosa; however, it can be a type of serious allergic or inflammatory reaction in the deeper tissue layers of the gastrointestinal tract. although there is no treatment consensus on eosinophilic gastroenteritis, several studies report good results with steroids in dosages from 20 to 40 mg/day, for 6 to 8 weeks [7-10]. in some case studies, leukotriene modifiers such as montelukast or mast cell stabilizers such as sodium cromoglycate have been proposed to be helpful for symptomatic improvement. antihistamines such as ketotifen or immunosuppressants such as mycophenolate mofetil are also used in the treatment of eosinophilic gastroenteritis; however, their therapeutic effects are not clear and would require more studies. although rare, eosinophilic gastritis should be considered in the differential diagnosis of patients with gastrointestinal symptoms and peripheral blood eosinophilia. lymphoma of the stomach, gastric cancer, and crohn disease involving the stomach may demonstrate endoscopic features similar to those of eosinophilic gastritis. gastrointestinal parasitic infection should be considered in patients with abdominal discomfort, weight loss, and peripheral eosinophilia. in particular, infestations by hookworms, ascaris, strongyloides, toxocara, trichuris, and intestinal capillaria should be considered in patients from endemic areas. in this case, the concentration of the antigen-specific ige to the a. simplex was 0.57 moreover, the serology for toxocara antibodies (igg) was positive. however, this is not clinically significant in korea because koreans often consume raw fish, which causes repeated exposures to anisakis. in addition, she had not had contact with any animals, including dogs and cats, at least for several years. moreover, she was treated with albendazole for 5 days, and her symptoms had become worse. after steroid treatment, the symptoms disappeared and the eosinophil count decreased to the reference range. moreover, the follow-up egd showed regenerative epithelial tissue with peeling off, of the necrotic tissue. she had undergone several egds that showed diffuse necrotic change in the fundus, cardia, and upper body. the etiology includes thromboembolism and occlusion of major arterial supply, ingestion of corrosive agents, volvulus of the stomach, endoscopic hemostatic injections, and infectious gastritis. in this case thus, the possible cause of gangrene could be infection, and she was treated with empirical intravenous antibiotics (cefoperazone and metronidazole). however, she was treated with broad-spectrum antibiotics during the next 5 days, and her symptoms became worse. the necrotic portion of the gastric high body is very vulnerable site of retching injury. this retching injury is called prolapse gastropathy syndrome, a clinical syndrome involving the invagination of part of the gastric mucosa into the lower esophagus. direct trauma to the mucosa occurs when the gastric mucosa becomes incarcerated through the lower esophageal sphincter. in addition, the endoscopic findings and histopathologic results were not compatible to prolapse gastropathy syndrome. on the basis of the clinical picture, laboratory findings, and therapeutic results, we concluded the diagnosis of eosinophilic gastritis presenting as necrotizing gastritis in our patient. this case highlights the reality of eosinophilic gastritis presenting as necrotizing gastritis, and that endoscopy and histopathological examination of the biopsies are the most useful tools for the diagnosis of eosinophilic gastritis presenting as necrotizing gastritis. eosinophilic gastritis should be considered in the differential diagnosis in patients with necrotic gastritis who do not respond to empirical treatment.

eosinophilic gastroenteritis is very rare disorder that is characterized by eosinophilic infiltration of the gastrointestinal tract in the absence of any definite causes of eosinophilia. it is associated with various clinical gastrointestinal manifestations, and depends on the involved layer and site. we report a case of eosinophilic gastritis presenting with severe necrosis. the symptoms disappeared immediately after beginning steroid treatment, and the eosinophil count decreased to the reference range. the patient showed eosinophilic gastritis characterized by necrotic change such as necrotizing gastritis. it is a unique presentation of eosinophilic gastritis. to the best of our knowledge, no case of eosinophilic gastritis characterized by necrotic change such as necrotizing gastritis has been previously reported in korea.

PMC4676670

pubmed-31

the skin is the largest organ of the body and protects the organism against external physical, chemical, and biological insults such as wounding, uvb radiation, and microorganisms. this major barrier resides in the upper layers of the epidermis (for review see segre, 2006). the epidermis is the upper part of the skin that is continuously renewed. the basal layer, or stratum basale, of the epidermis contains proliferating keratinocytes (fig. 1). upon withdrawal from the cell cycle, these basal keratinocytes detach from the basement membrane and undergo a terminal differentiation program to become corneocytes in the outer layers of the epidermis. the cells reinforce their cytoskeletal keratin filament network, and adjacent cells interact via many desmosomes, a specialized type of cell junction, to resist physical trauma. in the stratum granulosum, the keratinocytes become more flattened and express certain proteins such as profilaggrin and loricrin, which aggregate to form the typical keratohyalin granules of the stratum granulosum. in addition, lipids are produced and stored in lamellar bodies. at the final stage of differentiation, the keratinocytes lose their organelles, including the nucleus, and become the dead, flattened corneocytes of the stratum corneum. during cornification, proteins are cross-linked at the inner side of the cytoplasmic membrane to form a cornified envelope (for review see candi et al., 2005). in the transitional layer between the stratum granulosum and the stratum corneum, lipids are extruded to form a water-repelling envelope around the cornified envelope, thereby assuring an adequate permeability barrier function of the mammalian epidermis. improper formation of these envelopes results in an impaired epidermal barrier that can not protect against dehydration, uvb, and infection. the signaling cascades involved in epidermal barrier formation are largely unknown, but the many proteases that seem to be involved are currently being intensively studied. see introduction for details. since the cloning of caspase-14 in the late nineties, it has become clear that this protease is a unique member of the caspase family. unlike apoptotic caspases, which evolved in common ancestors such as hydra, echinodermata, insects, nematodes, and chordates, caspase-14 has so far been found only in terrestrial mammals (lamkanfi et al., 2002). in contrast to the ubiquitously expressed other members of the caspase family, caspase-14 is expressed and activated mainly in the epidermis and is absent from most other adult tissues (eckhart et al. recently, caspase-14 was found to be involved in epidermal barrier formation (denecker et al., 2007). in this review, we discuss current knowledge of the expression, regulation, and function of caspase-14. the expression pattern of caspase-14 is unique among the caspases, as it is present mainly in cornifying epithelia, such as the epidermis, the hassall's bodies of the thymus, and the forestomach of rodents (lippens et al., 2000, 2005; in skin, caspase-14 is expressed only in the differentiating and cornifying layers of the epidermis and the hair follicle (lippens et al., 2000; this is consistent with the observation that, in vitro, caspase-14 is only expressed in differentiating but not in proliferating keratinocytes (lippens et al. remarkably, nail matrix keratinocytes that differentiate into specialized nail corneocytes, the building blocks of the nail plate, do not express caspase-14 (jager et al. in addition, caspase-14 is not expressed in the noncornifying keratinocytes of the sweat gland or the mouth epithelium (lippens et al., 2000; ultrastructural analysis demonstrated that spatial distribution of caspase-14 in the epidermis and hair follicles is strongly conserved among several mammalian species (alibardi et al. caspase-14 was found to be associated with the nucleus, the keratohyalin granules, and the desmosomes, whereas in corneocytes, caspase-14 was found in the cytoplasm and was associated with corneodesmosomes (a modified version of desmosomes) and nuclear remnants. these observations suggested a role for caspase-14 in nuclear degradation during cornification, but nuclear degradation was not affected in caspase-14deficient mice (denecker et al., 2007). the expression of caspase-14 in the hassall's bodies of the thymus and in the forestomach of rodents is somewhat expected, as they are cornifying structures and express the typical late differentiation markers, such as profilaggrin and loricrin, which are also found in the epidermis (laster and haynes, 1986; favre, 1989; jarnik et al., 1996). protein expression of caspase-14 has also been reported in several noncornifying tissues (lippens et al., 2003; krajewska et al., 2004, 2005; kam et al., 2005 however, these observations should be interpreted carefully, as we have recently shown that the reported expression of caspase-14 in such tissues can be the result of aspecific staining (denecker et al., 2007). remarkably, so far caspase-14 has been found only in terrestrial mammals but not in birds or reptiles. whereas birds and reptiles have a stiff, dry, scaly epidermis, mammals have a soft stratum corneum because of the larger amounts of histidine-rich late differentiation markers (e.g., profilaggrin; alibardi, 2003). interestingly, profilaggrin is a direct substrate of caspase-14 (denecker et al., 2007). this could indicate that the occurrence of a soft stratum corneum and the caspase-14 gene are associated during evolution. although the expression of caspase-14 is very restricted, little is known about the transcriptional regulation of its gene. in vitro, caspase-14 is only expressed when keratinocytes are forced to differentiate by growing them postconfluently or in suspension or by adding vitamin d3 (eckhart et al. in contrast, adding ca at high concentrations to the medium, a method frequently used to induce differentiation, did not induce caspase-14 expression (eckhart et al. retinoids, which suppress keratinocyte differentiation, down-regulate caspase-14 expression (rendl et al., these results indicate that transcription factors that are specifically active during terminal differentiation are required to regulate caspase-14 expression. whether caspase-14 expression levels can be regulated at the posttranscriptional level is not known. down-regulation of several differentiation-associated genes by retinoids has been shown to be mediated by the transrepression of activator protein 1 (ap-1)mediated gene activation (fisher and voorhees, 1996). indeed, the caspase-14 promoter contains at least two potential ap-1binding sites (unpublished data). the green tea phenol ()-epigallocatechin-3-gallate (egcg) is a potent activator of ap-1 and has been shown to up-regulate caspase-14 in a p38- and jnk-dependent way (hsu et al., 2005, 2007). ap-1 alone is probably not sufficient to drive caspase-14 expression because tnf and 12-o-tetradecanoyl-phorbol 13-acetate, two potent activators of ap-1 in keratinocytes (arnott et al., 2002), did not induce caspase-14 expression in keratinocytes (lippens et al., 2004). differentiation-dependent expression of caspase-14 in keratinocytes could also result from a strong transcriptional repression in proliferating keratinocytes. this possibility is supported by the observation that mice deficient in nuclear receptor corepressor hairless (hr) had 510-fold higher levels of caspase-14 and profilaggrin mrna, starting from postnatal day 6 and progressing during development (zarach et al., 2004). these alterations in gene expression were detected mainly in the keratinocytes of the utricle, an abnormal pouch-like structure at the upper part of the hair follicle. increased gene expression occurred before the morphologically distinct utricle could be identified, indicating that the up-regulation was probably a cause rather than a consequence of utricle formation. it would be interesting to analyze both caspase-14 activation and filaggrin processing in these mice. multiple mutant hr alleles in mice and in humans show phenotypic variations that include congenital hair loss, skin wrinkling, and papular rash (cichon et al., 1998; 1998). whether caspase-14 overexpression is important for the observed phenotypes could be addressed by generating epidermis-specific caspase-14 transgenic mice or by crossing the hr mice with caspase-14deficient mice. procaspases consist of a prodomain, a large subunit (p20), and a small subunit (p10). activation of caspases is induced by dimerization, (auto-)proteolytic cleavage at asp residues, and/or conformational changes (lamkanfi et al., 2003). so far, maturation of caspase-14 by proteolytic cleavage into p20 and p10 subunits has been consistently observed only in cornifying epithelia such as the epidermis and the rodent forestomach (lippens et al., 2000; although some investigators suggested that caspase-8 and -10 can activate caspase-14 in vitro (ahmad et al., 1998; van de craen et al., 1998), this could not be confirmed by others (lippens et al. furthermore, caspase-14 is probably not proteolytically activated by a caspase in vivo, as other caspases are not activated during epidermal differentiation (eckhart et al. 2000; raymond et al., 2007), and caspase-14 is not processed at an aspartate residue like other caspases but is processed at ile in man and presumably at leu in the mouse (chien et al., 2002). alignment of the protease-sensitive loop between the p20 and p10 subunits of the known mammalian procaspase-14 amino acid sequences (fig. 2) reveals a conserved hydrophobic patch that is n terminal of the caspase-14 cleavage site. this patch contains p1-preferred amino acids of elastase-like serine proteases, such as val, ala, leu, and ile (mallory and travis, 1975; vered et al., 1985; takahashi et al., 1989), suggesting that a serine protease with elastase-like properties could be involved in caspase-14 activation (unpublished data). all together, these data indicate that during skin homeostasis, the caspase-14activating protease is not a caspase, separating caspase-14 activation from the apoptotic and inflammatory caspase cascades that could be detrimental to epidermal integrity. the precise epidermal layer in which caspase-14 is processed and activated is unknown because antiserum specifically recognizing activated caspase-14 is not yet available. however, the following findings indicate that activation of caspase-14 occurs at the interface between the granular and cornified layers of the epidermis or early during cornification. first, both the proform and activated form of caspase-14 can be found in total epidermal extracts, whereas in the cornified layer only activated caspase-14 is found (fischer et al., 2004). second, caspase-14 activation coincides with stratum corneum formation both during embryonic development and in organotypic skin cultures (eckhart et al. this implies that the main biological function of caspase-14 is exercised in the stratum corneum, as proven by the phenotype of the caspase-14deficient mice (see the next section). sequence analysis indicates that a hydrophobic patch in the protease-sensitive loop is conserved. only part of the alignment is shown here, including the c-terminal part of the p20 subunit, the protease-sensitive loop, and the n-terminal part of the p10 subunit. the darker the yellow, the more the amino acids are conserved between species. the catalytic qacrg box is delineated with a green box. the conserved hydrophobic patch is delineated with a red box, and the cleavage site in human caspase-14 is indicated with a red arrow. the alignment was performed using clustalw (mega version 3.1; kumar et al., 2004) and identification of caspase-14 substrates has been hampered for a long time by the unavailability of enzymatically active caspase-14. however, it was recently shown that proteolytically processed caspase-14 requires high concentrations of kosmotropic salt to be active in vitro, such as sodium citrate, in addition to proteolytic cleavage between the p20 and p10 subunit (mikolajczyk et al., 2004). these salts induce both dimerization and ordering of active site loops by partial desolvation of the protein to a more compact, catalytically active protease. the cellular environment of the stratum corneum of the epidermis probably favors caspase-14 activity in a similar way. indeed, the water content decreases from 45% at the transitional layer to 1525% at the skin surface (warner et al., 1988; caspers et al., 2001). human caspase-14 preferentially accommodates tryptophan or tyrosine in the s4 subsite, whereas mouse caspase-14 is more tolerant, with almost equal preferences for -branched and aromatic amino acids (mikolajczyk et al., 2004). for example, both human and mouse caspase-14 efficiently cleave the fluorescent peptide substrate wehd-amc, but only mouse caspase-14 cleaves ietd-amc as efficiently (fischer et al., 2004; mikolajczyk et al., these substrate preferences would classify human caspase-14 as an inflammatory caspase and mouse caspase-14 as an inflammatory and apoptotic initiator caspase (thornberry et al., 1997; thornberry, 1998). however, human caspase-14 can not proteolytically activate the inflammatory cytokines pro 2004), and there are no data supporting a direct role for caspase-14 in apoptosis (lippens et al., 2000; denecker et al., 2007). whether the substrate preferences of human and murine caspase-14 observed in vitro on peptide substrates also occur in vivo is not clear. importantly, profilaggrin, a major structural protein in the differentiating epidermis, has been shown to be a physiological substrate of caspase-14 (denecker et al., 2007). identification of additional substrates and determination of the cleavage sites will provide more insight into the preferred recognition sequence of caspase-14 in the context of a protein. keratinocytes can die by two different processes: apoptotic cell death induced by damaging agents such as uvb, chemicals, and cytotoxic cytokines or by a continuous process of differentiation leading to the formation of corneocytes. these processes are clearly distinct pathways executed by different players (for review see lippens et al., 2005), but the role of caspases in these two cell death programs has long been controversial. although almost all procaspases are constitutively expressed in the epidermis, only caspase-14 has consistently been shown to be activated during epidermal cornification (eckhart et al., 2000b; lippens et al., 2000; raymond et al., 2007). in addition, knockouts for apoptotic caspases were not reported to have a phenotypic skin anomaly except for caspase-3deficient mice, in which keratinocyte differentiation is delayed in the embryo but normalized at birth (okuyama et al., 2004). however, others could not confirm the activation of caspase-3 during embryonic epidermal development (fischer et al. although they are not activated during cornification, apoptotic caspases, in contrast to caspase-14, become activated during uvb-, staurosporine-, tnf-, and tnf-related apoptosis-inducing ligand induced apoptosis of keratinocytes and, thereby, play a role in the apoptotic cell death of keratinocytes (for review see lippens et al., 2005). thus, we conclude that apoptotic caspases are not involved in the physiological keratinocyte cell death program leading to cornification. furthermore, caspase-14deficient epidermal cells can undergo classical apoptosis, which genetically demonstrates that caspase-14 is dispensable for the apoptosis of keratinocytes. during development, caspase-14 protein expression is detectable from embryonic day (e) 15.5 on, and its processing is observed from e17.5 (hu et al., 1998; van de craen et al., 1998; fischer et al., 2005), which coincides with stratum corneum formation and establishment of the epidermal barrier. however, no differences in outside-in barrier formation of the skin of caspase-14deficient mice during embryogenesis were observed (denecker et al., 2007). furthermore, caspase-14deficient mice were born at the expected mendelian ratios, were fertile, and had long survival rates. detailed analysis of caspase-14deficient mice indicated that caspase-14 has an important role in cornification, hydration, and uvb protection. the skin of caspase-14deficient mice was shinier, characterized by deeper skin lines, and had larger scales (denecker et al., 2007) even though the shape and size of the cornified envelopes themselves were not altered. biochemical analysis indicated that caspase-14 was responsible for the correct processing and degradation of (pro)filaggrin, as epidermis lacking caspase-14 was characterized by an altered profilaggrin processing and staining pattern (fig. 3) and by the presence of aberrant keratohyalin granules, the profilaggrin storage granules. profilaggrin is a large, insoluble protein consisting of a calcium-binding a domain, a b domain, and several tandem repeats of filaggrin units. in the transitional layer 4), which aid in the bundling of keratin intermediate filaments and formation of the cornified envelope (for review see candi et al., 2005). subsequently, filaggrin is deiminated (conversion of arginine to citrulline by elimination of the imino group of arginine by peptidylarginine deiminases), causing its release from keratin and allowing its degradation into free hygroscopic amino acids that act as natural moisturizing factors of the stratum corneum (scott and harding, 1986; rawlings and matts, 2005). therefore, filaggrin plays an important role in skin hydration. although the filaggrin unit was detected in caspase-14deficient epidermis by western blot analysis, lower molecular weight filaggrin fragments were also present (denecker et al., 2007). immunofluorescence analysis showed that in these mice, filaggrin immunoreactive fragments accumulated in the upper layers of the stratum corneum (fig. this indicates that the correct degradation of filaggrin into free amino acids was affected in caspase-14deficient skin. interestingly, caspase-14 was found to be associated with keratohyalin granules in the stratum granulosum and to remain cytoplasmic in the stratum corneum (alibardi et al., 2004), which could correlate with its possible involvement in the generation of free amino acids. expression of caspase-14 and (pro)filaggrin in wild-type and caspase-14deficient skin. immunofluorescence staining for caspase-14 (red) and (pro)filaggrin (green) on paraffin sections of 5.5-d-old skin of both wild-type (+ /+) and caspase-14deficient (/) mice (denecker et al., 2007). fluorescence microscopy was performed on a cellm system (olympus) with an upright microscope (bx61; olympus). a specific dapi emission band-pass filter (450470 nm) and a gfp emission band-pass filter (510550 nm) were used. image acquisition and processing were performed with the cellm software using a cooled ccd camera with a 1,344 1,024 pixel resolution. image intensity scaling and color conversion were completed in imagej (national institutes of health). caspase-14 is expressed mainly in the spinous, granular, and cornified layers of wild-type mice and is absent in caspase-14deficient mice. (pro)filaggrin is expressed in the granular layer and in the lower cornified layer in wild-type skin. in caspase-14deficient skin, additional filaggrin caspase-14 protects the skin against uvb photo damage and water loss and is involved in the processing of (pro)filaggrin. caspase-14 expression starts in the spinous layer (indicated in shades of red), and cleavage into its p20 and p10 subunits occurs at the transition of the granular to the cornified layer. caspase-14 is active in the dehydrating environment of the cornified layer, where it has an important function in formation of the epidermal barrier leading to protection against uvb and water loss (denecker et al., 2007). profilaggrin is a large structural molecule consisting of an n-terminal a domain and a b domain followed by multiple filaggrin repeats and a unique c-terminal sequence (for review see candi et al., 2005). profilaggrin undergoes many posttranslational modifications, eventually leading to release from the keratin intermediate filaments (see the section on the function of caspase-14 for details). in the lower stratum corneum these amino acids compose 40% of the natural moisturizing factors present in the stratum corneum and are important for maintaining epidermal hydration (rawlings and matts, 2005). in caspase-14deficient skin, accumulating filaggrin fragments are present (denecker et al., 2007), indicating that an unidentified protease (asterisk) cleaves the filaggrin monomer into these fragments and that caspase-14 is responsible for the further processing and degradation of these fragments into free amino acids. as it is very unlikely that caspase-14 is directly responsible for degradation of the filaggrin fragments into free amino acids, we propose two possible mechanisms: (1) caspase-14 could first cleave these filaggrin fragments, leading to further degradation into free amino acids by another endo- and/or exopeptidase; or (2) caspase-14 could directly or indirectly (by inactivating an inhibitor) activate an endo- and/or exopeptidase that further processes the smaller filaggrin fragments. kg, keratohyalin granule; kif, keratin intermediate filament; nmf, natural moisturizing factors; sb, stratum basale; sc, stratum corneum; sg, stratum granulosum; ss, stratum spinosum; tg, transglutaminase. these results, together with the finding that caspase-14 can directly cleave (pro)filaggrin in vitro, demonstrate that caspase-14 has a critical role in the correct processing of (pro)filaggrin during cornification. whether the degradation of other differentiation-associated proteins is also affected in caspase-14deficient mice first, caspase-14 may cleave the filaggrin fragments and, thereby, expose cleavage sites that can be recognized by other endo- and/or exopeptidases for further degradation. second, caspase-14 may be the activator of an endo- and/or exopeptidase that cleaves and degrades filaggrin. direct degradation of filaggrin fragments into free amino acids by caspase-14 can be ruled out, as caspases only cleave after aspartate residues. the lack of filaggrin processing into free hygroscopic amino acids in caspase-14deficient mice may lead to the reduced epidermal hydration and increased trans-epidermal water loss observed in these mice (denecker et al., 2007). these results point to an important function of caspase-14 in the maintenance of epidermal hydration. although a profilaggrin-deficient mouse has not been generated, it has been demonstrated that flaky tail (ft/ft) mice, which have an autosomal recessive mutation in the flaky tail gene (probably the profilaggrin gene), lack a functional filaggrin monomer (presland et al., 2000). these mice have been proposed as a model for the filaggrin-deficient skin disease ichthyosis vulgaris because they have dry, flaky skin and irregular scales of variable size. the importance of filaggrin has been underscored recently by human genetic studies demonstrating that loss-of-function mutations in the profilaggrin gene are the primary cause of the skin disease ichthyosis vulgaris (smith et al., 2006), which is characterized by silvery scales on the abdomen and palmar hyperlinearity. 2006), possibly as a result of a defect in epidermal barrier function that allows the increased entry of allergens and infectious agents. two of the important functions of the skin are prevention of water loss and protection against environmental stress, such as protection against uvb radiation, which are essential for terrestrial life. the development of caspase-14deficient mice revealed that the absence of caspase-14 enhances sensitivity toward uvb-induced photo damage and apoptosis of the skin (denecker et al., 2007). importantly, this is not caused by cell-autonomous differences in dna damage sensitivity and apoptosis between wild-type and caspase-14deficient keratinocytes. instead, the uvb-filtering capacity of the stratum corneum is severely reduced in caspase-14deficient skin, as higher levels of cyclobutane pyrimidine dimers are detected immediately after uvb irradiation. this indicates that caspase-14 has an indispensable role in the photoprotective function of the stratum corneum. interestingly, topical application of egcg, an inducer of caspase-14 expression, has been shown to be photoprotective (elmets et al., 2001). how caspase-14 alters the structural and biochemical properties of the stratum corneum is currently under investigation. caspase-14 was shown to be expressed at the protein level in several cancer cell lines (pistritto et al., 2002; koenig et al., 2005; krajewska et al., 2005). in addition, caspase-14 mrna, together with keratin 1 and profilaggrin mrna, was decreased in murine uvb-induced squamous cell carcinoma, possibly reflecting reduced differentiation in the tumor (rundhaug et al., 2005). furthermore, in some cases, caspase-14 protein expression was associated with highly differentiated cornified areas of lung squamous cell carcinoma and cervix carcinoma (koenig et al., 2005). however, caspase-14 activation in tumors has not been shown, and so it might not be responsible for the tumor phenotype. presumably, the ectopic caspase-14 expression is caused by the changed transcriptional activity in these epithelial tumors. mutations in the caspase-14 gene have not been found in human carcinomas except very rarely in colorectal tumors, which most probably were not the cause of altered caspase-14 expression (koenig et al., 2005; we as well as other investigators demonstrated that caspase-14 expression is substantially down-regulated in psoriatic lesions but is unaffected in the nonlesional epidermis (lippens et al., 2000, 2004; psoriasis is an autoimmune disease characterized by the uncontrolled proliferation of keratinocytes and impaired cornification, which results in the aberrant presence of nuclei in the cornified layer, also called parakeratosis. although caspase-14 is absent in these parakeratotic regions, this is probably not the cause of the development of parakeratotic plaques, as caspase-14deficient mice did not show spontaneous parakeratosis (denecker et al., 2007). more likely, caspase-14 down-regulation results from the impairment of terminal differentiation or up-regulation of transcriptional repressors. the absence of caspase-14 in psoriatic plaques may lead to the formation of a defective barrier and, therefore, to the aggravation of psoriatic lesions. treating the parakeratotic plaques of patients with a vitamin d3 analogue results in the up-regulation of caspase-14 and likewise, in the flaky skin (fsn/fsn) mouse model of psoriasis, topical egcg treatment causes the up-regulation of caspase-14 and the amelioration of psoriasis (hsu et al., 2007). interestingly, the expression of junb is strongly down-regulated in psoriatic lesions, and inducible epidermal deletion of both junb and c-jun in mice results in a psoriatic phenotype (zenz et al., 2005). because caspase-14 might be regulated by these transcription factors, it would be interesting to elucidate whether caspase-14 is down-regulated in junb/c-jun deficient mice. recent evidence sheds light on the crucial role of caspase-14 in the skin, but several major questions remain. activation of caspase-14 occurs most probably at the interface between the granular and cornified layer, implicating a role for caspase-14 in the stratum corneum. it is now clear that the caspase-14activating protease is not a caspase but probably an epidermis-specific serine protease with elastase-like properties. this is not surprising, as it has been known for a long time that serine proteases are of major importance in epidermal homeostasis. determination of the in vitro conditions for caspase-14 activity that mimic stratum corneum conditions, together with the generation of caspase-14deficient mice, led to the identification of (pro)filaggrin as the first known physiological caspase-14 substrate. importantly, caspase-14 seems to be involved in the correct processing of filaggrin preceding its degradation into free hygroscopic amino acids, which might explain its role in the prevention of water loss from the epidermis. proteomic approaches could lead to the identification of additional caspase-14 substrates, which would contribute to understanding the role of caspase-14 in the skin. caspase-14 also protects against uvb-induced damage, which means that it is involved in the establishment of the biochemical or structural properties of the stratum corneum as a uvb filter. how caspase-14 establishes the uvb-filtering capacity of the corneum is not completely understood. an extensive biochemical analysis of caspase-14deficient epidermis could reveal these mechanisms. whether caspase-14, its activating protease, or its substrates could be used as therapeutic agents or as targets to improve formation of the epidermal barrier is a challenging research goal.

caspase-14 is a unique member of the evolutionarily conserved family of cysteinyl aspartate specific proteinases, which are mainly involved in inflammation and apoptosis. however, recent evidence also implicates these proteases in proliferation and differentiation. although most caspases are ubiquitously expressed, caspase-14 expression is confined mainly to cornifying epithelia, such as the skin. moreover, caspase-14 activation correlates with cornification, indicating that it plays a role in terminal keratinocyte differentiation. the determination of in vitro conditions for caspase-14 activity paved the way to identifying its substrates. the recent development of caspase-14deficient mice underscored its importance in the correct degradation of (pro)filaggrin and in the formation of the epidermal barrier that protects against dehydration and uvb radiation. here, we review the current knowledge on caspase-14 in skin homeostasis and disease.

PMC2234247

pubmed-32

kelch-like ech-associated protein 1 (keap1)-nuclear factor e2-related factor 2 (nrf2) is a major cellular pathway that protect normal cells against oxidative and xenobiotic damage.1 nrf2 is an essential transcription factor for antioxidant and detoxification genes and is crucial for the chemopreventive effect of various phytochemicals against carcinogenesis. representative chemopreventive agents that induce nrf2 include carotenoids, curcumins, cyclic lactones, diterpenes, dithiolethiones, epithionitriles, flavonoids, indoles, isothiocyanates, organosulfides, and phenols.2,3 accumulating evidences have demonstrated that phytochemicals can protect cells from oxidative stress related diseases including inflammatory, cardiovascular, and neurodegenerative diseases and cancer, through nrf2-dependent responses. paradoxically, recent researches demonstrated the dark side of nrf2.4 cancer cells acquire a growth advantage by eliminating keap1-mediated negative control of nrf2, which leads to activation of the nrf2-dependent defense response.4 constitutive upregulation of nrf2 has been found in many types of cancers, including skin, breast, prostate, lung, head and neck, and endometrial cancer.5 overexpressed nrf2 provides a growth advantage for cancer cells by protecting those cells from oxidative stress and anticancer agents, thus contributing to chemoresistance.5 in this review, we summarize the therapeutic development of nrf2 inhibitors that enhance the efficacy of the current cancer treatments. nrf2 is a basic leucine zipper transcription factor with 7 domains from neh1 to neh7. neh1 has dna binding motifs.6 neh3, neh4, and neh5 have domains involved in the transactivation of nrf2 target genes by binding coactivators. neh2 has a major regulatory domain with 2 binding sites for keap1 named etge and dlg (fig. keap1 has 5 functional domains including kelch, intervening region (ivr), broad complex/tramtrack, bric-a-brac domain (btb), an n-terminal region (ntr), and a c-terminal region (ctr).3 cysteine residues of ivr play as a sensor for reactive oxygen species (ros). the btb domain has a cys151 residue and is responsible for nrf2 ubiquitination through binding to cullin e3 ubiquitin ligase (cul3)-based ubiquitin e3 ligase (fig. 1a). under unstressed conditions, nrf2 forms a complex with keap1, leading to polyubiquitination and subsequent degradation by the 26s proteasome (fig. 2a).7 upon exposure to a variety of stressors, including ros, toxicants, and carcinogens, nrf2 is released due to modification of the cysteine residues of keap1 (fig. 2b).8 two mechanistic models have been proposed for the regulation of nrf2: the hinge and latch model and the keap-cul3 dissociation model.810 in the hinge and latch model, 2 binding motifs of the neh2 domain in nrf2 have different affinities.9 for the ubiquitination, both the dlg and etgf motifs should be occupied by keap1 proteins. ros modify the cysteine residues of keap1, leading to the release of the dlg motif (latch) without changing the etgf motif (hinge) on neh2. another model is the keap-cul3 dissociation model in which oxidative stress disrupts the keap1-cul3 e3 ligase interaction without changing the conformation of keap1.8 nrf2 stabilization with de novo synthesis increases the cytoplasmic level of nrf2. nrf2 translocates into the nucleus and binds to an antioxidant response element (are) as a heterodimer with musculo-aponeurotic fibrosarcoma (maf) to recruit a transcriptional coactivator and promote thetranscription of various cytoprotective genes that are associated with antioxidant and detoxification enzymes. upon recovery of cellular redox homeostasis, keap1 translocates to the nucleus to dissociate nrf2 from the are resulting in degradation of nrf2.11 nrf2 binds to ares in promoter regions of various genes that regulate the cellular response to oxidative and xenobiotic stress. nrf2 target genes include antioxidant genes and phase ii enzymes such as heme oxygenase-1 (ho-1), nad(p)h: quinone oxidoreductase 1 (nqo1), glutathione s-transferase (gst), and glutathione peroxidases. the are-bearing genes that are regulated by nrf2 in humans have been studied using microarray analysis of genes induced by chemopreventive agents or knockout of keap1 in human cell lines.2 these genes can be categorized into 5 groups: antioxidant genes (gclc, gclm, gpx2, gsr, slc7a11, srxn1, and txnd1), nadph-generating enzymes (g6pd, me1, and pgd), metal-binding proteins (fth1, ftl, mt1, and mt2), drug-metabolizing enzymes and drug transporters (akr1b1, akr1b10, akr1c1, akr1c2, akr1c3, akr1c4, cbr1, gstm3, mrp2, nq1, and ptgr1), and stress response proteins (gadd45, hmox1, hsp40, and hsp70).2 knockdown of keap1 in human keratinocytes resulted in upregulation of 23 mrnas while akr1b1, akr1b10, and akr1c1 were induced to the greatest extent, showing increases of between 12- and 16-fold indicating that aldo-keto reductases could be useful biomarkers for nrf2 activation.12 as keap1 is responsible for inhibitor kappa b (ib) kinase beta (ikk-) ubiquitination, upregulation of some of these genes could be attributable to the increase of nuclear factor-kappa b (nf-b) activity.13 expression profiling in mice has revealed that nrf2 regulates approximately 100 genes and that two-thirds of nrf2-target genes are not involved in detoxication or antioxidation. instead, many of the target genes are associated with inflammation and immunity proteins.2 the aryl hydrocarbon receptor (ahr) is a transcription factors that mediates the biological effects of its xenobiotic ligands including dioxin.14 the ahr activates the transcription of nqo1 and other genes through the xenobiotic-responsive element (xre).15 ahr induces the direct binding of nrf2 to promoters or indirectly produces reactive intermediates that trigger the nrf2 signaling pathway.16 the cross-talk between nf-b and nrf2 is more complex. several chemopreventive agents, such as sulforaphane, epigallocatechin-3-gallate, and curcumin, induce nrf2 signaling with concomitant repression of nf-b.14 the nf-b p65 subunit re-presses the nrf2-are pathway by preventing creb binding protein (cbp) from binding nrf2 through competitive interaction or by promoting the recruitment of histone deacetylase 3 (hdac3), a corepressor, to the are.17 in chondrocyte apoptosis, shear-induced cyclooxygenase-2, an nk-b target gene, can suppress phosphatidyl inositol 3-kinase (pi3k) activity, causing a reduction in thenrf2-mediated transcriptional response.18 in contrast, nrf2 inhibits the activity of nf-b by attenuating phosphorylated ib. nrf-2 target genes, including ho-1 have been reported to negatively affect nf-b activation.19,20 p53 has been reported to suppress the nrf2-dependent transcription of antioxidant response genes by directly interacting with are-containing promoters. previous studies have suggested that the function of this negative regulation is to prevent the formation of antioxidant environment, which could hinder the induction of apoptosis by p53.21 in contrast, direct targets of p53 could upregulate nrf2 activity. studies have shown that p21 inhibits the degradation of nrf2 by interacting with the dlg motif, leading to upregulation of antioxidant genes.22 these dual functions of p53 toward nrf2 indicate that p53 may decide the fate of damaged cells whether to repair the damaged cells or apoptosis. nrf2 is also involved in autophagy by inducing p62 which is a receptor for autophagic degradation. as p62 could block binding between nrf2 and keap1, p62 contributes to the activation of nrf2 target genes in response to oxidative stress creating a positive feedback loop.23 the association of nrf2 overexpression and chemoresistance has been reported in many cancers including non-small cell lung cancer, stomach cancer, endometrial cancer, and osteosarcoma.2427 overexpression of nrf2 target genes was also found in a variety of solid tumors.28 ho-1 overexpression was found in brain cancer, prostate cancer, and renal cancer.29,30 nqo1 is known to be overexpressed in hepatoblastoma, colon cancer, breast cancer, and non-small cell lung cancer.31 several mechanisms have been proposed for the activation of keap1-nrf2 signaling in cancer. mutation of keap1 can result in accumulation of nrf2 in nucleus through decreased degradation or inhibition of nuclear export.28 in non-small cell lung cancer, heterozygous mutation of keap1 (8% of patients) was reported to be sufficient for nrf2 overexpression.32 in addition to lung cancer, mutations in the keap1 gene were found in various cancers including breast (2%), colon (8%), gastric (11%), liver (9%), ovary (19%), and prostate (8%).3336 mutations in the neh2 domain of nrf2 were found in lung (11%), cancer (6%), and head and neck cancers (25%).37,38 all of these mutations were somatic mutations. mutation of egfr, kras, braf, myc, and the bcr-abl fusion can activate nrf2, resulting in enhancement of ros detoxification and other oncogenic roles of nrf2, including chemoresistance.3941 furthermore, posttranslational modifications can activate the keap1-nrf2 signaling pathway. hypermethylation of the keap1 promoter was found in 47% of lung cancer patients and this feature was associated with poor outcome.42 epigenetic regulation of keap1 was also found in malignant glioma, colon cancer, and breast cancer.4345 in addition to the upregulation of cytoprotective genes, constitutive expression of nrf2 may confer a survival advantage to cancer cells by promotion of cell proliferation, chemoresistance and inhibition of apoptosis. an active pi3k-akt pathway augments the nuclear accumulation of nrf2 which then re-directs glucose into the anabolic pathway to increase metabolism, indicating the reinforcement of metabolic reprogramming by nrf2.46 nrf2 could mediate cell proliferation with dual regulation through epidermal growth factor receptor (egfr) signaling and keap1 interactions.39 in cells with the keap1 gene mutation (a549 cells), activated nrf2 promotes cell proliferation independent of egfr signaling. therefore, egfr tyrosine-kinase inhibitors are intrinsically ineffective in these types of non-small cell lung cancer.39 cancer metastasis and tumor progression requires the epithelial-mesenchymal transition (emt) and the loss of e-cadherin is considered to be a main event in emt. in hek293 cells, complex of e-cadherin and beta-catenin could bind to c-terminus of nrf2 preventing nuclear translocation of nrf2.47 as e-cadherin inhibits nrf2-mediated transcription, loss of e-cadherin could promote nrf2 translocation and confer an additional survival advantage to cancer cells.47 the keap1-nrf2 pathway is involved in the inhibition of apoptosis by interacting with p53 and b cell lymphoma-2 (bcl-2). p53 inhibits the activation of nrf2 target genes by direct interacting with are-containing promoters or activating p21.21 considering that p53-induced apoptosis requires the accumulation of ros, increased activity of antioxidant genes by nrf2 in cancer cells can inhibit p53 dependent apoptosis. bcl-2 can repress cell death by dimerization with bcl-2-associated x protein (bax) and the bcl-2 homology 2 (bh2) domain of bcl-2 is required for this heterodimerization.48 it was found that keap1 binds to the bh2 domain and facilitates the ubiquitination of bcl-2 leading, to bax accumulation and enhanced apoptosis. thus, mutations in the keap1 binding site for bcl2 are responsible for the anti-apoptotic effect as well as overexpression of nrf2.49 moreover, it has been reported that nrf2 can directly activate the transcription of bcl-2 and bcl-xl.50,51 many studies have reported the association between nrf2 upregulation and chemoresistance in various cancers, including gastric cancer, osteosarcoma, non-small cell lung cancer, endometrial cancer, bladder cancer, and neuroblastoma.2427,52 platinum drugs generate electrophilic molecules that damage cancer cells. chemoresistance to these drugs can be explained by high expression of antioxidant nrf2 target genes.4 another mechanism of chemoresistance is the induction of the drug efflux pump family, which includes the mdr, by nrf2.53 some drugs, including hdac inhibitors, oxaliplatin, and proteasome inhibitors can induce nrf2 and thus decrease the effectiveness of chemotherapy.5456 in light of the data presented in this review, nrf2 is an attractive molecular target for the inhibition of cancer. in contrast to nrf2 activators, including numerous phytochemicals, only a small number of nrf2 inhibitors have been identified (table). trigonelline, a coffee alkaloid, efficiently decreased basal and tertiary butylhydroquinone (tbhq)-induced nrf2 activity in chemoresistant pancreatic carcinoma cell lines (panc1, colo357, and miapaca2) which have high nrf2 activity. along with inhibiting nrf2 the sensitivity of all cell lines to anticancer drugs and tumor necrosis factor-related apoptosis inducing ligand (trail)-induced apoptosis was enhanced by trigonelline.57 chrysin (5,7-dihydroxyflavone) is a natural flavonoid that is found in many plant extracts. in doxorubicin resistant hepatocellular carcinoma cell line (bel-7402/adm), chrysin significantly reduced nrf2 expression by downregulating the pi3k-akt and erk pathways.58 in that study, chrysin restored chemosensitivity by downregulating the nrf2-downstream genes such as ho-1, akr1b10, and mrp5. apigenin (4,5,7-trihydroxyflavone) is a natural flavone that is present in many fruits and vegetables and has various biological activities, such as anti-inflammatory and antioxidant properties. in a study using bel-7402/adm cell lines, apigenin reduced nrf2 expression as well as the expression of nrf2-downstream genes. when cells were treated with doxorubicin, apigenin showed a synergistic anti-tumor effect on bel-7402/adm cell lines, resulting in reduced cell proliferation and more substantially induced apoptosis.59 brusatol is a quassinoid that is extracted from brucea javanica. brusatol reduced nrf-2 protein level without changing the keap1 level and sensitized various cells including hela, mda-mb-231, and a549 to chemotherapeutic agents such as carboplatin, 5-fluorouracil, etoposide, and paclitaxel.60 the anticancer effect of brustalol mediated by nrf2 was confirmed in a xenograft model indicating that brusatol could be used to combat intrinsic resistance. ascorbic acid is an antioxidant that can suppress the level of nrf2. in the imatinib-resistant chronic myelogenous leukemia cell line kcl22/sr, binding of nrf2 to the are of the gamma-glutamylcysteine synthetase gene promoter was much stronger than in the parental imatinib-sensitive cell line kcl22.61 furthermore, addition of ascorbic acid to kcl22/sr cells resulted in a decrease in nrf2-dna binding and partial restoration of imatinib sensitivity to kcl22/sr.61 luteolin (3,4,5,7-tetrahydroxyflavone) is a polyphenolic flavonoid found in high concentrations in celery, green pepper, and parsley. in a study using a cell-based are-reporter assay, luteolin was found to be a potent nrf2 inhibitor.62 in that study, luteolin significantly sensitized a549 cells to the anticancer drugs oxaliplatin, bleomycin, and doxorubicin indicating the potential application of luteolin as a natural sensitizer in chemotherapy. constitutively expressed nrf2 can promote cancer cell proliferation and protect cells against oxidative stress and therapeutic agents. it must be kept in mind that chemopreventive agents including various phytochemicals can induce chemoresistance and tumor progression by activating the keap1-nrf2 pathway. however, it will be necessary to understand the molecular regulation of nrf2 and identifying the individualized status of nrf2 expression.

nuclear factor e2-related factor 2 (nrf2) is a transcription factor that upregulates expression of a battery of genes to combat oxidative and electrophilic stress. modification of kelch-like ech-associated protein 1 (keap1) by reactive oxygen species stabilizes nrf2 by escaping from degradation. nrf2 then binds to antioxidant response elements (ares) on the promoter region of various genes. activation of the keap1-nrf2-are pathway plays critical roles in the chemopreventive effect of various phytochemicals. however, nrf2 can protect cancer cells from oxidative stress and promote cell proliferation. moreover, recent studies reveal that activation of the nrf2 pathway is critical for resistance to chemotherapeutic agents. the aim of this review is to provide a molecular basis for the use of nrf2 inhibitors in overcoming chemoresistance.

PMC4204167

pubmed-33

sepsis is the major cause of death in the intensive care unit. despite improvement of antibiotics treatment and supportive techniques, the mortality of septic shock increases to approximately 60%., it not only helps doctors to make an early diagnosis of sepsis, but also predicts outcomes. there have been some biomarkers and cytokines used in both the clinical practice and laboratory including soluble triggering receptor expressed on myeloid cells-1 (strem-1), procalcitonin (pct), n-terminal probrain natriuretic peptide (nt-pro-bnp), c-reactive protein (crp), interleukin-6 (il-6), and interleukin-10 (il-10). trem-1 is a recently discovered member of the immunoglobulin superfamily of receptors that is specifically expressed on the surfaces of neutrophils and monocytes. strem-1 is a soluble form of trem-1 and is upregulated when exposed to infectious diseases. pct is a polypeptide consisting of 116 amino acids and is the precursor of calcitonin; it was proven useful to identify nonsystemic inflammatory response syndrome and was firstly used in sepsis. nt-pro-bnp is a biologically inactive form that is cleaved from the prohormone probrain natriuretic peptide (pro-bnp) by proteolytic enzymes before secretion. crp is a widely used biomarker to discriminate the inflammatory response to sepsis. il-6 and il-10 are important proinflammatory and anti-inflammatory cytokines during sepsis course.. indicated that strem-1 was more accurate than pct and crp in the diagnosis of sepsis, but others showed that the prognostic utility of serum strem-1 in septic shock was inferior to that of pct. the purpose of the study was to compare the prognostic value of biomarkers and cytokines versus clinical severity scores and improved death risk prediction. a total of 102 patients with sepsis from single centre hospital intensive care unit were enrolled from december 2010 to august 2012 according to the 2001 international sepsis definition conference. exclusion criteria included: age younger than 18 years, preexisting thyroid disease and lung cancer that influence procalcitonin levels, patients with acute coronary syndromes and renal dysfunction, and patients staying in icu less than 24 hours. the study was approved by the hospital's ethics committee and either the patients or their relatives provided informed consent. demographic and disease data of patients included age, gender, chief complaints for admission, vital signs, length of stay in icu, infection sites, microorganisms, routine blood test results, liver and kidney functions, coagulation indicators, blood gas analysis, acute physiologic assessment and chronic health evaluation (apache) ii scores, and sequential organ failure assessment (sofa) scores. these were recorded on 3 days (days 1, 3, and 5). serum was collected at these same time points and pct, strem-1, nt-pro-bnp, crp, il-6, and il-10 levels were determined in the end. pct was measured using an enzyme-linked fluorescence analysis kit (elfa, vidas brahms pct kit, biomerieux sa, france). strem-1 was determined using a double antibody sandwich elisa (quantikine human trem-1 immunoassay elisa kit, r&d systems, minneapolis, mn, usa). nt-pro-bnp was measured with an available immunoassay analyzer (elecsys 2010; roche diagnostics, mannheim, germany). crp was determined using scattering using a nephelometric assay (dade-behring, sa paris, france). il-6 and il-10 were determined using elisa (immulite; diagnostics products corporation, los angeles, ca). quantitative data with normal distributions are given as means standard deviations (sd). quantitative data that were not normally distributed were summarized as medians (interquartile ranges) and compared by nonparametric tests (mann-whitney u test). we made a logarithmic conversion for the nondistribution data when we did dynamic comparison in figure 2. proportions were used to express qualitative data and the differences in proportions between groups were compared using a chi-square test. we compared the characteristics of survivors versus nonsurvivors using univariate analysis and used receiver operating characteristics (roc) curves to evaluate prognostic value of the biomarkers and cytokines predicted 28-day mortality. those variables with p values less than 0.05 on univariate analysis were then entered into a multivariate logistic regression analysis to further identify the independent predictors of 28-day mortality. there were no significant difference in age and sex of these two groups (p>0.05). the apache ii and sofa scores of patients in the nonsurvival group were higher than those of patients in the survival group (p=0.000, p=0.000, resp.), (table 1). serum pct, strem-1, il-6 levels of patients in the nonsurvival group were significantly higher than those in the survival group on day 1 (p<0.001). there were no differences in nt-pro-bnp and crp, il-10 levels between the two groups (p>0.05) (table 2). the roc analysis showed that the accuracy of the pct, strem-1, il-6, apacheii, and sofa scores on day 1 for the prediction of 28-day mortality was moderate (auc>0.7, p<0.01), whereas the accuracy of nt-pro-bnp, crp, and il-10 was low (auc<0.7, p>0.05) (figure 1). comparing auc of pct, strem-1, and il-6, we found that there was no significant difference of auc between strem-1 and pct (p=0.2910), and the auc of the two markers were higher than that of il-6 (p>0.05). meanwhile, there was no significant difference of auc between apacheii and sofa scores (p=0.3753). the auc of apacheii and sofa scores were higher than those of strem-1 and pct (p<0.05) (table 3). the baseline day 1 variables that were found to be significantly different between survivors and nonsurvivors on univariate analysis (pct, strem-1, il-6, apacheii, and sofa scores) were entered into a logistic regression model. among these variables, three variables remained independently associated with 28-day mortality: strem-1, pct, and sofa score (table 4). median serum biomarkers and cytokines levels were determined on days 1, 3, and 5 and were compared between the survival and nonsurvival groups. serum pct, strem-1, and il-6 levels in the nonsurvival group were higher than those in the survival group on days 1, 3, and 5 (p<0.01). there was no difference in nt-pro-bnp levels on day 1 (p>0.05), but later the nt-pro-bnp levels in the nonsurvival group were higher than those in the survival group on days 3 and 5 (p<0.05). there were no differences in crp and il-10 levels on days 1, 3, and 5. serum pct, strem-1, il-6, and nt-pro-bnp levels showed a decrease trend in the survival group (p<0.05), but there was no decrease tendency in the nonsurviving group for these four biomarkers; strem-1 even had a increase trend (p<0.05). serum crp levels in both surviving and nonsurviving groups had decrease tendency (p<0.05) (figure 2). recently, pct, strem-1, crp, and nt-pro-bnp cytokines were widely used to diagnose sepsis and reflect the severity, but the results were not the same. meanwhile, there were few studies to put so many biomarkers in one study, particularly how to combine the biomarkers, and clinical severity scores remained unclear. the present study showed that the serum levels of strem-1 and pct in nonsurvival group were higher than those in the survival group; meanwhile, they decreased in survival group, but stayed in high levels even increased in the nonsurvival group during sepsis time course. many previous studies have shown that dynamic changes in strem-1 levels could predict survival and mortality of patients at the early stage of sepsis [10, 11]. strem-1 is widely used to diagnose sepsis [7, 12]. in the present study, serum strem-1 levels of patients in the nonsurvival group were significantly higher than those in the survival group on day 1; it decreased in survival group, but it even increased in the nonsurvival group. some studies failed to find the association between strem-1 and poor outcome [8, 13]. at a cutoff of 252.05 pg/ml, strem-1 measurements yielded a sensitivity of 85.7%, specificity of 75.7%, positive predictive value of 70.6%, negative predictive value of 88.2%, and an accuracy of 79.4% for differentiating nonsurvivors from survivors. pct is normally produced in the c cells of the thyroid gland; plasma pct levels in healthy humans are approximately 550 pg/ml in normal state; its half-time is about 2233 hours in serum. many tissues and cells except thyroid gland produce and release that pct on systemic inflammation. several previous studies reported pct could serve as a useful tool to distinguish sepsis from systemic inflammatory response syndrome [15, 16]. on the other hand, pct could reflect the severity of sepsis and outcome. a study by christophe clec'h and coworkers found that serum pct on day 1 was significantly higher in patients with than without septic shock. meanwhile, among patients with sepsis, pct concentrations were significantly higher in those who died than in the survivors, at all four measurement time points. similar results were drawn from other investigations [16, 18]. very few studies failed to find the prognostic value. at a cutoff of 10.64 ng/ml, procalcitonin measurements yielded a sensitivity of 76.2%, specificity of 81.7%, positive predictive value of 53.5%, negative predictive value of 67.8%, and an accuracy of 61.8% for differentiating nonsurvivors from survivors. nt-pro-bnp has been found to be a useful markers in the diagnosis, management, and prognosis of patients with congestive heart failure and was secreted into blood in response to atrial or ventricular wall stretch. a recent meta-analysis suggested that an elevated nt-pro-bnp level may prove to be a powerful predictor of mortality in septic patients. in our study, there was no difference in nt-pro-bnp level between groups on day 1, but the nt-pro-bnp levels in the nonsurvival group were higher than those in the survival group on days 3 and 5. meanwhile, serum nt-pro-bnp level showed a decreased trend in the survival group, but there was no decrease tendency in the nonsurvival group. we concluded that nt-pro-bnp may predict sepsis 28-day mortality in different stages. one research demonstrated that elevated serum nt-pro-bnp value represented an independent predictor for poor icu outcome in the presence of clinical severity scores; the cut-off in admission nt-pro-bnp that best predicted outcome was 941 pg/ml. crp is an acute phase protein and a sensitive systemic marker of inflammation and tissue damage. the secretion of crp begins within 46 h after stimulus, doubles every 8 h thereafter, and peaks at 3650 h. the role of crp in sepsis prognostic value seemed different. in our study, there was no significant difference between survivors and nonsurvivors during the three measures, similar to previous study, indicating that crp was just an inflammatory biomarker and failed in reflecting sepsis severity. have reported that crp levels in severe sepsis were lower than those in sepsis, suggesting that crp levels did not reflect the severity of sepsis. il-6 and il-10 are important proinflammatory and anti-inflammatory cytokines in sepsis. in our study, serum il-6 levels of patients in the nonsurvival group were significantly higher than those in the survival group on days 1, 3, and 5. the above results showed that il-6 had the prognostic utility for sepsis, whereas il-10 did not show the power. surez-santamar and coworkers enrolled 253 hospitalized septic patients; they found that il-10 and il-6 were the best predictors, whereas pct showed only moderate predictive value for mortality. another study investigated the prognostic value of il-6, pct, and crp in critically ill patients during the first increase of fever; only il-6 levels were significantly higher in nonsurvivors compared with survivors, in which prognostic value was superior to pct and crp. in contrast, kawczyski and polakowska indicated that the predictive value of il-10 plasma concentration was better than that of il-6. to sum up, strem-1, pct, and il-6 serum values attribute to the prognosis of sepsis during the time course. the dynamic changes of biomarkers and cytokines were more meaningful for predicting the sepsis procession. schneider and coworkers retrospectively analyzed the relationships between serum pct, il-6, and apacheii score and prognosis of 220 patients on the first day after operation. they found that pct was the sole independent predictor of 28-day mortality, in which prognostic ability was superior to those of il-6 and apacheii score. zhang et al. suggested that serum strem-1 levels reflected the severity of sepsis more accurately than those of pct and crp and were more sensitive for dynamic evaluations of sepsis prognosis. facing the results, we wonder which was the best predictor and how to combine them together and which was more valuable compared to clinical severity scores. apache ii and sofa scores have been widely used to validate mortality risk stratification. in our study, we used roc and logistic regression model to search for the best predictor. based on roc analysis, strem-1 and pct showed the equal prognostic ability (0.792 for pct, 0.862 for strem-1, p=0.291), whereas their prognostic utility was inferior to that of apacheii and sofa scores which had equal power to predict outcome (0.923 for apacheii score, 0.953 for sofa score, p=0.375). logistic regression model showed that serum strem-1, pct, and sofa score were the independent predictors of 28-day mortality, which was supported by other result. as far as we know, the interrelationship between strem-1, pct, nt-pro-bnp, cytokines, and clinical severity scores for mortality prediction in general icu patients has not been previously evaluated. our research firstly discovered that strem-1 and pct had the equal prognostic ability for sepsis mortality and were superior to other parameters. previous study has indicated that the iteraction of trem-1 and interact adaptor protein dap12 can stimulate neutrophil and monocyte-mediated inflammatory response via the triggering and release of pro-inflammatory cytokines and chemokines. strem-1 increases quickly when exposued to infection, and its half-time is short. in bacterial infections, serum pct levels start to rise at 4 h after the onset of systemic infection and peak at between 8 and 24 h; it decreased 50% every 24 hours along with therapy. in contrast, crp rises slowly and peaks 36 h after an endotoxin challenge. the mechanism of nt-pro-bnp release in sepsis is complex, and kinetics characteristic is unknown. il-6 and il-10 rise quickly and peak at 24 hours and maintain a short time. the patients admitted to icu often delayed more than 24 hours, either crp or cytokines serum concentration was unable to reach the peak at the period of sepsis. of course, the exact roles of biomarkers and cytokines in sepsis process are not clear, and need to be further studied. firstly, our study chosen a part of sepsis biomarkers and did not put all biomarkers in the research. of course, it was a costly and unnecessary task to do so. secondly, every biomarker has its own dynamic characteristics; meanwhile the patients were not in the same sepsis stages in the study; thus the explanation for the results would be influenced. thirdly, we excluded patients with previous heart diseases history, but we did not perform the ucg to evaluate cardiac function. finally, the sample size of the study was small and larger studies are needed. in summary, elevated serum strem-1 and pct levels provide superior prognostic accuracy to other biomarkers. combination of serum strem-1 and pct levels and sofa score can offer the best powerful prognostic for sepsis mortality. in the future, in order to improve the accuracy of the prognosis of sepsis, the combination of novel biomarkers and traditional markers of sepsis, reflecting different aspects of sepsis, is an attractive advice and is worthy of further investigation.

objective. to investigate the prognostic significance of serum soluble triggering receptor expressed on myeloid cells-1 (strem-1), procalcitonin (pct), n-terminal probrain natriuretic peptide (nt-pro-bnp), c-reactive protein (crp), cytokines, and clinical severity scores in patients with sepsis. methods. a total of 102 patients with sepsis were divided into survival group (n=60) and nonsurvival group (n=42) based on 28-day mortality. serum levels of biomarkers and cytokines were measured on days 1, 3, and 5 after admission to an icu, meanwhile the acute physiology and chronic health evaluation ii (apache ii) and sequential organ failure assessment (sofa) scores were calculated. results. serum strem-1, pct, and il-6 levels of patients in the nonsurvival group were significantly higher than those in the survival group on day 1 (p<0.01). the area under a roc curve for the prediction of 28 day mortality was 0.792 for pct, 0.856 for strem-1, 0.953 for sofa score, and 0.923 for apache ii score. multivariate logistic analysis showed that serum baseline strem-1 pct levels and sofa score were the independent predictors of 28-day mortality. serum pct, strem-1, and il-6 levels showed a decrease trend over time in the survival group (p<0.05). serum nt-pro-bnp levels showed the predictive utility from days 3 and 5 (p<0.05). conclusion. in summary, elevated serum strem-1 and pct levels provide superior prognostic accuracy to other biomarkers. combination of serum strem-1 and pct levels and sofa score can offer the best powerful prognostic utility for sepsis mortality.

PMC3941582

pubmed-34

worksites in glass and metal works, foundries, rolling mills, in the vicinity of hardening furnaces, rotary kilns in the cement industry, high-temperature laboratory furnaces, as well as firefighting, are associated, in addition to common mechanical hazards (e.g., due to splashes of molten metals and slag), sparks and direct contact with open fires, with hazards caused by emission of harmful optic radiation within the visible (vis) and infrared (ir) spectrum range. excessive exposure to ir radiation leads to dryness of the mucous membranes, burns of the eyeball and development of cataracts. protection against thermal hazards due to excessive amounts of ir radiation reaching the eyes is provided by protective optic filters installed in spectacles, goggles or face protections. besides protecting the user against hazardous ir radiation, such filters should ensure that the object/area of work can be seen clearly. if the level of ir radiation is very high, the use of filters reflecting radiation is recommended, as it allows the temperature increase in the filter itself to be reduced. the filters currently used for the protection of the face and eyes against hazardous ir radiation are of metallic reflective type, made of glass or organic material (mainly polycarbonate) bases coated with a single metallic layer for reflectance of ir radiation or absorption filters. absorption filters are manufactured from dyed glass obtained in a mass staining process by introduction of a factor modifying the radiation within the vis and near infrared (nir) spectrum range passing through the filter. the effect of a protective optical filter obtained as a result of this process is based on absorption of a large proportion of optic radiation, whereas only a small proportion of radiation the filter is exposed to is reflected. this is an undesirable phenomenon, contributing to deterioration of the filter user's comfort. reflective metallic filters are obtained by deposition of a metallic layer of copper (cu) or gold (au) on mineral or organic bases. such filters take advantage of the characteristic properties of the metals used in their production, i.e., significant reflection coefficient for ir radiation and significant transmittance for the vis spectrum. despite the fact that the technique of deposition of interference layers has been known for many years the application of thin film ir filters in eye protections to block the previous papers published by the authors of this study contain descriptions of the structure of interference filters designed for application in eye protections highly effective in reduction of ir radiation with high level of transmission of the vis spectrum. the authors investigated also the optical and mechanical parameters of the newly developed protective optic filters manufactured with the use of interference technologies. the analysis of the results of the conducted tests demonstrated numerous advantages of interference technology application in the construction of protective filters, including: improved resistance to damage of the external protective filter surface (cracking, scratching etc.) as well as high reflection coefficients for the nir spectrum. the aim of the present study was to compare the optic properties of protective filters manufactured by taking advantage of the interference technology and those coated with a single metallic layer reflecting ir radiation. the coefficients characterizing their optic parameters, allowing to assess the protective properties within the vis and ir spectrum range were compared. the following coefficients were analysed: luminous transmittance (v),ir transmittance within the 7801400 nm range (a),ir transmittance within the 7802000 nm range (n),ir reflectance within the 7802000 nm range (r n),ir absorptance within the 7802000 nm range (a n),shade number (n). luminous transmittance (v), ir transmittance within the 7801400 nm range (a), ir transmittance within the 7802000 nm range (n), ir reflectance within the 7802000 nm range (r n), ir absorptance within the 7802000 nm range (a n), the level of protection (shade number) is determined on the basis of luminous transmittance (the higher protection level correlates with the higher level of ir radiation blocked and appropriately lower vis light transmittance). protection level consists of a code number (e.g., 4) for ir filters and a shade number (e.g., 3, 5, 7) calculated according to the following equation: (1) where v=luminous transmittance. for the particular level of protection, the mean ir transmittance within the 7801400 and 7802000 nm ranges as well as the ir reflectance and absorptance within the 7802000 nm range are determined. for comparative analysis of the metallic reflective filters available in the market versus the interference filters developed by the authors, the following samples were prepared for the tests: off the shelf metallic reflective filters on a polycarbonate base (flat parallel plate of 50 mm diameter and 2 mm thickness with a metallic layer of copper (cu) protection levels: 4-3, 4-5, 4-7);interference filters on a polycarbonate base (flat parallel plate of 50 mm diameter with interference coating made up of the following materials: aluminium, h4 latio3 substance and silicon dioxide). off the shelf metallic reflective filters on a polycarbonate base (flat parallel plate of 50 mm diameter and 2 mm thickness with a metallic layer of copper (cu) protection levels: 4-3, 4-5, 4-7); interference filters on a polycarbonate base (flat parallel plate of 50 mm diameter with interference coating made up of the following materials: aluminium, h4 latio3 substance and silicon dioxide). the protection levels, luminous transmittance and symbols used for marking the tested filters are presented in table 1. characteristics of the filters.symbolluminous transmittance v (%) protection levelfilter typecu 4-314.1334-3metallic reflectiveitf 4-39.0824-3interferencecu 4-52.0214-5metallic reflectiveitf 4-51.4704-5interferencecu 4-70.3534-7metallic reflectiveitf 4-70.1604-7interferencenote: protection level consists of code number (4) for infrared filters and shade number (3, 5, 7) calculated according to equation (1). note: protection level consists of code number (4) for infrared filters and shade number (3, 5, 7) calculated according to equation (1). optical properties of the filters were measured with a cary 5000 spectrophotometer (varian, australia). the spectrophotometer enabled transmittance or reflectance of optical radiation within the vis and nir spectrum to be recorded with 1 nm steps and 0.001% resolution level. spectral transmittance and reflectance measurements were carried out within the wavelength range of 7802000 nm. the quartz halogen lamp was used as a source of radiation. to detect transmitted or reflected radiation the photomultiplier tube and lead sulphide detector were used for vis and nir spectrum, respectively. spectral transmittance was measured with incident radiation falling normally on the filter surface in the visual centre (defined as in). for spectral reflectance the measurements were taken every second owing to the high absorption of investigated filters within the nir region. since the minimum averaging time of the cary 5000 spectrophotometer was 0.0125 s, the values of transmittance or reflectance were measured 125 times for a given wavelength. the aforementioned coefficients were calculated from the following equations [1012]: luminous transmittance (2) where ()=spectral transmittance, v()=spectral visibility function of the average human eye for daylight or night vision, s d65()=spectral energy distribution of standard illuminant d65; ir transmittance within the 7801400 nm range (3) where ()=spectral transmittance; ir transmittance within the 7802000 nm range (4) where ()=spectral transmittance; ir reflectance within the 7802000 nm range (5) where ()=spectral reflectance; ir absorptance within the 7802000 nm range (6) where r n=ir reflectance within the 7802000 nm range, n=ir transmittance within the 7802000 nm range. luminous transmittance (2) ir transmittance within the 7801400 nm range (3) ir transmittance within the 7802000 nm range (4) ir reflectance within the 7802000 nm range (5) ir absorptance within the 7802000 nm range (6) the factor before integrals corresponding to the value of the measurement step at the measurement of spectral characteristics. transmittance and reflectance characteristics of the studied filters.note: cu 4-3=metallic reflective filter; itf 4-3=interference filter; cu 4-5=metallic reflective filter; itf 4-5=interference filter; cu 4-7=metallic reflective filter; itf 4-7=interference filter. table 2 presents the values of coefficients determined on the basis of equations (3)(6). infrared transmittance within the 7801400 and 7802000 nm range, infrared reflectance within the 7802000 nm range and infrared absorptance within the 7802000 nm range. infrared transmittance a and n (%) infrared reflectance rn (%) infrared absorptance an (%) symbola 7801400 (nm)n 7802000 (nm)rn 7802000 (nm)an 7802000 (nm)cu 4-31.4860.94283.51615.542itf 4-31.0030.56382.08417.353cu 4-50.2160.13487.31912.547itf 4-50.0380.00691.0378.957cu 4-70.0040.02388.30811.669itf 4-70.0040.03994.3125.649 optical radiation within the 7801400 nm spectrum range passes through the anterior compartment of the eye, the lens and reaches the retina. exposure to an excessive amount of radiation from this range can result in burns of the retina and, in extreme cases, its permanent damage., chronic long-term exposure may lead to the development of cataracts, referred to as ir or glass worker's cataracts. in view of the above the results presented on the graphs in figure 1 and the determined transmittance values (see table 2) indicate unequivocally more effective blocking of ir radiation provided by interference filters. the values of a and n for interference filters are lower even by an order of magnitude than those obtained for the metallic reflective filters. for instance, the mean value of ir transmittance within the 7802000 nm range for interference filters with protection level 4-5 (one of the most common protection levels used in the metallurgic industry) equals 0.006% and is significantly lower than the maximum acceptable value specified in, which is 10.6%. for all the investigated filters, the values of r n exceed 60%, which classifies them in the group of filters with increased ir reflectance. the levels of the ir absorptance for metallic reflective filters and interference filters of 4-3 and 4-5 protection class are similar. for the highest of the investigated protection levels 4-7 the above findings are important for selection of a filter appropriate for the radiation source. the selection of filter protection level is dependent on the temperature of the radiation source. the 4-7 level is used for radiation sources whose temperature exceeds 1600 k (e.g., occurs in the metallurgical industry). with such high temperatures, reduction of filter heating as a result of exposure to ir radiation is important. as it follows from comparative analysis of the optic properties of filters taking advantage of the interference technology and filters coated with a single metallic layer reflecting ir radiation, the interference filters provide a more effective blocking of ir radiation in comparison with the currently used protective filters.

the paper analyses the selected optical parameters of protective optic filters used for protection of the eyes against hazardous radiation within the visible (vis) and near infrared (nir) spectrum range. the indexes characterizing transmission and reflection of optic radiation incident on the filter are compared. as it follows from the completed analysis, the newly developed interference filters provide more effective blocking of infrared radiation in comparison with the currently used protective filters.

PMC4960498

pubmed-35

the synthetic modification of proteins enables the construction of biomolecular hybrids that can be used to study protein function, deliver potent therapeutics to cellular targets, and build new materials. the synthesis of these constructs requires a suite of chemoselective bioconjugation reactions that proceed under mild, aqueous conditions in the presence of the native functional groups that are present on protein surfaces. the most common methods for protein modification target the nucleophilic side-chains of lysine and cysteine. however, these strategies can result in complex product mixtures, as lysine is typically found in high abundance on the protein surface and uniquely reactive cysteine labeling sites can be difficult to install in many instances (such as thiol proteases and proteins produced via the eukaryotic secretory pathway, for example). many newer approaches for the site-selective modification of proteins involve the introduction of artificial amino acids with reactivities that are orthogonal to those of the native amino acids. along these lines, a number of powerful methods have been developed for the selective modification of azide, alkyne, alkene, carbonyl, and aniline moieties. however, the difficulty of introducing a non-canonical amino acid can limit the application of these methods. complementary approaches rely on the site-selective modification of native amino acids by enzymes. in addition, a reliable method for the modification of c-terminal thioesters with n-terminal cysteines, termed native chemical ligation, has been developed by kent and co-workers. this method has been used for the semi- and total synthesis of complex protein substrates, including the chemical synthesis of a single glycoform of human erythropoietin. as an alternative strategy, we and others have developed methods for the selective modification of the n-terminal amino group. methods that target the n-terminus can offer significant advantages for bioconjugate preparation, as they can be used for a wide range of protein targets produced by virtually any expression system. conceptually powerful as they are, however, these methods can be hampered by long reaction times, often require large excesses of reagent, and/or involve at least two-steps for the attachment of synthetic molecules. we have therefore sought to develop new techniques that can achieve n-terminal modification with similarly high positional selectivity, but with significantly improved efficiency. herein, we report an oxidative coupling pathway that can preferentially modify the n-terminus of proteins with fast kinetics. peptide substrates were first used to screen reaction conditions and identify the site of modification. a peptide panel with varying n-terminal residues was then evaluated to determine the sequence specificity of the reaction, leading to the identification of proline as the optimal n-terminal amino acid. the reaction was next applied to protein substrates, showing similarly high levels of conversion when an n-terminal proline residue was present. this mild bioconjugation reaction enables the facile, rapid modification of proteins to create a well-defined and stable linkage in a single position, and thus should be useful for many different applications in chemical biology and the construction of biomolecular materials. we have previously reported the chemoselective coupling of aniline moieties on proteins to electron-rich aromatic rings, such as o-aminophenols, at slightly acidic ph (6.06.5). these reactions require the addition of naio4 or k3fe(cn)6 as a terminal oxidant, with the latter reagent exhibiting improved compatibility with glycoproteins and substrates with free sulfhydryl groups. the use of ferricyanide as the oxidant also yields a single reaction product (1), whereas periodate leads to the formation of a ring contracted species as a competing pathway. the ferricyanide-based reactions are presumed to involve an o-iminoquinone as the reactive intermediate, as suggested in scheme 1, or could involve the corresponding o-quinone after imine hydrolysis. taken together, the oxidative coupling strategies have demonstrated excellent functional group compatibility and the ability to join large unprotected biomolecules at low concentrations, as demonstrated for the coupling of peptides, polymers, and nucleic acids to specific locations on viral capsids and antibody fc domains. while these coupling reactions were found to be highly aniline-selective under the conditions used, several studies have reported the reaction of o-aminophenols and o-catechols with native amino acids, dating back to 1949. in addition, recent work by messersmith has shown the ability of proteins to be coupled to o-quinone moieties present on polydopamine-coated surfaces. these reports suggested that secondary coupling pathways could be developed to achieve the modification of native amino acids with o-aminophenols (scheme 1), and thus initial experiments were designed to identify the optimal reaction conditions for achieving this with complex molecules. in our previous work, we noted that low amounts of background reactivity could be observed in aniline-based oxidative coupling reactions when higher ph conditions (> ph 6.5) were used. in an initial effort to characterize this alternative reaction pathway, conditions and reaction times were first screened to increase the reaction yields for peptides that did not contain aniline groups. angiotensin i and melittin were used as substrates, as they contain many reactive amino acids, including lys, arg, his, trp, and tyr. the peptides were exposed to 2-amino-p-cresol using k3fe(cn)6 as the oxidant. the reaction ph was varied from 5.5 to 8.5, and the reaction mixtures were analyzed using maldi-tof ms (supporting information figure s1). the level of modification increased with the basicity of the reaction, with near quantitative modification of angiotensin i after 20 min at ph 7.5 and higher. throughout these initial investigations, it was noted that angiotensin and melittin showed significant differences in reactivity, with angiotensin consistently demonstrating better conversion. ms/ms analysis of the angiotensin product was used to identify the participating residue, and revealed that the n-terminal amino group was responsible for the observed reactivity (supporting information figure s2). as further confirmation of the site-selectivity, several peptide substrates were screened for reactivity (figure 1a and supporting information figure s3). consistent with the n-terminal reaction selectivity, the only peptide that did not react had a pyroglutamate in this position, and therefore no free amino group. (a) modification of commercially available peptides was monitored by maldi-tof ms. (b) a positional scan of the n-terminal amino acid was evaluated. peptides with the sequence xadswag were tested for reactivity with 2-amino-p-cresol. the reactions were run with 100 m peptide, 200 m aminophenol, and 5 mm ferricyanide at ph 7.5 and analyzed by lc ms. shown is the average percent modification with error bars representing the standard deviation of three reactions. the same peptides were used for a screen of coupling partner equivalents (c) and a time screen (d). most buffers did not alter the reactivity, but imidazole and buffers containing a morpholine or piperazine ring (pipes, hepes, hepps, and mops) significantly impeded the reaction (supporting information figure s4). this is possibly due to small amounts of buffer impurities that react competitively with the oxidized intermediate. the time course of the reaction was also investigated (supporting information figure s5). in addition, it was found that the peptides could be modified using naio4 as the oxidant, or 4-methylcatechol as the coupling partner (supporting information figures s67). both of these reactions showed the same dependence on ph; however, moderate levels of modification were still observed at acidic ph (5.56.5) when using these alternative coupling conditions. these observations suggested that the peptides were reacting with the o-quinone intermediate formed in situ from either the catechol or the iminoquinone precursor (after imine hydrolysis). given the differential reactivity observed on peptide substrates, we synthesized peptides with varied n-terminal residues (xadswag) to determine the specificity of the reaction. the base sequence was selected to increase the mass of the peptide, impart water solubility, and include a tryptophan residue for quantitation using uv monitoring. the peptides were synthesized on the solid phase using standard fmoc synthesis, cleaved from the resin, and purified by hplc. after purification, the peptides were resuspended in phosphate buffer, ph 7.5, adjusted to a concentration of 1 mm, and stored at 20 c until use. to assess the effect of the n-terminal residue on reactivity, the peptides (100 m) were reacted with 2 equiv of 2-amino-p-cresol (200 m) in the presence of k3fe(cn)6 (5 mm) in phosphate buffer, ph 7.5 (figure 1b). ms (see supporting information figure s8 for representative ms data for the modified peptides). most n-terminal amino acids showed good-to-high levels of conversion (6090%), but proline stood out as the only residue that showed nearly complete modification (90100%). a second observation of this screen was the fact that tryptophan, tyrosine, and methionine residues were not oxidized by the ferricyanide reagent, consistent with our previous report of oxidative coupling with this oxidant (see supporting information figure s8). however, free cysteine residues can be oxidized to various species, potentially including disulfides and sulfenic acids, and thus it is recommended that they be protected as disulfides before oxidative coupling is attempted (vide infra). to optimize the reagent ratios (specifically the equivalents of o-aminophenol), the peptides (100 m) were reacted with 110 equiv of the o-aminophenol (1001000 m) in the presence of ferricyanide (10 mm). after 30 min, the reactions were quenched with excess tris(2-carboxyethyl)phosphine (tcep). it was demonstrated that conversion was highest using 25 equiv of the coupling partner (figure 1c). using more than 5 equiv of this was most likely due to the ability of the aminophenol to react with itself at higher concentrations (1 mm). consistent with this, when using 10 equiv of the o-aminophenol, a byproduct was observed with a mass that corresponded to the condensation of 3 aminophenols (344 da). we also investigated the differences in coupling rates for representative n-termini. the reaction of 2-amino-p-cresol with three different peptides was monitored over the course of 1 h (figure 1d). the peptides (100 m) were reacted with 2 equiv of the o-aminophenol (200 m) in the presence of ferricyanide (5 mm), and aliquots were quenched with excess tcep at the indicated time points. the proline terminal peptide not only reached the highest level of conversion, but also did so in a significantly shorter time than the other termini. despite efforts to optimize conditions for all n-termini the reaction of n-terminal amines with o-aminophenols was characterized using small molecule mimics. the methyl esters of phenylalanine (h-phe-ome) and proline (h-pro-ome) were coupled to 2-amino-p-cresol using ferricyanide at ph 7.5. the crude products were characterized using two-dimensional nmr and high-resolution mass spectrometry. the primary amine of h-phe-ome formed p-iminoquinone product 2, which was analogous to the one formed with aniline coupling partners (scheme 1, supporting information figure s9). however, the secondary amine of proline prevented the formation of the p-iminoquinone tautomer, and thus favored o-quinone product 3 (scheme 1, supporting information figure s10). given the different linkage obtained with proline, we verified the stability of the product to a variety of conditions. the proline terminal peptide, padswag, was first modified with 2-amino-p-cresol. after purification, the modified peptide (100 m) was exposed to reductants, nucleophiles and acidic and basic ph (10 mm additives or buffer). no loss of product was observed under any of the conditions tested, demonstrating the hydrolytic stability of the product (supporting information figure s11). the ability of the linkage to withstand these conditions renders this method quite useful for the construction of biomolecular materials for a variety of applications. with a view toward in vivo applications, current efforts are examining the stability of the linkage in blood plasma, as well as evaluating the intrinsic immunogenicity of the o-quinone group. in the process of characterizing the reaction products, it was observed that the colored products absorbed light at wavelengths greater than 500 nm (with max between 505 and 525 nm depending on the amine coupling partner). as the starting coupling partners and ferricyanide did not absorb at these wavelengths, this unique absorbance provided a means to monitor the reaction progress. the different amine coupling partners (p-toluidine, h-pro-ome, and h-phe-ome) were reacted with 4-methylcatechol in the presence of 10 mm ferricyanide, and the absorbance of the resulting solution was monitored at 520 nm to determine the relative rates of reactivity (figure 2a; for unnormalized data see supporting information figure s12). the catechol substrate was used for these studies to simplify the reaction pathway by eliminating the imine hydrolysis step. the reactions were run under pseudo-first order conditions with 0.1 mm catechol and 1 mm amine coupling partner. when the reaction was carried out at ph 6.0, the reaction with the proline analogue reached completion nearly as rapidly (2 min), but the reaction with the primary aliphatic amine of phenylalanine required longer reaction times (10 min). (a) amine coupling partners were reacted with 4-methylcatechol as a model substrate. reactions were run under pseudo-first order conditions with 100 m catechol, 1 mm amine, 10 mm ferricyanide in 50 mm phosphate buffer. (b) a peptide containing both an n-terminal proline and a p-aminophenylalanine residue (pad(paf)swag) was tested for reactivity with 2 equiv of 2-amino-p-cresol at ph 6. the remainder of the reaction was purified and then reacted with 2 equiv of the aminophenol at ph 7.5 and analyzed by lc ms. by quantifying product formation by absorbance, we were also able to measure the second-order rate constant for the proline-based coupling (supporting information figure s13). the reaction of 1 equiv of h-pro-ome (100 m) with 1 equiv of 4-methylcatechol (100 m) and 100 equiv of k3fe(cn)6 (10 mm) was performed in triplicate at 25 c. the second-order rate constant for the coupling was determined to be 44 4 m s. while proline reacted rapidly with the electron-rich coupling partner, the small molecule studies indicated that aniline should react faster. the rate for the aniline reaction was too fast under these conditions to determine the second-order rate constant accurately. given the differences in reactivity observed at ph 6.0 and 7.5, we hypothesized that it would be possible to modify the aniline side chain of p-aminophenylalanine (paf) and the n-terminal proline amine sequentially. to test this hypothesis, we synthesized a peptide containing both reactive moieties (pad(paf)swag). only one modification was observed when the peptide was reacted with 2-amino-p-cresol at ph 6.0 (figure 2b). ms/ms analysis of the modified peptide confirmed that the single modification occurred on the aniline side chain (supporting information figure s14). after this step, the peptide was purified and subsequently reacted with 2-amino-p-cresol at ph 7.5. reaction at the higher ph enabled a second modification of the peptide substrate, at the n-terminal proline residue. the differential reactivity was investigated by comparing the reactivity of a protein containing a paf residue to proteins without the artificial amino acid. the paf residue was introduced into the coat protein of bacteriophage ms2, which self-assembles into a spherical, hollow protein shell. myoglobin and a mutant of the tobacco mosaic virus (tmv) coat protein were used as native protein substrates. reactions with 2-amino-p-cresol were either performed on the isolated, individual proteins or with the aniline containing protein mixed with the native protein substrate (supporting information figure s15). addition of the aniline containing protein to the native protein decreased the n-terminal reactivity, indicating that the aniline residues react more rapidly than n-terminal residues with the o-aminophenols. in addition, ms2 showed significantly higher reactivity at all of the phs tested, confirming preference for aniline residues. the oxidative coupling reaction with n-terminal amino groups was first tested on proteins with native n-termini. several proteins were reacted with o-aminophenol-functionalized 5 kda peg under the optimized reaction conditions (figure 3). the native proteins showed moderate levels of reactivity, which could be attributed to inaccessible n-termini or simply to the less reactive n-terminal residues. to test if proline terminal proteins were more reactive, a proline residue was introduced to the n-terminus of gfp and the tobacco mosaic virus (tmv) coat protein. the n-terminus of tmv was also slightly extended from the native sequence (addition of pag). the proline-gfp was treated with a variety of conditions to determine the specificity of the reaction (figure 4a). only at basic ph in the presence of both the o-aminophenol substrate and the oxidant was modification observed. additionally, the proline-terminal variant showed significantly improved reactivity compared to that of the wild-type n-terminus. these high levels of modification were maintained even when only 12 equiv of the o-aminophenol peg was used. the site of modification was confirmed to be the n-terminal proline by lc ms/ms analysis of a tryptic digest of proline-gfp modified with 2-amino-p-cresol (supporting information figure s16). (a) the n-terminus of several proteins was pegylated using o-aminophenol-functionalized 5 kda peg and ferricyanide. (b) modification of wild type proteins with 5 kda o-aminophenol-peg was monitored by sds-page. (a) a proline was introduced to the n-terminus of gfp. the proline terminal variant showed much higher levels of modification than the wild-type protein. the band doubling is due to a gel artifact, and appears in all lanes. (b) mutants of tmv were reacted with 5 equiv of 2-amino-p-cresol and 1 mm k3fe(cn)6 for 30 min and analyzed by lc ms. reaction conditions for both native and proline terminal proteins were optimized by evaluating reactivity with myoglobin and proline-gfp. the reaction time, buffer, and ph were screened (supporting information figures s17 and s18). similar to the results obtained with peptide substrates, the reaction reached its highest level of conversion after about 1530 min. in addition, most buffer salts tested were compatible with the reaction with the exception of buffers containing morpholine (mops) or piperazine moieties (hepes), as was observed with peptide substrates. these buffers decreased the level of modification slightly, but did not completely inhibit reactivity. the effect of reaction ph was tested using both k3fe(cn)6 and naio4 as the oxidants. little reactivity was observed at acidic ph, and the level of conversion increased between ph 7.0 and 8.0. at higher reaction ph (8.0) a second modification was observed, indicating that lysines may also participate in the reaction. however, it is also possible that under more forcing conditions, such as higher reaction ph or increased concentration of aminophenol substrate, the aminophenol reacts with both itself and the n-terminal amino group resulting in double modification of the n-terminus. n-terminal mutants of tmv were also evaluated for their reactivity with o-aminophenols. the tmv monomers assemble into well-known double disk structures, displaying 34 copies of the n-terminal groups on their peripheries. two n-terminal mutants (pag and ag) were reacted with 5 equiv of 2-amino-p-cresol (100 m) and 1 mm k3fe(cn)6 for 30 min. ms demonstrated that the proline terminal mutant reached nearly complete conversion, while the alanine terminal mutant showed low levels of modification under these coupling conditions (figure 4b, see supporting information figure s19 for wider mass range and ion series). the compatibility of the reaction with cysteine residues was also tested using tmv. a single cysteine residue (s123c) was introduced into the tmv coat protein with a proline n-terminus (pag s123c tmv). this mutant was reacted with 2-amino-p-cresol and analyzed by lc the cysteine residue also reacted with the o-aminophenol, resulting in two modifications. however, it was found that the n-terminal proline could be modified selectively if the cysteine was first capped (figure 5a, b, see supporting information figures s2122 for wider mass range and ion series). to do this, the cysteine residue was protected as a disulfide bond by reaction with 5,5-dithiobis(2-nitrobenzoic acid) (dtnb, ellman s reagent). after the oxidative coupling step the disulfide bond was readily reduced by tcep, leaving the free cysteine and the modified n-terminus. alternatively, the cysteine residue was modified with a maleimide, followed by modification at the n-terminus with an o-aminophenol reagent. a) pag s123c tmv was reacted with small molecule substrates and analyzed by lc cysteine residues were protected as a disulfide using ellman s reagent (dtnb) before oxidative coupling. subsequent reduction of the disulfide resulted in selective modification of the n-terminus. the n-terminal proline was then modified with a rhodamine-functionalized o-aminophenol. this strategy allowed for the direct, dual modification of the protein at both the cysteine residue and the n-terminus. two fluorophores paired for frster resonance energy transfer (fret, alexa fluor 488 c5-maleimide and o-aminophenol functionalized rhodamine b) were thus conjugated to tmv using this strategy to create a templated array of chromophores for light harvesting applications. the free cysteine was first quantitatively labeled with an alexa fluor maleimide (supporting information figure s22). the n-terminal proline was then coupled to a fluorescent o-aminophenol resulting in 50% modification of the tmv monomers with both fluorphores (figure 5b). complete modification of the n-terminus was not observed as tmv precipitated from solution with increasing levels of modification with the rhodamine dye. in current experiments, we are using this dual-labeling strategy to introduce more soluble chromophores. the oxidative coupling reaction was also compared to the reaction of protein amines with activated esters. this acylation methodology is commonly employed, and can be targeted to the n-terminus by controlling the reaction ph in some cases. the reactions were compared on creatine kinase, a protein with a native proline n-terminus. reaction with 15 equiv of o-aminophenol peg resulted in good levels of modification of creatine kinase (5060%), while reaction with 15 equiv of n-hydroxysuccinimide (nhs) peg resulted in low levels of modification (525%, figure 6). only when a vast excess of the nhs peg was used were moderate levels of modification achieved. as was the case with proline-gfp, some over modification was observed under the oxidative coupling conditions when using five or more equivalents of aminophenol. this could result from dimerization of the oxidized species before protein coupling, but has yet to be characterized due to the low abundance of this product. in any case, lowering the reaction ph slightly or using fewer equivalents of the o-aminophenol substrate prevented the over modification from occurring. the modification of the n-terminus of creatine kinase with aminophenol peg was compared to the reaction of creatine kinase with nhs peg. in this study, we have identified conditions for the oxidative coupling of o-aminophenols to n-terminal amino acids. proline residues work particularly well with this strategy, and are therefore strongly recommended when using it. these groups can be introduced readily in n-terminal positions using site-directed mutagenesis and escherichia coli expression, especially since the methionine residue resulting from the start codon is cleaved when proline is in the second position. the fast kinetics of the reaction allow it to be successful even at low reagent and substrate concentrations, and suggest that it can be used for sterically demanding bioconjugations. the oxidative coupling strategy reported here offers two distinct advantages over other n-terminal labeling methods. first, the modification occurs in a single step and does not require initial oxidation of the n-terminus. second, the fast second-order kinetics allow for low concentrations of the coupling partners to be used. however, to achieve high levels of modification on protein substrates, proline was required as the n-terminal residue. this new protein modification strategy is currently being explored in our lab for the generation of protein-based materials. in the larger context, new techniques for the introduction of a single functional group in a specific position on a protein surface are always in demand. the ability of the n-terminal oxidative coupling method to achieve this in a single, brief reaction step is highly advantageous, and the fact that it can be combined with cysteine modification chemistry provides new opportunities for complex bioconjugate synthesis.

the synthetic modification of proteins plays an important role in chemical biology and biomaterials science. these fields provide a constant need for chemical tools that can introduce new functionality in specific locations on protein surfaces. in this work, an oxidative strategy is demonstrated for the efficient modification of n-terminal residues on peptides and n-terminal proline residues on proteins. the strategy uses o-aminophenols or o-catechols that are oxidized to active coupling species in situ using potassium ferricyanide. peptide screening results have revealed that many n-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive. when applied to protein substrates, the reaction shows a stronger requirement for the proline group. key advantages of the reaction include its fast second-order kinetics and ability to achieve site-selective modification in a single step using low concentrations of reagent. although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after n-terminal coupling is complete. this allows access to doubly functionalized bioconjugates that can be difficult to access using other methods.

PMC4353012

pubmed-36

alcohol withdrawal syndrome (aws) is characterized by varied symptoms that range from mild to severe intensity depending on several factors including the quantity, frequency and duration of alcohol intake, and the number of prior withdrawal episodes, as well as individual differences in the vulnerability [14]. symptoms usually present themselves within 6 to 24 hours after cessation of alcohol intake [5, 6]. subtyping of the aws has been attempted in the past, as gross conceptualized and proposed 3 constellations of alcohol withdrawal symptoms: factor 1 hallucinogenic that consisted of nausea, tinnitus, visual disturbance, pruritus, parasthesia, muscle pain, agitation, sleep disturbance, tactile hallucinations, and hallucinations which are auditory or visual or both; factor 2 affective and physiological that consisted of anxiety, depression, tremor, and sweats; and factor 3 delirium that consisted of clouding of the sensorium, impairment of consciousness, and impairment of contact with the observer. a cluster analytic study identified three different symptoms clusters of alcohol withdrawal, namely, cns excitation, adrenergic hyperactivity, and delirium. several rating instruments have been used to measure severity of alcohol withdrawal. among them, the most commonly used observer-rated scale is the 10-item clinical institute withdrawal assessment-alcohol, revised (ciwa-ar). it has been proposed that alcohol withdrawal symptoms in ciwa-ar appear multidimensional. a pubmed search supplemented with the study by pittman et al. was to explore the relationship between awsc and ciwa-ar, for which they carried out study on 127 male inpatients of alcohol dependence with principle components factor extraction with varimax rotation of ciwa-ar, a self-rated alcohol withdrawal symptoms checklist (awsc) and on combined items of ciwa-ar and awsc. they found three, five, and seven factor solution, respectively, for ciwa-ar, awsc, and combination of ciwa-ar and awsc. the analysis of ciwa-ar was done on 7 items as 3 items had zero variance. the first factor (variance 23.9%) was tension/anxiety which consisted of anxiety, agitation, and tactile disturbances. the second factor (variance 22.9%) was autonomic arousal which consisted of paroxysmal sweats, tremor, and headache or fullness in head, whereas the third factor (variance 17.4%, eigenvalue less than 1) was nausea and vomiting which consisted of a single item, nausea, and vomiting. in our setup, we use ciwa-ar as part of the measure for the management of alcohol withdrawal symptoms. it is generally observed that alcohol withdrawal symptoms fluctuate in presentation and severity across time. the present study was carried out to explore the dimensionality of this scale in an attempt to identify a set of underlying factors that exist and can explain the interrelationships among various manifestations of acute alcohol withdrawal symptoms. the knowledge of these underlying factors may enhance our understanding of aws and better prediction of complications thus management plans. this was a cross-sectional hospital-based study, conducted at centre for addiction psychiatry, central institute of psychiatry, ranchi, india, a tertiary care referral centre during may 2005 to june 2006. study sample included 201, only male fulfilling icd-10 dcr (world health organization) for alcohol dependence with currently withdrawal state, aged between 18 and 60 years, admitted within 24 hours of abstinence and patient himself or his guardian consenting for the study. information on patient's demographics, treatment history, past history, and family history was obtained from interviews with patients and accompanying person. detailed physical and neurological examinations were done to exclude any comorbid general medical condition, comorbid other psychiatric disorder, and any other comorbid substance use disorders except caffeine and tobacco. the sociodemographic data sheet included age, marital status, religion, community, education, and economic status. whereas clinical variables recorded were age of onset of drinking alcohol, duration of dependence, past history of detoxification, number of previous detoxification, past history of withdrawal seizure, past history of delirium tremens, family history of alcohol or substance dependence, degree of relationship if family history of alcohol or substance dependence is present, and family history of mental illness. it is the most widely used and studied 10-item alcohol withdrawal monitoring scale, which excludes vital sign abnormalities. it was developed from the 18-item clinical institute withdrawal assessment for alcohol and it has been studied across various geographic locations. it has a good reliability, validity, and it is considered as one of the most widely used alcohol withdrawal assessment scale for symptom-triggered therapy. each sign and symptom item of ciwa ar is evaluated on a 07 point likert scale except for one item orientation and clouding of sensorium, which is scored on a 04 point likert scale. a score of 8 points or less indicates mild withdrawal and patients scoring less than 10 do not usually need additional medication for withdrawal. patients admitted with alcohol dependence syndrome with acute withdrawal were evaluated with ciwa-ar immediately after admission then every six hours, as a routine protocol of the ward. detailed physical examination, mental status examination, and planned screening laboratory investigation were done to ensure conformity of study criteria. the patients get admitted with varying lengths of abstinence, ranging from hours to days, so we initially took all the ratings of all the patients and arranged them as rating at first 6 hours of abstinence and at every six hours like at 6, 12, 18, 24, 30, 36, 42, 48, 52, 58, and 64 hours till ciwa-ar scoring reaches below 10. the averages of each rating of ciwa-ar scores were computed to see the severity of withdrawal symptoms across time span of abstinence. many patients were admitted after overnight, 12 to 18 hours of abstinence, so we included the patients who were admitted with at least 24 hours of abstinence. meanwhile management and medications were continued as per ward protocol and no adjustment for study purpose was done. the standard detoxification protocol included thiamine supplementation, benzodiazepines either lorazepam or diazepam, and correction of fluids and electrolytes if any and other symptomatic treatment of associated conditions like dyspepsia or concurrent injury, wound, and infections. the collected data on 201 patients was statistically analyzed, using statistical package for social sciences (spss, inc., exploratory factor analysis (maximum likelihood method) was carried out to identify factor structure on all items of ciwa-ar for day three. kaiser-meyer-olkin measure of sample adequacy and bartlett's test of sphericity were also done to assess appropriateness of conducting factor analysis. two criteria for retaining the number of components were considered: kaiser's criterion to retain eigenvalues greater than unity and cattell's scree plot inspection for the point of inflexion. the mean age of the group was 37.18 (sd 9.35, range 1869) years. most of them were married (83.3%), educated (90%), residing in urban background (64.7%), belonging to middle socioeconomic status (60.3%), and earning a livelihood (80.4%). the mean age of starting alcohol was 21.63 (sd 4.99) years, whereas mean duration of dependence on alcohol was 6.49 (sd 5.06) years. 22.5% had history of withdrawal seizures, in their course of alcoholism, and only 2% reported history of delirium tremens in the past. family history of substance dependence was present in 33.3% of the total sample, out of which alcohol dependence was present in 56.9% of subjects and cannabis dependence in 8.3%. the item frequency and mean of all six hourly ciwa-ar ratings were calculated; the mean scores of ciwa-ar at 24 hours and at 36 hours are shown in table 2. the mean ciwa-ar score at 24 hours was 13.32 (sd 9.27) and 20.4 (sd 9.09) at 36 hours. based on the frequency variance and total ciwa-ar score we decided to carry out factor analysis with the 10 items of ciwa-ar as on scoring at 36 hours of abstinence. bartlett's test of sphericity was significant (2=1.044, df=45, p<.001), indicating that a factor analysis is appropriate. factor analysis (extraction method-maximum likelihood) with the 10 items of ciwa-ar for day three, resulted in initial three factors with eigenvalues greater than unity. the scree plot was also showing clear inflexion, supporting three factors (figure 1). therefore, three factors were retained, which captured 68.74% of variance. following varimax rotation with kaiser's standardization, the factors and their item loadings, with absolute values greater than 0.1, are shown in table 3. none of the items loaded on more than one factor. the first factor named as delirious factor, which had highest loading from tactile disturbances (.999), followed by auditory disturbances, orientation and clouding of sensorium, and agitation. it explained 34.34% of variance and showed good internal consistency (cronbach's alpha=.91). the second factor named as autonomic factor reflected four-item loading, highest from anxiety, followed by paroxysmal sweats, tremor, and headache or fullness in head. it explained 24.25% of variance and showed moderate internal consistency (cronbach's alpha=.66). the third factor named as nonspecific factor reflected two-item loadings, nausea, and visual disturbances that explained 10.04% of variance and a cronbach's alpha of .26. we examined the factor structure of the ciwa-ar in a population of adult men hospitalized to a tertiary psychiatric institute for treatment of alcohol dependence. the ideal study of aws could have been the assessment before starting medication, but this was practically not possible for many reasons. firstly, many of the patients come for admission after 12 to 18 hours of abstinence and severe withdrawal, so keeping them drug free was ethically not possible. thus natural aws presentation and its severity were masked by routine benzodiazepam administration and thiamine supplement. secondly, many other patients were referred from primary care centers with initial management, including long acting benzodiazepines like diazepam that masks the aws. thirdly, many other patients came even before onset of withdrawal and in a state of intoxication; the aws was not fully evolved in terms of range of symptoms and severity. for all these reasons, the mean ciwa-ar score for the initial 24 hours of abstinence (first day) of admission was only 13.32 (sd 9.27). later the sequential rating found more prominent withdrawal symptoms reaching highest mean score of 20.4 at thirty-six hours then gradually decreased. as drug free aws was not possible, we consider that the higher mean score of ciwa-ar represents the aws better than low score. also as both aws medications and alcohol itself are cns depressant and act in a similar way, either medication or alcohol intake should not make much difference in clinical picture. so we decided to proceed for factor analysis with highest mean ciwa-ar scoring at the 36th hour. the severity of withdrawal symptoms and appearance of complete sets of withdrawal symptoms at the 36th hour may have been influenced by plasma half-life of benzodiazepams being used for detoxification. in this study, choice of medication was with treating team of the hospital; however, only either intermediate acting lorazepam or long acting diazepam was used for this purpose. there was no use of short acting benzodiazepines, which causes varying and rebound withdrawal symptoms across different time frame with its dosing and changing plasma concentration. another more important influencing reason for varying withdrawal symptoms across different time frame could have been the dose of benzodiazepines, but we did not interfered with any medications or dosing and it was continued as per ward protocol to ensure naturalistic conditions. however, the equivalent benzodiazepines mean dose at 36 hour was 30 mg of diazepam per day. but this equivalent dose may not be accurate as many patients were on oral medications and others were on parenteral benzodiazepines. we excluded any other comorbid substance use disorders or polysubstance dependence but a few patients may also have undisclosed benzodiazepine or organic inhalant abuse or addiction. we also excluded any comorbid general medical condition especially epilepsy for that reason, patients on antiepileptic either taking it regularly or skipping will modify the alcohol withdrawal symptoms. for that matter any psychotropic drugs causing cns depression or any effecting stimulants will alter the withdrawal symptoms. most of the patients needed proton pump inhibitor drugs like pantoprazole or omeprazole for the alcohol induced dyspepsia, peptic ulcer disease, or gastroesophageal reflux disease, but these medications do not impose any effect on alcohol withdrawal symptoms. in a previous study by pittman et al., the mean ciwa-ar score for day one was 13.2 (sd 3.7) which was similar to our study, 13.32 (sd 9.27) at 24 hours; they continued with analysis on data collected on the first study day to exclude medication effects. but we analyzed the data as of 36 hours of abstinence, showing higher mean ciwa-ar score and all ten items had variance above 10%, thus representing fully developed withdrawal syndrome. however we could not control the medication effect used to control the withdrawal symptoms for ethical reasons, the used medications were benzodiazepine and thiamine supplementation for all the patients. two items of ciwa-ar, namely, auditory disturbances and tactile disturbances were very infrequent in our sample (9.5 and 10%) on day one which increased to 55.1 and 65.7% at 36 hours, other items scoring raised on day two like agitation, orientation and clouding of sensorium, visual disturbances, and paroxysmal sweats; few other items remained unchanged on day one and two like anxiety and tremor; however scoring of only this item headache or fullness in head improved on day two from 28.9 to 12.9% (table 2). this indicates the importance of time duration of abstinence to study the alcohol withdrawal symptoms. we found a three-factor solution based on rotated eigenvalues and scree plot analysis. the first factor explained 34.34% variance and consisted of four items, namely, tactile disturbances, auditory disturbances, orientation and clouding of sensorium, and agitation. this factor appears to represent perceptual abnormality and delirium and may be considered as subclinical spectrum of delirium. however, the only other factor analytic study of ciwa-ar by pittman et al. found these items (except agitation) were infrequent in their sample and were not included in analysis. the difference may be due to the time span of withdrawal on which data was collected. study analyzed ciwa-ar for day one and also our data on day one showed very low variance for these items. this suggests that ratings on alcohol withdrawal symptoms on the very first day may miss certain set of symptoms, which appears on and around day two and are characterized by perceptual abnormality and delirium like picture. further progression of alcohol withdrawal, we could not found beyond 36 hours, may be due to effects of continued medications for withdrawal suppression. surprisingly, visual disturbances item did not have high loading on this factor, whereas tactile and auditory disturbances had maximum factor loading (0.999 and 0.873, resp.). the etiological basis for these two disturbances includes cns rebound excitation which alters the perceptual quality. furthermore, some contribution of nutritional and specific vitamin deficiencies, such as thiamine and folate, and associated peripheral neuropathy probably add to these perceptual disturbances. the other two items, orientation and clouding of sensorium and agitation with factor loadings of 0.851 and 0.777, respectively, represent delirium. the perceptual alteration and delirium being loaded in a single factor may have some predictive association and hence need to be studied for management plan. the second autonomic factor explained 24.25% of variance and consisted of four items: anxiety, paroxysmal sweats, tremor, and headache and fullness in head with factor scores of .998, .660, .528, and .245 respectively. cronbach's alpha for this factor was 0.66 showing adequate internal consistency. within this factor this factor may be a result of the practice of not using adrenergic medication on routine basis at our institute. all items (paroxysmal sweats, tremor, and headache or fullness in head) were loaded similarly. we had additional anxiety to this factor; hence, we share the name of this factor with pittman et al. as autonomic. the third nonspecific factor explained 10.04% of variance and consisted of two items: nausea and vomiting and visual disturbances, with low factor scores of .51 and .27, respectively. there are three proposed physiologic bases for the symptom manifestation of alcohol withdrawal symptoms: cns excitation, adrenergic hyperactivity, and delirium, which may be attributed to different neurotransmitters and they respond selectively to different pharmacotherapy. the cns excitation may be secondary to deficiency in gaba activity, whereas increase in cns epinephrine level causes adrenergic hyperactivity. the nmda receptor hypersensitivity and overactivity of certain subtypes of nmda receptors are associated with delirium. though we did not find a factor structure of aws in accordance with very strict pathophysiological manifestation of either autonomic, or cns excitation or psychological/affective, or perceptual/hallucinogenic in our sample, the obtained factors suggest existence of subgroups of patients with different set of symptoms. this was expected as alcohol withdrawal manifests by simultaneous involvement of all mechanisms rather than any mutually exclusive mechanism. this simultaneous involvement of several other neurotransmitters besides gaba and nmda, like noradrenaline, acetylcholine, and dopamine as well as hormones and electrolytes [17, 18] will affect the symptom presentation. additionally our data analysis had ciwa-ar ratings of day two (36 hours) of admission with suppression of withdrawal symptoms from detoxification medications, that is, benzodiazepines. this would have differential effect on withdrawal symptoms manifestation in terms of gaba suppression only and selective unopposed action on other neurotransmitters or lacking adrenergic activity. though avoiding pharmacological suppression or modification was not possible on ethical grounds to understand completely natural unmodified withdrawal manifestation. there may also be possible influence of different types of alcoholic beverage and percent content of alcohol. strength of our study includes large sample size and not interfering with any medications or management strategies thus providing setting of naturalistic conditions. the use ciwa-ar is the most widely accepted alcohol withdrawal assessment scale and selection of abstinent hours was important to allow time for full appearance of symptoms, even though under cover of detoxification medication. there was for better coverage and inclusion of withdrawal symptoms at 36 hours as indicated by total ciwa-ar score and item frequency. these results have wider implications for the recognition and management of aws, particularly, for better understanding and identification of the symptom profiles for and differential management plans across subtypes of aws. the use of adrenergic antagonists may have a valuable role in addition to benzodiazepines, in a set of patients with autonomic features. one of the limitations in our study is that it includes male only patients; however, gender can be an important issue in aws presentation and its severity. even studies found that sex hormone affects the aws by modulating the function of the gaba-a receptor. also low levels of testosterone are associated with symptoms like indecision, excessive worrying, fatigability, and lassitude. another limitation of this study is patients sample with severe aws requiring inpatient management, thus limiting generalizability of our findings across gender and to mild to moderate severity cases. another limitation was that diagnosis was made clinically with icd-10 dcr criteria for alcohol dependence with currently withdrawal state, but not by a validated clinical interview. it is known that autonomic arousal is an important mechanism in aws; thus, other physiological measures and biological markers for objective assessment may be included in future studies. further studies may also be carried out including cases of mild to moderate severity and in both sex to uncover the differences. also, factor analysis depends heavily on the population studied; therefore, studies on different population may be required to generalize our findings. for the clinical practice, it is advisable not to overdepend on rating scales and it must not replace a thorough clinical evaluation of the patient's medical status in prediction of those at risk of severe alcohol withdrawal. the acute alcohol withdrawal symptoms was most severe at 36 hours of abstinence in our sample. this study finds multidimensionality of alcohol withdrawal symptoms as measured with ciwa-ar; we found three factors explaining 68.74 percentage of variance and named as delirious, autonomic and nonspecific .

objective. to identify the underlying factor structure of alcohol withdrawal syndrome, as measured with ciwa-ar. methods. exploratory factor analysis was conducted on the items of ciwa-ar. on 201 alcohol-dependent male patients seeking treatment for alcohol withdrawal at 36 hours of abstinence. results. a three-factor solution was obtained that accounted for 68.74% of total variance. first factor had loading from four items (34.34% variance), second factor also had four items (24.25% variance), and the third had two items (10.04% variance). conclusions. factor analysis reveals the existence of multidimensionality of alcohol withdrawal as measured with ciwa-ar and we found three factors that can be named as delirious, autonomic and nonspecific factors.

PMC4006624

pubmed-37

choroid plexus papilloma (cpp) is a rare benign tumor accounting for 0.5% of all intracranial tumors that is more common in childhood, consisting of 1.5 to 6.4% of pediatric intracranial tumors.1 2 the majority of cpps occur during the first 2 years of life, with 12.5 to 20% (10 to 12%) reported in infants under 1 year of age.1 3 there have been some reports of calcified cpp, cpp in an adult, cpp in the cerebellopontine angle (cpa), and atypical form of cpp.4 5 6 7 however, a case combining with these four features adult, dense calcification, atypical form, and cpa is considered to be extremely rare; such a case has never been encountered among the 8,540 skull base tumors on which the senior author (t.f.) has operated in the past 20 years. we report an adult case of densely calcified, atypical cpp originated from the cpa without connection to the forth ventricle. a 41-year-old man presented with a chief complaint of headache, dizziness, and difficulty with gait that had been gradually worsening for 8 months. neurological examination revealed blurred vision, nystagmus with lateral gaze, and mild right facial weakness. intermittent difficulty with balance and gait were also present; however, no motor weakness was observed. computed tomography (ct) of the head without contrast revealed a high-density multilobular mass in the right cpa causing mass effect and slight ventricular enlargement (fig. magnetic resonance (mr) images demonstrated a trilobed solid mass that measured 4.1 cm in maximum diameter (fig. the mass was extra-axial in location and revealed isointensity on both t1-weighted images (t1-wi) and t2-weighted images (t2-wi). ct and mr images did not reveal any connection between the tumor and the fourth ventricle. the surface of the tumor was roughly irregular and looked grayish pink. since the tumor was friable, gelatinous, vascular, and adherent to surrounding structures, it was hard to separate from cranial nerves. cranial nerves vi, vii, viii, ix, and x were enveloped in the tumor mass, which included calcification. the patient was discharged after recovery from the dysphagia by appropriate rehabilitation. computed tomography without contrast revealed a high-density multilobular mass in the right cerebellopontine angle. magnetic resonance images demonstrated a trilobed solid mass that was in an extra-axial location. the tumor revealed isointensity on both t1-weighted images (upper left) and t2-weighted images (upper right). homogeneous intense enhancement was identified (axial in lower left, sagittal in lower right). cpp reported in this article primarily occurred in the cpa with close proximity to the foramen of luschka lacking continuity of the fourth ventricle. the majority of choroid plexus tumors (cpts) originate in the ventricular system, 43 to 67% in the lateral ventricle, 24 to 39% in the fourth ventricle, and 9.5 to 11% in the third ventricle, respectively.8 9 the common site of origin differs in each pediatric and adult group. in the pediatric age group, 60 to 80% of such tumors occur in the lateral ventricle, especially in the trigone, because the vascular stalk of the choroid plexus in the lateral ventricle is usually attached to the inferior portion of the trigone. on the other hand, most cpp in adults occurs within the fourth ventricle.9 10 11 a small number of the tumors have been reported to be located in extraventricular regions, the cpa, or the cerebellar hemisphere or the suprasellar cistern.8 10 12 cpp within the cpa was first described by cushing in 1917.13 to date, cpp within the cpa has been reported in 7 to 9% of all cpts and is most exclusively found in adults.9 11 14 15 it is divided into two categories according to the origin of the tumor; one is primary extraventricular cpts and the other is a result of a direct extension of fourth ventricular tumors through the foramen of luschka.15 because the tumor of our case revealed no connection from the fourth ventricle, it is considered to originate from small choroid tufts that normally project from each recess at the foramen luschka into the cpa or from ectopic choroidal islets unconnected with the choroid plexus.16 the cpp of our case indicated dense calcification with ventricular enlargement. the bulbous tufts extended from the fourth ventricular choroid plexus through the foramen of luschka have been known as the bochdalek flower basket, which could be identified in 75% on ct and 96% on mr imaging.17 calcification of the region was found in 38% on normal imaging anatomy.17 in pathological states, calcification of cpp was reported in 4.1% on plain skull radiographs and 23.8% on ct examinations in patients of all 9 the feature of the calcification was commonly described as a stippled or patchy configuration that may include some degrees of hemorrhage.8 9 10 dense calcification was exceptionally reported by sarkar et al.2 the rate of hydrocephalus in association with cpp is 90% and the rate increases up to 100% in malignant cpps.8 the cause of hydrocephalus was considered a combination of cerebrospinal fluid (csf) overproduction (four to five times that in healthy persons), obstruction of csf pathways by the tumor mass, impaired csf absorption, increased protein content of csf around the tumor, subarachnoid scarring or granulations related to recurrent bleeding from the tumor, elevated intraventricular pulse pressure, and adhesions around the exit foramina of the fourth ventricle caused by highly proteinaceous or hemorrhagic csf.2 10 18 the finding of tumor calcification in the cpa generally required consideration of differential diagnosis with papillary or psammomatous meningioma, melanotic schwannoma, and myxopapillary ependymoma. among these tumors, association of ventricular enlargement with the finding of calcification might suggest the possible presence of cpts. gross total removal was performed in our case of atypical cpp, although the tumor adhered to adjacent structures. in the 2007 world health organization classification of tumors of the central nervous system, cpts are classified into cpp as grade i, atypical cpp as grade ii, and choroid plexus carcinoma (cpc) as grade iii. among cpts, total removal of cpts is recommended and seems to be feasible since a significant prognostic factor was relevant to the extent of tumor removal, and no definitive evidence of the beneficial effect of radiotherapy has been reported6 18 19 20. however, it is not always possible because of tumor bleeding and firm adhesion to adjacent structures, especially cranial nerves. the rate of complete resection of benign cpp was 80% and the 5-year survival rate indicated is nearly 100%. the majority of survivors are neurologically intact.19 because of the good prognosis, the preservation of cranial nerve functions should be primarily considered in surgery for benign cpp. as for the atypical cpp, the rate of total removal was decreased to 50 from 63%.11 21 atypical cpp showed a recurrence rate of 29%, which was much higher than the 6% seen in cpp. in addition, seeding within the central nervous system, spontaneous pulmonary metastasis, or rapid regrowth of atypical cpp were also reported.7 22 23 total or gross total removal should be advocated, especially in malignant cpts even though the tumor encases the cranial nerves. the finding of calcification in the cpa should suggest the possible presence of cpp, especially if ventricular enlargement is present.

choroid plexus papilloma is a rare benign tumor accounting for 0.5% of all intracranial tumors. the majority of choroid plexus papillomas occur during the first 2 years of life in the ventricular system. moreover, dense calcification of the tumor is uncommon. we report an adult case of densely calcified, atypical choroid plexus papilloma that originated from the cerebellopontine angle without connection to the forth ventricle. because the case involves a rare combination of four features adult, dense calcification, atypical form, and cerebellopontine angle each characteristic associated with the choroid plexus papilloma was discussed. the finding of calcification at the cerebellopontine angle in an adult should suggest the possible presence of choroid plexus papilloma.

PMC3836973

pubmed-38

multipath signals occur in numerous microwave and rf applications when an unwanted portion of the original transmission propagates along any alternate path and ultimately couples to the receiver distorting the amplitude and phase of the desired signal [14]. if the amplitude of the multipath signal is sufficiently large, its impact can be considerable. in multistatic radar and communication systems, these types of interference are most often caused by reflections of either the main beam or side lobes with objects near or actually in the beam path. classic examples include main beam propagation close to the earth's surface with associated reflections off of the ground or water (figure 1(a)). various approaches can be used to filter or compensate for these reflections through time-gating and signal time synchronization. the potential for interference from multipath signals increases substantially in near-field applications (figure 1(b)), especially in situations where the receiving and transmitting hardware are integrated. commercially available multichannel network analyzers (e.g., zvt8 by rohde&schwarz; munich, germany) utilize robust strategies to minimize these signal coupling problems. we are developing multichannel transceiving arrays for medical microwave imaging which exploit near-field concepts to produce electrical property maps (permittivity and conductivity) of tissues of interest [8, 9] and have addressed the issue by incorporating (a) dedicated mixers for each channel, (b) additional solid state switches for isolation, (c) double- and triple-braided coaxial cables, and (d) compartmentalized rf circuitry. the implementation has proven effective for our application achieving channel/signal isolation greater than 130 db. an alternative data acquisition strategy integrates a commercial, 2-port network analyzer with an electronic switching network to feed an array of antennas [1012] which is effective but also has limitations because (i) dynamic range is constrained by the provisions of the network analyzer, (ii) two-way signal loss is incurred through the network, and (iii) the switching matrix has relatively poor cross-channel isolation. equally important in near-field imaging is the multiple paths a signal can take within the imaging zone. figures 2(a) and 2(b) show a photograph of our clinical breast imaging tank and a schematic diagram of the antenna configuration, respectively. in this situation, the array of 16 monopole antennas surrounds the breast and can be moved to multiple vertical positions. the antennas and target are submerged in a solution of glycerin and water which is lossy over the operating frequency range (700 mhz3 ghz). early empirical tests have indicated that reflections off the tank side walls do not impact the desired signals for an array with the antennas mounted on a 15.2 cm diameter circle. likewise, analysis of the monopole beam patterns as a function of frequency has shown that artifacts are minimal when the array approaches the liquid interface at the top of the tank. with respect to reflections off the bottom surface, the base of the tank was at least 1.8 wavelengths (at the lowest frequency) below the active sections of the antennas in our initial clinical installation when the array was located at its lowest position during an exam. since minimal multipath signals resulted from reflections off the top liquid surface, symmetry would suggest that the same should be true for the base of the tank (figure 1(b)). however, surface waves can cause multipath signals that can be especially difficult to eliminate in near-field systems. their excitation can be complex, but their propagation characteristics along two material interfaces, whether planar or along cylindrically shaped structures, have previously been studied in depth [1520]. surface waves can readily propagate at the interface of two dielectric materials or one conductor juxtaposed directly with a dielectric. their propagation and attenuation characteristics are nominally determined by the electrical properties of the two materials. in addition, their amplitude decays exponentially away from the interface in the perpendicular direction as a function of the lossiness of the complementary materials. it should be noted that these investigations have stemmed partially from our efforts to perform microwave tomographic images on patients in an actual mr scanner for the purpose of exploiting the refined spatial resolution of the mr along with the more specific nature of the tissue dielectric properties. the mr bore is quite small and places a significant constraint of space for the microwave system. this was where we first encountered multipath signal corruption which subsequently led to this study. in the following sections, we discuss the theoretical underpinnings of these modes for the geometries present in our system. we demonstrate cases from our current imaging system, where the measurements indicate corruption of the desired signals from multipath signals associated with the base of the tank. we then show experiments that allow us to partially isolate the effects to surface waves propagating along other pathways. we realize that there are a number of propagation modes around the antennas, their feedlines and the tank surfaces, of which the surface waves are only one possible contributor, but understanding these contributions is important. we present an initial strategy for minimizing the effects of these signals which may be instructive for designing other near-field imaging systems, including simulations confirming the earlier theoretical discussion and validating our feedline design strategy. a challenging situation occurs when a portion of the transmitted signal propagates along an unwanted path and recombines with the original signal at the receiver. time-gating strategies can sometimes be effective when the nature of the multipath signal is well understood [1, 2]. the potential influence of a multipath signal when the original transmission is a continuous wave can be written as (1)resultant signal=acombcos(t+comb), where (2)comb=acombtan1(b1a1),acomb=a12+b12,a1=[adecosde+ampcosmp],b1=[adesinde+ampsinmp]. here, ade, amp, de, and mp are the desired and multipath signal amplitudes and phases, respectively, is the operating frequency, and t is time. for example, if the magnitude of the multipath signal is 25 db below that of the desired signal, the maximum possible amplitude and phase errors in the resultant signal would be 0.48 db and 3.22, respectively. for a 15 db multipath signal, these values increase to 1.42 db and 10.24. clearly, the resultant phase and amplitude errors can become very significant for multipath signals that are on the same order of magnitude as the desired signals. it should be noted that there can be many multipath contributions with a range of amplitude and phase contributions. this single contributor analysis serves to give a flavor that the unwanted effects can be significant and is generalizable to multiple sources. the first surface mode to be considered involves propagation along the interface between the tank base and coupling liquid. following the analysis by stratton, figure 3 shows a plane wave in region 1 impinging on an interface (x=0) with region 2. in this case, the magnetic field (hy) is only oriented in the y-direction (out of the page). the complex relative dielectric properties in the two regions are 1=1 j1 and 2=2 j2, respectively. the classic surface wave solution occurs when the reflection coefficient is zero (at the brewster angle), which is complex-valued in this instance. if a is the amplitude of the incident plane wave, then the magnetic component of the incident and transmitted waves can be represented as (3)hy=ae[jh1xjz], x>0,hy=ae[jh1xjz], x<0, where h1+=k1 and h2+=k2 are needed to satisfy the wave equation, k1 and k2 are the wave numbers for the two regions, and is the propagation constant. the wave impedances for the two regions are given by (4)z1=h1k1z1=h1k1z011j1,z2=h2k2z2=h2k2z012j2, where z1 and z2 are the free space impedances in the corresponding regions, and z1 and z2 are the associated wave impedances [21, 22]. this yields (5)h1=h2, where (6)=21=2j21j1. from these relationships, we can solve for (7)h1=k01(1)12,h2=k012(1)12,=k01(1)12. in this situation, we are primarily interested in surface waves propagating along the outside of a coaxial cable, and their associated attenuation as a function of distance after the mode has been sufficiently established. for this analysis, we will consider the case of a coaxial line which is abruptly terminated by an open end (figure 4). a coaxial cable supports a tem-mode electromagnetic field which is incident on the cable opening. part of the signal is partially reflected into the cable, while a second portion is transmitted as a surface wave propagating along the outside of the surrounding cable. the fields transmitted into the surrounding space can be determined from the distribution at the coaxial opening which can be found by solving the integral equation for the radial component of the electric field over the opening: (8)14+jcabe(,0)kc(,)d =j1abe(,0)kc(,)d0cosejkrrd, where c and 1 are the complex-valued permittivity of the coaxial cable insulator and the surrounding dielectric materials, respectively, a and b are inner and outer coaxial radii, respectively, is the operating frequency in radians, and k is the wavenumber in the coupling liquid, where 0 is the free space magnetic permeability. and are the radial cylindrical coordinates within and outside of the coaxial cable, respectively, is the angular coordinate within the coaxial cable, and r is defined as r=2+2-2cos. the variable kc(,) is represented as (9)kc(,)=jn=0n()n()an2n, where (10)n()=y0(na)j1(n)j0(na)y1(n),n={kc2n2,kc>njn2kc2,kc>n, an2=22n2[j02(na)j02(nb)1], n>0,a02=lnba. the eigenvalue n are solutions of the characteristic equation: (11)y0(na)j1(nb)=j0(na)y1(nb), where jn and yn are bessel functions of the first and second kind of order n, respectively, and kc is the wavenumber inside the coaxial line. once e(, z=0) is determined, then electric and magnetic fields can be found at any point (, z) in the surrounding medium from (12)e(,z)=1abe(,0)0(jk+1r) zcosejkrrdd, or (13)e(,z)=1abe(,0) 0[1(jk+1r)]cosrejkrrddh(,z)=j1abe(,0) 0cosejkrrdd, where (14)r=z2+2+22cos. from these equations it follows that the electromagnetic fields inside the surrounding medium decay approximately as e. figure 2(a) shows the illumination tank used in our current clinical breast imaging system. each monopole antenna consists of an exposed length (3.8 cm) of 2.2 mm diameter semirigid coaxial cable with only the center conductor and insulating teflon layer intact. for mechanical robustness, the coaxial feed line is enclosed in a 6.4 mm diameter rigid stainless steel tube, and the active section of the antenna is covered with an accompanying length of a delrin cylinder acting as a protective radome. the space between the copper coaxial outer conductor and the stainless steel sleeve is sealed at either end with silver epoxy to eliminate wave propagation along the gap. the antennas have a nominal return loss of 10 db over the bandwidth of 7003000 mhz. the black delrin fittings at the antenna/tank base contain hydraulic seals through which the antenna feeds pass to allow vertical motion of the array while eliminating any coupling fluid leakage. the 16 antennas are positioned on a 15.2 cm diameter circle, and both sets of 8 interleaved antennas are supported by individual mounting plates under the tank which provide independent motion of the array in groups of 8. the tank is fabricated out of plexiglas with an inner wall diameter of 27.3 cm and thickness of 1.3 cm, and the base has a thickness of 2.5 cm. all connecting cables are double-braided to minimize stray radiation. in these experiments, the antennas were positioned at heights close to the tank base (that were not used in any clinical exams) to study the multipath phenomenon in detail. figures 5(a), 5(b), and 5(c) show three illumination tanks with different heights, arrays of monopole antennas, and coaxial feed lines that were fabricated from the same plexiglas and had identical diameters and wall/base thicknesses as figure 2(a) tank. the feed lines passed through holes in the base of each tank and were fastened to sma flange connectors which were bolted to the tank floor. the tanks were filled with an 80: 20 glycerin/water mixture with the liquid level 1.5 cm above the antenna tips. in figures 5(a) and 5(c), the feed lines were both 10 cm long; however, the latter was bent in a serpentine shape such that the top height of the feed line was only 5 cm above the tank floor. the feed line in figure 5(b) was straight and was only 5 cm long. in these experiments, we used plexiglas for the tank materials with a dielectric constant of r=2.7 that was effectively lossless in this frequency range. the dielectric properties of the 80: 20 glycerin/water bath are plotted in figure 6 as a function of frequency. utilizing the clinical system described in section 2.2, a+5 dbm signal was transmitted at multiple frequencies over the 7002500 mhz range from a single antenna and received at the remaining 15 antennas. this sequence was repeated for the array positioned at multiple heights above the tank base. receive antenna amplitudes are plotted for representative frequencies in figure 7. at 900 mhz, the measured levels are high for the receivers closest to the transmitter (relative receiver numbers 1, 2, 14, and 15) compared to the rest of the array and do not change dramatically with changes in antenna height. however, the amplitudes are considerably lower for the more distant receive antennas. for antenna heights 7 cm or greater (above the tank floor), the attenuation follows a smooth curve hitting a maximum at antenna 8 (which is furthest away from the transmitter being located on the opposite side of the array). at antenna heights 5 cm and lower, the behavior is consistent with the three frequencies shown in figure 7. given that the distance from the antennas to the tank side walls did not change during the experiments, and that the antennas were sufficiently far from the liquid/air interface at all times for any array heights (10 cm in the worst case), the corruption of these most distantly received signals appears to be caused by multipath propagation associated with antenna tip proximity to the base of the tank most probably due to reflections off of the tank base or surface wave propagation along the dielectric interfaces. in this set of experiments figures 8(a) and 8(b) show the received signals for a single transmitter over a range of frequencies for straight feed line lengths of 10 cm and 5 cm (tanks in figures 5(a) and 5(b)), respectively. for the longer (10 cm) lines in figure 8(a), the field patterns appear well-behaved like those in the previous section, when the array was positioned at the largest heights above the tank floor. however, the patterns for the shorter (5 cm) line lengths in figure 8(b) exhibit corruption of the signals resulting from the longer propagation distances similarly to when the array heights were closest to the base of the tank for the clinical system (in figure 7). these corrupted signals are nominally between 50 to 70 dbm and occur in uneven patterns relative to the same signals from the longer feed lines which reach 80 to 90 dbm at the furthest antenna. for the shorter propagation distances (i.e., the signals received at antennas 14 and 1215), the attenuation patterns from the shorter and longer feed lines are similar (figure 8(b) versus 8(a)). these results suggest that the unwanted multipath signal effect is the same in both the experimental tanks and the clinical system tests in the previous section and depends on the antenna height from the base of the tank. however, in figure 8(c), the antenna feed length is exactly the same as in figure 8(a), but is curved such that the antenna tip is the same height above the tank floor as the antennas in figure 8(b) (which led to signal corruption); yet, the measurement results emulate those in figure 8(a) (which are not corrupted). here, the active part of the antennas is still positioned on the same 15.2 cm diameter circle as in the other two tanks. these findings show that the principle signal corruption observed in figure 8(b) is not due to reflections off the tank floor; otherwise it would have appeared in figure 8(c) since the antennas are at the same position above the base for both figures 8(b) and 8(c). it should be noted that the signal disruptions for this situation appear more substantial than that for the antennas used in the clinical system as discussed in section 3.1. this is very likely due to differences in the designs of the hydraulic seals, feedline shielding, and antenna radome for the other system. more likely, the multipath signals in figure 8(b) (with short feed lines) result from surface waves traveling along the outside of the coaxial lines, across the plexiglas/liquid and/or plexiglas/air interface and back up the outside of the receiver coaxial feed. the theoretical considerations in section 2.2 indicate that the attenuation along the coaxial lines is far more substantial than from the planar tank-base surface wave modes. figure 9(a) shows a plot of attenuation as a function of frequency for 15.2 cm of plexiglas/liquid surface waves and indicates that very little attenuation of a surface wave propagating along this interface occurs at the frequencies we used. thus, in our experiments, the real source of surface wave attenuation comes from propagation along the outside of the coaxial lines. figure 9(b) shows plots of the attenuation that results from single 5 and 10 cm lengths of feed line in the coupling fluid. the theoretical predictions of attenuation along the two 5 cm antenna feed lengths (transmit and receive) and the path along the plexiglas base/liquid interface are approximately 70 db ({ 2 34 db}+2 db) for the 1 ghz case (values interpolated from figures 9(a) and 9(b)). given a transmit power level of+5 dbm, the resultant 65 dbm multipath signal for the shorter line would easily corrupt the desired 81 dbm signal (figure 8(a)). only when the feed lines are nearly doubled in length, and the associated surface wave attenuation increased accordingly is the corruption of the desired signals reduced to an acceptable level. along with the analytical discussion in sections 2.1 and 2.2, we have also performed simulations of the configurations described in section 2.4. figures 10(a), 10(b), and 10(c) show the 900 mhz electric field magnitude distributions for the long and short, straight antenna feed and the longer, serpentine structure, respectively. these simulations were computed using ansys (burlington, ma, usa) hfss version 13.0. for all cases, there is a reasonably broad antenna pattern emanating outwards from the active part of the antennas, and this feature is reasonably similar for all feed line types. for the straight feeds especially the longer one, it is clear that there are considerable surface currents generated along the coaxial lines. for the shorter straight one, there is a high degree of field strength along the coaxial line within the plexiglas volume below the horizontal interface. the fields along the interface generally agree with our previous notion that the surface waves preferentially propagate within the lower-loss medium (in this case the plexiglas) as can be seen by the substantially greater amplitudes directly under the line. the results for the serpentine feedlines are consistent with previous results in that the field strength in the plexiglas is lower than that for the short, straight line case. figure 11 shows a plot of the field strength just below the liquid interface from a point directly underneath the antenna extending 140 cm to the right for all three corresponding plots. the field values are considerably less for the longer straight line but are also less for the serpentine cases compared to the short, straight cases. for the serpentine case, there seems to be some signal coupling between the lower feedline bend and the plexiglas. as discussed in section 1, surface waves do decay exponentially from the surfaces, and given the proximity of the feedlines and the liquid/plexiglas interface, some coupling is expected. the potentially debilitating effects of unwanted multipath signals is a critical consideration in translating near-field microwave imaging approaches into clinical and commercial systems. for our noncontacting antenna approach, surface waves (relative to signal reflections from the imaging tank walls) appear to cause the biggest effects as they propagate along the outside of the transmitting coaxial line across the illumination tank floor and back up the coaxial feed lines of the receivers. when the imaging tank is deep and the transmitting/receiving antenna tips are sufficiently far above the tank base, the surface wave signals are adequately suppressed relative to the transmissions through tissue. the results presented here indicate that 9-10 cm of distance along the feed line is adequate. however, reducing the tank depth is of interest for practical reasons and is essential in some settings and appears possible because reflections from the floor of the tank are still too small to degrade the measured signals propagating through tissue. indeed, we found that antenna tip distances as little as 5 cm from the tank floor maintain receiver signal fidelity across the array provided the surface wave contributions are attenuated through an equivalent feedline length approaching 10 cm. these findings are significant because they indicate that the antenna array and imaging tank geometry can be altered substantially by manipulating the shape of the antenna feed line, which can be exploited to ensure sufficient surface wave attenuation. there are certainly other mechanisms for multipath propagation including coupling of fields from the feedlines directly to portions of the breast tissue outside the immediate plane of propagation and are certainly good topics for further investigation.

microwave imaging techniques are prone to signal corruption from unwanted multipath signals. near-field systems are especially vulnerable because signals can scatter and reflect from structural objects within or on the boundary of the imaging zone. these issues are further exacerbated when surface waves are generated with the potential of propagating along the transmitting and receiving antenna feed lines and other low-loss paths. in this paper, we analyze the contributions of multi-path signals arising from surface wave effects. specifically, experiments were conducted with a near-field microwave imaging array positioned at variable heights from the floor of a coupling fluid tank. antenna arrays with different feed line lengths in the fluid were also evaluated. the results show that surface waves corrupt the received signals over the longest transmission distances across the measurement array. however, the surface wave effects can be eliminated provided the feed line lengths are sufficiently long independently of the distance of the transmitting/receiving antenna tips from the imaging tank floor. theoretical predictions confirm the experimental observations.

PMC3332071

pubmed-39

the main cause of treatment failure and death in cancer patients is metastasis the formation of secondary tumors in organs distant from the original neoplastic cell tissue. adjuvant therapy of proven efficacy is not currently available for cancer patients; therefore, the search for new targets of therapeutic reagents is required to prevent both proliferation and metastases. during metastasis, it is known that the mode of invasion is one of the markers of the malignancy and prognosis of cancer. the receptor for advanced glycation end-products (rage), a multiligand member of the immunoglobulin superfamily of cell surface molecules, interacts with distinct molecules implicated in homeostasis, development, and inflammation. rage binding by ligands such as advanced glycation end-products (ages) triggers the activation of key cell signaling pathways, thereby reprogramming cellular properties. in addition, several reports have suggested that rage is associated with cancer malignancy [4, 5]. the advanced stage of the glycation process (one of the posttranslational modifications of proteins) leads to the formation of ages and plays an important role in the pathogenesis of angiopathy in diabetic patients, aging, and neurodegenerative diseases [69]. a growing body of evidence suggests that the interaction of glyceraldehyde-derived ages (glycer-ages), but not glucose-derived ages (glc-ages), with rage elicits oxidative stress generation in numerous types of cells (vascular wall cells, mesangial cells, schwann cells, and cortical neurons), all of which could contribute to the pathological changes seen in diabetic vascular complications of alzheimer's disease [1013]. we have recently found that glycer-ages stimulated the growth and migration of cultured human melanoma cells and that anti-rage antibodies inhibited the tumor formation and lung metastasis of melanoma cell xenografts and subsequently improved survival in athymic mice. however, the effects of glycer-ages on other cancer cells remains poorly understood, and the molecular mechanisms behind their effects have not been clarified. in the present study, we examined the effects of glycer-ages on cultured human lung adenocarcinoma a549 cells and showed that glycer-ages enhanced their malignancy rather than their proliferation. n-acetyl-l-cysteine (nac) was purchased from sigma-aldrich (st. louis, mo, usa). briefly, 25 mg/ml of bsa (a0281, sigma-aldrich) were incubated at 37c for 7 days under sterile conditions with 0.1 m glyceraldehyde (ga; nakalai tesque, kyoto, japan) and 5 mm diethylenetriamine-pentaacetic acid (dojindo, kumamoto, japan) in 0.2 m phosphate buffer (ph 7.4). the modified albumin was then purified by pd-10 column (ge healthcare, buckinghamshire, england) chromatography and dialysis against phosphate-buffered saline (pbs). control unglycated bsa was incubated under the same conditions except for the absence of glyceraldehyde as a negative control. protein concentrations were determined with the dc protein assay reagent (bio-rad, richmond, ca, usa) using bsa as a standard. the preparations were tested for endotoxin using the pyrotell-t test (seikagaku bio-business, tokyo, japan), but no endotoxin was detected. human lung adenocarcinoma a549 and hepatocellular carcinoma hep3b cells were grown in dulbecco's modified eagle's medium (dmem; sigma-aldrich) supplemented with 10% fetal bovine serum (fbs; equitech-bio, kerrville, tx, usa) under standard cell culture conditions (humidified atmosphere, 5% co2, 37c). yamagishi, and its mock vector-transfected hep3b cells were maintained in 10% fbs/dmem in the presence of 700 g/ml g418 (roche, mannheim, germany). the cells were washed with ice-cold ca and mg free pbs (pbs (-)) and subjected to lysis buffer (1% tritonx-100/nonidet p-40, 10 mm sodium fluoride, 1 mm sodium orthovanadate, 5 mm sodium pyrophosphate, 2 mm egta, 5 mm edta, and 1 protease inhibitor cocktail (complete, mini; roche)). subsequently, the cell lysates were incubated on ice for 5 min and centrifuged at 10,000 g for 10 min at 4c. their protein concentrations were then measured using the bradford assay (bio-rad). cell lysates (30 g of proteins/lane) dissolved in sds sample buffer (62.5 mm tris-hcl (ph 6.8), 2% sds, 10% glycerol, and 0.01% bromophenol blue) containing 5% 2-mercaptoethanol (2-me) were boiled for 3 min at 95c, separated by sds-page, and then electrotransferred onto pvdf membranes (millipore, billerica, ma, usa). biotinylated markers (cell signaling, beverly, ma, usa) were used as molecular weight markers. the membranes were blocked for 1 h using 5% skimmed milk in pbs containing 0.05% polyoxyethylene sorbitan monolaurate (pbs-t). after being washed twice with pbs-t, the membranes were incubated with goat anti-rage antibody (n-16) or mouse anti--actin antibody (santa cruz, santa cruz, ca, usa) for 1.5 h. subsequently, the membranes were washed twice with pbs-t and incubated with anti-goat igg antibody (santa cruz) or anti-mouse ig's antibody (biosource, camarillo, ca, usa) and anti-biotin antibody (cell signaling) for 1 h. after being washed a further two times with pbs-t, the immunoreactive proteins were detected with ecl plus western blotting detection reagents (ge healthcare) using a luminescent image analyzer (las-1000uvmini; fujifilm, tokyo, japan). the density of the bands was analyzed using a multi gauge version 3.0 (fujifilm). the cells (710 cells/cm) were seeded in various plates or culture dishes (bd biosciences, franklin lakes, nj, usa) and incubated for 24 h. the control unglycated bsa and glycer-ages treatments were carried out in 0.1% fbs/dmem for 48 and 72 h, and cell viability was determined by the wst-1 assay. after removing the medium, 100 l/well of 10% fbs/dmem and 10 l/well of wst-1 solution (5 mm wst-1, 0.2 mm 1-methoxy-5-methylphenazinium, and 20 mm hepes buffer (ph 7.4)) (dojindo) were added, and the cells were incubated for 2 h. absorbance was measured at 450 nm and 650 nm using a microplate reader (labsystems multiskan ascent, model no. 354; thermo fisher scientific, kanagawa, japan), and the net difference (a450 a650) was used to express cell viability. the migration and invasion capacity were evaluated in 24-well transwell chambers and biocoat matrigel invasion chambers (bd biosciences), respectively. in both assays, the upper and lower culture compartments were separated by polyethylene terephthalate filters with a pore size of 8 m. ten percent fbs was used as a chemoattractant in the lower chamber compartments. before starting the invasion assay, the matrigel matrix of the chambers was reconstituted by adding serum-free dmem for 2 h. in the migration assay, 1 10 cells in serum-free dmem with control unglycated bsa or glycer-ages (100 g/ml) were added into each of the upper chambers for 20 h. in the invasion assay, 2 10 cells in serum-free dmem with control unglycated bsa or glycer-ages were added to each of the upper chambers for 48 h. then, the nonmigrating or noninvading cells on the upper side of the chamber membranes were removed using cotton swabs. the cells that migrated to or invaded the opposite side of the chamber were counted. the active form of rac1 was detected with the ez-detect rac1 activation kit (pierce, rockford, il, usa). this assay uses the ability of a glutathione s-transferase (gst) fusion protein corresponding to the p21-binding domain (pbd) of human p21 activated protein kinase 1 (pak1) to specifically bind to the active gtp-bound and not the inactive gdp-bound forms of rac1 and cdc42. cell lysates were incubated with gst-pak1-pbd and swellgel immobilized glutathione discs for 1 h at 4c, leaving an aliquot for measuring the levels of total rac1. the bound proteins were eluted in 2 sds sample buffer containing 5% 2-me for 5 min at 95c and characterized by western blotting using monoclonal anti-rac1 antibody. rac1 activity was determined by densitometric quantification of the pulled-down rac1-gtp level and normalizing it to the level of total rac1 detected in the sample lysates. total rna was isolated from the cells with isogen (nippon gene, tokyo, japan), and 50 ng of rna were reverse transcribed into cdna with primescript rt reagent kit (takara, shiga, japan) using a geneamp pcr system 9700 (perkin-elmer applied biosystems, foster city, ca, usa). real-time pcr was performed with sybr premix ex taq using a smart cycler ii system (takara). the reaction mixture (25 l) contained 1 sybr premix ex taq, 0.2 m pcr forward primers, 0.2 m pcr reverse primers, and 10 ng of cdna as a template. the primers used were as follows: tgf-: 5- gcg tgc taa tgg tgg aaa cc -3 and 5- cgg agc tct gat gtg ttg aag a -3, mmp-2: 5-agt ctg aag agc gtg aag-3 and 5- cca ggt agg agt gag aat g -3, and -actin: 5- tcc acc tcc agc aga tgt gg -3and 5- gca ttt gcg gtg gac gat -3. all processes were performed according to the manufacturer's instructions. -actin was used as an endogenous control gene in order to normalize target gene expression values. the cells were seeded and incubated for 24 h. the control unglycated bsa and glycer-ages treatments were carried out in serum-free dmem for 48 h. the conditioned medium was collected and used with a mmp-2 biotrak activity assay system (ge healthcare). all experiments were performed in duplicate and repeated at least two to three times, with each experiment yielding essentially identical results. the significance of differences between group means was determined by one-way analysis of variance. to investigate whether rage proteins are present in human lung adenocarcinoma a549 cells, we carried out western blot analysis using anti-rage antibody (n-16). rage proteins of different molecular weights were detected in a549 cells (figure 1). in full length rage cdna-transfected human hepatocellular carcinoma hep3b cells, the major band (57 kda) (indicated by an arrow in figure 1) represents the full-length rage protein. likewise, the full-length rage protein was also detected in a549 and mock transfected hep3b cells. no bands were detected in a neutralization experiment using blocking peptide (data not shown) so the other bands may represent deglycosylated rage proteins. we examined the effect of glycer-ages on the cell viability of a549 cells. cell viability was determined after 48 h incubation with control unglycated bsa or glycer-ages. twenty, 50, and 100 g/ml of glycer-ages significantly decreased cell viability to 83, 64, and 54%, respectively, (figure 2(a)). in contrast, control unglycated bsa had no effect. furthermore, glycer-ages significantly attenuated cell proliferation (figure 2(b)); however, no cell death was induced by glycer-ages according to cell death detection elisa (data not shown). we examined the effect of glycer-ages on cell migration and invasion. in the migration assay, the glycer-ages-treated cells showed significantly (2.7 times higher) greater migration than the control unglycated bsa-treated cells (figure 3(a)), and in the invasion assay, the glycer-ages-treated cells showed significantly (3.3 times higher) greater invasion than the control unglycated bsa-treated cells (figure 3(b)). when glycer-ages were added to the lower chamber, there was no effect on the results of the invasion assay (data not shown). these results suggest that glycer-ages-rage interaction is necessary for cell migration and invasion. rho family small gtpases are widely accepted to be key regulators of actin reorganization and motility. interestingly, rage signaling has been shown to result in the activation of rac1 [1720], and the activation of rage by ages has been shown to induce the generation of reactive oxygen species (ros). the glycer-ages-induced rac1 activity was about 2 times higher than that of the control unglycated bsa, and it was suppressed by pretreatment with n-acetyl-l-cysteine (nac), an ros scavenger (figure 4(a)). likewise, the glycer-ages-enhanced migration capacity was also significantly suppressed by pre-treatment with nac (figure 4(b)). thus, glycer-ages-rage interaction enhances migration capacity by activating rac1 via ros generation. members of the matrix metalloproteinase (mmp) family are widely accepted to be key regulators of tumor invasion [2224]. in particular, mmp-2 and -9, which are called gelatinases, are key enzymes for degrading type iv collagen and are thought to play critical roles in tumor invasion and metastasis. we examined the expression of transforming growth factor- (tgf-) and mmp-2 mrna and assessed mmp-2 activity with the mmp-2 biotrak activity assay system. neither the mrna expression of tgf- or mmp-2 was significantly increased by the addition of glycer-ages at 24 h (figures 5(a) and 5(b)). pro-mmp-2 activity was also not significantly altered by the addition of glycer-ages at 48 h (figure 5(c)). however, glycer-ages only increased mmp-2 (the activated form) activity to 120% (figure 5(d)). rage, which is a multiligand receptor, affects diseases such as cancer and diabetes through its ligands. several reports have suggested that rage and amphoterin are closely associated with invasion and metastasis in cancer cells [26, 27]. ages have been implicated in the development and progression of diabetic angiopathies, including skin dermopathy. however, it is still unclear which ages subtypes play a pathogenetic role and which ages receptors mediate the effects of ages on cells. we have provided direct immunochemical evidence for the existence of six distinct ages structures within the ages-modified proteins and peptides that circulate in the sera of diabetic patients. recently, we found that glycer-ages perform a diverse range of biological activities on vascular wall cells, mesangial cells, schwann cells, and cortical neurons [1013]. we also found that glycer-ages, but not glucose-derived ages (glc-ages), significantly stimulated the growth and migration of human melanoma cells. furthermore, the tumor formation of melanoma cells xenografts in athymic mice was prevented by treatment with anti-rage antibody. in tumor bearing-mice, survival rates were prolonged, and spontaneous pulmonary metastases were inhibited by treatment using anti-rage antibody. in addition, glycer-ages were present in the beds of human melanoma tumors, whereas they were hardly detected in normal skin. these results suggest that glycer-ages are involved in the growth and invasion of malignant melanoma through their interactions with rage. recently, we found that glycer-ages have the strongest binding affinity for rage [28, 29] and subsequently elicit oxidative stress generation and evoke vascular inflammation, thereby implicating them in accelerated atherosclerosis in diabetes [10, 11]. taken together, glycer-ages-rage interactions play an important role in the progression of melanoma cells to malignancy. however, the effects of glycer-ages on other cancer cells remain poorly understood, and the molecular mechanisms behind these effects have not been clarified. in the present study rage includes full-length, c-truncated, and n-truncated rage. the rage antibody (n-16) used in this study, which recognizes the n-terminus of rage, detects full-length and c-truncated rage. indeed, full-length rage (57 kda) was detected in a549 cells. the other bands may have represented the de-glycosylated form or c-truncated rage. glycer-ages are associated with cell cytotoxicity, but glc-ages are not [21, 31, 32]. in mesangial cells, intracellular ros produced by glycer-ages were found to induce apoptotic cell death. however, no a549 cell death induced by glycer-ages was observed in this study. it is thought that the cytotoxicity of glycer-ages depends on the sensitivity of their target cells to ros. furthermore, glycer-ages enhanced the migration and invasion activity of the a549 cells, both of which are prominent features of cancer malignancy. glycer-ages induced the invasion of a549 cells across matrigel, indicating that matrix degradation and migration mechanisms had been stimulated in these cells. at the molecular level, glycer-ages-induced phenomena resemble the effects of long-term oxidative stress or tgf-. both oxidative stress and tgf- are key regulators of the malignancy rather than the proliferation of cancer cells [3335]. tgf- strongly induces mmp-2 expression in a549 cells; however, mrna expression of tgf- was not induced by glycer-ages, and the mrna levels of mmp-2, which is produced by tgf-, did not change. in long-term oxidative stress, mori et al. showed that intracellularly produced ros activated rac1 and enhanced the invasion capacity of tumor cells by activating mmp [33, 3739]. a recent report showed that sustained exposure of cells to h2o2, but not one time exposure to h2o2, increased pro-mmp-2 activation through the induction of membrane type 1-mmp (mt1-mmp) expression. indeed, our results also showed that glycer-ages enhanced the migration capacity of a549 cells by activating rac1 via ros and increased their invasion capacity by activating mmp-2. however, glycer-ages only increased mmp-2 (the activated form) activity to 120%. it is suggested that the activation of other mmp such as mmp-13 may also participate in the above-mentioned changes, and future studies are necessary to clarify this matter. in conclusion, we demonstrated that glycer-ages enhanced the migration and invasion of a549 cells rather than their proliferation. these results suggest that glycer-ages play a critical role in cancer malignancy and are potential targets for therapeutic intervention.

the receptor for advanced glycation end-products (rages) is associated with the malignancy of cancer. a recent study has suggested that glyceraldehyde-derived ages (glycer-ages) enhanced the malignancy of melanoma cells, but glucose-derived ages did not. however, the effects of glycer-ages on other cancer cells remain poorly understood, and the molecular mechanisms behind the above-mentioned effect have not been clarified. the present paper aimed to examine the effect of glycer-ages on cultured lung cancer a549 cells. rage was expressed in a549 cells. glycer-ages significantly attenuated cell proliferation. furthermore, glycer-ages enhanced the migration capacity of the cells by activating rac1 via ros and also increased their invasion capacity. we demonstrated that glycer-ages enhanced the migration and invasion of a549 cells rather than their proliferation. these results suggest that glycer-ages play a critical role in the malignancy of cancer rather than its proliferation and are potential targets for therapeutic intervention.

PMC2902753

pubmed-40

according to numerous estimations, 15% to 29% of patients with cataract have 1.5 diopters (d) of refractive astigmatism.1,2 cataract surgeons usually prefer to treat cataract and correct refractive (spherical and astigmatic) disorders at the time of the surgery. corneal incision (astigmatic keratotomy) and peripheral corneal relaxing incisions (pcris) were performed to treat these disorders.3,4 the main drawbacks of these approaches are that the outcome depends on multiple factors as the patient s age, the depth and length of the incision, complications related to wound healing, epithelial defects, or induction of dry-eye symptoms. these parameters affect the visual outcome in a unpredictable way so the corneal incisions are not considered a reliable method for astigmatism correction. toric intraocular lens (iols) implantation was introduced in the 1990 s as an option for astigmatism correction in cataract patients. initially they presented the disadvantage of postoperative rotation that decreased the visual outcome.58 new toric iol designs (acrysof toric iol; alcon, fort worth, tx), approved by the us food and drug administration (fda) at the end of 2005, have been found to be more stable and appear to be the preferred iol for correcting preexisting astigmatism in conjunction with cataract surgery.9,10 the acrysof iols are available in three options: t3, t4, and t5 of astigmatic correction 1.5, 2.25, 3.00 d, respectively (at the iol plane). the aim of this study is to report the clinical results of acrysof toric iols implantation for preexisting astigmatism correction and compare the postoperative rotation of the iols one and six months postoperatively. this prospective study included eyes that had cataract surgery at the papageorgiou general hospital, thessaloniki, greece, between may 2008 and december 2008. the study was conducted according to the tenets of the declaration of helsinki and patients gave informed consent after the nature and intent of the study had been fully explained to them. inclusion criteria were: cataract, age 70 years or younger, and preoperative regular corneal astigmatism greater than 1.00 d. exclusion criteria were: glaucoma, corneal disease, previous corneal or intraocular surgery, macular degeneration or retinopathy, and history of ocular inflammation. each patient had a complete ophthalmologic examination, including visual acuity (va), slit-lamp examination, intraocular pressure (iop) measurement, and dilated fundus examination. topography (magellan mapper; nidek technologies, padova, italy), automated refractometry (rk600; reichert, inc., depew, ny), and ultrasonic immersion biometry (ocuscan rxp; alcon) were performed to determine the appropriate iol spherical power. cylindrical power and axis placement to achieve emmetropia were calculated using an online toric iol calculator program (available from http://www.acrysoforiccalculator.com/). the 0180 axis was marked with the patient in a sitting position to avoid cyclotorsion using the nuijts/lane preoperative toric reference corneal marker (ae-2791tbl; asico, westmont, il) (figure 1). intraoperatively, the desired implantation axis was marked using an intra-op toric axis marker ii (ae-2794; asico) (figure 2). a foldable iol was implanted in the capsular bag through a 2.75 mm limbal incision on 110. the toric iol was injected with a monarch-ii injector (alcon) and placed around 1015 off axis before the ophthalmic viscosurgical device (sodium hyaluronate 1%) was removed. after ophthalmic viscosurgical device removal, the iol was rotated to its final position by exactly aligning the toric reference marks with the limbal implantation axis marks. all surgeries were performed by the same experienced surgeon (it) using topical anesthesia and a standard divide-and-conquer phacoemulsification technique. because of the intraoperative marker design and the pen mark fading during the operation in several cases, it was difficult to assess the proper alignment of the iol after its placement. because of this difficulty, an image was captured from the real-time streaming recording of the surgery and was assessed blindly by a second operator using a commercially available software (screen protractor; iconico, ny, usa) (unpublished data). to overcome this difficulty we consider that the use of the marker set consisting of beveled degree gauge (ae-1590; asico) and maloney astigmatism axis marker (ae-2741; asico) is more appropriate for the whole procedure because it allows maximum visibility. the on-screen assessment of the axis remains a useful tool for postoperative evaluation of the iol location (figure 3). measurements of visual acuity (using the snellen opto-type at a six-meter distance), iop, and comprehensive slit lamp examination were performed at one-day and one- and six-month postoperative visits. at the one- and six-month follow-up, a digital photograph and corneal topography (only at the one-month visit) were obtained to estimate the postoperative rotation of the iol using the software tools mentioned above (figure 1). outcomes of interest included uncorrected va, cylindrical astigmatism power before and after iol implantation, and the possible rotation of the iol one and six months after the operation (when the initial desired place was at 0). absolute values of the rotation used for the analysis after detecting rotation was not under investigation. completed data forms were analyzed with microsoft excel 2007 (microsoft corporation, redmond, wa) and spss software (version 16.0; spss inc., chicago, il). twenty-nine eyes of 19 patients (mean age 63.03 5.42 years) were enrolled in this study. uncorrected va was found to be 5/10 or more in 26 of 29 eyes (89.7%) and 8/10 or more in 19 of 29 patients (65.5%). preoperative and postoperative corneal topography showed significant reduction of refractive astigmatism in all eyes after the surgery. mean power of the astigmatism was 2.38 0.91 d (range 1.55 d) preoperatively and 0.64 0.61 d (range 02.5 d) postoperatively (figure 4). one month postoperation, the mean toric iol axis rotation was 2.2 1.5 (range 0.67.8). six months postoperation, the mean toric iol axis rotation was 2.7 1.5 (range 0.98.4). the later rotation occurred between one and six months, and was found to be more than one degree (1.1) only in one eye (3%) and in all other cases was less than this value (figure 5). it is known that one degree of deviation causes 3.3% reduction of the iol cylindrical power. calculated reduction of the desired correction (in diopters) because of the observed rotation (mean value, one and two standard deviations) for the three iol models is shown in table 1. no eye had secondary surgery to reposition the iol axis within the six-month postoperative period. the results of our study corroborate previous studies,11 which demonstrates that proper selection and preoperative examination of patients followed by uncomplicated iol implantation of one-piece hydrophobic acrylic toric iols results in acceptable stability and visual outcome. in particular, the postoperative rotation of toric iols appears to occur in the early postoperative period (1 month) and remains constant later than six months. the lens rotates until a fibronectin and collagen adhesion develops between the iol and the posterior capsule, which prevents any further rotary motion.12 the amount of cylindrical correction reduction due to postoperative rotation was not large enough13 to affect the expected end result in final uncorrected distance va. acrysof one-piece hydrophobic acrylic toric iols implantation shows satisfactory stability, acceptable clinical results, and is an exceptional option for correction of refractive astigmatism.

purpose: to present clinical results of toric intraocular lens (iol) implantation for preexisting astigmatism correction and determine the time of any postoperative rotation. patients and methods: twenty-nine eyes of 19 patients underwent uncomplicated phacoemulsification and were implanted with an acrysof toric iol. uncorrected visual acuity, residual astigmatism, and postoperative rotation of the iol were estimated one and six months after the operation. results:uncorrected visual acuity was 0.5 in 26 of 29 eyes (89.7%) and 0.8 in 19 of 29 patients (65.5%). the mean toric iol axis rotation was 2.2 1.5 (range 0.67.8) one month postoperation and 2.7 1.5 (range 0.98.4) six months postoperation. conclusion:implantation of one-piece hydrophobic acrylic toric iols appears to have acceptable stability, which encourages visual outcome and emerges as an attractive alternative for correction of refractive astigmatism.

PMC2850825

pubmed-41

song is a complex, learned motor skill that involves the precise coordination of vocal and respiratory musculature in order to produce highly stereotyped renditions of a memorized song model. two pathways contributed to this behavior are identified in the songbird forebrain: the motor pathway that is required throughout life for normal song production, and the anterior forebrain pathway (afp) which is necessary for song learning and plasticity [13]. hvc (used as the proper name) is the beginning nucleus of both pathways and projects to the robust nucleus of the arcopallium (ra) and afp. the motor pathway is from hvc to ra, and then ra projects to brainstem nuclei controlling the vocal and respiratory muscles. during song, ra neurons in adult birds generate a highly stereotyped sequence of bursts [5, 6] and also receive input from lateral magnocellular nucleus of the anterior nidopallium (lman), the output nucleus of afp. notably, recordings from lman neurons projecting to ra revealed highly variable spiking activity across song renditions, suggesting that lman may act as a source of variability. these results indicate that synaptic activity in lman-ra synaptic transmission is crucial to song plasticity [8, 9], but the detailed mechanism is unclear. it has been observed that ra received excitatory glutamatergic inputs from hvc and lman. the former is mediated by a mixture of n-methyl-d-aspartate (nmda) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic-acid- (ampa-) type receptors [4, 10, 11], and the latter is mediated almost exclusively by nmda-type receptors. lman-ra synaptic transmission is necessary for the variability of song in adult zebra finch, suggesting that the activity of nmda receptor on the lman-ra synapses might induce the activity pattern change of ra neurons and change the bursts that control the vocal and respiratory muscles. the control of gain, which refers to modulation of a neuron's responsiveness to input, is critically important for normal sensory, cognitive, and motor functions [1215]. gain control is achieved through a divisive process and is observed as a change in the slope of the input-output relationship. such gain changes are frequently observed in the responses of cortical neurons and are thought to play an important role in neural computations. modulations of gain have been observed in the enhancement of neural responses by attention [12, 16] and can also be induced pharmacologically. in a neuron model, nmda receptors caused such membrane potential fluctuations between a hyperpolarized down-state and a depolarized up-state, which may gate the information and gain modulation. here, we test the gain change of ra projection neurons after exogenous nmda was applied to selectively activate nmda receptors, using whole-cell current clamp recording in adult male zebra finch slices. brain slices were prepared from adult male zebra finches (taeniopygia guttata) (> 90 days old) obtained from commercial suppliers as previously described [19, 20]. brains were dissected into ice-cold, oxygenated (95% o2 and 5% co2) slice solution. slice solution consists of (in mm) sucrose 248, kcl 5, nahco3 28, glucose 10, mgso47h2o 1.3, and nah2po4h2o 1.26 [21, 22]. coronal brain slices (250 m thick) containing ra were cut with a vibrating microtome (ma752, usa) and collected in artificial cerebrospinal fluid (acsf) that had been warmed to 37c and subsequently allowed to cool to room temperature. slices were allowed to recover in the holding chamber for at least 1.5 h and were equilibrated to room temperature before recordings were made. standard acsf consists of (in mm) nacl 125, nahco3 25, nah2po4h2o 1.27, kcl 2.5, mgso47h2o 1.2, cacl2 2.0, and glucose 25 and was adjusted with sucrose to a final osmolarity of 350 mos. for recording, a slice was transferred to a submerge-type chamber where it was continuously exposed to acsf, saturated with 95% o2 and 5% co2 at room temperature (2226c) at the rate of 2.0 ml/min. ra and the surrounding tissues were distinguishable under bx51wi microscope connected with a dic-ir video camera (olympus, japan). at high magnification (40x), ra neurons were visualized and the recordings were made from healthy cells. recording pipettes were fabricated from borosilicate glass (sutter instruments, usa) using a flaming-brown puller (model p-97, sutter instruments, usa) and were filled with the pipette solution containing (in mm): k meso4 120, nacl 5, hepes 10, egta 2, mg-atp 2, and na3-gtp 0.3 (ph 7.3-7.4, and 340 mos). the recording pipettes, which had resistances ranging from 3 to 5 m, were positioned using an integrated motorized control system (sutter instruments, usa). the potential changes after application of nmda were corrected during the input-output stimulation protocols. the series resistance (rs), between 15 m and 30 m, was compensated using the bridge balance. all agents were applied by changing the bath perfusate from standard acsf to modified acsf in which various drugs were simply added. unless indicated otherwise, the clampfit 9.2 (axon instruments, usa), mini 6.0 (synaptosoft inc., fort lee, nj, usa), and originpro 8.0 (originlab, nothampton, ma, usa) were used for analysis. events with peak amplitude of 50 mv or higher and a rise time of about 0.5 ms were detected automatically, and the results were analyzed with origin pro 8.0. the steady-state spike rate was estimated by counting the number of spikes in 1 second, and the result was plotted versus the intensity of the injected current (f-i relationship). the slope of the f-i relationship (referred to as gain) was estimated by linear fitting. slope parameters were estimated separately for individual neurons and that figures 4 and 5 contain mean slope values averaged for the whole groups of neurons. the amplitude of ahp was quantified as the difference between spike threshold and the lowest point of ahp. throughout means were compared using paired two-tailed student's t-test or one-way anova followed by scheffe's multiple comparison. stable whole-cell recordings were obtained in 160 ra projection neurons from 51 adult male zebra finches. a neuron with the resting membrane potential<50 mv and an overshooting action potential was considered viable. ra projection neuron was identified by a larger soma, time-dependent inward rectifier induced by hyperpolarizing current and spontaneous regular spike firing or by depolarizing current pulses. the input-output relationship was examined by injecting current pulses from 0 to 90 pa with an increment of 10 pa and an interval of 15 s, and they were lasted for 1 s. traces of action potentials in response to 1 s current steps of 10, 50, and 90 pa were shown in figure 1(a). the slopes of the f-i relationships were 246.92 28.00 hz/na before nmda application and increased to 325.49 73.34 furthermore, the input resistances were decreased by 20 m nmda in ra projection neurons (p<0.05, n=6) (figure 2). the afterhyperpolarization (ahp) of action potential underlies the modulation of firing frequency. to test the effect of nmda on spike ahp comparison of spike shape is shown in figure 3(a) with 90 pa current injection before and after nmda application. in average, there was no significant change in the amplitude of ahp after nmda application (p>0.05, n=5) (figure 3(b)). then we used 50 m dl-apv, a nmda receptor antagonist, to check the selectivity of nmda receptor, and we found that the slope of f-i was not changed by dl-apv alone (p>0.05). the slope was 255.78 66.69 hz/na and 269.34 56.96 hz/na (p>0.05) before and after application of 50 m dl-apv (figure 4), respectively. nmda increased the f-i slope 1.32 times (p<0.05); however, the gain modulation induced by nmda was completely abolished by dl-apv (figure 4). previous studies reported that ra projection neurons received glutamatergic and gabaergic inputs [23, 24], which might increase background synaptic activities and modulate the gain of neurons [25, 26]. to determine the effect of ampa receptor and gaba receptor on gain modulation by nmda, we examined the effects of nmda in the presence of cnqx, an antagonist for ionotropic glutamate ampa receptors, or/and bicuculline methiodide (bic), a competitive antagonist of gabaa receptors, respectively. bic (10 m) accelerated the depolarization by nmda application, but bic alone had little effect on the f-i slope. in the presence of bic, the mean initial slopes were 276.46 59.4 and 332.6 40.02 hz/na before and during application of nmda. the f-i slope was increased from 1.32 to 1.39 in the presence of nmda and bic (p>0.05); that is, there is an increase of 105.30% over the nmda treatment (figure 5; p>0.05, unpaired t-test). cnqx (10 m) decreased the membrane potential and abolished the spontaneous spikes of ra projection neurons sometimes but did not affect the slope of f-i relationship. in the presence of cnqx, nmda application hardly changed the f-i slope of ra projection neurons no matter whether bic was present or not (figure 5; p>0.05). these results indicated that the ampa receptor-dependent excitability played a crucial role in the gain modulation by nmda. the slope was 0.97 or 1.11 in the presence of cnqx or cnqx and bic, respectively. the gain induced by nmda was significantly decreased by cnqx (p<0.05). in this study, we showed that nmda increased the gain of ra projection neurons in adult zebra finch. nmda-induced gain increase was limited to firing frequencies less than 40 hz. the effect of nmda on gain was mediated through nmda receptors and might involve synaptic activities. in songbirds, we found that nmda increased the slope of f-i relationship in ra projection neurons, which was completely blocked by dl-apv, a nmda receptor selective antagonist, suggesting that nmda increased the sensitivity of ra projection neurons. the pharmacology of nmda receptors involved in gain modulation has been examined in mammals. in cat visual cortex, nmda increased the gain of the contrast-response (c-r) curve of the neuron in layers ii/iii and v/vi. in mouse, the thalamocortical neurons in the dorsal lateral geniculate nucleus play a key role in the generation of firing patterns through nmda receptors. the neural activity in the afp of songbirds can direct moment-by-moment changes in the primary motor areas responsible for generating song. singing-related activity in lman is more variable in pattern across repeated renditions during undirected song than directed song [28, 29]. reported that the fundamental frequency of the syllable was shifted by stimulating the lman-ra pathway, which is mediated by nmda receptors. our results indicated that the activity of nmda receptor changed the sensitivity of ra projection neuron. ra, as a premotor nucleus, occupies an important position in the song system, integrating information from both pathways. ra projection neurons are responsible for transmitting song information to midbrain and brainstem vocal and respiratory structures. temporal pattern of spike bursts in ra projection neurons is associated with the timing of acoustic features of birdsong. precise timing of individual spikes has a close relationship with stereotypic behavior, which suggests that the song is represented in ra by a temporal code [5, 31]. thus, the modulation of nmda receptors in the activities of ra projection neurons could be very important for the plasticity of birdsong. previous studies in slices demonstrated that nmda receptor achieves the nonlinear, multiplicative behavior seen in gain modulation by several mechanisms. mel presented results from compartmental modeling studies showing that dendrites containing nmda-type synaptic conductances and voltage-dependent na, k, and ca channels can impose nonlinear interactions between nearby synapses that give rise to approximate multiplications [32, 33]. in thalamocortical cell of the rat and cat dlgn, nmda receptors contribute to the burst firing generated by the low-threshold ca potential and depend on the membrane potential range over which this type of firing occurs. in substantia nigra dopamine neurons, nmda receptor-mediated synaptic conductance generates transient high-frequency activity by rapidly but transiently overwhelming the conductance's underlying action potential ahp and/or engaging postsynaptic voltage-dependent ion channels in a manner that overcomes the limiting effects of ahp. but our results showed that there was no significant change in spike ahp after nmda application. so the gain induced by nmda in ra projection neurons may not be mainly modulated by the change of spike shape, but by synaptic activities. the change in membrane potential has been shown to play an important role in the firing pattern regulation by nmda receptor, supporting the idea that depolarization acutely alters firing frequency. it has been shown that simple excitability or inhibition alone can induce a multiplicative gain change in the cortical neurons. in this model, one set of synaptic inputs provided purely excitatory drive to a target neuron, and another set of combined excitatory/inhibitory inputs (or balanced inputs) provided the modulation. in this setup, changing the firing rates of the balanced inputs produced an approximately multiplicative gain modulation of the response to the purely excitatory drive. here, blockade of the excitatory or inhibitory transmission alone had no effect on the f-i slope of ra projection neuron, implying that simple excitability or inhibition alone can not induce a multiplicative gain change. however, our results did not exclude a role of spontaneous synaptic activity on gain modulation in vitro, which, due to the loss of synapses in slice, was not sufficient to produce a significant change in gain. ampa receptors are required for the gain modulation in our study, because blocking excitatory transmission mediated by ampa receptors almost abolished the effect of nmda on gain modulation. two reasons might be involved: one is the two states of nmda-base mechanism due to the special anatomical structure of ra. ra projection neurons receive inputs from two areas, hvc and lman. both are excitatory glutamatergic but have distinct postsynaptic properties. hvc-ra is largely mediated by ampa receptor, whereas lman-ra is almost completely mediated by nmda receptor. ampa/nmda receptor composition of different neuronal pathways is the foundation of gating and gain modulation. nmda receptor-rich pathway can gate ampa-rich input and increase the gain of a neuron responding to the input of ampa-rich pathway. the second is related to the intrinsic properties of nmda receptors, which are uniquely voltage dependent. except binding with neurotransmitter blockade of main excitatory inputs mediated by ampa receptor with cnqx hyperpolarized the membrane potential, which caused nmda receptors not activated. therefore, the ampa receptor-mediated excitatory transmission is essential for the gain modulation induced by nmda. it is well known that the nmda receptor plays a critical role in many physiological and pathological processes, such as learning, memory, neurodegeneration, epilepsy, and ischemia. the nmda receptor-mediated synaptic transmission satisfies the associative property of hebbian learning and in fact plays a critical role in its cellular model, long-term synaptic plasticity. nevertheless, pharmacological agents which block nmda receptors impair a variety of brain processes, suggesting that nmda receptor transmission also plays an important role beyond long-term memory and participates in shaping the dynamic activity of neural networks. in song system, the expression pattern of nmda receptor is related to the development of song learning [21, 3740]. recent results show that the nmda receptor on the lman-ra synapses generates variability across different song renditions, thereby facilitating reinforced learning of songs. how nmda accomplishes this role is not clear. our results showed that nmda application increased the gain of ra projection neurons, in which ampa receptor was necessary. the ampa receptor-mediated excitability is derived only from hvc [10, 11]. therefore, nmda receptor in lman-ra synapse might regulate hvc-ra synaptic transmission, which implicated that nmda receptor is more effective to modulate the syllable online. these findings suggest a mechanism by which nmda receptor can selectively modulate behaviorally relevant excitatory inputs.

the song of zebra finch is stable in life after it was learned successfully. vocal plasticity is thought to be a motor exploration that can support continuous learning and optimization of performance. the activity of ra, an important pre-motor nucleus in songbird's brain, influences the song directly. this variability in adult birdsong is associated with the activity of nmda receptors in lman-ra synapses, but the detailed mechanism is unclear. the control of gain refers to modulation of a neuron's responsiveness to input and is critically important for normal sensory, cognitive, and motor functions. here, we observed the change of gain in ra projection neurons after exogenous nmda was applied to activate nmda receptors using the whole-cell current clamp recording. we found that nmda substantially increased the slope (gain) of the firing rate-current relationship in ra projection neurons. the ampa receptor-dependent excitability played a crucial role in the modulation of gain by nmda. these results suggested that nmda receptors may regulate the dynamics of ra projection neurons by input-output gain.

PMC3363989

pubmed-42

adolescents in sub-saharan africa face high exposure to human immunodeficiency virus (hiv) infection. despite all efforts to prevent the disease, prevalence estimates in sub-saharan unaids (i.e., the joint united nations programme on hiv and aids) priority countries range from 1.3 to 15.6% among young women and 0.56.5% among young men aged 1524 years (unaids, 2011). higher prevalence estimates are associated with certain regions (e.g., eastern and southern africa) and subpopulations (e.g., adolescent orphaned and/or married girls; birdthistle et al., 2008; operario, underhill, chuong,&cluver, 2011; pascoe et al., 2010). in addition, united nations children s fund (unicef) estimated there were 2 million adolescents aged 1019 years living with hiv globally in 2009 (unicef, 2011). kenya and zimbabwe were among countries with the highest numbers of infected adolescents with an estimated 136,000 (prevalence estimate 1.5 %) 1 and 104,000 (prevalence estimate 3.1 %) adolescents living with hiv, respectively (unicef, 2011). the true measure of effectiveness for such interventions is reduction in hiv and other sexually transmitted infections (stis). however, very few adolescent prevention studies have used biomarker data as outcomes (michielsen et al., 2010) because of concerns about the difficulties in collecting these data in resource poor settings (jukes, simmons,&bundy, 2008), even when power analyses suggest that moderate intervention effects could be detected. behavioral prevention scientists have instead relied on self-reported sexual behavior, which is subject to social desirability bias and likely to differ between study arms (cowan&pettifor, 2009; kirby, obasi,&laris, 2006). widespread access to testing and treatment facilitated by the president s emergency plan for aids relief (pepfar; http://www.usaid.gov/what-we-do/global-health/hiv-and-aids) have greatly enhanced opportunities to successfully incorporate biomarker procedures into adolescent hiv prevention research designs, since infected individuals identified through testing offered by studies may access free or subsidized hiv treatment and care (stringer et al., 2006; wools-kaloustian et al., 2006, 2009) moreover, including biomarker outcomes can greatly improve the credibility and legitimacy of hiv prevention research. to address the issue of feasibility, we present two case studies of prevention trials with biomarker data collection among adolescents in kenya and zimbabwe. together these studies provide a valuable opportunity to examine the ethical and practical challenges of biomarker procedures within hiv prevention intervention clinical trials in developing countries. specifically, we describe the challenges faced by investigators in the two cases in developing and implementing ethical procedures for informed consent, biomarker testing, and disclosure of test results. we also present the practical challenges of collecting biological samples in resource-limited countries and compare sample collection by blood spots relative to venipuncture. in addition, we examine difficulties with analyzing biological samples when the performance of assays in certain populations is unclear. we also provide specific suggestions for further research and development in collecting and analyzing biomarker data for adolescent prevention research. the primary goal of hiv prevention among adolescents (ages 1019 years) is to reduce new infections and related sexual risk factors [world health organization (who), 2006). although the hiv prevalence of adolescents ages 15 and older is monitored through demographic and health surveys (dhs)2 in most sub-saharan africa countries, little is known about the prevalence of hiv in adolescents under 15 years. moreover, these adolescents are rarely tested for hiv in clinical settings unless they present in poor health or with recurrent infections (ferrand et al., 2009). although a small number of recent randomized controlled trials have used biomarkers (baird, mcintosh,&ozler, 2010; cowan et al.; pronyk et al., 2006; ross et al., 2007), most adolescent hiv prevention studies have relied on changes in self-reported sexual behavior to assess intervention effectiveness (see reviews: cowan&pettifor, 2009; michielsen et al., 2010), with audio computer-assisted self-interviews intended to improve veracity (mensch, erulkar,&hewett, 2003). however, biomarker comparisons have suggested that self-reported measures have low validity (cowan et al., 2002; gavin et al., 2006; mensch, hewett, gregory,&helleringer, 2008; palen et al., 2008; plummer et al., 2004). for example, in one study, only one of 16 youth with hiv admitted to ever having sex, and four of the 16 also had either chlamydia or gonorrhea (cowan et al., 2002). inclusion of a highly prevalent and reliable biomarker of sexual activity in conjunction with self-reported sexual behavior can help to improve reliability of this outcome measure. hiv infection is not the most useful biomarker of adolescent sexual activity by itself because of the relatively low prevalence among younger adolescents and the potential for transmission through perinatal and non-sexual blood-borne routes (amornkul et al., 2009; ferrand et al., 2009; stover, walker, grassly,&marston, 2006). herpes simplex virus type 2 (hsv-2), the primary cause of genital herpes, is a commonly used biomarker of sexual activity because the presence of hsv-2 antibodies is highly associated with past sexual behavior (tobian et al., 2009; van de laar et al., 1998). in contrast to other stis such as chlamydia, gonorrhea, syphilis and trichomoniasis, infection with hsv-2 is life-long, and, once established, there is currently no treatment to eliminate it (geretti, 2006; gupta, warren,&wald, 2008). hsv-2 prevalence in the adult general population in sub-saharan africa is high, ranging from 30 to 80% in women and from 10 to 50% in men, as assessed by detection of hsv-2 antibodies [amornkul et al., 2009 ;, 2005; munjoma et al., 2010; national aids&sti control programme (nascop), republic of kenya, 2009; tobian et al., 2009; weiss, 2004]. prevalence estimates for adolescents are only available through community-based studies and are lower. one study in zimbabwe among adolescent females aged 1519 years reported that 12% of participants tested positive for hsv-2 (birdthistle et al. a study in western kenya found hsv-2 prevalence to be 9% for females and 4% for males aged 1314, and 28% for females and 17% for males aged 1519 years (amornkul et al., 2009). another study conducted in zimbabwe among adolescents whose mean age was 15 years reported a prevalence estimate of 0.2 %, suggesting a later stage of sexual debut (cowan et al. 2012) evaluated the use of hsv-2 as a biomarker of sexual debut among young people aged 1024 years. following a comprehensive review of the literature, the authors concluded that the use of hsv-2 as a biomarker for sexual debut is limited because of its low transmissibility and the fact that not all potential sexual partners are infected with the virus. building on these findings, our study describes the challenges of collecting hiv and hsv-2 biological data as biomarkers for sexual activity among sub-saharan african youth in prevention trials. we present two clinical trial case studies to examine important issues related to adolescent biomarker data collection in two clinical trials in kenya and zimbabwe (hallfors et al., both studies were school-based, cluster randomized trials testing school support (including payment of school tuition and school uniforms) as a structural hiv prevention intervention for adolescent orphans. in both studies, we collected blood samples from participants for hiv and hsv-2 testing, but they differed as follows: we collected samples by venipuncture with results disclosed to participants and their guardians in kenya, whereas in zimbabwe, we collected samples by finger pricks for dried blood spots (dbs) and results were not disclosed. the kenya sample (n=837) comprised young adolescent male (52 %) and female (48 %) orphans in grades 7 (61 %) and 8 (39 %) enrolled in school in siaya district, nyanza province. their mean age was 14.9 (sd 1.5; range 1121 years); 22% reported ever having had sex. among girls, hiv and hsv-2 prevalence was 1% (n=10) and 3% (n=28), respectively. among those with positive hiv or hsv-2 test results, 70% (n=7) and 64% (n=18), respectively, reported never having had sex. the original sample for the zimbabwe study was orphan 6th grade girls (n=328) in manicaland province primary schools; surveys were administered annually during 20072010 (hallfors et al., 2011). after securing a project renewal grant, we collected biomarker specimens and a final survey in march through june 2012, when most participants were 1617 years old. from the original sample, we located 287 (88 %) for the 2012 survey. twenty-three percent reported ever having sex; 18% had ever been married and/or pregnant. hiv and hsv-2 prevalence was 4% (n=12) and 6% (n=16), respectively. among those with positive hiv or hsv-2 test results, 50% (n=6) and 44% (n=7), respectively, reported never having had sex. although the protection of adult participants in hiv prevention research is well developed (national institute of mental health collaborative hiv/std prevention trial group, 2007), we found that clear guidance regarding hiv and other sti testing and disclosure is lacking for children and adolescents. as an example, our two clinical trials were peer-reviewed by two different nih scientific review panels; one set of reviewers thought that it was an ethical imperative to disclose hsv-2 as well as hiv results to adolescents, whereas the other was concerned that disclosure of biomarker results might lead to stigma and abuse. these divergent opinions led to discrepant protocols for disclosure, with one trial disclosing and the other not disclosing test results to participants. we used the national hiv testing and counseling (htc) guidelines [central statistical office (cso)&macro international inc., 2007; nascop, republic of kenya, 2008] for hiv in both countries. given the lack of national guidelines for adolescent sti testing in kenya [ministry of health (moh), republic of kenya, 2006), we developed a protocol in conjunction with the siaya district aids and std control office that closely followed hiv guidelines. study protocols were reviewed and approved by irbs in the us, kenya and zimbabwe. the kenya study was approved by the ministry of education and siaya district education office; school head teachers identified orphans and organized meetings in which moi university research collaborators and a district education officer explained the study to students and their caregivers. caregivers and students we invited a total of 923 orphans in grades 7 and 8 in 2011 to participate; 849 (92 %) consented and 837 (91 %) completed both the student survey and biomarker testing. following national guidelines, we required kenya study participants under age 18 to have an adult guardian accompany them for hiv rapid testing (nascop, republic of kenya, 2008). in some instances, however, the guardian was not available because of work, other commitments, or illness. in such cases we permitted a relative, teacher or neighbor to stand-in for the guardian if they had a note from the guardian or if research staff were able to speak with the guardian by phone. given the sensitive nature of hiv testing, the senior counselor interviewed all unclear or undocumented cases to determine whether or not to proceed with specimen collection and htc. we visited clinics several times to give participants the opportunity to return for testing with their guardian or authorized proxy. although not required, all participants ages 18 and older were accompanied by an adult for support in case of a positive hiv test result. in accordance with national guidelines, we gave referrals to publicly funded hiv care and treatment centers to all adolescents with positive hiv test results, while hiv negative participants were counseled on risk reduction (nascop, republic of kenya, 2008). we disclosed results to both guardians and participants if the latter were under age 18 and to the participants alone if they were 18 years old according to the study protocol. in zimbabwe, with permission from the provincial education officer, the consortium principal investigator and a research associate held meetings at study schools to explain the continuing study to parents/guardians, participants, and key school staff. we obtained written parent/guardian consent and participant assent for continued voluntary participation in the study, including separate checkbox consent for survey and blood sample collection. guardians raised few concerns, although they did ask why we were not disclosing test results. participants who were married or 18 years or older provided their own consent. in deference to local cultural norms and to prevent potential spousal abuse, we prepared a second consent form for husbands to provide consent for their wives participation. most of the wives gave their own consent for the survey and did not ask their husbands for consent. in four cases, the husband signed a consent form and in five additional cases, the husband refused and the wife did not take the survey. although asking husbands for permission for their wives study participation is not consistent with us culture, it appears to be a useful protocol accommodation for some young wives in rural zimbabwe. we consistently relied on local researchers and key informants to make such culturally sensitive decisions. after 4 months, we found 88% of the original sample who consented to the survey; 85% consented to blood specimen collection. of those not surveyed, two participants had died in childbirth, 2% refused, and approximately 10% of the sample could not be found. of those not consenting to blood specimen collection (n=8), six were married and refused for religious reasons or husband s objection. two were consented for the survey by school officials who refused to provide additional consent for the blood collection, and no guardian was available to give permission. hiv testing is technically far more advanced for widespread use in sub-saharan africa than hsv-2 testing. rapid hiv antibody tests are now widely used and accepted (moh, republic of kenya, 2006), and sequential algorithms for retesting have been adopted in kenya and zimbabwe for rapid testing (cso&macro international inc., 2007; nascop, republic of kenya, 2008; who, 2004). rapid hiv tests meet high performance standards, sample collection procedures are simple, tests are easy to perform, and interpretation of test results is reasonably clear. in contrast, hsv-2 serologic testing presents some important challenges. although a number of hsv-2 type-specific serologic tests are available, only a limited number have been tested among sub-saharan african populations. these include the herpeselect hsv-2 enzyme-linked immunosorbent assay (elisa; focus diagnostics, cypress, ca) and kalon hsv-2 elisa (kalon biological ltd., guildford, uk; biraro, mayaud, morrow, grosskurth,&weiss, 2011). these tests require skilled laboratory staff, specialized equipment, and a constant source of electricity. they are appropriate for use with large batches of samples as in sentinel surveys and are inexpensive, costing about $3 per test. although the literature reports variable performance of these tests with respect to sensitivity and specificity in sub-saharan populations, kalon is often found to be superior to focus herpeselect (see biraro et al., 2011 for a review of the performance of hsv-2 tests in sub-saharan africa; 2008; ngayo, friedrich, holmes, bukusi,&morrow, 2010; smith et al., 2009; van dyck et al., 2004). when using the manufacturer s instructions (index cutoff value=1.1 for both tests), kalon tends to have lower sensitivity but higher specificity than focus herpeselect (delany-moretlwe et al., 2010; gamiel et al., 2008; ngayo et al., 2010; smith et al., 2009; van dyck et al., the low specificity of focus herpeselect is particularly problematic when disclosing results to research participants because of an unacceptably high rate of false positives when prevalence is low (gamiel et al., 2008; mark et al., 2008; ngayo et al, 2010; van dyck et al., 2004). increasing the assay cut-off values is one strategy for improving the performance of both the kalon and focus herpeselect tests. a number of studies have shown that increasing the cut-off value for focus herpeselect to 3.5 increases specificity, although sensitivity is necessarily reduced (delany-moretlwe et al., 2010; smith et al., other studies similarly improved performance at cut-off values of 3.2 and 3.3 (delany-moretlwe et al., 2010; gamiel et al., 2008; ngayo et al., although findings are mixed, increasing the cut-off value for the kalon test to 1.5 has also been shown to increase specificity, with an attendant reduction in sensitivity (gamiel et al., 2008; ngayo et al. in our studies, the kenya laboratory used the kalon assay, and the zimbabwe laboratory used the focus test. at the time of our studies, neither assay had been validated for dbs by the manufacturer in african populations. the efficiency of dbs for hsv-2 testing with each assay must be evaluated in specific populations prior to use (hogrefe, ernst,&su, 2002). because the kalon test had not been validated for hsv-2 testing with dbs samples, specimens in the kenya study were obtained by venipuncture. the zimbabwe laboratory had validated the focus test with dbs (mudzori&mutsogoro, 2006). in both studies, we used a modified algorithm to interpret results: samples with initial index values<0.9 were reported as negative and no further testing was conducted. samples with initial index values>2.5 were defined as high positive and were reported as positive without additional testing. samples with initial index values from 1.5 to 2.5 were considered low positive, and those with initial index values between 0.9 and 1.5 were considered equivocal; all low positives and equivocals were retested in duplicate. if both retest results had index values>1.5, the final result was reported as positive. if both retest results were<0.9, the final result was negative. if retest results were equivocal (0.91.5) or discordant, the final result was indeterminate. although test performance was improved by increasing the cut-off and expanding the range of results for repeat testing, the performance characteristics of tests to detect antibodies to hsv-2 are imperfect. based on a recent systematic review of hsv-2 antibody test performance in sub-saharan africa (biraro et al., 2011), sensitivity for the kalon test used in kenya was estimated at 94% and specificity at 92% with the higher cut-off value of 1.5. the predictive values of all diagnostic tests are strongly influenced by the prevalence of infection in the population, and in low prevalence situations, even a very good test will have a poor positive predictive value (ppv). using the sensitivity and specificity values for the kalon test under the testing conditions employed in our kenya study, the ppv was estimated at 26% with a 3% hsv-2 prevalence. that is, we can be confident that about one in four participants with positive test results truly had hsv-2 antibodies present in their blood sample; however, as many as three in four could have had a false positive test result. in kenya, we conducted sample collection and htc primarily in local public health facilities near the schools. eighteen facilities participated. in two sites, with permission from the school headmasters and district education officer, we collected blood samples and conducted htc at the school. venipuncture was conducted by six skilled phlebotomists trained in htc. to avoid obtaining two separate blood specimens (finger prick for rapid hiv testing and venipuncture for hsv-2 testing) the process, including counseling, blood draw, hiv testing, disclosure of results took on average 20 min. the process was longer for participants with discordant results who needed a tie-breaker test and for participants with hiv positive results. we offered all participants and their guardians a snack either before or after specimen collection. we stored blood samples for hsv-2 testing in labeled vacutainer tubes in a cooler box without ice and transported them to a local laboratory within 24 h for serum processing. sera were stored at the local laboratory in a freezer at 20 c until transport on ice weekly to the hsv-2 testing laboratory. the logistics of specimen collection, processing and transport for this study were challenging but feasible. in comparison, we did not require guardians to be present because hiv results were not provided to participants. sampling and dbs preparation were much less obvious to others nearby, helping to safeguard participant privacy and reduce concerns about witchcraft or other conspiracy theories regarding hiv/aids and blood, which circulate in sub-saharan african communities (tenkorang, gyimah, maticka-tyndale,&adjei, 2011). blood from the finger prick was dropped on five circles on a dbs filter card labeled with the participant s study i d. some participants may have been dehydrated or not very well nourished, making it difficult to fill all circles, but adequate samples were collected from all participants. specimens were dried, stored with a desiccant, and transported to the testing lab at ambient temperatures. dbs samples are easy to store and transport and require no processing or refrigeration (see table 1 for a summary of the study procedures). table 1comparison of biomarker testing procedures in two research studieskenyazimbabweethical considerationswritten guardian consent if<18/adolescent assentwritten guardian consent/adolescent assentresults disclosed to guardian and adolescentno results disclosedreferrals for carenot applicablecollecting and analyzing biological samples methodvenipuncturedried blood spot hiv testrapid testrapid test hsv testkalon elisafocus elisafeasibility of collection methods time20 min3 min temperature for specimen transportcooler with no iceambienttesting costsreference labreference lab bench fee, supplies, storage, labor us$ 5,784bench fee, supplies, labor us$ 8,000field site lab sample processing, labor, supplies, storage us$ 3,611additional fee us$ 480 other supplies us$ 100 transportation us$ 375 total us$ 9,919total us$ 8,480 elisa enzyme-linked immunosorbent assay comparison of biomarker testing procedures in two research studies elisa enzyme-linked immunosorbent assay given the experience we have described, we conclude that it is feasible to obtain hiv and hsv-2 biomarker data for adolescent hiv prevention intervention studies. in particular the us pepfar program has accomplished considerable staff training in sub-saharan africa; it has also stimulated the development of well-equipped laboratories, as well as useful tools for testing and protocols for improving test sensitivity, specificity, and cost (stringer et al., 2006). in addition, it has stimulated the development of national guidelines (particularly for adults) for the disclosure of results. for hiv incorporation of hsv-2 serology results poses considerable challenges, particularly if results are disclosed to participants. based on our experience, there are two reasons why we do not recommend disclosing hsv-2 serology results to adolescent human subjects in sub-saharan africa. first, in populations with low or moderate prevalence of infection, as might be expected for adolescents, the potential for false positive test results is substantial in combination with relatively minor imperfections in test specificity. hsv-2 infection is often a silent disease, insofar as only 1025% of people with antibodies for the condition are aware that they have genital herpes (fleming et al., 1997; leone, fleming, gilsenan, li,&justus, 2004; sizemore, lakeman, whitley, hughes,&hook, 2006). adolescents without symptoms may deny ever having had sexual intercourse3, further complicating the clinical picture and raising concerns about false positives with the potential for psychological and social harm related to participation in the study. second, local departments of health and sti authorities in sub-saharan african countries were much less familiar with hsv-2 pathology and treatment than with hiv and some other stis. even in cases where subjects acknowledge sexual exposure or genital herpes symptoms, local health clinics may not be prepared to provide appropriate treatment or care. given these vexing problems, the wisdom of disclosing hsv-2 results to adolescents after testing within a clinical prevention trial is questionable. in particular, decisions about whether to inform adolescent participants in prevention trials about hsv-2 test results should be made only after prevalence data are available and the predictive values of the test can be accurately assessed. in light of the feedback from the nih study section that raised concerns about possible stigma and abuse of hiv-positive participants in zimbabwe, as well as our own concerns, we recommend that more research be conducted to study the consequences of disclosure to adolescents in sub-saharan africa. a review of the literature indicates that sub-saharan adolescent prevention studies with biomarker outcomes have taken a variety of approaches to disclosure. in some studies, counselors disclosed participants hiv results immediately after rapid testing (baird, garfein, mcintosh,&ozler, 2012; dalal et al., 2012; medley et al., 2012). in other studies, participants could choose to receive their results after testing, either immediately or a few weeks later (amornkul et al., 2009; jewkes et al., 2006; ross et al., 2007). in still other studies, the research did not provide test results but voluntary counseling and testing (vct) was made freely available for study participants (birdthistle et al. none of these studies, however, examined whether adolescents experienced any untoward outcomes with testing and disclosure, whether hiv positive youth enrolled in care or took steps to prevent transmission, whether hiv negative youth benefited from prevention counseling and engaged in subsequent testing, or if adolescents and their guardians felt it was appropriate to learn their status in the context of a research study. from our kenya study, the problem of stigma did not appear to be a major obstacle to adolescent testing and disclosure in our study area. perhaps this is because hiv prevalence is among the highest in the country, and few families have been unaffected by hiv/aids. moreover, high hiv prevalence has drawn research, education, and hiv counseling and testing campaigns to the area. for example, in our kenya clinical trial, more than 50% of participants (all in grade 7 and 8) reported that they had previously been tested for hiv, mostly through a home-based testing initiative of the kenya medical research institute (kemri) and us centers for disease control and prevention (cdc). although our study provided hiv test results to youth, we relied on phlebotomist/nurse counselors to refer youth and their guardians for care, following national guidelines. the onus of making contact with the clinic was left up to the individual and was not tracked by the system of care; and follow up to make sure that hiv positive youth accessed care was well beyond the scope of our study. since it is unknown whether youth experience significant mental trauma and social prejudice, and whether they actually engage in the system of care, we recommend that further research be conducted to examine the perceptions and behaviors of youth and particularly newly diagnosed youth after disclosure of their test results to ascertain the consequences of this aspect of research participation and whether it can be improved. this is particularly important for adolescents, since results are disclosed in the presence of their guardian. regarding procedures for collecting biomarkers, we conclude that finger-sticks and dbs are overwhelmingly superior to venipuncture in sample collection efficiency and reduced burden on participants and community institutions. manufacturer validations of dbs for hsv-2 are urgently needed, however, for this procedure to be more widely accessible to researchers. because blood collection for dbs is minimally invasive, this procedure has excellent potential for widespread acceptability and consequent high participation in school and community research sites. in addition, rapid hiv testing in sub-saharan africa is typically conducted by finger prick, making this the preferred way for trained african health workers and counselors to participate in research data collection (who, 2004). given the complex decisions required for biomarkers, we recommend that behavioral prevention scientists interested in using biomarker data collaborate from the early planning stages with biomedical scientists who have expert knowledge in hsv-2, as well as hiv, laboratory tests and testing procedures. we found that team members with such expertise provide valuable assistance in selecting qualified in-country laboratories, developing appropriate budgets, protocols, quality assurance and algorithms for interpreting results, and helping research staff to bridge communication with in-country laboratory staff. further, we recommend that hsv-2 test kit manufacturers determine optimal cut-off standards for sub-saharan populations, and that researchers who conduct hsv-2 testing explicitly define the testing cutoffs they use in publications. there is now a substantial literature documenting validity problems when using manufacturers cutoffs with african populations potentially inflating prevalence findings (gamiel et al., 2008; ngayo et al., 2010; van dyck et al., 2004 this suggests the need for action to improve assay validation practices on the part of manufacturers as well as methodological details from researchers documenting their findings. when conducting collaborative international research, it is important to recognize that alternative ethical systems exist. thus, for international research we recommend inclusion of team members who are knowledgeable about local contexts. indeed, successful collaborative research partnerships respect local cultures, values and practices; negotiate effectively within these systems; and incorporate into study designs ethical practices that are appropriate and sensitive to local cultural contexts (e.g., in our case, providing for husband consent for married participants; christakis, 1992; emanuel, wendler, killen,&grady, 2004). behavioral prevention scientists traditionally have relied on self-reported sexual behavior survey item measures to evaluate adolescent hiv prevention interventions. our experiences conducting research with orphan adolescents in kenya and zimbabwe suggest that collection of hiv and sti test results even in rural, resource-poor settings in sub-saharan africa is a feasible addition to the behavioral research toolkit. the use of sti biomarkers can greatly improve the validity of findings from adolescent behavioral intervention trials. research is urgently needed to examine the risks and benefits of hiv testing and disclosure of test results in the context of a research study for adolescents. further development and implementation of sti biomarker assessment techniques particularly pertaining to using dbs is needed to advance hiv prevention science.

self-report of sexual behavior among adolescents is notoriously inconsistent, yet such measures are commonly used as outcomes for human immunodeficiency virus (hiv) prevention intervention trials. there has been a growing interest in the use of hiv and other sexually transmitted disease biomarkers as more valid measures of intervention impact in high hiv prevalence areas, particularly in sub-saharan africa. we examine the challenges, benefits, and feasibility of including hiv and herpes simplex virus type 2 (hsv-2) biomarker data, with details about different data collection and disclosure methods from two adolescent prevention trials in kenya and zimbabwe. in kenya, whole blood samples were collected using venipuncture; adult guardians were present during biomarker procedures and test results were disclosed to participants and their guardians. in contrast, in zimbabwe, samples were collected using finger pricks for dried blood spots (dbs); guardians were not present during biomarker procedures, and results were not disclosed to participants and/or their guardians. in both countries, prevalence in the study samples was low. although the standard of care for testing for hiv and other sexually transmitted infections includes disclosure in the presence of a guardian for adolescents under age 18, we conclude that more research about the risks and benefits of disclosure to adolescents in the context of a clinical trial is needed. notably, current serological diagnosis for hsv-2 has a low positive predictive value when prevalence is low, resulting in an unacceptable proportion of false positives and serious concerns about disclosing test results to adolescents within a trial. we also conclude that the dbs approach is more convenient and efficient than venipuncture for field research, although both approaches are feasible. manufacturer validation studies using dbs for hsv-2, however, are needed for widespread use.

PMC3996329

pubmed-43

nanobiomaterials are characterized by constituent particles and/or surface features less than 100 nm in at least one dimension. starting with photolithography and dry etching in the 1980 's to high-resolution electron beam lithography and other technologies in the 1990 's, nanotechnology allows for making surface structures for cell engineering and has led to an increasing application in healthcare over the last decades. nanolayers are used to enhance the surface biocompatibility of polymeric drug delivery systems, control the release of substances such as antibiotics or growth factors, act as gene-delivery vehicles, or serve as robust light emitters for cellular labeling and tracking [semiconductor nanocrystals, quantum dots (qds)]. nanotechnology is also applied to modify and improve the surface structure in orthopaedic implants to promote their osseous integration. however, there are also side effects of nano- and microparticles in vivo. micro- and nanoparticles released by friction of articulating partners from artificial joints are a major reason for aseptic implant loosening in orthopaedic surgery and may lead to severe peri-implant osteolysis (particle disease). in addition, nanoparticles can induce or promote allergic or inflammatory reactions or influence hemolysis and blood coagulation [57]. although the cytocompatibility of a biomaterial is strongly influenced by its chemical composition, surface topography plays a crucial role for cell-surface interactions. material surface properties have been studied intensively, but still lack from reliable data about cytocompatibility. especially, the superordinate principles of cellular responses to surfaces with a defined topography are not well known and poorly understood. because many variables influence cellular interactions to surface structures, it is difficult to draw conclusions and formulate general principles for nano- and microstructured surfaces. this review summarizes recent data of effects by nano- and microstructured biomaterials and particles in vitro designed for orthopaedic application to get a solid framework outlining the critical interactions that govern the cytocompatibility. because biomaterials in orthopaedics are predominantly applied on bone, this review is focussed on the interactions of osteoblasts and bone-marrow-derived cells with structured biomaterials. osteoblasts and osteoclasts are mainly responsible for the osteointegration of nanostructured biomaterials in orthopaedics. osteoblasts derive from mesenchymal progenitor cells which are localized mainly in the bone marrow and periosteum. they are characterized by cuboidal and flat morphology (diameter about 20 m), present a large amount of rough endoplasmatic reticlum and a large golgi apparatus, and are potent to produce osteoid, a collagen i rich matrix. in addition, these mononuclear cells are also responsible for osteoid calcification (hydroxyapatite). typical marker proteins for osteoblasts are cbfa1/runx2, osteocalcin, osteopontin, osteonectin, bone sialoprotein (bsp), osteoprotegerin (opg), collagen i, and alkaline phosphates (alp). figure 1 gives a brief summary of the expression of several markers during osteoblast differentiation. osteocytes act in a paracrine and mechanosensory manner, and can activate osetoblasts and osteoclasts. the latter cell type derived from the hematopoietic line, has multiple nuclei and is responsible for bone resorption. its ruffled border is flanked by a sealing zone which facilitates local acidification and removal of bony matrix such as ca, h3po4, and h2co3 by endocytosis. osteoclasts express high levels of tartrate-resistant acid phosphatase (trap) and cathepsin k. the interaction between osteoblasts and osteoclasts is complex. during differentiation, the ostoblast progenitors express receptor activator of nuclear factor ligand (rankl) and macrophage colony-stimulating factor (m-csf) which are strong stimuli for osteoclastogenesis. in contrast, osteoprotegerin (opg) is a potent inhibitor of osteoclasts. moreover, the interactions between osteoblasts and osteoclasts in vivo are regulated by several hormones and cytokines, including parathyroid hormone (pth), calcitonin, and il-6. it is generally accepted that the three-dimensional surface topography (size, shape, surface texture) is one of the most important parameters that influence cellular reactions [2, 1119]. although many studies have investigated cellular reaction to different surface pattern, the significance of macro structure studies on bone cell behavior is questionable since in vivo adhesion structures (e.g., cell membranes, basement membranes) are comprised of much smaller nanometer scale features [20, 21]. the immature bone is characterized by an average inorganic grain size of 1050 nm whereas mature bone has an average inorganic grain size of 2050 nm (25 nm in diameter). considering these parameters, modern implants for bone application have been designed with a smooth surface at the nanometer level. it was surprising that some of these have induced the formation of peri-implant fibrous tissue and implant loosening in vivo, while other implants with a higher degree of roughness showed significant better osteoconductive properties [2325]. there are various methods to modify the degree of roughness as well as surface energy and topography in orthopaedic implants. typically applied techniques to enhance the degree of roughness and promote the osteointegrative properties of biometals (e.g., ti, cocrmo, ss) are chemical etching or anodization and also sand-blasting, sputter-coating, and machine-tooling. the lack of knowledge in cellular reaction to nanostructered biomaterials is based to a great extent on the difficulty in varying surface chemistry and topography independently. moreover, the use of different cell lineages and culture conditions makes it difficult to compare results from different investigators [2631] (table 1). there is also a lack of consensus concerning the proper representation of implant surface topography. one major misunderstanding is the practice of defining a surface by its manufacturing process instead of concisely defining the topographic measurements [17, 33]. considering these limitations for interpretation, the following review gives an overview of cellular reactions to surface structures of different orthopaedic biomaterials. the first step after exposure of any biomaterial to a biological environment results in the rapid adsorption of proteins to its surface. the composition, type, amount, and conformation of adsorbed proteins regulate the secondary phenomena such as cellular adherence and protein exchange [3537] and also following cellular reactions such as migration, proliferation, and differentiation. the potency for biomaterials to adsorb proteins is influenced by its physiochemical characteristics such as surface energy or hydrophobicity, and is also dependent on the local environment (ph, concentration of ions, composition and functional groups of proteins, strength of solution, temperature) (vroman effect) (figures 2, and 3). for inorganic nanocrystals and microstructured surfaces there are at least two approaches to change their hydrophobic surfaces: a ligand exchange reaction can replace the original hydrophobic surface with bifunctional coupling molecules or an inorganic coating such as silica (1) or an encapsulation of nanocrystals in an amphiphile organic coating (2). the first phase of protein adsorption onto a biomaterial's surface is characterized by the attachment of small rapidly diffusing proteins, followed by a progressive replacement by larger proteins with a high affinity to the substrate. here, especially proteins with arg-gly-asp (rgd) containing sequences such as fibronectin or vitronectin act as cell receptors and have chemotactic or adhesive properties to bone cells. in addition, these rgd-peptides also have a strong effect on matrix maturation and biomineralization [4648]. after conditioning of a naked biomaterial by protein adsorption, cells attach rapidly on the protein-coated surface. besides the influence of proteins, the cellular attachement to a nanostructed surface is also influenced by its physiochemical properties, especially by the outer functional groups [30, 50, 51]. schweikl et al. showed on self-assembly monolayers that the osteoblast proliferation on hydrocarbon chains, terminated by ch3, was as high as on amino groups (nh2) and hydrophilic oxidized surfaces, but significantly lower on fluorocarbon (cf3) groups. mller et al. showed that 3-aminopropyl triethoxysilane (apts) presents amine functional groups which allow for grafting rgd tripeptides and that the rgd-apts hybrid promotes cell adhesion, spreading, and cytoskeletal organization. here, the zetal potential (differences in potentials between the surface of a tightly bounded layer and a diffuse layer) and the interfacial tension (wettability) of a surface is crucial [54, 55]. it was demonstrated for cpti surfaces that the contact angle (ca), parameter for wettability, increases linearly with the average roughness when the angles were higher than 45, but decreases linearly with roughness when the angle was less than 45. recent data examining osteoblast response to controlled surface chemistries indicate that hydrophilic surfaces (high number of polar components) improve cell attachment and matrix synthesis and also the osteogenic potency compared to hydrophobic surfaces [5759]. compared ti alloys and cocr alloys towards protein absorptive properties and cell attachment with an osteoblast precursor cell line. they found no significant differences between ti alloys and cocr, but significantly greater cell adhesion rates for the ti implants and concluded that cell adhesion is a result of higher hydrophilicity of ti alloys. in contrast, other data showed that a low degree of wettability promotes protein adhesion and also cellular attachment to a biomaterial, and mller et al. found no direct correlation between the wettability of the material surface and the osteoblast attachment and proliferation rate. also qu et al. found no significant differences of cell attachement on various titanium surfaces with different degrees of wettabilities (hydrophobic acid-etched, coarse-blasted large grit acid-etched, hydrophilic modified acid-etched, and modified coarse-blasted large grit, acid-etched) on mg68 cells. heating (oxygen/atm) or peroxide treatment of biometals result in a thicker oxide layer and a more hydrophilic surface.. showed that heat-treated titanium surfaces changed the wettability (more hydrophilic) but does not significantly affect the fibronectin and albumin adsorption as well as the initial osteoblast precursor cell attachment in vitro. emphasized that the rate of protein correlates more with changes in chemical composition than with changes in wettability in metal surfaces. they showed that a preheating of ti6al4v specimen does not only lead to a thicker oxide layer but also results in an enrichment of v and al within the surface oxide. in contrast, post-treatment with butanol after preheating reduces the content of v, but not in al, and significantly increases the rate of fibronectin adsorption up to 2040%. compared to the cellular attachment phase, the following adhesion phase lasts longer and involves various proteins and molecules (figure 2). as a link between cell and biomaterial, the interactions of a surface topography and serum proteins are crucial for the cytocompatibility of a biomaterial. especially, the adsorption of adhesion proteins, such as fibronectin and vitronectin, from serum containing solutions and integrin-mediated signaling has been demonstrated to mediate cell adhesion and spreading. it has been shown that nanotube or nanoparticle surfaces created by anodization have promoted osteoblast adhesion up to three times compared to unanodized ti. these results were confirmed by the group of webster and other investigators [6871] who demonstrated that the initial attachment of osteoblasts onto the surface of biometals such as cpti, ti6al4v, and cocrmo is enhanced by submicron to nanometer consistent particles compared to metals composed of respective micron particles. one possible explanation of this phenomenon is the higher amount of particle binding sites for osteoblast adhesion at the surfaces of nanophase metals compared to micron particle size metals. the theory of enhanced protein and cell binding capacities by larger surface areas/roughness degrees was also confirmed for porous ha materials. another example of the significance of surface structures for protein binding and osteoblast attachment is the helical rosette nanotubes (hrn) which can build self-assembly surface structures. it was demonstrated that a significant change of hrn coverage by heating correlated with the protein-binding and osteoblast adhesion potency in titanium surfaces [73, 74]. it is evident that not only the surface topography influences protein deposition and cell adherence but also proteins and cells modify the surface properties of a defined surface. based on a surface analysis of the different biometal specimen before and after cell cultivation, we showed previously that a cell attachment and/or protein precipitation increase the roughness in polished biomaterials (steel, ti6al4v, and cocr). for porous coated cocr surfaces, we found only slight and no relevant changes in roughness whereas cell cultivation onto sandblasted ti6al4v lead to a strong decrease in specimen roughness. both, the increase in roughness after cell culturing in the different biometals and the decrease in roughness of sandblasted ti6al4v could be explained by the dense cellular growth and accumulation of debris in depth of the structured surfaces and/or protein deposition as shown by other investigators [75, 76]. in addition, not only the amount but also the type of protein adsorption by a surface is crucial for cellular adherence and following reactions such as migration and differentiation. as an example, ti surfaces (ra: 0.370.01 m) adsorp fibronectin in higher concentrations compared to albumin, and fibronectin-coated ti surface promoted more osteoblast attachments in comparison to albumin-coated ti surfaces. these results correspond to the data of other authors who showed excellent osteoconductive properties after fibronectin adsorption onto a biomaterials ' surface [7880]. based on irm and tem analysis, the closest distance of cells to a surface (glass) was found to be approximately 10 nm [81, 82]. historically, results from chicken fibroblasts have lead to a classification of three different types of separation. (1) focal contacts (fc): approximately 1015 nm separation from the substrate under the peripheral regions of the leading lamellae (appearing black in tem). fc act as an interface between intra and extracellular components and occur linearly beneath the associated cytoplasmic stress fibres [83, 84]. they are tenacious adhesion sites that remain attached to the substratum even when cells are forcibly detached, indicating their function as anchorage structures. (2) close contacts: corresponding to approximately 30 nm separation (broader grey areas in tem). (3) greater separation: corresponding to approximately 100140 nm (white regions in tem). it is evident that not only fc appear soon after cellular attachment but also that (-catenin-positive) adherence junctions occur within 14 hours for grooved ti-based substrates. these observations underline the high significance of an early intercellular communication soon after adherence to a surface. the mechanisms of initial cellular adherence to a surface are different from long-term adherence as shown by a lack of statistical correlation between short-term adhesion (strength of cell attachment and early adhesion) and long-term adhesion (strength of cell-matrix interface) forces [14, 15, 86].. showed that the cultivation time has an influence on the long-term adhesion in biometal surfaces according to td (t)=atb, a being independent of b (td: time-dependend adhesion index, a: surface-dependent parameter, b: substrat-independent exponent, 0.5+/-0.03). for polylactides (plla), it was shown on oct-1 osteoblast-like cells that cell adhesion but not the proliferation could be enhanced by nanoscale and microscale roughness compared to smooth surfaces. in addition, there is evidence that fc show a dynamic behavior which allows for cellular migration and motility. linear plla fibres with length scales of 0.52 m, constructed by electrospinning, have shown cellular contact guidance and enhanced osteoblastic differentiation. here, cell morphology revealed that cells grown on fibres had smaller projected areas than those on planar surfaces. also other polymers such as plga have been shown to be effective in enhancing osteoblast differentiation in vitro. diener et al. demonstrated on mg-63 osteoblastic cells that fc adhesion was smaller on ti and ss than on collagen-coated glass coverslips and that all fc showed a mobility of focal adhesions. however, anselme et al. found higher adhesions on ti6al4v substrates than on noncollagen-covered glass samples, and emphasized that substrates with various surface compositions but with the same surface topography did not induce significant differences of adhesion. based on the knowledge of protein adsorption and its effects on cellular attachment and adherence, a selective surface coating of nanostructured surfaces with rgd or collagen proteins offer a promising solution to improve the number of osteoblasts adhered on artificial surfaces [53, 95102]. imprinting surfaces technology with deposition of specific protein-recognition sites can help to promote osteoblastic growth and differentiation [103106]. protein-recognition can be based on a protein-ligand binding and/or electron donor-acceptor interactions or other types of binding forces. integrin 51 and 5 v3 subunits competitively bind to rgd-sites of fibronectin [107, 108]. dependent on the surface topography and chemistry of the biomaterial, fibronectin undergoes changes in structure including modulation in functional activity and shift in integrin binding capacity. based on the data of self-assembled monolayers, it was shown that integrin subunits show selective binding capacities to different terminal groups. integrin 51 shows a strong affinity to oh and nh2 surfaces, whereas 51 and 5v3 bind also to cooh but show poor binding capacities on ch3 surfaces [109113]. furthermore, some data show that oh and nh2 surfaces can up-regulate osteoblast-specific gene expression but also matrix mineralization compared with cooh and ch3 functional groups [47, 112]. cell migration and proliferation is the attachment following phase between the cell and the material surface. it is evident for designing nanostructured implants that cells use the nanotopography of a substrate for orientation and migration [117119]. although it is known that bone cells align along defined substrate morphologies (contact guidance), the detailed relation between ordered nanotopography and cell behavior remains unknown in detail. for the first time, in 1964 it was shown that convex surfaces enhance cellular overlap, while grooves minimize cellular overlap. as pre-requisite to reach a defined cell colonization during directed tissue formation, structured nanophase surfaces lead to a predictable osteoblast orientation and migration on these surfaces [17, 121, 122]. interaction between the ecm and associated changes in the orientation of the cytoskeleton are crucial for cell metabolism of cells and morphology due to actin-myosin tension structures. anisotropic topographies (e.g., topographical grooves, chemically patterned stripes, or curved surfaces of a fibre) are potent to exert morphological as well as physiochemical features on cells at the same time, indicative for the complex environmental influence on cells. focal contacts are important structures for cellular adherence onto a surface but may also delay migration and mobility of the cells. it was shown that bone-derived cells (mg63 cells) respond to a nanoscale roughness by a higher cell thickness and a delayed appearance of focal contacts. especially, nanoporous ti-oxide surfaces promote cellular spreading and induce numerous filopods and osteoblastic differentiation [124, 125]. on electrochemically microstructured hexagonal pattern, mg63-cells go inside 30100 m but not in 10 m cavities. most authors report a parallel orientation of cells cultured on polished (smooth) surfaces [57, 114, 126] (figure 4). another method to not only enhance cellular adherence but also to promote osteoblastic differentiation and biomineralization of biometals is a surface anodization, for example, by -glycerophosphate sodium and calcium acetate [6671]. cellular adhesion via fc may strengthen the linkage between cell and ecm and also impair the ability to dynamically remodelling the ecm and influence the migration rate. for collagen-coated coverslips, focal adhesion of mg-63 osteoblastic cells moved with a speed of 60 nm/min, whereas the speed was reduced in ti and more in ss surfaces. another study on nb2o5-coated polished cpti samples showed that mc3t3-e1-osteoblast migration was fastest on smooth surfaces (ra=7 nm), whereas adhesion strength, spreading area, and collagen-i synthesis were promoted by intermediate roughness (ra=15 nm). however, it was surprising that higher degrees of roughness (ra=40 nm) were rather peaked and reduced the speed of adhesion process in the same study. besides the surface properties of a biomaterial, the cellular migration rate is dependent on the cell type and its differentiation stage. a higher migration rate is associated with a lower level of osteoblast differentiation. cells with a low motility are characterized by a strong formation of fc while motile cells form less adhesive structures. it was found that mature osteoblasts spread out and form a greater number of fc when settled on smoother surfaces. although cellular spreading is higher on smoother surfaces, some data indicate that the alp-expression is higher for rough isotropic surfaces (electro-erosion, acid-etching, sandblasted) compared to smoother substrates (machine tooling, polishing). considering recent publications, there is no or only week statistical significance that there is a difference between the initial number of adherent cells and following proliferation of cells cultured onto a biometal or ceramic nano-/microscale surface in vitro. however, some authors emphasize that the influence of functional chemical groups for cellular migration and proliferation are stronger than general surface properties such as wettability. especially a tio2-layer seems to promote cellular growth and proliferation on nanostructured biometals [128, 129]. other examples for a promotion of cell-to-bone contact in vitro and also in vivo are machine-etched ti-surfaces (e.g., osteotite), defined sand-blasted implants [124, 125, 131], and hydroxyapatite (ha) coatings, for example, by plama-spray techniques [132134]. recent studies investigating the response of adherent cells to nanography surfaces indicate that different cell phenotypes have different levels of sensitivities [117, 135137]. here, osteoblasts react to features as low as to the 10 nm dimensions, which is comparable in size to a single collagen fibre. moreover, the qualitative and quantitative kinetics in gene and protein expression is strongly influenced by topography and physiochemistry of a defined surface. microporous ha surfaces seem to promote a high number of fc and increased levels of alp but short actin stress fibres compared to nonmicroporous ha surfaces [72, 139]. there is also evidence that ti and ha surfaces can activate early intracellular signalling pathways as shown by expression of relevant molecules such as -and 1-integrin, fak, erk followed by c-jun and c-fos genes for proliferation and alp for differentiation [139, 140]. however, hallgren et al. found no significant histomorphometric and biomechanical differences between nanopatterned and control implants.. showed that microfabricated discontinuous-edge surfaces (des), repeated open square boxes with a depth of 10 m, alter osteoblast adherence and migration but enhance cell multilayering, matrix deposition and mineralization when compared to smooth controls. in contrast to our data, anselme et al. found higher proliferation rates on ss compared to ti6al4v. however, bigerelle et al. demonstrated that neither material composition nor surface roughness amplitude influence cell proliferation, whereas they found a very significant influence on manufacturing process and surface topography for long-term adherence and proliferation in vitro. our in vitro results confirm the well known osteogenic in vivo properties of ti implants, which may be based on surface factors observed on its outer tio2-layer [143146]. mller et al. demonstrated the ability of osteoblasts to grow into an open-porous ti implant (metal foam) and li et al. also demonstrated that mc3t3-e1 cells attach to and are able to divide well in the inner surface of a highly porous trabecular ti6al4v implant. some in vitro studies demonstrated an enhanced total protein and collagen production, as well as increased alp activity of osteoblasts cultured on nanoparticulate metals (cpti, ti6al4v, and cocrmo) indicating advantages for nanostructured surfaces for osteointegration [1, 149, 150]. based on the data of redey et al., it can be concluded that the low attachment and collagen production rates are related to a low wettability of a nanosurface. nanotextured surfaces of ti surfaces prepared by chemical etching have upregulated the expression of bsp and op. as demonstrated by qu et al., the expression of the bone-associated genes such as alp, oc, type-i-collagen, osteoprotegerin, and glyceraldehyde-3-phosphate-dehydrogenase is promoted by modsla ti surfaces. some data also suggest that fluoride-modified ti surfaces can stimulate osteoblastic differentiation compared to unmodified titanium surfaces [151, 152].. showed in their in vitro experiments that nanophase biometals induce significantly greater calcium and phosphorus deposition by osteoblasts and also allow for calcium and phosphorous precipitation from culture media without osteoblasts in contrast to microphase ti6al4v and cocrmo. furthermore, the authors found advantages in mineral precipitation without osteoblast for tial4v but no differences in dependency to the type of ti (wrought, microphase, or nanophase). it was evident that the increased calcium and phosphorus mineral content correlated to greater amounts of underlying aluminium content on ti6al4v surfaces. although some data indicate that nanostructured ti alloys promote non-cell-mediated ca/po4-mineral deposition from culture media compared to cocrmo substrates, the greatest cell-dependend calcium and phosphorus mineral deposition occurred on nanophase cocrmo. it is evident that micropattern collagen films or scaffolds promote not only cellular adhesion but also allow for an osteoblastic differentiation and biocalcification in vitro [153155]. for ha- and dcpp-coated, ti surfaces the ca/p ratio influence the biomineralization rate in vitro. besides the osteoblast-promoting effects of defined substrates and surface topographies, some data also allocate an inflammatory response induced by nano- or microstructured biomaterials. it was shown in many studies that cell-biomaterial interactions can activate macrophages which results in the synthesis of proinflammatory agents such as tnf, ifn, il-1 and -6, rankl and no [157159]. some data have shown proinflammatory effects of different biomaterials which increase with the degree of surface roughness. here, macrophage inflammatory protein-1, tnf, monocyte chemoattractant protein-1, and members of the interleukine and leukotriene family play a crucial role in biometal-induced inflammations [160164]. most studies report about an enhanced expression of pro-inflammatory cytokines and chemokines by cells attached to rougher surfaces. some data also indicate that anionic and neutral hydrophilic surfaces increase macrophage-monocyte apoptosis and reduce macrophage fusion to modulate inflammatory responses to implanted materials. however, adverse cellular effects seen with metallic implants may also be attributed to corrosion products or to the separation of metal ions (fe, cr, ni) which may have a major impact on cellular survival and differentiation [166168]. those studies which suggest that a cell-mediated metal ion release by biometals that did not affect the cell viability or proliferation are characterized by short cultivation periods or other conditions which limit the reliability of data [169171]. up to date, only few authors report about no significant influence of the cellular adherence and expression of osteoblast proteins by different biometals and surfaces such as alp expression [172, 173]. in contrast to the great opportunity enhancing biocompatibility and osteogenic potency of surfaces applied on bone by nanotechnology, micro- and nanoscaled particles released by friction of artificial joints can induce severe inflammation and may lead to osteolysis and implant failure [174, 175] (figure 5, table 2). there is a wide range in particles size and morphology produced by simulators for artificial joints. particles released from metal-metal (crcomo alloys) are predominantly chromium oxide particles or cocrmo with varying ratios of co and cr. they show a round to oval morphology and also a substantial number of needle-shaped particles were found during the first circles. emphasize the importance of particle size as a critical factor in osteoblasts proliferation and viability in vitro. some data indicate that in contrast to ti-surfaces nano- and mircoparticles induce an inflammatory response although titanium is one of the biometals with the highest degree of cytocompatibility. as shown by miyanishi et al., the release of vegf may play a crucial role in the pathogenesis of ti-induced osteolysis. some data indicate that phagocytosis of ti particles is not a precondition for an inflammatory response such as a release of tnf or il-6 in cultured macrophages. it is evident that a binding of the macrophage cd11b/cd18 (macrophage mac-1 receptors/receptor of complement cr3bi, can also bind to icam-1 and icam-2) by integrin-specific antibodies also increased the release of tnf and il-6 in macrophages. this finding also suggests that the complement system plays a role in the pathogenesis of particle-induced inflammation, too. especially, uhmwpe particles with a size range of 0.11.0 m have been shown to be most reactive for macrophage activation and cytokine secretion in bone marrow cells [179, 180]. however, not only the particle size but also the particle volume (number) is a critical factor for particle-mediated release of cytokines by macrophages. green et al. demonstrated for pe that the cell-particle ratios of 1: 100 (size 0.497.2 m) and 1: 10 (size: 0.494.2 m) induced significant stronger release of tnf and il-1 in macrophages. the authors conclude that especially particles in the phagocytosable size range of 0.310 m appear to be the most biologically active ones. the latter statement was also confirmed for silicon carbide (sic) particles and biometals such as cpti, ti6al4v and uhmwpe [184, 185]. granchi et al. investigated the in vitro effects of al2o3 and uhmwpe particles in an osteoblast-osteoclast co-culture system. both particles did not affect either cell viability or tnf and gm-csf release, whereas il6 release was dependent on the particle concentration. uhmwpe particles increased the release of rankl from osteoblasts and induced large amounts of multinucleated trap-positive giant cells in an osteoblast-osteoclast co-culture system. also, carbon-based particles with low wear factors such as p25-cvd showed a high degree of cytocompatibility in vitro. howling et al. demonstrated on fibroblasts and monocytes that p25-cvd particles<100 nm were significantly less cytotoxic to both cell types than cocr metal wear particles. while the classical water-suspendable nanoc60 nanocrystal is apparently cytotoxic to various cell lines, the closely related fully hydroxylated, c60(oh)24, is nontoxic, thus producing no cellular response. also, functionalized single-walled carbon nanotubes are nontoxic to cells in culture [198200]. there is evidence that not only particle size and chemical content but also the concentration strongly influence cellular reactions in vitro. wilke et al. showed a positive correlation between the release of proinflammatory cytokines (il-6, -1, and tnf) and amounts of ti6al4v-particles (10, 10, 10, and 10 particles/ml) by human bone marrow cells over 2 weeks. some in vitro data also indicate that ti particles induce a stronger fibroblastic differentiation signal than uhmwpe in monocytes and other cells [182184].. showed that particles of high-density polyethylene (hdp) and ti6al4v induced significantly more proinflammatory mediators (il-1, il-6, tnf) and bone resorption compared to al2o3 and zro2 in vivo. based on these data, it can be assumed that ceramics show a high degree of cytocompatibiltiy. for ha especially, particles with a size <53 m inhibit cellular proliferation, especially in osteoblasts and lead to a decrease in tgf1 and a significant increase in pge2 and ldh concentration, but did not influence the tnf or alp titer in vitro. it could be concluded that larger ha particles may be compatible with bone cells while smaller-sized ha particles can both activate the osteoclasts and decrease the cell population of the osteoblasts in vitro. numerous variables influence the biocompatibility and osteogenic potency of nanostructured biomaterials in vitro and in vivo. besides the locotypical environment in vivo or in vitro, the surface structure and the composition of a biomaterial affects cellular attachment, adherence, proliferation and migration, and also differentiation and survival of defined cell types. here, information about typical parameters such as chemical composition, surface structure (topography, geometry, roughness, particle size), surface energy, hydrophobicity, and the degree of solubility in aqueous solutions of a biomaterial will help to value and grade a defined implant concerning its osteblast promoting potency. considering recent publications, we could assume some general principles of cytocompatiblity and cell-surface interactions in nano- and microstructured surfaces. (1) wettability of a nanosurface influences significantly protein adsorption, which is a prerequisite of cellular adherence in serum containing solutions. (2) nanostructured surfaces enhance the surface area of biomaterials and promote cellular adherence. (3) the chemical outer functional groups of a nanosurface significantly influence cellular migration, proliferation, and differentiation but direct correlations between distinct parameters and cell functions are not entirely cleared. (4) the formation of fc underly a dynamic process and influence the motility and migration of cells. (5) a higher degree of differentiation is corresponding to a decreased cellular motility. (6) phagocytable particles with a size <10 m induce the strongest cellular response with regard to releasing inflammatory cytokines. (7) although ti has a high degree of cytocompatibility in vitro, phagocytable ti particles can induce a fibroblastic differentiation.

cell-surface interactions play a crucial role for biomaterial application in orthopaedics. it is evident that not only the chemical composition of solid substances influence cellular adherence, migration, proliferation and differentiation but also the surface topography of a biomaterial. the progressive application of nanostructured surfaces in medicine has gained increasing interest to improve the cytocompatibility and osteointegration of orthopaedic implants. therefore, the understanding of cell-surface interactions is of major interest for these substances. in this review, we elucidate the principle mechanisms of nano- and microscale cell-surface interactions in vitro for different cell types onto typical orthopaedic biomaterials such as titanium (ti), cobalt-chrome-molybdenum (cocrmo) alloys, stainless steel (ss), as well as synthetic polymers (uhmwpe, xlpe, peek, plla). in addition, effects of nano- and microscaled particles and their significance in orthopaedics were reviewed. the significance for the cytocompatibility of nanobiomaterials is discussed critically.

PMC2233875

pubmed-44

the success of valproic acid (vpa, 2-propylpentanoic acid), a short branched chain fatty acid, for the therapy of absence seizures, partial seizures, tonic-clonic seizures, bipolar disorder, schizoaffective disorder, social phobias, neuropathic pain and for the prophylaxis and treatment of migraine headaches has spurred investigation for its use in the treatment of other conditions. treatments for encephalopathy secondary to hyperammonemia as a side-effect of vpa treatment remain empirical and restoration of possible hepatic carnitine deficiency remains controversial. while some authors have found a positive correlation between plasma ammonia level and vpa dose or concentration others have found none. her elevated vpa levels were associated with elevated ammonia levels as expected of a positive correlation. in contrast, later on in the patient's clinical course her raised venous ammonia levels were found to be slowly down-trending in the presence of sub-therapeutic serum vpa. the present case report is about a 25-year-old hispanic female with a history of depression since age of 12 years, bipolar disorder, borderline personality disorder, purging type bulimia and history of suicide attempts at age 12 was admitted to the intensive care unit after attempted suicide by consuming an unknown number of vpa pills. this patient had recently started taking vpa (depakote) as a mood stabilizer at a dose of 250 mg at bed time about 3 days earlier, after a failed therapy. patient was taking paroxetine 20 mg for depression; this was incremented to 40 mg at the time vpa was started. she reported that the change to vpa made her listless, lowered her mood, decreased her motivation, worsened her depression and caused suicidal thoughts. she overdoses herself on vpa which she took with alcohol. after ingesting the pills she notified her family who then brought her to the emergency room. on admission, her vitals were stable (blood pressure: 118/72 mm hg; temperature: 98.7f; pulse: 82 beats/min; oxygen saturation: 99% on room air). on initial examination she looked disheveled, although drowsy, was oriented to person, place and time. the complete blood count and comprehensive metabolic panel that was recorded initially and subsequently were unremarkable [table 1]. subsequently, she was administered oral lactulose once and then 1400 mg of levocarnitine orally every 6 h for the next 2 days. her vpa levels which peaked after admission continued to trend down over the next 4 days, the ammonia levels also peaked subsequent to admission then showed an initial dip followed by a very slow down-trend [figure 1a]. the normalized log-linear plot of vpa serum concentration over time showed a down-trending linear first-order kinetics with a calculated t=15.08 h [figure 1b] (expected t for depakote: 9-16 h following oral dosing regimens of 250-1000 mg. the serum ammonia normalized log-linear plot in contrast showed two different decay kinetics: initial sharp down-trend for vpa levels greater than 90.9 g/ml but much slower downtrend for vpa levels less than 90.9 g/ml down into the subtherapeutic range of 8.5 g/ml. patient continued to complain of lethargy for the next 4 days which subsequently alleviated. at 14 days laboratory values of the patient at the time of admission (a) valproic acid (vpa) and ammonia levels as a function of time (days). vertical axes: straight line (left): vpa levels; dashed line (right): venous ammonia levels. (b) logarithm of normalized vpa and ammonia levels as a function of time in days. the normalization for each compound, for levels at each recorded time point (x) was to its recorded maximum level (xmax). the normalized log-linear plot of vpa showed linear first-order decay kinetics with a calculated t=15.08 h (r=0.994) recognition of symptoms of hyperammonemia remains a challenge especially in psychiatric patients. in a review of 11 case reports of symptomatic hyperammonemia in a psychiatric setting it was noted that acute hyperammonemic encephalopathy in psychiatric patients may present in various ways, sometimes with subtle clinical features. the previously reported spectrum of clinical features included: improvement in mood, worsening of psychiatric symptoms, mild complaints of fatigue and delirium. in our patient, hyperammonemia secondary to vpa overdose caused listlessness, lowering of mood, decreased motivation, worsening depression and suicidal ideation. we observed presence of vpa in serum caused increased venous ammonia levels, which eventually trended down to normal after its cessation. increasing vpa levels in serum was associated with increasing venous ammonia levels initially [figure 1]. the peak concentration of ammonia noted was likely a decrement from the real peak based on the time to peak of depakote tablets being 4 h (the patients initial vpa levels were measured at 2 h after she allegedly consumed the vpa while the next blood draw was 9 h later). we also made the following observations: (1) raised venous ammonia levels may remain symptomatic for a significant period of time beyond discontinuation of vpa. (2) existence of more than one concurrent mechanism with different decay kinetics in the metabolism of ammonia in presence of high or low concentrations of serum vpa. unmasking of an inherent urea cycle metabolic disorder could have amplified the differential decay rates of venous ammonia. the possibility of more than one mechanism causing hyperammonia secondary to vpa use is suggested by the different proposed mechanisms of vpa metabolites causing hyperammonemia, these include: (1) propionic acid inhibiting the urea cycle enzyme carbamoyl phosphate synthetase i; (2) valproyl-coa or a closely related compound as a proximate inhibitor of mitochondrial ureagenesis; (3) valproyl-carnitine ester causing a relative carnitine deficiency and (4) 2,4-dien-vpa inhibiting b-oxidation. although, we can not speculate on the mechanisms responsible for the different kinetics of decay of ammonia levels in the presence of down-trending vpa levels we can confidently state that non-elevated hepatic enzymes and hyperammonemia despite carnitine replacement indicated that hepatic damage and carnitine deficiency had very little role in causing the slowly down-trending elevated ammonia levels in the presence of subtherapeutic serum vpa levels. hyperammonemia and psychiatric disturbance secondary to valproate use in our case showed a score of 9 by a naranjo algorithm, indicating strong causality with the drug and placing it in causality category of definite. our assessment of a high adverse reaction probability scale was based on the fact that there are previous conclusive reports of hyperammonemia secondary to vpa use with similar psychiatric features as presented in this case report, hyperammonemia occurred only after vpa use and eventually trended down to normal levels with discontinuation of vpa, patients psychiatric disturbance correlated with hyperammonemia, the patient symptomatically improved with drug discontinuation, there are no alternative explanation of hyperammonemia and altered mood in this case, the serum levels of vpa were elevated with concentrations known to be toxic and that toxic effects observed were more severe with high dose and less severe when secondary hyperammonemia decreased. this case report suggests that hyperammonemia, which is known to occur in about 50% patients treated with vpa, may have more than one concurrent etiologic mechanism with different decay kinetics which are not related to hepatic damage or carnitine deficiency.

valproic acid (vpa) has successfully been used in the therapy of a number of conditions including absence seizures, partial seizures, tonic-clonic seizures, bipolar disorder, schizoaffective disorder, social phobias, neuropathic pain and migraine headaches. there is a high rise in number of cases of toxicity due to overdose of vpa. hyperammonemia, a common side-effect of vpa, is caused by several proposed etiologies, reported as having uncertain correlation with vpa dose or concentration. we present here a case of a 25-year-old female patient with a past history of psychiatric complaints, presented with elevated serum vpa levels associated with elevated venous ammonia levels subsequent to vpa overdose. later in the presence of sub-therapeutic serum vpa levels her venous ammonia levels remained raised and slowly down-trending. vpa levels and ammonia levels were found to be normal after 14 days. patient was treated with levocarnitine. her liver enzymes were never elevated. different decay kinetics of venous ammonia in presence of high and low concentrations of vpa indicates that vpa can cause symptomatic hyperammonemia via more than one concurrent etiological mechanism. in this patient, the mechanisms causing hyperammonia secondary to vpa use were not related to hepatic damage or carnitine deficiency.

PMC4071718

pubmed-45

intramammary infection (mastitis) is the most common reason for the use of antimicrobials in dairy cows. antimicrobials have been used to treat mastitis for more than fifty years, but consensus about the most efficient, safe, and economical treatment is still lacking. the concept of evidence-based medicine has been introduced to veterinary medicine and should apply also to treatment of mastitis. the impact on public health the aim of this article is to review current treatments of mastitis during lactation and seek for evidence-based, best practice treatment recommendations for bovine mastitis. penetration of substances into milk when administered parenterally or absorption and distribution throughout the udder when infused intramammarily (imm) depends on their pharmacokinetic characteristics. these are lipid solubility, degree of ionization, extent of binding to serum and udder proteins, and the type of vehicle. antimicrobial treatment of dairy cows creates residues into milk, and residue avoidance is an important aspect of mastitis treatment. the activity of macrolides, tetracyclines and trimethoprim-sulphonamides has been shown to be reduced in milk. selecting a substance with a low minimum inhibitory concentraton (mic) value for the target pathogen the antimicrobial should have bactericidal rather than bacteriostatic action, because phagocytosis is impaired in the mammary gland. however, activity in vitro does not guarantee efficacy in vivo when treating bovine mastitis. antimicrobial resistance amongst mastitis pathogens has not yet emerged as a clinically relevant issue, but geographical regions may differ in this respect. the biggest problem is the widespread resistance of staphylococci, particularly staphylococcus aureus, to penicillin g. cure rates for mastitis caused by penicillin-resistant strains of s. aureus seem to be inferior to those of mastitis due to penicillin-susceptible strains. it is not known if this is due to pharmacologic problems of the drugs used, or virulence factors possibly linked to -lactamase gene of the resistant isolates. using an in vitro -lactamase test for determining resistance to penicillin g of staphylococci before treatment coagulase-negative staphylococci tend to be more resistant than s. aureus and easily develop multiresistance. mastitis causing streptococci have remained susceptible to penicillin g, but emerging resistance to macrolides and lincosamides has been detected. antimicrobial susceptibility of coliform bacteria varies but normally is not a limiting factor for therapy. an important question regarding the treatment of mastitis is whether the antimicrobial should accumulate in the milk or in the udder tissue. the target site may depend on the causative agent: streptococci are known to remain in the milk compartment, but s. aureus penetrates udder tissue and causes deep infection (table 1). the advantages of this route are high concentrations of the substance achieved in the milk and low consumption of the antimicrobial as the drug is directly infused into the diseased quarter. for example, concentration of penicillin g in milk after imm administration is 100-1000 times as high as the concentration after systemic (parenteral) administration. a disadvantage of the imm administration is uneven distribution throughout the udder and the risk of infecting the quarter when infusing the product via the teat canal. efficacy of imm treatment varies according to the causative pathogen, with the best therapeutic response being shown for mastitis caused by streptococci, coagulase-negative staphylococci, and corynebacterium spp .. where to target antimicrobial therapy in clinical mastitis due to different pathogens the systemic route of administration has been suggested to be more efficient than imm for the treatment of clinical mastitis as antimicrobials theoretically have better penetration of the udder tissue by this route. however, it is difficult to attain and maintain therapeutic concentrations in milk or udder tissue following systemic administration. many commonly used broad-spectrum antimicrobials such as oxytetracycline, trimethoprim-sulphonamide and ceftiofur, it is difficult to produce and maintain therapeutic concentrations in the milk. macrolides would have ideal pharmacokinetics, but clinical studies have failed to demonstrate efficacy when used for the systemic treatment of clinical mastitis. in streptococcal mastitis, spiramycin and tylosin have shown reasonable efficacy. one additional problem for the bovine practitioner is that the recommended dosage for many antibiotic preparations for adult cattle may be too low when pharmacological aspects are considered, but residue studies have been carried out using the approved dosages. repeated intramuscular injections of large volumes of antibiotics can be irritating and can not be recommended from the animal welfare point of view. one substance used for systemic treatment is penicillin g, which as a weak acid penetrates poorly into the mammary gland, however, due to the very low mic values of susceptible organisms, therapeutic concentrations can be achieved in milk. penethamate is a more liphophilic penicillin g formulation and diffuses better than penicillin g procaine into milk. the efficacy of systemic treatment with penicillin g or penethamate has been shown in clinical trials. combinations of penicillin and aminoglycosides should not be used, as there is no scientific evidence demonstrating a better efficacy for the combination and aminoglycosides are known to produce long-lasting residues. the only type of mastitis where systemic treatment would be clearly advantageous may be mastitis caused by s. aureus. in severe mastitis due to coliform bacteria, parenteral administration of antimicrobials has been suggested to combat bacteraemia. the general benefit of antimicrobial treatment in coliform mastitis has been questioned, but systemic antimicrobial treatment is recommended in cases of severe escherichia coli mastitis with heavy bacterial growth in the udder. fluroquinolones and cefquinome have shown efficacy in experimental trials and ceftiofur in a clinical field trial. there is no evidence that administering bactericidal antimicrobials to cows with severe coliform mastitis causes the release of massive amounts of endotoxin. finally, the antimicrobial used for systemic treatment of mastitis must be approved for dairy cattle. for example, penicillin g procaine or fluoroquinolones are not approved for dairy cattle in the united states. treatment of mastitis should be targeted towards the causative bacteria whenever possible, but in acute situations, treatment is initiated based on herd data and personal experience. rapid or on-farm bacteriological diagnosis would facilitate the selection of the most appropriate antimicrobial. treatment protocols and drug selection for each farm should be made by veterinarians familiar with the farm. the use of on-farm written protocols for mastitis treatment can promote judicious use of antimicrobials. therapeutic response of the cows can be monitored using individual somatic cell count data if available, or using the california mastitis test, and with bacteriological samples in herds with contagious mastitis. in general, the use of narrow-spectrum antimicrobials is preferable (table 2). first choice antimicrobials for treating mastitis caused by streptococci and penicillin-susceptible staphylococci are -lactam antimicrobials, particularly penicillin g. broad-spectrum antimicrobials such as third or fourth generation cephalosporins should not be used as first alternatives for mastitis, as they may increase emergence of broad-spectrum -lactam resistance. systemic treatment is recommended in clinical mastitis due to s. aureus and in severe cases of coliform mastitis, preferably in combination with imm treatment. too short a duration of standard treatment is probably an important reason for poor cure rates in mastitis therapy. a longer treatment improves cure rates, and duration of treatment should generally be extended in mastitis caused by s. aureus and streptococcus uberis. clinical mastitis should be treated for at least three days; this recommended treatment duration is longer than label treatments in many countries. all mastitis treatment should be evidence based i.e., the efficacy of each product and treatment length should be demonstrated by scientific studies. treating subclinical mastitis with antimicrobials is generally not economical during lactation because of high treatment costs and poor efficacy. in a study with a large number of subclinical mastitis cases, the overall bacteriological cure rate for antimicrobial treatment was 75% and that for no treatment 68%. the marginal benefit applied for streptococcal mastitis only; in mastitis due to s. aureus, antimicrobials were equal to no treatment. treatment of subclinical mastitis will not affect the incidence of mastitis in the herd unless other preventive measures are taken. studies on treating cows based on high somatic cell counts have generally shown that no effect on milk production has been achieved in herd problems caused by very contagious bacteria such as s. aureus or streptococcus agalactiae treatment of subclinical mastitis is advised .

treatment of mastitis should be based on bacteriological diagnosis and take national and international guidelines on prudent use of antimicrobials into account. in acute mastitis, where bacteriological diagnosis is not available, treatment should be initiated based on herd data and personal experience. rapid bacteriological diagnosis would facilitate the proper selection of the antimicrobial. treating subclinical mastitis with antimicrobials during lactation is seldom economical, because of high treatment costs and generally poor efficacy. all mastitis treatment should be evidence-based, i.e., the efficacy of each product and treatment length should be demonstrated by scientific studies. use of on-farm written protocols for mastitis treatment promotes a judicious use of antimicrobials and reduces the use of antimicrobials.

PMC3339349

pubmed-46

the gram-negative coccobacillus pasteurella multocida is normally found in the oral cavity of dogs and cats. it is a recognised cause of wound infection as a result of bites, licking or scratching. bacteraemia and endocarditis are uncommon complications and, from literature reviews, are found to be associated with prior animal trauma, a second source of infection (e.g. cellulitis) and high mortality rates. we herein report a case in which a pet owner developed p. multocida endocarditis and was subsequently treated via opat (outpatient parenteral antimicrobial therapy) service with an excellent clinical outcome. species identification of p. multocida was performed using traditional microbiological methods and confirmed with 16s rrna sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof). a 38-year-old man was admitted to our hospital with a 2 days history of fever, malaise and cellulitis around a mitrofanoff abdominal catheter. multiple comorbidities included marfan syndrome (with metallic aortic valve inserted sixteen years before) and congenital urological abnormalities that led to the mitrofanoff appendicovesicostomy and a recent renal transplant, five years and six months before admission respectively. he was on steroids and tacrolimus as immunosuppressive therapy. on presentation, the patient was febrile, hypotensive and tachycardic, had a normal leukocyte count (5.0910/l) but raised creactive protein (crp) 45 mg/l with mild anaemia (hb of 10.1 g/dl from a baseline of 14.6 g/dl) and renal impairment (creatinine 163 umol/l and urea 8.1 mmol/l). the cellulitis around the mitrofanoff abdominal catheter was noted and a swab was taken for culture. his chest x-ray did not show any relevant consolidation and abdominal ultrasound noted two small abdominal collections that had already been noted post-transplant and had since decreased in size. the patient was documented to be penicillin allergic (previous skin rash), so was initiated on ertapenem iv 1 gr/od. the cellulitis improved and the patient was clinically well within 48 hours of commencing antibiotics. the blood culture set (aerobic and anaerobic bottles) taken on admission was positive with p. multocida but the abdominal swab grew only mixed anaerobes. urine culture was negative and all subsequent blood cultures taken after starting ertapenem were negative after 5 days of incubation. transesophageal (tee) echocardiography revealed the presence of a vegetation (0.57 0.2 cm) above the atrium of the right coronary artery. a previous transthoracic echocardiogram (tte), around one month prior to admission, was reported as negative. therefore, the patient was treated following our local protocol for infective endocarditis: he was switched to ceftriaxone iv 2 gr/od, a picc line was inserted in the right arm and he was discharged under the opat (outpatient parenteral antimicrobial therapy) service to complete six weeks iv antibiotics as an outpatient. on follow-up, two repeat ttes showed clear aortic valve. we reviewed all the previous reported cases of p. multocida endocarditis in the literature through pubmed and cross-references search. to our knowledge this is the twenty-first reported case of endocarditis but the first to be reported in a penicillin allergic patient. as with previous cases, the diagnosis of infective endocarditis was made according to duke criteria: one major criterion (evidence of endocardial involvement) and three minor criteria (predisposing heart condition, fever and microbiological evidence with positive blood culture). laboratory isolation and identification of p. multocida was performed using standard microbiological techniques: api-20ne, api-20e (biomerieux) and the phoenix automated system (bd). interestingly, both api profiles reported a low confidence value for identification as p. multocida to the species level (cv 53%) and the phoenix assay only identified the isolate to genus level as pasteurella spp. thus, confirmation of species identification was performed using 16s rrna sequencing and maldi-tof protein profiling: 16s rrna sequence was performed using pcr parameters detailed in maskell na et al. (2006) and we obtained a 584bp fragment sharing 99% sequence homology to that of p. multocida sub species septica. maldi-tof profiling was performed using a bruker daltonics microflex platform with recommended -cyano-4-hydroxycinnamic acid matrix under pre-defined conditions as specified by bruker. the maldi-tof mass spectra for the clinical isolate was analysed against maldi-tof mass spectra for 3,438 species of bacteria located in the bruker daltonics flexanalysis 3.0 database. the clinical isolate was identified as p. multocida with a confidence value of 2.321 indicating highly probable species identification, with no differential bacterial species being reported. sensitivities were as follow (eucast breakpoints): penicillin mic (mg/l) was 0.19, ciprofloxacin 0.016, doxycycline 0.5, co-trimoxazole 0.032, ertapenem 0.016 and ceftriaxone<0.016, all being susceptible. of note the accuracy of previous reports in the literature has been questioned as their standard microbiological description was incomplete and pasteurella species may be misidentified as haemophilus spp. they both belong to the pasteurellaceae family and based on the genomic sequence it appears they diverged approximately 270 million years ago. are gram-negative coccobacilli generally present in the oral cavity of cats and dogs. to date twenty-one species have been identified and the species p. multocida can be further divided into three different subspecies (subspecies multocida, septicum and gallicida). the spectrum of infections varies from skin and soft tissue to central nervous system infections. ten out of twenty-one (47.6%) endocarditis cases described in the literature had close contact with animals detailed in the clinical history. our patient did not have a clear history of bites or scratches but he did have two cats at home and close contact with dogs at his sister's house. in fact, of the cases of p. multocida endocarditis reviewed, two out of twenty-one (9.5%) had cirrhosis as underlying condition. penicillin still remains the best antimicrobial agent for the treatment of virtually all forms of pasteurella infection. a fluoroquinolone, doxycycline, or co-trimoxazole should be considered for skin and soft tissue infections as an alternative for patients with intolerance to -lactams. there are no clear guidelines about the treatment of p. multocida endocarditis in penicillin-allergic patients. the patient was known to be penicillin allergic (previous rash) and ciprofloxacin and doxycycline were contraindicated as causing interaction with his anticoagulant treatment. we decided then to switch to ceftriaxone as this antibiotic is already vastly used in the treatment of endocarditis caused by other gram-negative organisms and is more cost effective. aminoglycosides have moderate to poor in vitro activity against pasteurella and probably should not be used, particularly given the paucity of clinical experience. finally, our patient improved within 48 hours of commencing antimicrobial therapy and surgical intervention was unnecessary. from the literature, only three other cases have been successfully treated with solely antibiotics and valve replacement was mandatory in seventeen out of twenty-one (81%) of cases described. clinicians may face various challenges, from achieving the correct species identification to chosing treatment regimens in penicillin-allergic patients. in our case new diagnostic techniques such as 16s rrna sequencing and maldi-tof were demonstrated to be invaluable tools for species confirmation. we propose that maldi-tof is a rapid, accurate method for the identification of p. multocida from bacterial cultures and can significantly reduce the time to diagnosis of p. multocida bacteraemia. furthermore we have shown that cephalosporins and carbapenems are probably good substitutes for penicillin in case of allergy.

pasteurella multocida is a rare cause of infective endocarditis with only a few cases described. this report involves a 38-year-old penicillin-allergic patient in an immunocompromised state with several co-morbidities. two molecular microbiological techniques, 16s rrna sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used to confirm the species identification as p. multocida. previous reports in the literature are also reviewed.

PMC3892627

pubmed-47

genital infection due to chlamydia trachomatis is one of the most prevalent bacterial sexually transmitted infections (stis). according to the who estimates, globally 92 million new cases of c. trachomatis infection occur each year and about two-thirds of these cases occur in the developing world, where diagnostic and treatment services are scarce [2, 3]. most epidemiological data on chlamydia trachomatis infection (cti) is from industrialized nations and reliable data from the resource poor developing nations is not available where the disease burden is concentrated. however, it is important to document laboratory-confirmed incidence and prevalence of cti from the developing world as well. the available indian data show a wide variation in ct prevalence with infection rates in indian women ranging from 3.3% to 33% depending on the population sampled [413]. infection with this agent is usually asymptomatic in up to 80% of women which makes diagnosis and detection all the more difficult. left undetected and untreated the infection may evolve into pelvic inflammatory disease and may result in serious sequelae, such as ectopic pregnancy and infertility [14, 15]. cti in women has also been linked to adverse pregnancy outcomes like recurrent miscarriage and preterm labor and may cause conjunctivitis, nasopharyngitis, and pneumonia in newborns by vertical transmission. because the infection is easily treatable with antibiotics, early detection and treatment of infected individuals are the key to prevent adverse sequelae among those infected and reduce c. trachomatis transmission. thus, it is important to screen adolescents and sexually active women for cti even if they are asymptomatic [17, 18]. but, in the developing countries with the exception of sporadic testing, screening for chlamydia is rarely done. epidemiological studies have also shown that untreated genital chlamydia infection can lead to an increased risk for heterosexual acquisition of hiv. hence, screening for cti done in high risk populations can assist in designing hiv risk reduction strategies. on the other hand, immunosuppression due to hiv may lead to more aggressive chlamydia disease conditions like pid in hiv seropositive women. thus, screening for cti in hiv seropositive women is highly recommended to prevent morbidity associated with the disease and devastating clinical consequences. different diagnostic modalities for detection of cti like serology, culture method, elisa for antigen and antibody, direct fluorescence assay (dfa) and nucleic acid amplification tests (naats) have been used in the last 20 years but none of them are 100% sensitive. table 1 shows the prevalence of cti detected by using different diagnostic techniques in new delhi population. polymerase chain reaction is an accurate, rapid, and reliable method for the detection of chlamydia trachomatis. real-time pcr has increasingly been used and is easier to perform and faster, and since it is performed in a closed system it is less prone to contamination than the conventional pcr. keeping the above background in mind this study was undertaken to generate reliable data regarding prevalence of cti in hiv-infected and hiv-uninfected women by real-time pcr, the most sensitive and specific test available currently for diagnosing genital chlamydia infection. the primary objective of this study was to establish the need for screening hiv seropositive women for cti. study subjects were recruited as follows: thirty adult hiv seropositive women with symptoms suggestive of rtis (study group a1),thirty adult hiv seropositive women without symptoms suggestive of rtis (study group a2),thirty age and sex matched adult hiv seronegative women with symptoms suggestive of rtis (control group b1),thirty age and sex matched adult hiv seronegative women without symptoms suggestive of rtis (control group b2). thirty adult hiv seropositive women with symptoms suggestive of rtis (study group a1), thirty adult hiv seropositive women without symptoms suggestive of rtis (study group a2), thirty age and sex matched adult hiv seronegative women with symptoms suggestive of rtis (control group b1), thirty age and sex matched adult hiv seronegative women without symptoms suggestive of rtis (control group b2). all the study subjects were enrolled from the integrated counseling and testing center (ictc) for hiv/aids, department of microbiology, maulana azad medical college, which is attached to the lok nayak hospital, new delhi. this study was conducted prospectively between july 2010 and january 2011 at the hiv molecular laboratory of the department of microbiology, maulana azad medical college. this was a cross-sectional analysis to determine the prevalence of chlamydia trachomatis infection by using real-time pcr in hiv seropositive and seronegative, symptomatic, and asymptomatic women visiting the ictc of our department. subjects were enrolled in this study following institutional ethical committee clearance and a written informed consent of all participants. each participant was interviewed using a questionnaire concerning general sociodemographic information, personal details, and clinical symptoms. subjects having one or more of the following symptoms were considered symptomatic for rti: vaginal discharge, vesicular and/or nonvesicular genital ulcers, inguinal bubo, lower abdominal pain, genital skin conditions, urinary burning or frequency, dysmenorrhea, menorrhagia, and intermenstrual bleeding. vesicular and/or nonvesicular genital ulcers, lower abdominal pain, genital skin conditions, urinary burning or frequency, dysmenorrhea, menorrhagia, and intermenstrual bleeding. each study subject then underwent a general physical, per abdomen, per speculum, and per vaginum examination. diagnosis of hiv infection was done by following the standard protocol at our ictc that employs pretest and posttest counseling and obtains informed consent before hiv testing. three different rapid tests were used to detect hiv-1 and hiv-2 antibodies (combaids (span diagnostics ltd.), retrocheck hiv (qualpro diagnostics), and tri-line (rapid diagnostics)) following the manufacturer's instructions. one endocervical swab was collected from all participants according to the instructions provided in the specimen collection and transport kit (amplicor std swab collection and transport set) for detection of chlamydia trachomatis by real-time pcr. genital c. trachomatis infection was diagnosed by using cobas taqman ct test, v2.0, an in vitro nucleic acid amplification test for the qualitative detection of chlamydia trachomatis dna in female endocervical swab specimens. specimens were processed using the amplicor ct/ng specimen preparation kit for manual specimen preparation and the cobas taqman 48 analyzer for automated amplification and detection (roche diagnostics). the age and gender profile of all the participants is shown in table 2. in both the hiv seropositive study group and the hiv seronegative control group the mean age of hiv-infected cases was found to be 30.92 5.7 years and in hiv-uninfected controls 28.52 6.9 years. table 3 shows the presenting complaints of the symptomatic women in both the hiv seropositive study group and the hiv seronegative control group. vaginal discharge and lower abdominal pain were the most common presenting complaints amongst the symptomatic participants. table 4 shows the sti/rti syndromes diagnosed in study subjects on per speculum examination. vaginitis was the most common syndrome diagnosed in both the study and the control groups. table 5 shows the correlation between presence of symptoms and cti in the study and the control groups. chlamydia trachomatis infection was more commonly diagnosed in the asymptomatic hiv seropositive study subjects as compared to the symptomatic hiv seropositive study subjects. in our study the prevalence of cti was higher in hiv seropositive women as compared to hiv seronegative women (or 4.214; 95% ci 0.45738.865) and among the hiv positive asymptomatic as compared to the hiv negative asymptomatic (or 2.111; 95% ci 1.6062.776), although the differences were not found to be statistically significant (table 6). in india stis/rtis and hiv/aids are major public health problems. incidence and prevalence data have a key role in control strategies for hiv and stis. comprehensive baseline information on the epidemiology of stis is essential for the design, implementation, and monitoring of successful control programs to reduce their incidence. routine surveillance of stis/rtis is not carried out in our country due to the lack of laboratory diagnostic facilities, limited resources, stigma, and discrimination associated with stis and poor attendance of sti patients, especially women, in sexually transmitted disease (std) clinics. taking the above facts into consideration, it is meaningful to have genuine laboratory-confirmed data on the incidence/prevalence of rtis/stis in india. as the asymptomatic nature of stis is well known, there is a need to adopt a specific strategy for the screening of the sexually active population in india to reduce the overall rate of stis, which would, in turn, reduce the risk of hiv infection. our study presents an insight into the prevalence data of genital chlamydia infections in hiv-infected and hiv-uninfected women visiting the ictc of new delhi's largest tertiary care hospital. the mean age of the hiv seropositive subjects in our study was 30.92 5.7 and that of the hiv seronegative control group was 28.52 6.9. this compares well with the findings of another study done in baroda, india, to look for the prevalence of rtis in hiv-infected women wherein the mean age for hiv positive women was 30 and that for hiv negative women was 27. the predominant age group in the hiv positive participants in our study was 2635 years. this association of chlamydia infection with younger age is consistent with studies from other developing countries [32, 33]. this finding supports the fact that young sexually active adults should constitute a priority target group in the sti control program. however, the findings of our study can not be generalized to all sexually active adults as the study has its limitations of a small sample size and the study participants belonging to a high risk group being recruited from the ictc of our department. 80% of our hiv positive study participants were married which is quiet similar to what has been reported by another study from sub-saharan africa in which 85.3% of the hiv-infected women were married. this finding highlights the importance of concurrently screening and treating spouses/sexual partners to decrease the sti burden in the country but this is challenging due to the lack of knowledge and cooperation from husbands especially in the indian setup. in the present study, vaginal discharge and lower abdominal pain were the 2 most frequently reported symptoms by the symptomatic women in both the study and the control groups, confirming the data reported by some previous indian studies [22, 29]. another notable observation of our study was that vaginitis was the most common clinical finding detected on per speculum examination in both the hiv positive (50%) and the hiv negative groups (56.67%) which is in accordance with the observations of garg et al. and balamurugan and bendigeri where a majority of women on clinical examination had vaginitis of 94.6% and 36.9%%, respectively [22, 35]. the present study also validates the fact that chlamydia infections are usually asymptomatic as 10% of the asymptomatic women were diagnosed with cti while only 3.3% of symptomatic women had lab-confirmed cti in the hiv positive study group. once again this emphasizes the importance of routine screening of at risk young sexually active women. in our study cti was detected in 6.67% (4/60) hiv-infected women and in 1.67% (1/60) hiv-uninfected women by real-time pcr although this difference was not statistically significant but the odds ratio was 4.214. a study from cuba reported c. trachomatis infection in 10% of hiv-infected cases and 6.6% of hiv-uninfected cases by nested pcr but the difference was not statistically significant while the odds ratio was 3.39 which is in concordance with our study. studies conducted by natividad-villanueva et al. in usa and seck et al. in senegal reported c. trachomatis infection in 3.33% and 2.1% of hiv-infected females, respectively [38, 39]. larger studies are required to validate our observation as our study was restricted to a small number of cases. this study clearly shows that cti is more prevalent among hiv-infected females (with or without symptoms of rti) as compared to hiv-uninfected females. our study also stresses the usefulness of screening asymptomatic hiv-infected and hiv-uninfected females for cti by risk assessment and diagnostic testing periodically to prevent the occurrence of adverse outcomes associated with the disease and also to check further spread of infection in the community. for countries like india that still do not currently have an active chlamydia screening program in place, randomized controlled trials are required to delineate the benefits of screening in the sexually active population as our study participants represent a high risk group. it is also important that any test adopted in a national screening program be used in the primary care setting by practitioners without the need for expensive training and equipment. however, since use of real-time pcr is not feasible in most hospitals in developing countries efforts should be made to develop a simple, cost-effective, sensitive, and specific point of care test to identify and treat women with cti for prevention of sequelae and hiv transmission.

reproductive tract infection (rtis)/sexually transmitted infections (stis) are recognized as a major public health problem, particularly due to their relationship with hiv infection. early detection and treatment of chlamydia trachomatis infection (cti) among hiv-infected and hiv-uninfected women may impact heterosexual hiv transmission. a total of 120 participants were enrolled: 30 hiv seropositive women with symptoms of rtis, 30 hiv seropositive women without symptoms of rtis, 30 hiv seronegative women with symptoms of rtis, and 30 hiv seronegative women without symptoms of rtis. one endocervical swab was collected from all participants and cti was detected by real-time pcr (cobas taqman ct test, v2.0). cti was detected in 4 (6.67%) hiv-infected women and in 1 (1.67%) hiv-uninfected woman (or 4.214; 95% ci 0.45738.865). vaginal discharge was present in almost half of hiv-infected and hiv-uninfected women; lower abdominal pain was present in 11 (18.3%) of hiv-infected and in 9 (15%) of hiv-uninfected women. this study showed that cti is more prevalent among hiv-infected females as compared to hiv-uninfected females. as the use of real-time pcr is not feasible in most hospitals, efforts should be made to develop a simple, sensitive, and specific test to identify women with cti for prevention of sequelae and hiv transmission.

PMC3870114

pubmed-48

hip fracture is a moderate musculoskeletal trauma that mainly affects the older population with comorbid conditions. the number will increase markedly in coming years due to the ageing of the population. comorbidity and the double trauma may dispose them to serious postoperative adverse outcomes and a high mortality dominated by cardiovascular events [25]. myocardial injury may be difficult to diagnose because of impaired communication, limitations of clinical manifestation and non-specific electrocardiographic (ecg) changes [6, 7]. the isoenzyme myocardium-specific creatine kinase (ck-mb) is expressed in the myocardium and ck in the skeletal muscle cells. these two enzymes have traditionally been analysed in plasma to distinguish myocardial injury and skeletal muscle injury [8, 9]. troponins have been shown to be more specific and sensitive to cardiac injury [10, 11]. increased plasma levels of troponin have also been reported in pulmonary embolism, septicaemia and following major orthopaedic and cardiac surgery. in those conditions high plasma troponin levels have been associated with severe adverse outcomes and increased mortality [7, 1215]. in this study we wanted to test the hypothesis that fatal outcome following hip fracture the study was approved by the regional ethics committee and national medical authorities and conducted in accordance with the helsinki declaration. a total of 302 consecutive patients over 75 years of age with dislocated hip fractures were enrolled in the study at elverum (ech) and buskerud (bch) central hospitals during the period 20052009. comorbidity was routinely assessed according to the classification of the american society of anesthesiologists (asa). a hemiprosthesis was inserted through a lateral incision and fixed with or without bone cement (landos titan or landos corail, depuy, warsaw, in, usa). thromboprophylaxis (low molecular weight heparins) was administered routinely preoperatively (on hospital admission) with dalteparin 5000 iu s.c. blood samples were obtained from an antecubital vein, collected preoperatively (1) and postoperatively [within 24 hours (0)] and on days one [2448 h (+ 1)] and four (+ 4). troponin t (tnt) and ck-mb were measured by electrochemiluminescence immunoassay (elica, roche, basel, switzerland and abbott, abbott park, il, usa). ck was measured by absorption photometry (roche, basel, switzerland and abbott, abbott park, il, usa). values for tnt were only measured at the ech study centre (n=146). differences between mortality groups were tested using a two-sample t test or chi-square test for continuous or categorical data, respectively. non-parametric mann-whitney tests were done for tnt due to skewed distribution. a linear mixed model for repeated measurements with a random intercept and bonferroni adjusted pairwise post hoc comparisons were used to analyse the biochemical markers with respect to mortality and repeated venous blood analyses. univariate and stepwise multivariate logistic regression analyses were used to estimate unadjusted and adjusted odds ratios with respect to survival at the three month follow-up. results from multivariate logistic regression were based on models including only significant terms from stepwise regression to maximise the number of patients included. there were 229 women and 72 men, with a mean age of 84.7 (sd 5.1) and 83.7 (sd 4.7) years, respectively. by three months, 59 of 302 (19.5%) had died, 62% women and 38% men. table 1demographic and clinical characteristics of patients at 3-month follow-upcharacteristicsalive (n=243)dead (n=59)p valuesage, years (mean sd)84.1 (5.1)86.2 (4.6)0.004sex female193 (79.4%)37 (62.7%)0.005 male50 (20.6%)22 (37.3%)mobility not mobile37 (15.8%)10 (17.2%)0.109 living aid23 (9.8%)5 (8.6%) crutches51 (21.8%)21 (36.2%) no aid123 (52.6%)22 (37.9%)asa score i10 (4.2%)0 (0.0%)<0.001 ii103 (42.9%)9 (15.3%) iii116 (48.3%)38 (64.4%) iv11 (4.6%)12 (20.3%)asa american society of anesthesiologistsminor deviations between total number of patients in categories compared to number alive or dead are due to missing data. missing data are assumed to be completely at random demographic and clinical characteristics of patients at 3-month follow-up asa american society of anesthesiologists minor deviations between total number of patients in categories compared to number alive or dead are due to missing data. missing data are assumed to be completely at random asa, male sex and age were significantly associated with mortality within three months (table 1). within one day after surgery, the plasma levels of ck and ck-mb increased nearly threefold (inverse for the ratio). on days 1 and+1, ck and ck-mb values were significantly higher among those who died compared to those who survived (p=0.001 and p=0.031, respectively) (fig. 1). on the fourth postoperative day, tnt plasma levels rose twofold in the mortality group and remained unchanged in the alive group. the plasma levels were significantly higher (p<0.05) in the mortality group at all sampling times except at day 0 (fig. 1mean and 95% confidence intervals for troponin t, creatine kinase (ck), myocardium-specific creatine kinase (ck-mb) and ratio of ck-mb to ck before surgery, perioperatively and 1 and 4 days after surgery for 3-month mortality. statistical significance (p<0.05) between mortality groups is indicated by an asterisk. statistically significant (p<0.05) differences between before surgery and during follow-up are indicated by the connecting lines. day 1 1 day before surgery, day 0 within 24 h after the operation, day+1 2448 h postoperatively, day+4 4 days after surgery mean and 95% confidence intervals for troponin t, creatine kinase (ck), myocardium-specific creatine kinase (ck-mb) and ratio of ck-mb to ck before surgery, perioperatively and 1 and 4 days after surgery for 3-month mortality. statistical significance (p<0.05) between mortality groups is indicated by an asterisk. statistically significant (p<0.05) differences between before surgery and during follow-up are indicated by the connecting lines. day 1 1 day before surgery, day 0 within 24 h after the operation, day+1 2448 h postoperatively, day+4 4 days after surgery the tnt plasma concentrations were split into three equal-sized data subsets, i.e. 0.01, 0.010.04 and>0.04 g/l, and the ratios of ck-mb to ck concentrations were split into two equal-sized data subsets, i.e. below and above 0.02./l before surgery correlated with three month mortality [odds ratio (or) 10.9, 95% confidence interval (ci) 2.254.0, p=0.003] (table 2). stepwise multivariate logistic regression with age, sex, asa category, levels of tnt and ratio of ck-mb to ck concentrations were performed. high tnt plasma concentration was associated with increased mortality (or 6.1 95% ci 623.1, p=0.008) at day four after surgery. no statistically significant association was found for the ratio of ck-mb to ck during the entire sampling period when adjusted for age, sex and asa (table 3). similar regression analyses were done for ck and ck-mb. on day+1, the ck-mb was associated with mortality (or 1.1, 95% ci 1.021.2, p=0.012). we found that our hypothesis, stating no predictive value from cardio-muscular plasma enzymes with regard to early mortality in patients with hip fracture, was false. table 2results from univariate logistic regression models predicting 3-month mortality (death)variablesunadjusted or (95% ci)p valuesage1.1 (1.01.1)0.004if male2.4 (1.34.4)0.006asa3.7 (2.26.1)<0.001if ck-mb/ck>0.02 day 11.6 (0.83.4)0.225 day 00.7 (0.31.6)0.413 day+11.5 (0.82.9)0.239 day+41.2 (0.62.4)0.620tnt day 10.011.0 (reference)0.010.041.5 (0.46.0)0.532>0.0411.0 (2.254.6)0.0030.011.0 (reference) day 00.010.041.5 (0.45.4)0.556>0.042.9 (0.711.0)0.1270.011.0 (reference) day+10.010.041.6 (0.47.4)0.512>0.046.0 (1.721.1)0.0050.011.0 (reference) day+40.010.043.3 (0.715.0)0.126>0.047.0 (1.925.6)0.003or odds ratio, ci confidence interval, asa american society of anesthesiologists, ck-mb/ck ratio of myocardium-specific creatine kinase to creatine kinase, day 1 1 day before surgery, day 0 within 24 h after surgery, day+1 2448 h postoperatively, day+4 4 days after surgerytable 3results from multivariate stepwise logistic regression models predicting 3-month mortality (death)variablesday 1day 0day+1day+4adjusted or (95% ci)p valuesadjusted or (95% ci)p valuesadjusted or (95% ci)p valuesadjusted or (95% ci)p valuesage1.1 (1.01.2)0.0061.1 (1.01.2)0.0061.1 (1.01.2)0.0011.1 (1.01.2)0.027if male2.4 (1.24.6)0.0132.4 (1.24.60.0132.7 (1.45.1)0.002asa3.3 (2.05.6)<0.0013.3 (2.05.6)<0.001ck-mb/cktnt 0.011.0 (reference) 0.010.041.9 (0.49.4)0.450 >0.046.1 (1.623.1)0.008or odds ratio, ci confidence interval, asa american society of anesthesiologists, ck-mb/ck ratio of myocardium-specific creatine kinase to creatine kinase, day 1 1 day before surgery, day 0 within 24 h after surgery, day+1 2448 h postoperatively, day+4 4 days after surgery results from univariate logistic regression models predicting 3-month mortality (death) or odds ratio, ci confidence interval, asa american society of anesthesiologists, ck-mb/ck ratio of myocardium-specific creatine kinase to creatine kinase, day 1 1 day before surgery, day 0 within 24 h after surgery, day+1 2448 h postoperatively, day+4 4 days after surgery results from multivariate stepwise logistic regression models predicting 3-month mortality (death) or odds ratio, ci confidence interval, asa american society of anesthesiologists, ck-mb/ck ratio of myocardium-specific creatine kinase to creatine kinase, day 1 1 day before surgery, day 0 within 24 h after surgery, day+1 2448 h postoperatively, day+4 4 days after surgery this prospective study on 302 elderly patients with hip fracture disclosed that 19.5% had died within three months. preoperatively obtained basic patient information was shown to be of particular importance to assess the risk of postoperative mortality. asa score on comorbidity, male sex and age correlated significantly with three month mortality, also described by other investigators [1719]. autopsy studies have shown that cardiovascular events are the main cause of death after hip fracture surgery [20, 21]. autopsies are rarely done today and the direct cause of postoperative death has not been possible to establish in this or in other recently conducted studies. in the elderly perioperative myocardial ischaemia is often clinically silent, without haemodynamic or notable ecg changes [2225]. over the years, biochemical plasma markers have been used to distinguish myocardial injury from skeletal muscle damage, an approach also adopted in this study. potential heart muscle damage was investigated in this study with ck-mb and tnt analyses. we found small and inconsistent differences for ck and ck-mb plasma levels between those who died and those who survived. the ratio of ck-mb to ck was decreased from hospital admission (day 1) to day 0 and stabilised, indicating that skeletal muscle damage dominated and that any myocardial injury remained undetected. this analysis suggests that ck and ck-mb are unspecific enzymes that are not distinct for skeletal and cardiac muscle damage following a hip fracture and are not feasible as prognostic markers of mortality, a finding consistent with other investigators. release of troponins into the circulation is considered to specifically reflect cardiac injury. in our study we analysed tnt and found that the plasma concentration was significantly higher on the fourth postoperative day in patients that subsequently died compared to those who were alive. g/l had a six times higher risk of dying vs those with normal plasma levels. this calculation was robust when correction was done for age, sex and comorbidity (asa score). these results fit with other reports that showed a second wave of troponin elevation several days after surgery which correlated with postoperative mortality [13, 29]. in summary, this study showed that basic clinical information on sex, age and comorbidity (asa score) and a high postoperative plasma concentration of tnt>0.04 g/l are robust predictors of three month postoperative mortality in the elderly undergoing hip fracture surgery. this information may be of importance for therapeutic and post-hospital health care intervention.

hip fracture, a moderate musculoskeletal trauma, is associated with a high postoperative mortality. most patients are elderly, with comorbid conditions and often with heart disease. the objective of this study was to find out if clinical parameters and analyses of specific muscle enzymes could predict three month postoperative mortality. a total of 302 patients above 75 years of age with hip fracture were consecutively enrolled. baseline information on age, sex and comorbidity assessed with the american society of anesthesiologists (asa) score was obtained before surgery. creatine kinase (ck), myocardium-specific creatine kinase (ck-mb) and troponin t (tnt) were analysed from venous blood, collected the day before surgery (1) and postoperatively, within 24 hours (0) and on days one (+ 1) and four (+ 4). the overall three month mortality was 19.5%. multivariate analyses showed that age, male sex and comorbidity (asa) correlated with mortality (p=0.027, p=0.002, p<0.001, respectively). surgery induced a two- to threefold increase of ck and ck-mb but without any correlation with mortality. however, high tnt levels>0.04 g/l correlated significantly with death (days 1,+1 and+4, p=0.003, p=0.005 and p=0.003, respectively). multivariate analyses, adjusted for age, sex and asa category, confirmed this correlation (day+4, p=0.008). thus, in elderly patients with comorbidities undergoing hip fracture surgery information on sex, age, asa category and postoperative laboratory analyses on tnt provide the clinicians with useful information on patients at risk of fatal outcome.

PMC3103949

pubmed-49

an infliction in the life expectancy figure of patients with sickle cell disease (scd) occurred around the middle of the 1970s (fig. this minimal increase in life expectancy coincided with my appointment at thomas jefferson university as the associate director of the newly created adult sickle cell center. the number of adults at that time was small and the transition from pediatrics to adult programs was at the age of 18 years. the trickle of patients increased gradually and we were faced with adolescent and young adult african american patients who were in a state of confusion. stripped from the protective sphere of the pediatric world and the empathy of their pediatric hematologists and the pediatric ancillary staff, they were in a state of fear, anxiety, depression and, worst of all, severe pain. the fact that most patients were barely educated, many without a high school degree, unemployed, mediocre health coverage, and dysfunctional family structure conferred a logarithmic dimension to the problem. the arrow indicates the infliction point where life expectancy of patients with sickle cell disease began to increase. the steady stream of admissions of patients with acute painful vaso-occlusive crises (vocs) to the emergency department (ed) and hospital were not welcome by most providers, hospital administration, the house and nursing staffs. there was subtle resentment of the patients that sometimes extended to the hematologists who showed compassion to the patients. soon labels such as drug addicts, drug-seeking behavior, and hospital hopping and frequent flyer emerged. listening to and believing the patients and keeping detailed records of ed and hospital admissions and the analgesics prescribed, revealed that most patients genuinely do not respond to a certain analgesic or a certain dose. increasing the doses of an analgesic or switching to another drug solved the problem in most patients. accordingly and with the approval of the institutional review board (irb), i issued an identification wallet-sized, plasticized card that was carried by patients and presented to the provider treating their voc in the ed, hospital or any other medical facility. information printed on both sides of the card included: 1) demographic data and a recent photograph; 2) hematological data including reticulocyte count; 3) medical data including the type of scd, its complications and co-morbidities if present; 4) all medications being taken by the patient and the recommended treatment of vocs including the name, dose, and the route of administration of the analgesics in question; and 5) my name and contact information for answering questions if needed. it was not expensive to issue these cards. a polaroid camera available at that time and a laminator were the only equipments needed to issue these cards. later on, information on the card was computerized and a printed copy was given to the patient. the patients were very compliant in carrying it as faithfully as they carry their medical cards. some providers liked it very much because it facilitated having a concise history about the patients. while this controversy was brewing, interesting developments in basic science were in progress to understand the pharmacodynamics and pharmacokinetics of opioids. in the 1970s, it was hypothesized that opioids have receptors to bind to and activate in order to relieve pain by blocking or minimizing the transmission of painful stimuli and raising the pain threshold. it did not take long after that to identify opioids as ligands that bind to stereospecific and saturable receptors in the central nervous system and other tissues [3, 4]. in addition, recent elegant studies [6-10] have revealed a helical structure of the opioid receptors, which forms pockets in which the corresponding opioid (ligand) fits snugly (fig. recognition is highly specific, such that only l-isomers of certain opioids exert analgesic activity. physiologically, by binding to receptors, opioids initiate a series of biochemical events including activation of g proteins, inhibition of adenylate cyclase, and extrusion of potassium ions, resulting in hyperpolarization of cell membranes [14-16]; this delays or prevents transmission of painful stimuli. thus, the riddle why some patients respond to one opioid but not another had a pathophysiologic explanation. (a) morphine-like molecule (yellow) in the deep pocket (blue) of the -opioid receptor. (b) -opioid receptors from an intimate pair when crystallized with a ligand (yellow) such as morphine. knowing how an opioid molecule (yellow) sticks in the pocket of its receptor (blue) parallel to the progress in the pharmacodynamics of opioids mentioned above, a concomitant advance in the pharmacokinetics of opioids was bubbling to the surface. phase i involves the cyp enzymes and phase ii metabolism conjugates the drug to hydrophilic substances, such as glucuronic acid, sulfate, glycine, or glutathione. morphine, hydromorphone and oxymorphone are metabolized by glucuronidation, whereas the majority of the other opioids are metabolized by the cytochrome p450 isoenzyme system. the net effect of an opioid depends on the availability of enzyme(s) to convert it into metabolites that could be active or inactive. briefly, the cyp2d6 genotypes are categorized into phenotypes based on the activity of the variant enzymes. ultrarapid metabolizers (ums) have greater than normal activity due to duplication or triplication, of active alleles [20-23], extensive metabolizers (ems) have normal enzyme activity, intermediate metabolizers (ims) have decreased enzyme activity, and poor metabolizers (pms) have absent or little enzyme activity. patients who are ums of fentanyl would rapidly convert it into inactive metabolites with minimal or absent analgesic effect requiring increasing the dose of fentanyl. on the other hand, patients who are pms of fentanyl would experience prompt relief with relatively small doses of fentanyl but higher doses could be toxic due to the accumulation of unmetabolized fentanyl. the cyp3a4 enzyme metabolizes more than 50% of all drugs; consequently, opioids metabolized by this enzyme have a high risk of drug-drug interactions. together, current data on the pharmacodynamics and pharmacokinetics of opioids show great variability of genotypes among patients and extreme variability in individual responses to opioids. determining the pharmacogenetics profile of each patient facilitates the choice of drugs that would be efficacious for that patient and avoid those drugs associated with harmful drug-drug interaction. this approach in diagnostics and therapeutics ushers in the dawn of a new field for the management of individual patients based on their unique pharmacogenetics, phenotypic and biomarker characteristics. this future approach is referred to as personalized medicine or, more recently, precision medicine. we hope that this methodology would be approved and sponsored by the insurance companies for patients with scd. in the meantime, listening, believing and respecting the patient with sickle cell pain should be maintained for now as the approach to individualized therapy.

in the 1970s, sickle cell pain was treated with trial and error approach by increasing or decreasing the dose of an opioid or switching from one analgesic to another. this approach was controversial with criticism and doubt about its usefulness. since then, advances in determining the structure of opioid receptors and the role of the cyp450 enzymes in metabolizing opioids revealed that these anatomic and metabolic findings are not the same in all persons, thus explaining the variability in response to opioids among patients. thus, the trial and error approach has a scientific basis after all.

PMC4817573

pubmed-50

the potential of rnai to silence any gene has made it an attractive therapeutic modality. however, the main obstacle to rnai in the clinic is delivery. to be effective, sirnas must be transported through the body, bind, and be taken up by target cells where they must traverse the plasma membrane and gain access to the cytosolic compartment, where the rnai machinery resides. to be useful, ease of formulation and administration, overall cost, and any associated toxicities are essential considerations. dcs are heterogeneous, with subsets defined phenotypically, functionally and by location (reviewed in ref. 2). a delivery vehicle that knocks down expression of specific genes in a distinct dc population(s) would be a valuable tool for targeting diverse diseases including cancers, infectious diseases, autoimmunity and as a vaccine component. therefore, a platform that delivers sirnas to specific dc subsets in situ would be useful for inhibiting or activating immune responses. lipid nanoparticles (lnps) are one of the most advanced platforms for sirna delivery to hepatocytes, and are under clinical evaluation for conditions that require hepatic gene silencing. these lnps typically contain ionizable cationic lipids (pka ~6.5) that bind nucleic acids via electrostatic interactions at low ph, but are charge neutral at ph 7.4. as a well-perfused organ furthermore, lnp uptake by hepatocytes is mediated by association with serum apoe leading to efficient uptake via low-density lipoprotein receptors in the liver. following cellular uptake of the lnp, the ionization of the lipid within acidic endosomes is thought to promote sirna escape to the cytosol. importantly, these lnps are associated with minimal toxicity, including little induction of proinflammatory cytokines following administration of physiologically relevant doses. in contrast to the liver, sirna delivery to extra-hepatic cells is challenging. in particular, immune cells such as dcs are relatively resistant to sirna uptake in vitro and in vivo. although designed for hepatic gene silencing, the efficacy of these lnps and their derivatives has been assessed for rnai-mediated gene silencing in macrophages (mos) and dcs in vitro and in vivo. lnp uptake and modest gene silencing was achieved at high doses of lnps (~ 5mg/kg). together with a complementary study, this work demonstrates the feasibility of gene silencing in immune cells using lnp technology. clearly, however, lnp formulations must be modified to achieve optimal gene silencing in immune cells. enhanced uptake of lnps by primary hepatocytes is mediated by apolipoprotein e binding to the neutral lnps. this results in recognition by receptors including the low-density lipoprotein receptor and scavenger receptors largely expressed on hepatocytes. the mechanism of uptake strongly suggests that retargeting of these particles to other receptors or other tissues is possible. indeed, akinc et al. demonstrated that lnps modified with n-acetylgalactosamine (galnac) were retargeted to the asialoglycoprotein receptor (asgpr) expressed by hepatocytes in vivo. for delivery to nonhepatic cells, we have shown in vitro that anti-transferrin receptor aptamers can be used to redirect similar lnps. more recently, liang et al. described a similar approach using aptamer-coated lnps to target osteoblasts in vivo. for uptake by immune cells, a recent study coated lnps with a full-length anti-cd4 antibody for sirna delivery to primary cd4 t cells. lnp uptake and gene silencing was observed in cd4 t cells in various organs including the spleen, lymph nodes, and blood. these studies demonstrate the potential for attaching exogenous ligands to lnps for delivery to hepatic, and importantly nonhepatic primary cells in vivo. drawing from these works, we reasoned that this lnp platform could be a useful foundation for the delivery of sirnas to dcs to modulate immune responses. to achieve this, we modified lnps using a single chain antibody (scfv) specific for the dc receptor dec205, a c-type lectin expressed at high levels on cd8 dcs. dec205 dcs mediate cross-presentation of antigen resulting in modulation of cd8 t-cell responses. therefore, inhibition of gene expression by dec205 dcs is a potentially powerful approach for regulating cd8 t-cell activation. using this approach, we assessed the ability of dec-lnps (lnps coated with scfv specific for murine dec205, containing sirnas specific for costimulatory molecules) to inhibit immune responses. injection of dec-lnps resulted in preferential uptake of the dec-lnps by splenic dec205 dcs. furthermore, when coadministered with adjuvant, lnps containing sirna targeting cd40, cd80, and cd86 reduced expression of these costimulatory molecules to levels similar to those observed in immature dcs. most importantly, dcs isolated from mice injected with a low dose (~0.6 mg/kg) of these dec-lnps, were able to suppress a robust mixed lymphocyte reaction (mlr), demonstrating the functional efficacy of this approach. interestingly, when we performed experiments using 2 ' fluoro (2'f) modified sirna, a formulation reported to be nonimmunostimulatory and nonimmunogenic, significant immune activation of the targeted dcs was detected. this effect was not observed in assays performed with human peripheral blood mononuclear cells (pbmcs) or monocyte-derived dcs (modcs) and could be mitigated through selective 2-o-methyl (2ome) modification of the sirna. overall, our study demonstrates the functionality of lnps modified for receptor targeting for sirna delivery to dcs. additionally our results suggest that specific cell targeting can mediate immunological responses to sirna formulations. while care needs to be taken in designing and evaluating targeted approaches using lnps, we found that 2ome modification of the sirna was sufficient to negate immune activation. our approach enhances the efficacy of sirna-mediated gene silencing by ~10-fold when compared to nontargeted delivery. therefore, this targeted strategy should prove effective for regulating multiple immune-mediated diseases. lnps containing sirnas specific either for cd80, cd86, cd40, or a control (nontarget) sequence were synthesized by extrusion using a mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine (dspc):dlindma: dspe-peg: cholesterol at a ratio of 15:40:5:40 (based on refs. murine anti-dec205 scfv with a c-terminal cysteine was attached to the lipid dspe-peg by a maleimide group (figure 1a, b). following synthesis, we subjected lnps to quality control to ensure consistency between batches. dynamic light scattering was used to determine diameter and polydispersity of lnps, and cryo-electron microscopy confirmed lnp size and their unilamellar structure (figure 1c, d). the efficiency of sirna incorporation and scfv binding to dspe-peg was also assessed (figure 1c, e). to show specificity of binding to dec205, scfv-lnps containing dy547-labeled sirna were cultured with either parental (dec205) or dec205 cho cells. as seen in figure 2a, at 4 c dec205 cells bind dec-lnps approximately fourfold better compared with nontargeted lnps (nt-lnps: lnps not coated with any scfv), or lnps coated with an isotype scfv (iso-lnps; cho-dec205 panel). when similar assays were performed at 37 c to allow cell uptake, cell staining increased by at least fivefold. little uptake was observed when lnps (targeted or nontargeted) were incubated with parental cho cells (cho panel). similar results were obtained when bone marrow-derived dcs (bmdcs), derived from wild-type mice (b6) were incubated with dec-lnps. we note that as dec205 is expressed at lower levels on bmdcs compared with cho-dec205, less dec-lnp binding and uptake was observed in bmdcs (~2-fold enhancement compared with nt- or iso-lnps; b6 bmdc panel). to further confirm target-specific uptake we performed experiments using bmdcs derived from dec205 mice, and no specific cell binding or uptake the dec205 antibody is internalized via receptor-mediated endocytosis, and is targeted to late endosomes and lysosomes. confocal microscopy confirmed that similar to dec205 antibody, dec-lnps were targeted to lamp-1 late endosomes or lysosomes (figure 2b). having demonstrated dec205-mediated uptake of dec-lnps in vitro, we investigated their localization in vivo. b6 mice were injected systemically (intravenous, i.v.) with fluorescently labeled scfv-lnps. as the dec205 receptor is endocytosed when it binds its ligand, we could not use this molecule to identify dec205 dcs. therefore, we used cd8, a protein that is coexpressed on dec205 dcs, as a surrogate marker for tracking these cells. more than 50% of cd11ccd8 cells took up dec-lnps, and uptake was largely restricted to the cd8 dc population, with little uptake observed by cd8 dcs (figure 3a, b). specificity was demonstrated by comparison of uptake of dec-lnps and iso-lnps. significant uptake was only observed in dec205 dcs following dec-lnp injection, and other splenic immune cells took up little dec- or iso-lnps (figure 3a, c). when dec-lnps were injected into dec205 mice, no uptake was detected (figure 3d). to show that dec-lnps were competent for specific gene silencing, we generated dec-lnps containing a sirna specific for cd80 (having identified the most effective sirna sequence; see supplementary figure s1) and injected into b6 mice. cd11cdec205 cells took up similar amounts of lnps following injection with dec-lnp containing either cd80- or control-sirna (lnp uptake panel, mean fluorescence intensity (mfi): dec-lnp-control=443 versus dec-lnp-sicd80=464). in mice injected with dec-lnp encapsulating cd80-specific sirna, cd80 protein expression approached basal levels, and was reduced by ~2-fold when compared with dec-lnp containing control-sirna (figure 4a). a 75% reduction in cd80 mrna was observed in dec205 dcs that had taken up dec-lnps containing cd80-specific sirna (compared with dec-lnps containing control sirnas; figure 4b). we confirmed that gene knockdown was rnai-mediated using 5 race to detect sirna-directed mrna cleavage products (figure 4c). sequencing of the polymerase chain reaction (pcr) fragment verified that it was derived from cd80 mrna and that the cut site corresponded with nucleotide positions 1011 of the sirna guide strand (not shown). interestingly, while the data in figure 4 demonstrate the ability of our dec-lnps to effectively knockdown cd80 expression in dcs in vivo, we observed significant dc activation following injection of dec-lnps containing control sirnas (figure 4a, right panel). this observation was surprising considering these experiments were performed using sirnas containing 2f modified rna, previously reported to have reduced immunostimulatory activity. to better assess this effect, we synthesized a series of luciferase-specific control sirnas (luc) containing 2oh rna (unmodified), 2f pyrimidines or selected 2ome modifications (see methods for modification strategy; based on refs. lnps were generated and their ability to stimulate immune responses using multiple assays was examined. first, b6 mice were injected with dec-lnps that contained either unmodified, 2f or 2ome modified luc-specific sirnas in the absence of any adjuvant. one day later, splenic dcs were analyzed for the expression of costimulatory receptors (supplementary figure s2a). as expected, dcs isolated from mice that had received unmodified sirnas demonstrated significant upregulation of cd40, cd80, and cd86. consistent with our previous results (figure 4a), animals treated with dec-lnps containing 2f modified sirnas also upregulated these costimulatory molecules. importantly, and similar to work by other groups, we found that 2ome modified sirnas induced minimal immune activation. to attempt to circumvent the need for a time and resource intensive in vivo assay, we wanted to determine whether an in vitro assay could be used to detect immunostimulatory sirnas. however, when we cultured b6-derived bmdcs with dec-lnps (containing cd80-specific sirnas) no difference between 2oh modified sirna, 2f or 2ome modified sirna was detected (supplementary figure s2b). we also used nt-lnps to assess the effects of various sirna formulations on human pbmcs and modcs using standard preclinical assays (supplementary figure s2c). pbmcs or modcs were cultured for 24 hours at which time cells were assessed for induction of apoptosis and culture supernatants were tested for the presence of proinflammatory cytokines (see methods). apoptosis was not observed under any conditions tested (data not shown). as expected, pbmcs cultured with the highest concentration of lnps containing unmodified sirnas elicited the secretion of several cytokines. 2f and 2ome luc sirnas induced production of one cytokine (il8), also at the highest concentration tested. no cytokine production was observed following culture of modcs with lnps encapsulating unmodified, 2f or 2ome sirnas. taken together, these experiments suggest that the assays tested show marked differences in sensitivity. surprisingly, bmdcs and modcs were the least receptive for detecting immunostimulatory sirnas. conversely, dec-lnp injection was useful for the identification of sirnas with immune-activating potential. from these results, we used sirnas containing selective 2ome substitutions for all subsequent studies. to target autoimmune diseases, reducing the expression of several costimulatory molecules therefore, we next determined the ability of dec-lnps that encapsulated 2ome modified cd86 specific sirnas, to reduce gene expression following i.v. administration (optimal sirna identified as previously; supplementary figure s1). as the sirnas were 2ome modified, mice were coinjected with lps to stimulate expression of costimulatory molecules. analysis of splenic dec205 dcs 1 day following dec-lnp injection showed reduction of cd86 protein to near steady-state levels (figure 5a). total rna was isolated from dec205 dcs that had taken up dec-lnps encapsulating either cd86 or control sirnas. an approximately 70% reduction in cd86 mrna levels was observed from dcs isolated from mice that received dec-lnps containing cd86 sirna in comparison to dcs derived from control sirna treated mice. to verify knockdown occurred via the rnai pathway, we next tested whether combining sirnas in dec-lnps would induce gene silencing equivalent to that observed with single sirnas. a combination of 2ome modified sirnas targeting cd80, cd86 and cd40 were incorporated into dec-lnps and injected into b6 mice along with adjuvant (lps) to activate the dcs. reduction of each of the targeted costimulatory molecules was observed, with cd80 and cd86 knocked down to basal levels (figure 5c). gene silencing was similar to that achieved following treatment with dec-lnps containing single sirnas (e.g., ~50% for cd86: compare cd86 dec-lnp, figure 5a with mix dec-lnp, figure 5c right panel). to determine functional relevance of this knockdown, we used the robust mlr assay. b6 mice were injected with dec-lnps encapsulated with either a mix of cd80, cd86, and cd40 or control sirnas. after 24 hours, splenic cd8 dcs that had taken up dec-lnps were isolated, irradiated, and cultured with splenic t cells derived from a balb/c mouse. maximal proliferation was observed when dcs were isolated from mice injected with lps only, or lps plus dec-lnps containing control sirnas. in contrast, proliferation of t cells cultured with dcs derived from mice injected with lps plus dec-lnps containing cd80, cd86, and cd40 sirnas was significantly reduced (figure 5d). currently, the most advanced lnp platforms use the ionizable cationic lipid dlindma, or its dlin-kc2-dma and dlin-mc3-dma derivatives. following systemic administration, these lnps accumulate in first pass organs, i.e., liver and spleen, making them attractive vehicles for hepatic gene knockdown. furthermore, these lnps have been shown to safely and effectively reduce expression of hepatocyte genes in clinical trials. however, there is only very limited data demonstrating targeted delivery of potentially clinically relevant lnps to nonhepatic cells. we have shown that conjugation of a scfv, specific for the dc receptor dec205, is sufficient to direct delivery of dlindma-formulated dec-lnps to splenic dcs. similar to conventional nt-lnps, dec-lnps incorporated sirnas with high efficiency (> 80%), and were of uniform diameter (~100 nmol/l). we found that coating lnps with only ~50 scfv was sufficient for dc delivery (figures 1, 2, and 3). we showed that dec-lnp uptake correlated well with dec205 receptor density: dcs>b cells, t cells and macrophages (dec205 expression is 1050-fold less than on dcs). use of dec205 mice further confirmed that uptake occurred via the dec205 receptor (figures 2a and 3d). we also showed that the intracellular pathway accessed by dec-lnps was consistent with the dec205 pathway (figure 2b). importantly, we demonstrated that targeted delivery resulted in effective rnai-mediated gene silencing of one, or several genes, to essentially basal expression levels (figures 4 and 5a c). this reduction in gene expression was sufficient to inhibit a robust mlr (figure 5d). coated lnps formulated with the cationic lipid ddab with full-length dec205 antibody for sirna delivery and demonstrated the ability to reduce expression of the costimulatory molecule cd40 in dec205 dcs. while some gene knockdown was achieved, this lipid is known for its adjuvant qualities, and is being developed for applications that require immune response stimulation. the lnps also proved relatively inefficient at sirna loading (10% compared with 85% for dlindma lnps). the sirnas used were also unmodified, which could serve as another possible source of immune stimulation. therefore, the potential for this formulation appears to be limited. more recently, ramishetti et al. utilized a full-length antibody targeting cd4 to enhance t-cell uptake and induce sirna-mediated gene silencing using a clinically relevant lnp formulation similar to the one we employed in our work. interestingly, both this study and the work from zheng et al. chose to utilize full-length antibodies for lnp targeting, which may limit their utility when considering the immunogenicity of whole antibodies and their rapid clearance from the circulation by fc-mediated uptake by macrophages. in fact, in preliminary studies, we coated lnps with full-length dec205 antibody and failed to observe uptake by dec205 dcs. using our dec-lnps mg/kg) comparable to that study which used n-acetylgalactosamine (galnac)-coated lnps to target hepatocytes via the asialoglycoprotein receptor (asgpr) in apoe knockout mice. in contrast, when the ability of nt-lnps to silence antigen presenting cells (apcs), including dcs, was investigated a dose of 5 mg/kg was required to achieve partial silencing: ~10-fold higher when compared with our dec-lnps. furthermore, the authors used the dlindma derivative, dlin-kc2-dma, which displays improved silencing ability in hepatocytes (relative ed50 dlin-kc2-dma: 0.1 mg/kg versus dlindma: 1 mg/kg). as these lnps were formulated for effective gene silencing in hepatocytes, biodistribution studies show that nt-lnps are found in the spleen, although at ~50-fold less than in the liver. while targeting ligands have been shown to have little effect on the overall biodistribution of nanoparticle formulations, they can enhance cell-specific uptake. therefore, while high doses of nt-lnps can confer partial gene silencing in apcs, at low lnp concentrations, we find that a targeted approach is required to achieve knockdown in a specific dc subset. injection of low-dose nt-lnps failed to result in detectable uptake/gene silencing. as the rnai pathway is resident in the cytoplasm, the intracellular pathway used following receptor ligation is an important consideration. we chose the well-characterized dec205 receptor for targeting our lnps. following binding, the internalized receptor is routed to late endosomes and associated cargo gains access to the cytoplasm. the dlindma lipid fuses with anionic phospholipids present in the endosomal membrane resulting in release of encapsulated cargo into the cytoplasm. however, it is likely that different routes of uptake will affect the efficiency of delivery and subsequent gene silencing. thus, a logical extension to our approach is to determine whether we observe improved gene silencing using other dc specific targeting agents and/or more effective dlindma derivatives. we are currently investigating the efficacy of dec-lnps formulated with the dlin-mc3-dma (ed50 0.03 mg/kg). having demonstrated that we could efficiently target sirnas to dcs, the lack of toxicity of these lnps and their cargo had to be established. we observed significant dc activation following injection of targeted lnps containing unmodified and 2f modified sirnas, and incorporation of 2ome modifications at distinct residues was required to maintain costimulatory molecules analyzed at basal levels (figure 4a and supplementary figure s2a). as injecting mice to determine immune stimulation is time and resource intensive, we assessed immune stimulation in murine and human tissue culture systems. murine bmdcs were refractory to lnp stimulation suggesting they would not serve as a useful surrogate for gauging immunogenicity of lnps. taken together, these assays demonstrate a requirement for in vivo analysis to uncover the stimulatory capacity of the lnps. recently, a human whole blood cytokine assay has been described that showed similar activation profiles elicited by lnps when compared with in vivo injection, potentially circumventing a requirement for cumbersome in vivo analyses. for the purposes of inhibiting a multifactorial immune response, such as that observed in graft rejection or autoimmunity, the simultaneous knockdown of several (co)stimulatory genes this approach has been used to demonstrate that combining antibodies targeting cd80 and cd86 can reduce disease severity in experimental autoimmune encephalomyelitis and in antigen-induced arthritis. transfer of dcs treated ex vivo with cd40, cd80, and cd86 antisense oligonucleotides (as-odn) into nod mice delayed type 1 diabetes (t1d) onset. a follow-up study demonstrated that multiple injections of microspheres containing cd40, cd80, and cd86 as-odns were sufficient to prevent t1d, possibly due to reduced expression of the costimulatory molecules by splenic dcs. we achieve significant reduction in expression of cd40, cd80, and cd86 on splenic dcs using our dec-lnps following systemic delivery, and ongoing studies are addressing the durability of gene knockdown and the potential use of these dec-lnps for treating autoimmune diseases. in summary, we have shown that a clinically relevant lnp can be modified for cell-specific delivery of sirnas. in vivo administration did not produce overt inflammatory responses, and we achieved significant rnai-mediated gene-specific knockdown at low sirna doses. these attributes make this an attractive platform to warrant further investigation as a potential therapeutic agent for various autoimmune diseases. furthermore, judicious selection of the ligand coating the lnp opens up the potential for targeting multiple cell types. mice. c57bl/6 (b6) and balb/c mice were purchased from taconic (hudson, ny). dec205 mice were bred to homozygosity on the b6 background (dec205), and validated by pcr and flow cytometry. mice were housed in the barrier facility at the institute for animal studies, albert einstein college of medicine (bronx, ny) according to institutional animal care and use committee (iacuc) guidelines. the single-chain fragment variable (scfv) antibodies constructed for dec205 (clone nldc145) and isotype (clone gl117) were cloned from antibody constructs, as previously described. the variable regions of the heavy and light chain sequences were fused with a flexible linker (g4s)6 separating them and cloned into a pet28a (emd millipore, billerica, ma) bacterial expression vector. further modifications to the constructs included a strep-tag-ii (wshpqfek) at the n-terminus and a 10-residue histidine tag (h10) with a short flexible linker (g4s) near the c-terminus. a c-terminal unpaired cysteine residue was introduced for scfv conjugation to the lipid dspe-peg-mal. the scfv constructs were transformed into bl21 (de3) plyss competent cells (promega, fitchburg, wi). the scfv dec205 or isotype were expressed in escherichia coli strain bl21 (de3) plyss as insoluble inclusion bodies. following bacterial growth in luria broth (lb) medium and induction of protein expression with isopropyl 1-thio-d-galactopyranoside cells were lysed, and insoluble protein pelleted by centrifugation. solubilized inclusion bodies were passed over a ni-nta column and the bound protein was eluted with 8 m guanidine hydrochloride. the purified proteins were refolded by rapid dilution in refolding buffer, and purified by size-exclusion chromatography using superdex 200 (ge healthcare life sciences, pittsburgh, pa). proteins were buffer-exchanged in phosphate-buffered saline (pbs) and concentrated. scfvs were tested for endotoxin contamination using a kinetic chromogenic limulous amoebocyte lysate (lal) assay (kinetic-qcl lal assay, lonza, walkersville, md). the free cysteine engineered onto the n-terminus of the scfv was reduced using tris-(2-carboxyethyl) phosphine hydrochloride (life technologies, carlsbad, ca). following buffer exchange into pbs, the reduced scfv was reacted with a 10-fold molar excess of alexafluor488 (af488)-c5-maleimide according to manufacturer instructions (life technologies, carlsbad, ca). polyacrylamide gel electrophoresis (sds-page) and coomassie staining and storm molecular phosphorimager scanning of the stained gel (ge healthcare, piscataway, nj). preparation of lnps. lnps were formed using the following lipids at a 15:40:5:40 mol %: dspc (avanti polar lipids, alabaster, al), cholesterol (chol; sigma-aldrich, mo), 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-n-(maleimide-(polyethylene glycol)-2000) (dspe-peg-mal, avanti polar lipids, alabaster, al), and 1,2-dilinoleyloxy-3-dimethylaminopropane (dlindma) synthesized by the chemical biology core facility at albert einstein college of medicine as previously described. a fluorophore conjugated to a lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-(lissamine rhodamine b sulfonyl) (dope-rhodamine b; avanti polar lipids, alabaster, al) was incorporated (0.1 %) to monitor lnp delivery. lnps were formed using a modified version of the spontaneous vesicle formation by ethanol dilution method. the lipid mixture was extruded (mini-extruder and 1 ml gas-tight syringes, avanti polar lipids, alabaster, al) sequentially with polycarbonate 200 and 80 nm pore size membranes. the extruded lipid mixture was mixed via rapid pipetting and vortexing with sirna, followed by dialysis into pbs. for attachment of scfv, lnps were mixed with reduced scfv at room temperature for 1 hour, and free maleimide groups were then quenched with -mercaptoethanol (me). tangential flow filtration (microkros filter module) with a 500 kda mwco (spectrum laboratories, rancho dominguez, ca) was used for buffer exchange into pbs and concentration of lnps. lnps were assayed for size and polydispersity by dynamic light scattering (dynapro plate reader, wyatt technology, santa barbara, ca). cryo-electron microscopy confirmed lnp size, geometry and lamellarity (analytical imaging facility; albert einstein college of medicine, bronx, ny). sirna incorporation was determined by quant-it ribogreen rna assay (life technologies, carlsbad, ca). to determine scfv conjugation efficiency, lnps were resolved on sds-page gels. a 3 kda difference in migration of the scfv was indicative of binding to dspe-peg-mal (unconjugated scfv ~30 kda, scfv-dspe-peg-mal ~33 kda). a cholesterol quantification kit (abcam, cambridge, ma) was use to determine the total lipid concentration. based on lipid concentration, the surface area per lipid head group (~0.6 nm), lnp diameter and their unilamellar structure, ~5060 scfvs are conjugated to each lnp under the conditions outlined above. sirna synthesis. modified and unmodified individual sirna sequences were synthesized on an expedite 8909 dna synthesizer (biolytic, fremont, ca) and purified by standard protocols, as described. dylight 547-phosphoramidite was attached to the 5 end of the sense strand for synthesis of fluorescently labeled sirna (dy547-sirna; glen research, sterling, va). sirnas were modified by 2f substitutions at every pyrimidine or by 2ome substitutions at the bolded nucleotides: cd40 sense 5-gaagauuaucccggucauauu-3; cd40 anti-sense 5-uaugaccg; ggauaaucuucuu-3; cd80 sense 5-gaauuaccuggcaucaauauu-3; cd80 anti-sense 5-uauugaugccagguaauucuu-3; cd86 sense 5-caacuggacucuacg acuuuu-3; cd86 anti-sense 5-aagucguagaguccaguuguu-3; control sense 5-uagcgacuaaacacaucaauu-3; control anti-sense 5-uugauguguuuaguc gcuauu-3. luciferase sense 5-gauuauguccgguuauguauu-3; luciferase anti-sense 5-uacauaaccggacauaaucuu-3. lnp binding, uptake, and intracellular localization. cho cells, cho cells that stably express dec205 (cho-dec), a dec205 b lymphoma line (a20 cells) and bmdcs, derived from b6 and dec205 mice, were used to assess lnp binding and uptake. cells were grown in complete medium (dulbecco's modified eagle's medium, 5% fbs, 10 mmol/l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 20 mmol/l l-glutamine). bmdcs were generated from bone marrow derived from the femur and tibia as previously described. adherent cells (a20, cho) were harvested using ethylenediaminetetraacetic acid to preserve receptor expression and were incubated at 4 c, 30 minutes with 500 nmol/l scfv-lnps (containing either 0.1% dope-rhodamine b or cy3-labeled sirnas). for uptake, cells were washed with fluorescence-activated cell sorting buffer (pbs containing 0.5% bsa, 0.1% na n3, 2 mmol/l edta) and analyzed by flow cytometry (lsrii, becton dickinson, franklin lakes, nj; flowjo, ashland, or). to monitor intracellular localization, a20 cells were incubated with scfv-lnps, containing sirnas labeled with dylight 547 (ge dharmacon, lafayette, co), 1 hour, 37 c. following one wash with pbs, cells were pipetted onto poly-l-lysine coated coverslips and allowed to adhere. adhered cells were fixed with 4% paraformaldehyde, washed with pbs and stained with anti-lamp1 (clone 1d4b; ebioscience, san diego, ca) and anti-eea1 (clone c45b10; cell signaling technology, danvers, ma). stained cells were mounted with vectashield mounting media containing 4,6-diamidino-2-phenylindole (dapi) (vector laboratories, burlingame, ca) and samples were visualized on a leica sp5 aobs confocal microscope (leica, buffalo grove, il). image processing (linear adjustments to brightness and contrast) was performed on image files using nih imagej software (bethesda, md). lnps (0.61 mg/kg) were injected (retroorbital (r.o.)) into b6 mice and 25 ng ultrapure lps (invivogen, san diego, ca) was injected r.o. one day later, spleens were harvested, collagenase treated to maximize dc yield, and single cell suspensions subjected to red cells lysis. cells were stained with cell surface markers and analyzed by flow cytometry to determine lnp uptake and expression of costimulatory molecules. antibodies were used for detection of the following cell surface markers: cd11c, cd8, dec205, cd40, cd80, cd86, cd19, b220, cd3, tcr, f4/80, cd11b, h2k, dcir2, cd49b, cd45.1. cell viability was monitored using a live/dead exclusion dye, i.e., live/dead blue (life technologies, carlsbad, ca). cells were acquired using an lsrii cytometer and data analyzed using flowjo software. to measure gene knockdown, cd11ccd8 splenocytes that had taken up lnps (i.e., sirna-dy547) were sorted by flow cytometry (facs aria iii, becton dickinson, franklin lakes, nj) into rnaprotect (qiagen, valencia, ca) to stabilize rna. total rna was isolated using the rneasy rna isolation kit (qiagen, valencia, ca), reverse transcribed using superscript iii (life technologies, carlsbad, ca) and random hexamers (idt, coralville, ia), according to manufacturer's instructions. quantitative pcr was performed using platinum taq polymerase (life technologies, carlsbad, ca) and sybr green to detect amplified products on an iq5 icycler (biorad, hercules, ca). reactions were performed in triplicate with the following primers: u6 forward: 5-ctcgcttcggcagcacatatacta-3; u6 reverse: 5-acgaatttgcgtgtcatccttgcg-3; cd40 forward: 5-cggctgtgcgcgctatg-3; cd40 reverse: 5-ggctgagaattcgcctgagtc-3; cd80 forward: 5-ctactctcttatcatcctgggcct-3; cd80 reverse: 5-cccggaagcaaagcaggtaatcct-3; cd86 forward: 5-gccacccacaggatcaattatcct-3; cd86 reverse: 5-aaagagagaggctgttggagatac-3. relative amounts of mrna were normalized to u6 mrna and primer specificity verified by melt curve analysis. to detect rnai-mediated cleavage products, total rna was isolated from cd11ccd8 splenocytes that had taken up lnps, i.e., sirna-dy547 (as for gene knockdown by qpcr), ligated at the 5 end to an adapter rna oligonucleotide and first strand cdna was synthesized using a gene-specific primer (gsp). pcr amplification using an adapter-specific primer and a nested gsp primer will amplify the cleavage product and identify the cut site which corresponds to position 10/11 of the sirna guide strand if mediated via rnai. the primers and oligonucleotide sequences used: adapter rna: 5-cgacuggagcacgaggacacugacauggacugaaggaguagaaa-3; gsp forward: 5-cgactggagcacgaggacactga-3; cd80 gsp reverse: 5-tgcgccgaatcctgccccaa-3; cd80 reverse nested: 5-atcaggagggtcttctgggg-3; cd86 gsp reverse: 5-agtaactgaagctgtaatctccttccaata-3; cd86 reverse nested: 5-ctgtgacattatcttgtgatatctgcatgt-3. analysis of immune stimulation. cd11c murine bmdcs (day 6; macs purified, miltenyi biotec, san diego, ca) were cultured with dec-lnps and monitored for activation (cd40, cd80, cd86) 1 day later by flow cytometry. in vivo stimulation was assessed by injection of dec-lnps r.o. into b6 mice, followed by analysis of activation markers (cd40, cd80, cd86) on splenic dcs after 24 hours by flow cytometry. the pbmc fraction was used directly or following purification of cd14 cells (stemcell technologies, vancouver, british columbia, canada), monocytes were cultured for 5 days in the presence of granulocyte-macrophage colony-stimulating factor and il4 to generate modcs. nt-lnps (5, 1, 0.2, 0.04, 0.008 mol/l) were cultured with pbmcs or modcs overnight and cell culture supernatants collected for cytokine analysis (ifn, il1, il2, il4, il5, il6, il7, il8, il10, il12, il13, tnf, granulocyte-macrophage colony-stimulating factor) using a luminex multiplex format (life technologies carlsbad, ca). cd11ccd8 splenic dcs were isolated by flow cytometry from b6 mice injected the previous day with dec-lnps containing either a 1:1:1 mix of cd40, cd80, and cd86 sirnas or control sirnas. splenic t cells from balb/c mice were isolated by macs using a pan t cell isolation kit ii (miltenyi, san diego, ca). macs sorted balb/c t cells were carboxyfluorescein succinimidyl ester labeled according to published protocols, and carboxyfluorescein succinimidyl ester-labeled t cells were cocultured with the irradiated b6 dcs. three days later, dilution of carboxyfluorescein succinimidyl ester was monitored by flow cytometry to determine t-cell activation.

due to their ability to knock down the expression of any gene, sirnas have been heralded as ideal candidates for treating a wide variety of diseases, including those involving undruggable targets. however, the therapeutic potential of sirnas remains severely limited by a lack of effective delivery vehicles. recently, lipid nanoparticles (lnps) containing ionizable cationic lipids have been developed for hepatic sirna delivery. however, their suitability for delivery to other cell types has not been determined. we have modified lnps for preferential targeting to dendritic cells (dcs), central regulators of immune responses. to achieve directed delivery, we coated lnps with a single-chain antibody (scfv; dec-lnps), specific to murine dec205, which is highly expressed on distinct dc subsets. here we show that injection of sirnas encapsulated in dec-lnps are preferentially delivered to dec205+dcs. gene knockdown following uptake of dec-lnps containing sirnas specific for the costimulatory molecules cd40, cd80, and cd86 dramatically decreases gene expression levels. we demonstrate the functionality of this knockdown with a mixed lymphocyte response (mlr). overall, we report that injection of lnps modified to restrict their uptake to a distinct cell population can confer profound gene knockdown, sufficient to inhibit powerful immune responses like the mlr.

PMC4754549

pubmed-51

type iii dysbetalipoproteinaemia is a kind of lipid metabolism disorders, caused by apo-e deficiency, which leads to accumulation of chylomicrons and very low density lipoprotein remnants in the plasma.123 lipid metabolism disorders are mostly manifested by xanthomas, which are known as yellowish cholesterol-rich material in large foam cells accumulating in the skins and tendons.14 these yellowish lesions may appear all over the body, on the palm of the hands, sole of the foot, tendons, and even on the eyelids.5 these lesions firstly described with or without hyperlipidaemia in association with monoclonal immunoglobulin. based on this fact, there are three forms of xanthoma: hyperlipaemic xanthoma, normolipaemic xanthoma and necrobiotic xanthogranuloma.6 xanthomatosis is usually associated with hyperlipidaemia, and morbidity and mortality of this condition are related to atherosclerosis and pancreatitis.7 hyperlipaemic xanthoma lesions are more polymorphic and can include tuberous, tendinous, palmar or eruptive xanthoma. verruciform xanthoma usually presents as a hyperkeratotic, cauliflower like, verrucous or papillomatous lesion.6 it initially was described as a unique clinicopathologic lesion of the oral mucosa and was subsequently reported in the skin. the epidermal-mucosal changes of hyperparakeratosis, neutrophilic infiltrate and dermal-submucosal foam cell reaction are distinctive features of these xanthomas.8 hereby, we present a rare type of xanthomas calling cauliflower xanthoma in a 43-year-old man with dysbetalipoproteinaemia. a 43-year-old man was presented to outpatient endocrine clinic of tabriz university of medical sciences, tabriz, iran, by his sister for evaluation of skin lesions. he had multiple lesions on shoulders and back, lateral and medial part of dorsal surface of the foot, medial and lateral malleus of both feet, and dorsal and palmar surface of both hands. siblings of the patient have been followed up in that clinic for clinically diagnosed type-iii dysbetalipoproteinaemia. this diagnosis was based on characteristic palmar, eruptive, tuberous and trauma site xanthomas with typical high levels of both total cholesterol and triglycerides in the plasma. history of coronary artery or other atherosclerotic disorders were negative in index case and siblings. there were not any other complaint, and as a greengrocer, he had an active lifestyle. he had a pathology report of skin lesion biopsy that was performed by a dermatologist few months ago, with presence of lipid laden macrophages consistent with diagnosis of xanthoma. fasting serum lipid levels reported as: total cholesterol=507 mg/dl, triglycerides=470 mg/dl and high density lipoprotein (hdl) cholesterol=41 mg/dl. he had normal fasting blood sugar, and normal thyroid, renal and liver function tests. physical examinations revealed numerous xanthomas resembling cauliflower on both knees [figure 1], unusual eruptive, tuberous xanthomas same as previous lesions, on the lateral malleus of both feet [figure 2]. cauliflower xanthomas on the knees and lateral malleus of both lower extremities cauliflower xanthomas on the lateral malleus of lower extremities there were lots of smaller xanthomas on the right shoulder [figure 3], and cauliflower like xanthomas on the extensor side of the left upper extremity, especially on the elbow [figure 4]. based on available information, the patient diagnosed as familial dysbetalipoproteinaemia (fdl) and the unique lesions on the lower limb called cauliflower xanthoma. although siblings of this case had the same disorder, they did nt develop such lesions. xanthomas on the shoulder and back of the neck xanthomas on the extensor side of the hand by searching in the medical data bases, we can not find a previous such a lesion. therefore, we decided to present this case without revealing patient's name, after taking his consent. considering the fact that the treatment with high dose statin was effective in correction of lipid abnormality and regression of skin lesions in his three siblings, we wanted to try the treatment procedure in this patient but he refused any intervention. fdl, also known as hyperlipoproteinemia type-iii or broad beta disease, is a rare inherited disorder characterised by improper metabolism of certain lipids, specially plasma cholesterol, triglyceride rich chylomicron and very low density lipoproteins (vldl) remnants.1 presence or absence of the symptoms of this disease depends on two major risk factors: genetic and diet.910 mutations in the gene for apolipoprotein e (apo e) are the main cause of this disease. replacement of an arginine by a cysteine in position 158 of the 299-amino acid chain of apo e5 is responsible for the defective binding of chylomicron and vldl remnants to cell receptors. thereafter, slower plasma clearance of these particles occurs, and results in the abnormal accumulation of lipids in the body.9 on the other side, diet has an essential role in the development of the disease. this means that with standard cholesterol diet, symptoms of fdl will not appear, even in the genetically susceptible person.10 xanthomas can be a symptom of fdl. xanthomas may also be the symptoms of a generalised histiocytosis, or a local fat phagocytosing storage process.11 they are yellowish lesions on the skins and tendons, macroscopically. these macrophages are filled with lipid droplets, which are dissolved and removed from tissue during histologic processing.8 fdl is mostly diagnosed by combination of clinical and laboratory findings.12 most cases are inherited as autosomal recessive trait. men are more susceptible for fdl probably because of protective impact of estrogen in women. most of the diagnosed patients were typically young males, with strong family history, characteristic skin lesions, high serum levels of cholesterol and triglycerides and confirming skin histology.113 most of these cases have premature atherosclerosis and other signs of ischaemic disorders.9 the patient in this article was a middle aged man with family history of dysbetalipoproteinaemia and high levels of serum lipids as follows: total cholesterol=507 mg/dl and triglycerides=470 mg/dl. he had a rare form of xanthoma all over his body, causing social problems for him. nicotinic acid, clofibrate, statins or gemfibrozil properly reduce cholesterol and triglycerides in people affected with dysbetalipoproteinaemia.1 the patient mentioned in this article refused receiving any medical intervention. he has just been advised to have low lipid diet, fish oil and regular moderate exercise in order to reducing serum lipid levels. there should be distinctive monitoring of fdl patients including regular checking of their serum lipids. controlling underlying disorders, and reducing excess calories, saturated fat and cholesterol, is the main aim of treating these patients. according to this article, cauliflower xanthomas could be a symptom of fdl, and it should be considered as a differential diagnosis while approaching to these lesions.

familial dysbetalipoproteinaemia (fdl) is an inherited disorder in which both cholesterol and triglycerides are elevated in the plasma, pre-disposing the people to coronary artery disease and peripheral vascular disease. the disease is mostly manifested by xanthomas, which have variable forms according to lipid amounts in the plasma of the blood. hereby, we report a 43-year-old man with fdl, presenting with a rare form of xanthomas calling cauliflower xanthoma all over the body.

PMC3821231

pubmed-52

the national cancer institute (nci) of the united states of america has proposed a reporting system for thyroid fine needle aspiration (fna) cytology, so-called bethesda system, which became an international standard of thyroid cytology [1, 2]. following this recommendation, the uk royal college of pathologists (the uk system) [3, 4] and italian societies of endocrinology and the italian society for anatomic pathology and cytology joint with the italian division of the international academy of pathology (the italian system) [5, 6] updated their diagnostic schema comparable with the bethesda system. in 2013, the japan thyroid association (jta) published guidelines for clinical practice for the management of thyroid nodules, including its diagnostic system for reporting thyroid fna cytology as shown in table 1 [79], using criteria similar to those used in the papanicolaou society recommendation, the bethesda system [1, 2], the uk system [3, 4], and the italian system [5, 6]. the jta reporting system of thyroid cytology further recommends risk stratification of follicular neoplasms (fn) into favor benign (low risk: lr), borderline (moderate risk: mr), and favor malignant (high risk: hr), which was adopted from the practice of high-volume thyroid centers in japan. this study examined observer variation in the subclassification of fn among 4 thyroid experts to validate the usefulness and limitations of this characteristic risk stratification of fn recommended in the jta guidelines. it is not our purpose to address in detail all the morphological issues of thyroid cytology. conventional smear samples of indeterminate diagnosis (n=389) were selected from files (n=3843) from the year 2005 at ito hospital, tokyo, japan, by one (k. kameyama) of the authors. inadequate samples or those with poor preparation and cases under clinical follow-up (no final histologic diagnosis) were excluded. there were 91 (23.4%) surgically treated patients with thyroid nodule under indeterminate cytological diagnoses. these patients visited ito hospital in 2005 and underwent diagnostic surgery or curative surgery in the subsequent years between 2005 and 2008. there were 48 cases of benign diagnoses, including 29 follicular adenomas (fas), 19 adenomatous nodules (an), and 43 malignant diagnoses, including 13 follicular carcinomas (ftcs), 24 papillary carcinomas (ptcs), 1 poorly differentiated carcinoma, 2 c-cell carcinomas, and 3 malignant lymphomas. twenty cases of cytological smear samples with follicular patterned lesions were randomly selected by one (k. kameyama) of the authors and they were further analyzed for subclassification of fn and diagnoses by 4 reviewers, as shown in table 2. the present study is a reproducibility study undertaken by 4 reviewers (kennichi kakudo, kaori kameyama, mitsuyoshi hirokawa, and ryohei katoh) who have special interest in thyroid pathology, all of whom are members of the clinical guideline committee of the jta. they were requested to subclassify fn following the new jta reporting system of thyroid cytology as shown in table 1. those 20 cases of smear samples (one representative smear sample each stained by the papanicolaou method) were circulated among the 4 reviewers without clinical information. the 4 reviewers examined cytological samples independently without exchanging opinions and were unaware of the original cytological diagnoses and final histologic diagnoses. because this study was conceived as an audit of cytology performance and the results are anonymized, institutional ethical committee permission was not required for its conduct. informed consent of all 4 reviewers was obtained for this study protocol. for statistical analysis of observer concordance, was first introduced as a measure of the level of agreement between pairs of raters and extended to multiple raters, known as composite [11, 12]. composite statistical analysis was performed by using data analysis and statistical software, version 13 (stata press, college station, texas, usa). values of can be interpreted as follows: 0.000.20, slight or very weak agreement; 0.210.40, weak to fair agreement; 0.410.60, moderate agreement; 0.610.80, substantial or good agreement; and values over 0.81, optimal or almost perfect agreement. there were 8 malignant cases (2 ftcs, widely invasive; 4 ftcs, minimally invasive; and 2 ptcs) and 12 benign cases (1 an and 11 fas). all diagnoses made by the 4 reviewers on the 20 follicular pattern lesions are shown in table 2. there were only 6 (30%) cases with a consensus diagnosis among all 4 of the reviewers: 3 in fa and 3 in ftc. diagnoses of fn and hr were made in only 7 cases (35%), including 4 malignant and 3 benign cases. composite statistical analysis clearly demonstrated good concordance for the hr diagnosis in malignant cases (= 0.7714) (table 3), while this was not found for the other subcategories in both benign and malignant lesions (< 0.4). it is of note that there was no interobserver variation in ftcs, widely invasive (n=2), and all 4 reviewers classified the 2 cases into the hr subcategory. it is clear that ftcs, widely invasive, have different degrees of cellular abnormality in our study. in contrast, there were significant disagreements among other types of malignancy and benign lesions, which clearly confirmed that the subclassification of fn is not a definite diagnosis but risk stratification useful for triage patients. concerning ftc, minimally invasive (n=4), there was only one concordant case among the 4 reviewers and all reviewers made a mr diagnosis in 1 case. there were disagreements between mr and hr in 1 case and between lr and mr in 2 cases. as for ptc (n=2), the 4 reviewers gave 2 diagnoses each, which included 3 lrs, 3 mrs, 1 hr, and 1 suspicious for ptc. however, it is of note that there was no single case in our 20 cases whose subclassification varied among all 3 subcategories. in benign final histologic diagnosis (n=12), there were 9 split diagnoses, including 6 cases between lr and mr and 3 cases between mr and hr, but no case between lr and hr. the incidence of lr diagnoses by the 4 reviewers in the 12 benign lesions was 31.3% (15/48) and that of lr diagnoses in the 6 cases of ftc was 16.7% (4/24) (p=0.186). the incidence of hr diagnosis in the 12 benign lesions was 10.4% (5/48) and that of hr in the 6 cases of ftc was 47.1% (10/24) (p=0.002). the incidence of malignancy of the hr was high at 60% for reviewer a, 75% for b, 75% for c, and 75% for d (6075%). the incidence of malignancy in the mr was lower than that of hr and was 33.3% for reviewer a, 35.7% for reviewer b, 25% for c, and 30.8% for d (2535.7%). the incidence of malignancy in the lr was lower than that in the hr and was 33.3% for reviewer a, 0% for b, 37.5% for c, and 33.3% for d (037.5%). although the risks of malignancy in the subcategories of fn differed among the 4 reviewers, it is of note that hr cytological diagnosis was significantly correlated with malignant histological diagnosis, and the risk of malignancy (6075%) was significantly higher than that in the other 2 subcategories (035.7%) in our study (p=0.002). although the standardization of terminology and diagnostic criteria is important for accurate communication among patients, clinical doctors, and cytopathologists [110], there are still differences among reporting systems in thyroid cytology as to how to interpret cytological diagnoses and how to decide on the clinical management of patients. in japan, cytopathologists attempted to use an internationally accepted reporting system, but it had to be modified to fit our practice. in japan, all patients with indeterminate cytology undergo further diagnostic procedures, without immediate surgery, to search for higher risk patients who should undergo surgery [79, 1315]. it is because the majority of thyroid carcinomas in indeterminate cytology are indolent and more conservative approaches, other than immediate diagnostic surgery, usually do not create any harm to the patient with malignancy [1620] and diagnostic surgery to all patients with indeterminate cytology results in risks of unnecessary surgery to the patients with benign nodules, more than 80% of the patients [79, 1922]. the proportion of malignancy found at thyroidectomy from patients with indeterminate cytology in this clinical setting will increase in number, and the malignancy rate of patient with indeterminate cytology in our study was calculated as 47.3% as shown in materials and methods. takezawa et al. from japan retrospectively analyzed their 1606 cytological samples using bethesda system, although it was written in japanese with english abstract, and they identified 115 (7.9%) cases of aus/flus (atypia of undetermined significance/follicular lesions of undetermined significance) and 61 (4.2%) cases of fn/sfn (follicular neoplasms/suspicious for follicular neoplasms). the resection rate of their aus/flus nodules was 30.4% (35/115 cases) and its malignancy rate was 88.6% (31/35 cases), and the resection rate of fn/sfn nodules was 36.0% (22/61 cases) and its malignancy rate was 72.4% (16/22 cases), which were very different from the ranges reported in most of the literatures from western countries using the bethesda system or the uk system [1, 2, 2023]. as it is clear, further triage of patients with indeterminate nodules reduces resection rates and increases malignancy rates in any diagnostic systems including the bethesda system. this clinical management was also true in some other countries [2427], as crippa and dina commented in a letter to an editor that thyroid cytology is the most important but not the only important deciding factor and therefore it must be integrated with other diagnostic procedures. we propose that future thyroid cytology classification schemes should reconsider clinical managements of indeterminate categories and how to reduce unnecessary surgeries for patients with benign thyroid nodules [79]. the risk stratification of follicular pattern lesions into 3 subcategories (cellular follicular lesion, fn favor benign, and fn favor malignant) was suggested by the papanicolaou society in 1996, but it did not become popular in thyroid cytology worldwide, apart from japan [8, 9]. there have been some reports in the literature on risk stratification of fn [10, 2834]. kelman et al. reported that 31/52 (60%) nodules with nuclear atypia consistent with fn were malignant and it was 4/9 (44.4%) in fn with atypia by goldstein et al.. reported that atypical proliferation was more often malignant than follicular group (53% versus 19%) in their indeterminate category (tir3/thy3). some researchers pointed out that the malignancy rate of fn without atypia is low and assessment later on could be an alternative approach [31, 32] and patients with high-risk cytological features such as nuclear overlapping (crowding) should be advised to have a surgical intervention [3133]. gerhard and da cunha santos using the papanicolaou society guidelines studied reproducibility between 2 observers in 97 diagnoses. they reported a substantial level of diagnostic interobserver (= 0.71) and intraobserver (= 0.66) reproducibility, although interobserver disagreement in the cytological diagnosis occurred in 23 cases (24.7%) and 18 (41.7%) of them were for fn. in an interobserver reproducibility study using the uk system, kocjan et al. reported that the statistic was very poor (0.11) for the thy3a category and that for thy4 was 0.17, in contrast to moderate to good agreement for thy1 (0.69), thy2 (0.55), thy3f (0.51), and thy5 (0.61). the observer variation of fn in our study occurred more often between lr and mr (9 cases, 45%), followed by between mr and hr (5 cases, 25%), but it is remarkable to note that none occurred between lr and hr. in other words, discordance is limited to between lr and mr or mr and hr, so we may conclude that the mr subcategory has an essential role in minimizing discordance between lr and hr. another choice of subclassification of follicular neoplasms would be two categories (low cancer risk and high cancer risk) instead of three categories (lr, mr, and hr), and this modification (two categories) is also described as acceptable in the reporting system recommended by the japan thyroid association [7, 8]. the second conclusion we may draw is that hr in the jta system is a powerful cytological subcategory to be used for the triage of patients for diagnostic surgery because the risk of malignancy of hr is high (6075%) equivalent to suspicious for malignancy category in the bethesda system, with good concordance among the 4 reviewers (= 0.7714). abele and levine reported their rate of indeterminate category to be 5% of 51,000 adequate fnas and they suggested that the national rate of 15% was in large part due to overdiagnosis. this significant difference in ratio of indeterminate categories may be due to experience of thyroid cytology and not patients ' background. clary et al. commented in their interobserver variability study that some pathologists make greater use of indeterminate categories such as follicular lesion, favor nonneoplastic or follicular lesion, and favor neoplastic lesion, whereas others show more definitive categorization into benign and neoplastic groups. cibas et al. also stated in their report on interobserver variability that cytopathologists with experience of thyroid cytopathology are more likely to make a definitive interpretation (i.e., benign or malignant). this tendency was seen in our present study in which reviewers b and d (whose indeterminate diagnosis rates are about 15% in their practice) made mr diagnosis more often (70% and 65%, resp.) and lr diagnosis less frequently (10% and 15%, resp.) than those of reviewers a and c (whose indeterminate diagnosis rates are about 5% in their practice). the difference in the prevalence of benign and fn/sfn may explain the different rates of lr (low risk) and mr (moderate risk) among reviews in our study, because incidence of one category may expand or contract depending on the rates of other categories. some fn/sfn lesions with benign pattern would be classified as benign by different authors and the incidence of fn/sfn of bethesda system in recent 7 series varied from 1.5 to 9.7% and that of benign category was between 54 and 77.4% summarized by ohori and schoedel. rapid development of molecular analyses on thyroid cytology may lead us possibly in the near future to more accurately identify patients who should be referred to surgery. until that time comes, thyroid fna cytology remains a main stay in the management of patients with thyroid nodules integrated with other clinical tests, such as ultrasound image diagnosis. as a conclusion the hr subcategory has a high predictive value of malignancy and good agreement among the 4 reviewers which is clinically helpful to triage patients for surgery. we believe that patients with cytological diagnosis of hr subcategory of fn in the jta system should be surgically treated especially if other risk factors coexisted. on the other hand, patients with lr or mr category therefore, surgical resection rate of indeterminate category is low in japan usually less than 50%, particularly in cases with fn.

background. the japan thyroid association recently published guidelines for clinical practice for the management of thyroid nodules, which include a diagnostic system for reporting thyroid fine needle aspiration cytology. it is characterized by the subclassification of follicular neoplasms, which is different from other internationally accepted reporting systems. materials and methods. this study examined observer variability in the subclassification of follicular neoplasms among 4 reviewers using papanicolaou-stained smear samples from 20 surgically treated patients with indeterminate cytology. results. the favor malignant subcategory had high predictive value of malignancy (risk of malignancy: 6075%) and good agreement among the 4 reviewers (= 0.7714). conclusion. these results clearly confirmed that the risk stratification of follicular neoplasms, which was adapted from cytology practice of high-volume thyroid centers in japan, can provide clinically helpful information to estimate the risk of malignancy and to triage patients for surgery.

PMC4334867

pubmed-53

plant cells need to maintain the functional integrity of their walls during cell morphogenesis and exposure to biotic/abiotic stress. the available evidence suggests that a dedicated plant cell wall integrity (cwi) maintenance mechanism exists (wolf et al., 2011). while our understanding of the mechanisms regulating stress responses and morphogenesis has increased significantly, our knowledge regarding the processes maintaining cwi is still limited. in the last years several reviews have been published on the plant cwi maintenance mechanism illustrating the increased interest in this area (humphrey et al., 2007; hematy et al., 2009; ringli, 2010; seifert and blaukopf, 2010). a recently published review focuses on cwi maintenance during plant cell wall morphogenesis (wolf et al., 2011). similarities between the yeast and plant cwi maintenance mechanisms have also been reviewed (hamann and denness, 2011). therefore, the available knowledge regarding cwi maintenance during plant development and in yeast will be covered here only briefly to provide a conceptual framework regarding cellular processes involved and to illustrate the degree of functional conservation between species. this review will focus on recent developments regarding the role of cwi maintenance during biotic stress responses. it will discuss how cwi maintenance could have a previously unrecognized role in the perception of and response to biotic stress. while both plant and yeast cells are enveloped by cell walls, certain important differences exist that affect the biological role and function of the plant cwi maintenance mechanism. in addition, plant cell walls are structurally and chemically more complex than the yeast cell wall. this means that in plant cells the sheer number of cell wall-related signaling events during development and plant environment interaction could disguise the activity of a dedicated plant cwi maintenance mechanism. the yeast cwi monitoring and maintenance network is quite complex, providing an indication of the possible complexity of the plant cwi maintenance network. by combining inputs from a turgor pressure sensor (sln1), mechano-perception (mid1/cch1), and dedicated cell wall damage (cwd) sensors (wsc1, 2, 3, mid2, mtl1) the yeast cwi maintenance network generates signals that permit highly specific responses to any challenge that impairs the functional integrity of the yeast cell wall. the available phenotypic and genetic data also implicate turgor pressure, mechano-perception, and cwd detection in plant cwi maintenance (hamann et al. interestingly, arabidopsis histidine kinase1 (ahk1) and cytokinin receptor1/arabidopsis histidine kinase4 (cre1/ahk4) can at least partially rescue a yeast strain with a loss of function allele in sln1 (urao et al. in addition, expression of the arabidopsis thaliana mid1 complementing activity 1 and 2 (mca1,2) genes rescues a mid1-deficient yeast strain suggesting that they could function as stretch-activated calcium channels (nakagawa et al. no functional homologues for the yeast cwd sensors have been identified in plants. in yeast, the signals generated by the sensors are relayed to the response genes via different signaling cascades involving calcineurin (mid1/cch1); rho1/mpk1 (wsc1, mid2), and ypd1 (sln1) (levin, 2005). transcription factors mediating the response are skn7, rlm1, and swi4/6 (levin, 2005). the response can involve activation of genes required for cell wall biosynthetic processes, remodeling of the cytoskeleton, and cell cycle progression. the available data suggest both organizational and functional similarities between the yeast and plant cwi maintenance mechanism while also highlighting how signals from mechanical and chemical sensors regulate jointly the cwd response. the plant cell wall is comparable to an exoskeleton surrounding the plant cell and providing both structural support and protection from biotic as well as abiotic stresses. it consists of cellulose microfibrils, pectin, hemicelluloses, proteins, and in certain cases lignin (somerville et al., 2004). plant cell walls are divided into primary (laid down during cell elongation/differentiation) and secondary (formed after cell morphogenesis is concluded) walls. in parallel, dependent on the presence of certain polysaccharides type i and type ii cell walls are distinguished (popper et al., 2011). cellulose microfibrils are the main load bearing elements, which are cross-linked to hemicelluloses and (in vitro) to pectin (dick-perez et al., 2011). hemicelluloses and pectin also form direct links creating a matrix in which the microfibrils are embedded like the steel mesh in a concrete wall. pectic polysaccharides like homogalacturonan (hg) are connected by calcium bridges between dimethyl-esterified parts of the molecules or through borate ester linkages in the case of rhamnogalacturonan ii (rg ii). they are targeted by pathogen-derived cell wall degrading enzymes (polygalacturonases), which generate oligogalacturonides (ogas) from hg (hahn et al., 1981; kars et al., 2005). biologically active ogas consist of chains of 915 galacturonic acid (gala) monomers and can function as signaling molecules (see below). during secondary cell wall formation monolignols (precursors for lignin) are secreted into the cell wall space and randomly cross-linked (vanholme et al., 2010). the cross-linking is dependent on the availability of reactive oxygen species (ros) generated by laccases and peroxidases. this process reinforces the wall against pathogen infection, waterproofs it, and increases structural integrity (tronchet et al., 2010; reduction of cellulose biosynthesis during primary cell wall formation through genetic or chemical means leads to lignin production (ellis et al., 2002; hamann et al., this highlights the ability of the cell to adapt to changes in cell wall composition and provides evidence for the existence of a cwi maintenance mechanism. plant cell walls are capable of adjusting their composition and structure in response to pathogen infection (dong et al., 2008). a mutation in (cellulose synthasea3, cesa3) a subunit of the cellulose synthase complex leads to ectopic production of lignin, i.e., replacement of a missing load bearing cell wall component by another one (cano-delgado et al follow up studies found that inhibition of cellulose biosynthesis during primary cell wall formation either through mutations like constitutive expression of vsp1 (cev1) and ectopic lignification1 (eli1) or inhibitors such as isoxaben results in transcriptional activation of stress response mechanisms; ectopic production of ethylene (et), salicylic acid (sa), jasmonic acid (ja), callose, and ros as well as changes in cell wall composition and structure (ellis et al., 2002; cano-delgado et al., 2003; manfield et al., 2004; hamann et al., the experiments also showed that the response to cwd consists of early and late stages reminiscent of the response to pathogen infection (denness et al., 2011). during the early stage, ros- and calcium-based signaling cascades are required for initiating the response to cwd (denness et al., 2011). interestingly, 1-aminocyclopropane-1-carboxylic acid (acc, an et precursor) and not et itself seems to be acting as signaling substance during the early response to isoxaben with inhibition of cell expansion being an active process and not simply an automatic consequence of cellulose biosynthesis inhibition (tsang et al., 2011). during the late stage, responses to cwd-like lignin deposition are initiated and the extent of lignin formation is apparently modulated by a negative feedback loop formed by ros and ja (denness et al., 2011). a combination of genetic and phenotypic analysis has implicated the nadph oxidases respiratory burst oxidase homologd and f (rbohd, f), the serine/threonine kinase oxidative signal inducible1 (oxi1), mca1, the receptor-like kinase (rlk) theseus1 (the1) as well as the ja biosynthesis genes allene oxide synthase (aos) and jasmonic acid resistant1 (jar1) in the signaling mechanism mediating the response to cwd in arabidopsis seedlings. interestingly, the cev1 mutation that affects cellulose biosynthesis during primary cell wall formation also causes enhanced resistance to infection by different powdery mildews (erysiphe orontii, e. cichoracearum, and oidium lycopersicum; ellis and turner, 2001). a screen for mutants causing resistance to powdery mildew infection provides further evidence of the close relationship between plant cell walls and pathogen resistance (vogel and somerville, 2000). three of the powdery mildew resistance (pmr) mutants that have been identified on the molecular level affect genes involved in cell wall biosynthetic processes. pmr4 encodes a callose synthase, pmr5 a gene of unknown function required for pectin production and pmr6 a pectate lyase (vogel et al. pmr4 resistance seems to be mediated via hyper-activation of sa signaling, whereas pmr5 and 6 resistance phenotypes are independent of ja, sa, and et signaling. mutations in irregularxylem1 (irx1/cesa8), 3 (irx3/cesa7), and 5 (irx5/cesa4) impair cellulose biosynthesis during secondary cell wall formation and cause enhanced resistance to the soil borne bacterium ralstonia solanacearum and the necrotrophic fungus plectosphaerella cucumerina (hernandez-blanco et al., 2007). mutations affecting cellulose biosynthesis during primary cell wall formation (cesa1, 3, 6) or other components of the secondary cell wall (pmr5, pmr6) did not cause enhanced resistance to the same pathogens. genetic analysis showed that the enhanced resistance in cesa4, 7, 8 is independent of ja, sa, and et-based signaling mechanisms. results from expression profiling experiments and genetic analysis using different abscisic acid (aba)mutants(aba insensitive 1-1;2-1;aba1-6) suggest that aba is mediating developmental and pathogen resistance phenotypes caused by the irx mutants. however, it remains to be determined if the aba involvement is direct or a secondary effect due to water stress caused by problems with xylem cell wall formation in the mutants. to summarize, these results suggest distinct resistance signaling cascades are induced by defects in primary and secondary cell wall formation as well as for different secondary cell wall components. they also highlight the direct impact of changes in cell wall composition/structure on the response to pathogen infection. the mode of action of the plant cell wall maintenance mechanism is not well understood. based on the knowledge from yeast, chemical and physical signals could act as indicators for the functional integrity of the plant cell wall either individually or jointly. by combining these different types of signal the plant cell would receive precise information regarding the state of its cell wall and the exact type of cwi impairment occurring. physical signals could be generated by stretching of the plasma membrane due to a weakened cell wall that can not resist the high turgor pressure levels within a plant cell or a plasma membrane that is displaced relatively to the cell wall. these events could be detected by stretch-activated or mechanosensitive channel proteins that lead to calcium influx into the cytoplasm, indicating cwd. sensor candidates could be encoded by members of the mechanosensitive channels of small conductance (mscs)-like (msl) gene family like msl 9 and 10 affect mechano-perception in protoplasts derived from arabidopsis root cells (haswell et al., 2008). another candidate of interest is the putative stretch-activated calcium channel mca1 is required for cwd-induced lignin deposition. interestingly, all isoxaben-induced cwd phenotypes can be suppressed by provision of osmotic support suggesting that changes in turgor pressure due to a weakened cell wall could result in signal generation via turgor pressure sensors (hamann et al., 2009; denness et al., 2011). while ahk1 and 4/cre1 can function as osmosensors in yeast and have been implicated in abiotic stress responses, no clear evidence exists implicating them in cwd perception in plants (urao et al., 1999; inoue et al., 2001; tran et al., 2007). in addition, ahk4/cre1 has been shown to function as a cytokinin receptor(inoue et al., 2001). therefore, the question that needs to be resolved at this point is if turgor pressure is a passive element in the process (generating cell wall fragments due to a weakened cell wall) or an active component that is being monitored and provides input into the process. the plant cell wall contains a large number of components that could generate chemical signals (ligands) indicative of cwd or general danger signals. the term damage associated molecular patterns (damps) has been coined to describe such ligands and the number of possible damps originating in plant cell walls is rather large (zipfel, 2009). here i will focus on the best-characterized group of signals, which are probably ogas. they can be generated through degradation of hg by pathogen-derived enzymes (kars et al. ogas have been shown to induce gene expression changes, stomatal closure, production of et, and ros as well as cell wall reinforcement (denoux et al., 2008; ferrari et al., 2008). a hybrid kinase consisting of the extra cellular domain of wall-associated kinase1 (wak1) and the intracellular domain of elongation factor tu receptor (efr) kinase can bind ogas and activate defense responses (brutus et al., 2010). wak1 belongs to a family of five wak genes encoding plasma membrane-localized ser/thr kinases that have been implicated in response to pathogen infection and regulation of cell elongation (kohorn et al., 2011). the effects of the chimeric wak1 kinase on pathogen resistance suggest that ogas and waks represent an in vivo ligand receptor pair. results from the analysis of a dominant active wak2 allele suggest the cwd signals perceived by waks could be relayed to downstream response genes through mapkinase6 (mpk6) (kohorn et al. interestingly, wak2 has also been implicated in regulation of invertase activity and turgor pressure during cell elongation (kohorn et al., 2006). however, there is currently no confirmation that waks are actively involved in cwi maintenance. in arabidopsis, more than 600 rlks have been identified and a large number of them have been implicated in developmental and stress response processes (shiu and bleecker, 2001). i will focus here on several kinases that have been implicated in cwd perception and/or pathogen response. most of the rlks implicated in cwi maintenance [the1, hercules1 (herk1), feronia (fer)] belong to the catharanthus roseus rlk1 (crrlk1)-like protein family, which has 17 members in arabidopsis. the1 was isolated as a suppressor of the cellulose-deficient cesa6 procuste (pre) mutant, which exhibits a hypocotyl elongation defect (hematy et al., 2007). subsequently it has been shown that the1 is required for cellulose biosynthesis inhibition-induced ros production and lignification in the root elongation zone (denness et al., 2011). the1, herk1, and fer have been implicated in brassinosteroid-induced cell elongation (guo et al., 2009; deslauriers and larsen, 2010). both fer and nortia/mildew resistance locus o 7 (ml07; a seven-transmembrane domain protein involved in powdery mildew resistance) are required for successful fertilization and resistance to infection by golovinomyces (syn. interestingly, ropgef (guanine-exchange factors) proteins have been identified as targets of fer activity (duan et al., 2010). ropgefs are required for the activation of rho gtpases, which in turn activate nadph oxidases like rbohd/f. these results suggest the same molecular components could mediate cell-cell interaction during development and plant heterotrimeric g-proteins (g, g, g) form a highly conserved signaling complex that has been implicated in signal transduction during development and stress responses in mammals, yeast, and plants (digby et al., 2006; temple and jones, 2007). in arabidopsis, five genes gpa1 (g), agb1 (g), agg1, 2, 3 (g1, 2, 3) encode the subunits of the complex (thung et al., 2011). recently, it has been reported that mutations in agg1, 2, and agb1 apparently cause enhanced susceptibility to infection with p. cucumerina (delgado-cerezo et al., 2011). a combination of metabolomic and microarray-based expression profiling studies of the mutants established that the pathogen phenotype is independent of sa, ja, aba, and et signaling cascades. interestingly, a large number of cell wall biosynthetic/modifying genes are mis-regulated in agbl and aggl2 plants. analysis of cell wall composition/structure in these plants found reduced xylose contents in the mutants compared to wildtype. to summarize, the available evidence supports the notion that the plant cell wall is an integral component contributing to pathogen response mechanisms and illustrates the influence of cell wall defects on infection. specific signaling cascades seem to mediate the response to particular cell wall defects, which in turn affect the response to necrotrophic or biotrophic pathogens. more importantly the data presented above allow correlation between certain types of cell wall defects, and not only signaling cascades but also resistance phenotypes. mutations in cesa4, 7, 8, agg1, 2, and agb1 affect resistance to necrotrophs and are independent of phytohormone-based signaling cascades. pmr5, 6 plants exhibit resistance to biotrophs and also do not rely on phytohormone-based signaling cascades. pmr4 affects resistance to biotrophs and resistance depends on the integrity of the sa signaling cascade. the cell wall composition/structure changes could prevent pathogen colonization simply because the infection machinery of the pathogen is too specialized to breach the chemically modified cell wall. another option is that the cell wall mutants cause defects similar to those occurring during infection by particular pathogens, i.e., simulate infection by necrotrophs or biotrophs. this would cause early/constant activation of the cwi maintenance/defense mechanism, which primes plant immunity thus making successful infection more difficult. the latter would explain both the specificity of the responses observed and dependence on particular signaling mechanisms. therefore, studies focusing on the effects of particular cell wall defects on pathogen resistance and the mode of action of the cwi maintenance mechanism could facilitate research into biotic stress response. the reason being, that by removing the potentially multiple effects of the pathogen during infection, they reduce the complexity of the interaction and should therefore allow novel insights into the mechanisms responsible for detection of infection and/or physical damage. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

plant cell walls provide structural support during development and represent together with the cuticle the first line of defense against biotic and abiotic stress. in recent years, evidence has accumulated that a dedicated plant cell wall integrity (cwi) maintenance mechanism exists. this mechanism monitors and maintains functional integrity of the cell wall during different biological processes. the available data suggest that it may represent a component of the stress response mechanisms underlying biotic and abiotic stress responses, which has not been identified previously as a distinct mechanism. here i will review the available evidence regarding the mode of action of the cwi maintenance mechanism and discuss its role in the context of biotic plant stress response mechanisms.

PMC3355559

pubmed-54

acute patellar dislocation is a common injury usually associated with a significant traumatic mechanism resulting in lateral displacement. vertical axis rotation following dislocation is a rare variant of this type of injury and can prevent closed reduction in the acute setting. a 32-year old gentleman presented with an irreducible patella dislocation following an unusual atraumatic mechanism. following attempts at closed reduction under sedation and regional nerve block, eventual open reduction and soft tissue reconstruction was required under general anesthetic. during the open reduction procedure, it was noted that the patella had dislocated into a lateral extra-articular position and rotated around its vertical axis. a review of the literature suggests dislocations such as the current presentation, are extremely rare and although have been described to occur with minor trauma, have never been described to occur following a largely atraumatic event. in such cases, closed reduction may be impossible even with adequate analgesia due to patella position and soft tissue obstruction. acute patellar dislocation is defined as the abrupt disruption in the relationship of the patella within the femoral groove. patellar dislocation is a relatively common occurrence and is usually managed in the emergency department with closed manipulation under sedation. however, when the patella itself rotates around its vertical axis during dislocation, surgical intervention is usually required. a 32-year old gentleman was admitted to our trauma department with a painful, immobile right knee. he sustained the injury externally rotating his knee whilst in slight flexion while he was moving from the driver s seat to the passenger s seat of his car. the first event was 15 years prior, when his knee twisted in flexed position while pushing a car. the second event was four years prior, when he sustained an injury while pushing a van, this time with the right knee fully extended. on this occasion, our patient had no other significant past medical or surgical history and explained his right knee was completely asymptomatic prior to this current event. on examination, there was an obvious deformity suggesting lateral displacement of the patella with a sulcus in the skin evident over the femoral groove. furthermore, the knee was held fixed in 15 degrees of flexion. due to patient positioning difficulties we were unable to obtain true ap and lateral views on x-ray. the radiographs demonstrated a laterally displaced and a mal-rotated patella in the vertical plane (see figs. 1 and 2). lateral radiograph of the right knee demonstrating rotation of patella our patient was subsequently taken to theatre where one final attempt of closed reduction was carried out under general anaesthetic and muscle relaxation. ultimately, this failed and an open reduction was performed. complete rupture of the medial patellofemoral ligament (mpfl) was identified with the patella situated lateral to the lateral femoral condyle, everted by approximately 100 degrees. the knee joint was washed out with normal saline and the medial patella retinaculum was repaired. after the repair, post-operative radiographs confirm the patella in a satisfactory position in the antero-posterior and lateral planes (see figs. 3 and 4). the patient was placed in an extension splint for comfort purposes immediately post-operatively. early mobilization was encouraged after 5 days and the patient was referred for early physiotherapy. at 3 months follow-up, our patient had no further episodes of dislocation, full range of knee extension and flexion, and normal patella tracking. a hypermobility assessment at this stage revealed a beighton score of 2 with extension beyond 10 degrees of both elbows only. intraoperative antero-posterior florous copy image following successful reduction lateral radiograph of the right knee following surgery in knee splint patellar dislocations can vary widely in their pattern of presentation however vertical axis rotation complicates only a small number of these cases. in 1844, cooper described the earliest case of patellar dislocation with vertical axis rotation. since then, only a handful of case reports describing vertical axis rotation of the patella have been published with the majority describing the location of the patella within the femoral trochlea (intra-articular) [2-11]. our case is rare as our patient had a less common variant of extra-articular patellar dislocation complicated by vertical axis rotation and by the fact, that the causative mechanism was largely atraumatic. previous descriptions postulate that the prominence of the lateral femoral condyle acts as a pivot point, which can cause the patella to rotate around on its vertical axis. in this case, however, the patient denies any trauma, and instead describes externally rotating the leg whilst extended as the mechanism of injury. it is almost certain that the previous dislocations in our patient s case had resulted in significant damage of the mpfl. with the patient reporting his injury taking place with the knee slightly flexed, the function of the mpfl in this case as the primary medial restraint during the first 20 degrees of flexion was likely to be inadequate. previous authors have described similar cases of extra-articular lateral patellar dislocation with vertical axis rotation and have noted in their subsequent reviews of the limited literature that intra-aritcular dislocations were usually related to far more significant trauma than extra-articular dislocations [3-10]. this was also the finding in a previous report highlighting the relative minor force resulting in an extra-articular dislocation. we feel this may be an indication of the chronic deficiency in the mpfl likely contributing to cases of extra-articular dislocation. on reviewing the previous cases of extra-articular dislocation in the literature, none had described a completely atraumatic mechanism and none had discussed previous dislocations or previous surgical history in their patients. detailed imaging can be difficult to obtain in the acute scenario; however in these cases ct imaging may clarify patella position and demonstrate mechanical engagement of the patella on the lateral femoral condyle. in one case report, ct imaging was utilized and confirmed avulsions of the vastusmedialis muscle and medial crus of the patella tendon with resultant impaction of the medial border on the patella onto the lateral femoral condyle. in this case, imaging led to the decision for open reduction being required, minimizing further attempts at closed reduction which could in theory result in further damage. it is difficult to be absolutely sure why attempts at closed reduction had failed in our particular case, but this is likely to be multifactorial. vertical axis rotation results in impaction of the lateral edge of the patella on the lateral femoral epicondyle which may result in a corresponding femoral defect resulting in a lodging effect. furthermore patient factors, namely obesity and large muscle mass around the knee made manipulation technically difficult in our case. open reduction under general anaesthetic is rarely necessary for routine patellar dislocations however there are some obvious advantages. these include; the possibility of direct visualisation of the obvious damage to the soft tissue structures contributing to or resultant from the dislocation. in our case it was likely the mpfl had been damaged previously and repair is likely to help in restoring central patella tracking and overall stability. in this case, the patient was noted as having a shallow trochlea which might have accounted for his increased tendency to dislocations. direct visual inspection may also help determine patients who are at risk of recurrent dislocations due to factors relating to their bony morphology and allow the surgeon to plan further management accordingly. lastly, visualisation of the articulating surfaces allows the surgeon to identify and address any osteochondral defects and corresponding loose bodies which may otherwise have contributed to long term future problems requiring eventual surgical intervention. from our experience with this case, difficulty in reducing a dislocated patella should alert surgeons to the possibility of an extra-articular dislocation with possible vertical axis rotation. in these scenarios, multiple attempts at reduction we were able to achieve adequate analgesia and sedation using local and regional anaesthesia, without successful reduction of the dislocation. although some authors have demonstrated successful closed reduction with the use of local anaesthetic and muscle relaxants, this is not always effective. furthermore, attempted closed reduction under general anaesthesia in our case was also unsuccessful, demonstrating how closed reduction can be futile in certain cases. we would recommend a low threshold for open reduction in such cases by a specialist knee surgeon who may assess and reconstruct damaged soft tissue damage in one operation vertical axis rotation is a rare entity complicating patella dislocations. in this rare event emergency physicians should be aware of this complication and refer to the relevant specialist for open reduction under general anaesthesia in order to minimize patient discomfort and potential articular surface damage.

introduction: acute patellar dislocation is a common injury usually associated with a significant traumatic mechanism resulting in lateral displacement. vertical axis rotation following dislocation is a rare variant of this type of injury and can prevent closed reduction in the acute setting. case presentation: a 32-year old gentleman presented with an irreducible patella dislocation following an unusual atraumatic mechanism. following attempts at closed reduction under sedation and regional nerve block, eventual open reduction and soft tissue reconstruction was required under general anesthetic. during the open reduction procedure, it was noted that the patella had dislocated into a lateral extra-articular position and rotated around its vertical axis. following patella reduction, the medial patellar retinaculum was repaired. examination under anesthetic revealed satisfactory tracking of patella following repair. conclusion:a review of the literature suggests dislocations such as the current presentation, are extremely rare and although have been described to occur with minor trauma, have never been described to occur following a largely atraumatic event. in such cases, closed reduction may be impossible even with adequate analgesia due to patella position and soft tissue obstruction. open reduction is essential in these situations.

PMC4845415

pubmed-55

in recent years, the concept of minimal intervention (mi) has prevailed in dentistry. therefore, the importance of diagnosing caries at an early stage has increased. in conventional procedures, the diagnosis of caries has mainly consisted of visual inspection and tactile assessment with probing. however, lussi reported that the sensitivity of detecting caries was 0.62 by visual inspection and 0.82 by probing. in addition, the pressure of probing can damage the demineralized fissure and increase the risk that caries progress [3, 4]. to promote mi, diagnosis without a probe has been recommended. the laser fluorescence-based caries detection device diagnodent (kavo, germany) has been introduced as an alternative. however, no single detection method for caries is sufficient; therefore, the combination of some detection methods has been recommended [57]. recent improvements in the personal computer have made the process of digital imaging more efficient and convenient. if the shape of caries can be quantified, and the relationship between the numerical value and the condition of the lesion can be demonstrated, this information would be helpful to diagnose dental caries. the authors of this paper have previously shown that the fractal dimension and proportion of the area of pit-and-fissure discoloration to the area of occlusal surface obtained by digital imaging were significantly correlated with the depth of the caries and the diagnodent values in extracted teeth. for assessment of the method as a diagnostic system, the ability of the diagnosis, such as the sensitivity, the specificity, and the accuracy, should be researched in clinical situation. the aim of this study was to assess the possibility of the clinical application of the diagnosis of occlusal caries using digital imaging by examining the sensitivity, the specificity, and the accuracy in comparison with the diagnodent values and the dentists ' diagnoses. one hundred teeth (36 premolars and 64 molars) with pit-and-fissure discoloration from 19 outpatients were examined at the clinic of oral diagnosis and general dentistry, dental hospital, tokyo medical and dental university. the occlusal surface of each tooth was washed with the robinson brush to remove dental plaque without any abrasive paste. then, pit-and-fissure discoloration was dried by air and measured three times using diagnodent. the mean scores were used as the diagnodent values of the teeth (dd). next, the occlusal surface of each tooth was photographed as large as possible with an intraoral digital camera (penscope, morita, japan). each image was stored in a personal computer using a video capture interface (pc-mdvd/u2, buffalo, japan). without knowing the dd, a dentist preliminarily diagnosed each tooth using visual inspection and tactile examination to decide which treatment plan would be appropriate (preventive or operative). the clinical diagnosis of preventive treatment for teeth was classified as co. on the other hand, carious lesions requiring operative treatment were removed in a conventional clinical way. if the resulting cavity preparation was limited in the enamel, then the clinical diagnosis was classified as c1. if the resulting cavity preparation reached the dentin, and sound tissue still remained between the cavity and the pulp chamber, then the clinical diagnosis was classified as c2. five dentists ranging from 3 to 15 years of professional experience examined the teeth after calibration of the criteria of the caries assessment conducted before the study; the calibration was done as follows; first, the five dentists examined 30 extracted teeth and decide which treatment plan would be appropriate (preventive or operative). at that time then, the teeth were sliced pallarel to the teeth axis and to the depth of lesion for each tooth was determined. at last, the dentists discussed to accord the treatment planning for each tooth referring the depth of the lesion. the digital photographs obtained were processed and analyzed using image analysis software (image j, nih, usa). first, each image was converted to an 8-bit gray-scale image, in which the density of grayness of each pixel was linearly scaled from min 0 (black) to max 255 (white). then, the occlusal surface in the image was isolated from the background using a density histogram of the image, and the area was measured. pit-and-fissure discoloration was also isolated from the occlusal surface using the density histogram, and the area was measured. the proportion of the area of pit-and-fissure discoloration to the area of the occlusal surface was calculated (pa). next, the image of the isolated pit-and-fissure discoloration was converted into a binary image, in which the density of pit-and-fissure discoloration was 0, and its background was 255, followed by calculating the fractal dimension of pit-and-fissure discoloration (fd). differences in fd, pa, and dd between each clinical diagnosis were analyzed using two-way anova and games-howell test to reveal the clinical diagnosis and the effect of the examining dentists. the correlation between the clinical diagnosis and each fd, pa, and dd was analyzed using spearman's correlation coefficient. discriminant formulas were obtained using discriminant analysis with the treatment plan (preventive/operative) as the objective variable and fd, pa, and dd as explanatory variables. sensitivity and specificity were calculated by applying fd, pa, and dd to each discriminant formula. the accuracy, ratio of the number of teeth showing accordance between the treatment plan decided by the dentists and the predictive treatment plan decided using the discriminant formula to the number of all the teeth, was also obtained. the entire process was approved by the ethics committee of the faculty of dentistry, tokyo medical and dental university (no. 317). fd, pa, and dd values corresponding to each clinical diagnosis are shown in table 1. the two-way anova revealed that the fd, pa, and dd were different among the clinical diagnosis (p<.01). on the other hand, the difference of the examining dentists did not affect the fd, pa, and dd. spearman's correlation coefficients between the clinical diagnosis and each fd, pa, and dd were 0.743, 0.700, and 0.652, respectively (p<.01). there were also significant correlations among fd, pa, and dd (p<.01). table 2 shows the discriminant formula, sensitivity, specificity, and accuracy of each explanatory variable. based on the discriminant formula of each explanatory variable, the thresholds of fd, pa, and dd between preventive and operative treatments were 1.20, 0.012, and 28.8, respectively. the sensitivity of fd was greater than that of pa, dd, and the combination of fd and pa. the specificity of pa was greater than that of fd, dd, and the combination of fd and pa. the accuracy of the combination of fd and pa was greater than that of fd, pa, and dd. previously, it was reported that the fractal dimension and the proportion of the area of pit-and-fissure discoloration to the area of occlusal surface were significantly correlated with the depth of the caries and the diagnodent values in extracted teeth. in this study, the fractal dimensions for c0, c1 and c2 in the former study were 0.97, 1.30, and 1.52, respectively. these results indicate that image analysis of molar pit-and-fissure discoloration was clinically useful for the diagnosis of caries. an increase of the proportion of the area of discoloration corresponded to a change of the volume of caries lesion, while an increase of the fractal dimension corresponded to a change of the shape of the lesion caused by caries progression. a fractal is a geometric shape, possessing characteristics of self-similarity or self-affinity, and widely observed in nature [10, 11]. recently, fractals have been in the spotlight in the field of medicine, and research has been introduced regarding its use in the field of diagnosis [1214]. for example, a point is described as the zero dimension; a straight line is described as the first dimension, and a plane is described as the second dimension. such decimal dimensions can be obtained by expanding the definition of the dimension as the rate at which the perimeter (or the surface area) of an object increases, and the measurement scale is reduced. several ways to measure the fractal dimension have been introduced. in this study, the authors used a simple way to determine the fractal dimension called box counting. in this method, a grid of squares is placed over the object, and the number of squares through which any part of the object passes is counted. this process is repeated with different grids having different sizes. the number of squares placed over the object versus the length of the side of the square are then plotted on log-log scale. when a regression line is obtained from the plots, the degree of uneven complexity of a boundary or a coast can be quantified using this approach. in this research, the fractal dimension of discoloration increased from 0.8 to 1.6 as the depth of the caries increased, which corresponded to a change in the shape of the discolored area from a point or a line to an area based on the progression of the caries. generally, the addition of valuables into discriminant formula is one of the ways to improve the accuracy, however, in this study, the accuracy of the combination of fd and pa was similar to that of single fd. therefore, further study to find other valuables is needed to improve the accuracy of this method by combination of valuables. thus, the accuracy of the diagnosis of occlusal caries using digital images of discolored areas was comparable to that of diagnodent; therefore, its clinical application as a diagnostic tool is possible. because this study was clinical, the final diagnosis of each examined tooth was not confirmed by a histological procedure but by a dentist's clinical examination. as mentioned above,, the results of two-way anova showed that the effect of the examining dentist on the dd, pa, and fd values was not significant. therefore, we considered that difference of the diagnosis among the dentists would be small. in the present study, to examine the possibility of digital imaging in the diagnosis of occlusal caries, diagnodent was used as a comparative pre-existing dental caries detection tool. there are other caries detection tools, such as fiber optic transillumination (foti), digital imaging fiber optic transillumination (difoti), quantitative laser or light fluorescence (qlf), and electrical conductive measurements (ecm). foti, difoti, and qlf have been tested in vivo, however, the number of clinical studies has still been small [15, 16]. on the other hand, a comparatively long time has passed since diagnodent was introduced to the market, and many findings have been reported. sheehy et al. reported that diagnodent had greater sensitivity and specificity than ecm. the correlation coefficient between diagnodent readings and the depth and the volume of caries lesions was reported to be 0.47. several researches have pointed out that diagnodent measurements are affected by other factors, such as hypomineralization, plaque, debris, staining and wetness [1921], while a high correlation between inter and intraobserver agreements was also mentioned [22, 23]. we employed diagnodent as a comparison because the diagnosis was provided as a number, the handling was easy, and, moreover, its clinical use has been discussed in other studies. in the present study, diagnosis using digital images of pit-and-fissure discoloration depended on the statistical relationship between the shape of discoloration and the depth of caries. the shape of the discolored area in the occlusal surface is, however, possibly affected by medication history, individual history, and lifestyle. consequently, diagnosis using digital images of pit-and-fissure discoloration is rather experimental. additionally, the procedure can not be applied to colorless lesions, such as acute caries. therefore, diagnosis using digital images of pit-and-fissure discoloration should not be used for definitive diagnosis. rather, initial diagnosis, screening such as mass examination would be suitable because of its good sensitivity of fd and, the convenience of the procedure. actually, the core of the diagnostic system is digital imaging processing by computers. if computer programing for all procedures was achieved, then screening of hundreds of examinees would be automated after photograph taking, which would make mass examination less time-consuming with low cost. for such automated uses of the computer, the process of extracting colors from the image must be improved; in this study, the threshold for the colored area was decided one by one by an observer's visual inspection. how to calculate the fractal dimension is also open to discussion. we used the box-counting method attached in the image analysis software, image j. the box-counting method is only suitable for self-similar profiles, not for more general, self-affine cases, that is, the fractal dimension using the box-counting method might be the approximate value. a special computer program must be developed to measure the fractal dimension more accurately, for example, the minkowski method or the richardson method [8, 10]. as mentioned above, the shape of the discolored area on an occlusal surface is affected by many factors. therefore, the thresholds or the discriminant formulas acquired from this research are not universal. further research is needed to determine the discriminant formulas to diagnose caries using the image analysis of molar pit-and-fissure discoloration .

the aim of this study was to assess the possibility of digital image analysis of pit-and-fissure discoloration in order to diagnose caries. digital images showing pit-and-fissure discoloration in 100 teeth of 19 patients were analyzed to obtain the fractal dimension (fd) and the proportion of the area of pit-and-fissure discoloration to the area of occlusal surface (pa). diagnodent values were measured (dd), and dentists ' diagnoses were also obtained. the sensitivity and specificity of fd, pa, dd, and the combination of fd and pa compared to the dentists ' diagnoses were calculated. the sensitivities of fd, pa, dd, and the combination of fd and pa were 0.89, 0.47, 0.69, and 0.86, respectively, and the specificities were 0.84, 0.95, 0.91, and 0.86, respectively. although further research is needed for the practical use, it is possible to use the analysis of digital images of pit-and-fissure molar discoloration as a diagnostic tool.

PMC2836524

pubmed-56

polyarginine peptides are known as one of the widely used classes of cell-penetrating peptides (cpps) and cellular delivery tools. it has been reported that the presence of the guanidine group in the side chain of arginine plays a key role for improved ability of arginine-rich peptides to cross the cell membrane. various systematic structural investigations have been performed to determine the required number of arginine residues and the length of the peptide for the optimization of cellular uptake. short polyarginine peptides containing less than six arginine residues did not exhibit significant cellular uptake in several previously reported investigations. thus, the presence of more than six arginine residues in the structure of polyarginine peptides is critical for their efficient cell-penetrating functions. however, several investigations were conducted to increase the cellular uptake of polyarginines by attaching the fatty acid to the n-terminal of the peptide. it has been previously reported that the acylation of the n-terminal by fatty acids can facilitate the intracellular uptake of polyarginines. for instance, katayama et al. synthesized acylated octa-arginines and discovered that the introduction of hydrophobic fatty acid enhanced the intracellular uptake of octa-arginine peptide and its conjugated ubiquitin. furthermore, lee et al. designed a class of lipopeptides carrying 715 arginine residues. among them, myristoylated-hendeca-arginine (c14r11) was found to be the most efficient cell-penetrating peptide. however, the fatty acylated polyarginine peptides that contain 715 arginine residues can potentially cause toxicity, and they can be easily degraded by proteases. moreover, linear peptides carrying l-form are not stable in serum and therefore have a limited application for in vivo studies (figure s2, supporting information). replacing l-form amino acids with d-form to improve the peptide stability leads to high cost production. thus, the synthesis and development of cyclic cpps containing short amino acid sequence with less toxicity is desired. herein, we designed acylated cyclic polyarginine peptides (acpps) containing five arginine residues and investigated their ability as cell-penetrating peptides. we compared acpps with a corresponding acylated linear polyarginine peptide (alpp) and a nonacylated cyclic polyarginine as controls. we hypothesize that the combination of acylation and cyclization of short polyarginine peptides having less than six arginine residues will increase the intracellular uptake and can generate peptides with molecular transporter properties. for convenience, square brackets [] and parentheses () phosphopeptide ptyr-glu-glu-ile (pyeei) is an optimal peptide ligand for binding to the src tyrosine kinase sh2 domain. in this study, we used negatively charged fluorescein-labeled phosphopeptide f-gpyeei as a model cell-impermeable compound. all peptides were synthesized by fmoc/tbu solid-phase peptide synthesis strategy either manually or using rainin ps3 synthesizer (protein technologies inc.). manual reactions were carried out in a glass reaction vessel with a sintered glass frit by mixing under nitrogen bubbling at room temperature. fmoc-l-amino acid building blocks, fatty acids, and preloaded h-arg(pbf)-2-chlorotrityl resin were used as starting materials. 2-(1h-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (hbtu), hydroxybenzotriazole (hobt), and n, n-diisopropylethylamine (dipea) in n, n-dimethylformamide (dmf) were used as coupling and activating reagents, respectively, for manual synthesis. in the case of using peptide synthesizer, 0.4 m n-methylmorpholine in dmf was used instead of dipea. piperidine in dmf (20%) was employed to deprotect fmoc group at each step. to cleave the linear peptide from the resin, a mixture of trifluoroacetic acid (tfa)/triisoproylsilane (tis)/water (92.5/5/2.5, however, in the synthesis of cyclic peptides, the side chain protected linear peptides were first cleaved from the resin by using a cleavage cocktail, containing 2,2,2-trifluoroethanol (tfe)/acetic acid/dichloromethane (dcm) (2:1:7, v/v/v). cyclization reaction was performed by employing a mixture of 1-hydroxy-7-azabenzotriazole (hoat) and n, n-diisopropylcarbodiimide (dic) in anhydrous dmf/dcm for 12 h. after solvent evaporation, the peptide was deprotected and cleaved from the resin by using a cleavage cocktail reagent r, containing tfa/thioanisole/1,2-ethanedithiol (edt)/anisole (90:5:3:2, v/v/v/v) for 23 h. the crude peptides were precipitated and washed with cold diethyl ether. to purify the crude peptides, we used a reversed-phase high pressure liquid chromatography (rp-hplc) system using shimadzu lc-8a preparative liquid chromatography on a phenomenex gemini c18 column (10 m, 250 21.2 mm) with a gradient 0100% of acetonitrile (ch3cn) containing 0.1% tfa (v/v) and water containing 0.1% tfa (v/v) for 1 h with a flow rate at 15.0 ml/min at the wavelength of 214 nm. as a representative example, the synthesis of dodecanoyl-[r5] is described here. h-arg(pbf)-2-chlorotrityl resin (660 mg, 0.35 mmol, 0.53 mmol/g) was swelled in dmf for 40 min by n2. fmoc-arg(pbf)-oh (681 mg, 1.05 mmol, 3 equiv) was coupled to the n-terminal of the resin, using hbtu (398 mg, 1.05 mmol, 3 equiv), hobt (142 mg, 1.05 mmol, 3 equiv), and dipea (366 l, 2.1 mmol, 6 equiv) in dmf (15 ml) by agitating the resin for 1 h using n2. after the coupling, the resin was washed with dmf, followed by fmoc-deprotection with piperidine in dmf (20%). the subsequent three fmoc-arg(pbf)-oh couplings and one dde-lys(fmoc)-oh (559 mg, 1.05 mmol, 3 equiv; dde=1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl) coupling was carried out in the same manner, respectively. after removing the fmoc group in side chain of lysine, dodecanoic acid (210 mg, 1.05 mmol, 3 equiv) was coupled using hbtu, hobt, and dipea. then dde protection group at n-terminal of peptide was removed by 2% hydrazine in dmf, followed by washing with dmf and dcm. the side chain protected linear peptides were cleaved from the resin by using a cleavage cocktail, tfe/acetic acid/dcm (2:1:7, v/v/v), for 1 h. the filtrate was evaporated, and the residue was dried overnight in a vacuum. the cyclization was conducted under a dilute condition with anhydrous dmf/dcm (5:3, v/v, 250 ml), using hoat (190 mg, 1.4 mmol, 4 equiv) and dic (240 l, 1.54 mmol, 4.4 equiv), and stirred for 12 h under nitrogen atmosphere. after cyclization, the solvent was evaporated, and the side chain deprotection was carried out by the addition of reagent r for 2 h. the crude dodecanoyl-[r5] was precipitated and washed with cold diethyl ether and purified by preparative rp-hplc system as described above. fluorescein-labeled peptides were synthesized with the same protocol before the attachment of fatty acid. we used fmoc-12-aminododecanoic acid instead of dodecanoic acid, and after removing fmoc group, 5(6)-carboxyfluorescein diisobutyrate (cfdi) was attached using 7-azabenzotriazol-1-yl-oxytris(pyrrolidino)phosphonium hexafluorophosphate (pyaop), hoat, and dipea. as an example, we started fluorescein-labeled peptide synthesis in smaller scale with h-arg(pbf)-2-chlorotrityl resin (208 mg, 0.11 mmol, 0.53 mmol/g). fmoc-arg(pbf)-oh (214 mg, 0.33 mmol, 3 equiv), dde-lys(fmoc)-oh (176 mg, 0.33 mmol, 3 equiv), and fmoc-12-aminododecanoic acid (144 mg, 0.33 mmol, 3 equiv) were used to couple each building block to the resin using hbtu (125 mg, 0.33 mmol, 3 equiv), hobt (45 mg, 0.33 mmol, 3 equiv), and dipea (115 l, 0.66 mmol, 6 equiv) in dmf. cfdi (170 mg, 0.33 mmol, 3 equiv) was coupled with amino group of 12-aminododecaonic chain using pyaop (172 mg, 0.33 mmol, 3 equiv), hoat (45 mg, 0.33 mmol, 3 equiv), and dipea. after removal of dde protecting group with 2% hydrazine in dmf and washing with dmf and dcm, side chain protected fluorescein linear peptides were cleaved from resin using tfe/acetic acid/dcm (2:1:7, v/v/v). the molecular weights of final products were confirmed by an axima performance matrix-assisted laser desorption/ionization-time-of-flight (maldi-tof) mass spectrometer (shimadzu corporation). dodecanoyl-(r6): maldi-tof (m/z) c48h96n22o8 calcd. 1108.7781; found 1109.7308 [m+h]. f-dodecanoyl-(r5): maldi-tof (m/z) c69h107n23o14 calcd. 1481.8368; found 1482.7586 [m+h]. w-dodecanoyl-[r5]: maldi-tof (m/z) c61h107n25o9 calcd. human ovarian adenocarcinoma (sk-ov-3), leukemia (ccrf-cem), and embryonic kidney (hek 293 t) cells were purchased from american type culture collection. the sk-ov-3 and hek 293 t cells were grown in eagle s minimum essential medium (emem), and rpmi-1640 medium (atcc, manassas, va) was used for ccrf-cem cells in a humidified atmosphere of 5% co2 at 37 c. both media were supplemented with fetal bovine serum (fbs, 10%) and penicillin streptomycin solution (1%, 10,000 units of penicillin and 10 mg of streptomycin in 0.9% nacl). cytotoxicity of peptides was examined by mts proliferation assay in two human cancer cell lines (sk-ov-3 and ccrf-cem) and one human normal cell line (hek 293 t). cells were seeded into 96-well plates (sk-ov-3 (5 10 cells/well), ccrf-cem (1 10 cells/well), and hek 293 t (1 10 cells/well)). then, the cells were incubated with complete media (100 l) overnight. different concentrations (0600 m) of peptide solution (10 l) were added to cells and incubated at 37 c with 5% co2 for 24 h. then, celltiter 96 aqueous solution (20 l) was added to each well and incubated for 14 h. the absorbance was measured at 490 nm using microplate reader. sk-ov-3 cells were grown in 6-well plates (2 10 cells/well) with complete emem media 24 h prior to the experiment. the fluorescein-labeled peptide stock solution (1 mm) was prepared in water and diluted in gibco opti-mem i reduced serum medium to obtain the final concentration of 5 m. the media were removed, and the mixture containing fluorescein-labeled peptide solution (5 m) was added. after 1 h incubation, trypsin edta solution was added to detach cells from the plate s surface and remove cell surface binding peptides. after 5 min, the complete media (2 ml) were added to neutralize the trypsin. finally, the cells were analyzed by bd facscalibur or facsverse flow cytometer using fitc channel. 5(6)-carboxyfluorescein (fam) was used as a negative control. for examination of the cellular uptake mechanism of f-dodecanoyl-[r5] at low temperature, the assay was carried out at 4 c to inhibit the energy-dependent cellular uptake pathways. the sk-ov-3 cells were preincubated at 4 c for 15 min and incubated with the fluorescein-labeled peptide for 1 h at 4 c. cells were collected and analyzed using flow cytometry with the previously described protocol above. the data collected at 37 c were used as the control. to perform the atp-depletion assay, cells were incubated with sodium azide (10 mm) and 2-deoxy-d-glucose (50 mm) for 1 h before adding the fluorescein-labeled peptide. during the incubation time (1 h), the fluorescein-labeled bicyclic peptide (5 m) was prepared in the opti-mem i reduced serum medium in the presence of sodium azide (10 mm) and 2-deoxy-d-glucose (50 mm). then, the cells were incubated with this solution for 1 h. the following sample preparation and flow cytometry analysis protocol was the same as described above. sk-ov-3 cells were seeded with complete emem on coverslips in 6-well plate (1 10 cells/well) and kept until 50% confluency. the media were removed, and cells were incubated with f-dodecanoyl-[r5] (10 m) and f-dodecanoyl-(r5) (10 m) in gibco opti-mem i reduced serum medium (life technologies, grand island, ny) for 1 h at 37 c. then cells were washed with 1 phosphate buffered saline with calcium and magnesium (pbs) for three times. the coverslips were mounted on microscope slides, and images were obtained using carl zeiss lsm 700 system with a 488 nm argon ion laser excitation and a bp 505530 nm band-pass filter. sk-ov-3 and ccrf-cem cells were seeded in 6-well plates (2 10 cells/well for sk-ov-3 and 1 10cells/well for ccrf-cem) and grown with complete emem media (rpmi-1640 for ccrf-cem) overnight. a mixture of fluorescein-labeled phosphopeptides f-gpyeei (5 m) and peptides (10 m) were prepared in opti-mem i reduced serum medium at room temperature and incubated for 15 min. then the cells were incubated with the premixed solution at 37 c with 5% co2 for 1 h. the sample preparation for facs analysis was carried out by previously mentioned protocol described before. in this assay, the acylated cyclic polyarginine peptides were synthesized by fmoc/tbu solid-phase peptide synthesis method. fmoc-l-arg(pbf)-oh was coupled on h-arg(pbf)-loaded 2-chlorotrityl resin in the presence of hbtu, hobt, and dipea in dmf. then dde-lys(fmoc)-oh was attached, and a fatty acid was coupled to the side chain of lysine. dde protecting group was removed by 2% hydrazine in dmf, and a cleavage cocktail containing tfe/acetic acid/dcm (2:1:7 v/v/v) was used for 1 h to cleave the side-chain protected linear peptides from the resin. cyclization of linear peptides was carried out in the presence of a mixture of hoat and dic in anhydrous dmf/dcm for 624 h. the side-chain deprotection of cyclic peptide was carried out by a cleavage cocktail reagent r for 2 h. the crude peptides were precipitated and purified with rp-hplc as described above. as a representative example, the synthesis of dodecanoyl-[r5] is shown in scheme 1. a corresponding acylated linear polyarginine peptide (alpp) was synthesized for comparative studies with the cyclic peptide (acpp). moreover, a cyclic polyarginine without fatty acid [r5] was also synthesized to investigate the effect of the fatty acid on cyclic peptide and its effect on molecular transporting efficiency. to investigate whether the peptide alone can enter into cells, fluorescein-labeled f-dodecanoyl-[r5] and f-dodecanoyl-(r5), where f=fluorescein, were synthesized for facs and microscopy investigations. chemical structures of synthetic peptides used in this study (f, fluorescein-labeled; [], cyclic peptide; (), linear peptide). the cytotoxicity of all peptides were tested in two different cancer cell lines, adherent (sk-ov-3) and nonadherent (ccrf-cem) cells, and a normal cell line (hek 293 t) using mts assay (figure 2). cyclic polyarginine [r5] without fatty acid was used to explore the effect of n-terminal acylation on cytotoxicity and cellular uptake. alpp (dodecanoyl-(r5)) and [r5] peptides showed consistently less cytotoxicity in all three cells compared to acpps (dodecanoyl-[r5] and dodecanoyl-[r6]). after 24 h incubation, acpps showed approximately 20% toxicity in cells at a concentration of 25 m in ccrf-cem cells. however, dodecanoyl-linear (r5) and [r5] did not exhibit significant cytotoxicity at 25 m and showed less than 20% toxicity at the concentration of 100 m. in sk-ov-3 cells, dodecanoyl-(r5) and [r5] showed more than 80% cell viability at the concentration of 25 m. in normal cells (hek 293 t), all peptides exhibited less than 5% toxicity at 25 m. this differential behavior of the peptides in normal, and cancer cells can be possibly rationalized through the interaction between polyarginine peptides and cell membranes. the membrane of cancer cells holds more negative charges compared to that in normal cells because of the presence of anionic lipids such as phosphatidylserine. therefore, polyarginine peptides can be interacted with cancer cells more effectively compared to normal cells. these data indicated that acpps are more toxic than alpp and a nonacylated cyclic peptide [r5], especially at concentration of 25 m. thus, a noncytotoxic concentration of 510 m was used in cell-based assays. comparison of cytotoxicity between cyclic and linear acylated polyarginine peptides and nonacylated cyclic peptide [r5] at various concentrations against ccrf-cem, sk-ov-3, and hek 293 t after 24 h. the intracellular uptake studies of fluorescein-labeled acylated cyclic and linear pp, f-dodecanoyl-[r5] and f-dodecanoyl-(r5), was carried out in sk-ov-3 cells by using flow cytometry and confocal laser scanning microscopy (clsm) methods. fluorescein (fam, f) alone was selected as a negative control. as it is shown in figure 3, the f-dodecanoyl-[r5] and f-dodecanoyl-(r5) showed approximately 13.7- and 10.3-fold higher cellular uptake than that of control 5,6-carboxyfluorescein (fam), respectively, in sk-ov-3 cells. the cellular uptake of f-dodecanoyl-[r5] was confirmed by clsm images (figure 4). f-dodecanoyl-[r5] showed higher fluorescence intensity compared to that of f-dodecanoyl-(r5) in sk-ov-3 cells. therefore, acpp f-dodecanoyl-[r5] was found to be a more efficient cell-penetrating peptide compared to its linear counterpart. as shown in figure 4, the fluorescence signal is extended through the whole cells, suggesting that f-dodecanoyl-[r5] can get localized in the nucleus as well as cytoplasm. comparative cellular uptake of f-dodecanoyl-[r5] and f-dodecanoyl-(r5) (5 m) in sk-ov-3 cells (1 h). confocal laser scanning microscope image of (a) f-dodecanoyl-[r5] and (b) f-dodecanoyl-(r5). the peptides were incubated for 1 h in sk-ov-3 cells at 10 m concentration. the mechanism of the cellular internalization of f-dodecanoyl-[r5] was investigated by a temperature control assay at 4 c along with atp depletion assay. these two assays have been widely used to examine the energy-dependent endocytosis. facs results showed that the intracellular uptake of f-dodecanoyl-[r5] was significantly reduced at 4 c, indicating that the mechanism of internalization was mainly dependent on the endocytosis pathways (figure 5). furthermore, atp depletion assay was performed to investigate receptor-mediated endocytosis. to induce atp depletion, sk-ov-3 cells were preincubated with sodium azide (10 mm) and 2-deoxy-d-glucose (50 mm) for 1 h prior to the experiment, and a similar concentration was maintained during the incubation (1 h) with f-dodecanoyl-[r5]. the results showed that the cellular uptake of f-acpp was inhibited in the presence of sodium azide and 2-deoxy-d-glucose, suggesting that receptor-mediated endocytosis is involved for the cellular uptake of f-acpp. in atp depletion assay, the basic cellular uptake of fam was higher compared to that in temperature control assay. however, this is evident that the intracellular uptake of f-dodecanoyl-[r5] was inhibited, and there was no significant difference between the cells treated with fam compared to those treated with f-dodecanoyl-[r5] in temperature control and atp depletion assays, suggesting that endocytosis is the major pathway for the cellular uptake of f-dodecanoyl-[r5]. cellular uptake of f-dodecanoyl-[r5] (5 m) in sk-ov-3 cells in temperature control assay at 37 and 4 c, and atp depletion assay with nan3 (10 mm) and 2-deoxy-d-glucose (50 mm) analyzed by flow cytometry. cells with no treatment were used as control. the ability of acpps as a molecular transporter was evaluated and compared by selecting a fluorescein-labeled phosphopeptide, f-gpyeei, as a molecular cargo. phosphopeptide, pyeei (ptyr-glu-glu-ile) is an optimal peptide template for the sh2 domain of src tyrosine kinase. several analogues of this peptide have been synthesized as potent ligands for this target. because of the presence of the negatively charged amino acid residues in the structure of the peptide including phosphorylated tyrosine moreover, the internalization of the negatively charged phosphopeptide in cancer cells by diffusion is more difficult because cancer cell membranes are composed of more negatively charged lipids. thus, cellular delivery of cell-impermeable negatively charged phosphopeptides is significantly challenging. we have previously reported different peptide-based carriers for the intracellular delivery of negatively charged phosphopeptides as model cell-impermeable drugs in several cell lines. in this study, the intracellular uptake of f-gpyeei was monitored in the presence and absence of synthetic peptides after 1 h incubation by flow cytometry. as it is exhibited in figure 6, the acpps (dodecanoyl-[r5] and dodecanoyl-[r6 ]) delivered the phosphopeptide more efficiently compared to alpps, dodecanoyl-(r5) and -[r5]. the intracellular uptake of f-gpyeei in the presence of dodecanoyl-[r5] and dodecanoyl-[r6] was enhanced by 3.4- and 5.5-fold higher than the uptake in the absence of acpps. however, dodecanoyl-(r5) and -[r5] only improved 1.3- and 1.4-fold intracellular uptake, respectively. the results showed that acylated and cyclized polyarginine peptides can deliver the phosphopeptide effectively. however, the intracellular uptake of the phosphopeptide did not improve significantly in the presence of either acylated linear polyarginine peptide or cyclic [r5]. these data suggest that a combination of acylation and cyclization would improve the molecular transporting efficiency of the polyarginine-based peptide (containing less than six arginines) for the intracellular delivery of a cell-impermeable phosphopeptide. this has been previously reported that the acylated linear octa-arginine increased the cellular uptake of molecular cargoes by just adding fatty acid to the n-terminal of octa-arginine. however, we discovered that both cyclization and acylation in a short penta-arginine can significantly improve the delivery of a cell-impermeable phosphopeptide in sk-ov-3 cells. cellular uptake of f-gpyeei (5 m) in the presence of dodecanoyl-[r5] and -[r5], dosecanoyl-(r5), and dodecanoyl-[r6] (10 m) in sk-ov-3 cell line. phosphopeptide delivery efficiency of dodecanoyl-[r5] were compared with known cpps (r7, crrrrrrr; tat, ygrkkrrqrrr). the major driving forces for the intracellular delivery are presumed to be structural rigidity through cyclization of the peptide and the interaction of the fatty acid with the cell membrane. it has been previously reported that the cellular uptake of the peptide can be increased due to the structural rigidity by cyclization of arginine-rich peptides. they proposed that the maximal distance between guanidine groups of arginine residue can lead to an efficient transduction of arginine-rich peptides. our investigations showed that dodecanoyl-[r6] is able to deliver more efficiently by 1.6-fold higher f-gpyeei uptake compared to that of dodecanoyl-[r5]. increasing the number of positively charged arginine residues can enhance the cellular uptake through ionic interactions with the negatively charged phosphopeptide and/or phospholipid in the cell membrane through ionic interactions. however, the higher number of arginine residue is not the only responsible element for the efficient cellular internalization. for example, it has been reported that polyarginine containing 11 amino acids (r11) showed higher cellular uptake compared to the polyarginine containing 13 amino acids (r13). at the same time, r11 was found to be a more potent transporter compared to r9 in prostate cancer cells. these investigations showed that an optimal number of arginine residues are required for the highest degree of functionality. however, the greater number of amino acid residues in cyclic peptides can decrease the structural rigidity, which lowers the ability of the peptide to get into cells. dodecanoyl-[r5] was also compared with several commonly cpps, such as cr7 and tat (ygrkkrrqrrr) peptides. the acpp improved the cellular uptake of the phosphopeptide by 1.4- and 1.8-fold higher than those of cr7 and tat, respectively (figure 6). these results revealed that although other peptides containing arginine can deliver the molecular cargo, acpp dodecanoyl-[r5] with a shorter peptide sequence than cr7 and tat can work as a molecular transporter with higher efficiency. chemical structures of acpps with different length of fatty acid chains (c8, c12, and c16). to investigate the effect of the chain length on the cell penetration potency, we synthesized octanoyl-[r5], dodecanoyl-[r5], and hexadecanoyl-[r5] (figure 7). acpps showed less than 20% toxicity in cells at the concentration of 25 m (figure 8a). the in vitro toxicity results showed that increasing the fatty acid chain length caused enhanced toxicity in cells as hexadecanoyl-[r5] was more cytotoxic than dodecanoyl-[r5] and octanoyl-[r5]. these data indicate that the fatty acid chain length could alter the interaction with the cell membrane and disturb the membrane integrity. on the basis of the cytotoxicity data, (a) cytotoxicity assay of cyclic polyarginine peptide-fatty acid conjugates against sk-ov-3 cells (24 h incubation). (b) cellular uptake of a phosphopeptide, f-gpyeei (5 m), in the presence of peptide-fatty acid conjugates (10 m) in sk-ov-3 cells. it has been previously reported that there was a relationship between the length of fatty acid in polyarginines and their cellular uptake, meaning that the optimal length of fatty acid is required for optimal functionality based on the peptide sequence and cell type. the results exhibited that the cellular uptake of f-gpyeei was improved in the order of octanoyl-[r5]<dodecanoyl-[r5]<hexadecanoyl-[r5] (figure 8b). the cellular uptake of f-gpyeei in the presence of hexadecanoyl-[r5] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[r5] and dodecanoyl-[r5], respectively. these data suggest that the length of the attached fatty acid chain in the structure significantly influences the efficiency of the peptide as a molecular transporter. sixteen-carbon chain length (c16) was found to be an optimized length for the intracellular delivery of f-gpyeei in sk-ov-3 cells. after acpps were found to act as cpp and molecular transporter, a systematic investigation was performed to modify the fatty acid to another hydrophobic moiety. w4-[r5], the whole fatty acid chain was replaced with four tryptophan residues. the second conjugate w-dodecanoyl-[r5] had one more tryptophan at the end of the dodecanoyl fatty acid chain. w4-[r5] enhanced intracellular delivery of f-gpyeei by 4.1-fold higher compared to that of dodecanoyl-[r5] in sk-ov-3. however, w-dodecanoyl-[r5] improved the uptake of f-gpyeei by 1.3-fold higher compared to that of w4-[r5] in ccrf-cem cells (figure 9). thus, it is not straightforward to compare the cellular uptakes in sk-ov-3 and ccrf-cem. however, the results could be assessed indirectly by relative comparison with the cellular uptake by w4-[r5]. these data suggest that the presence of hydrophobic tryptophan moieties could enhance the molecular transporting efficiency. in other words, appropriate modification of hydrophobic moiety in cpps can increase the drug delivery ability of acpp. thus, cyclic nature, short length of polyarginine, and hydrophobic segments were found to be critical elements to generate a peptide with an optimized molecular transporter efficiency. cellular uptake assay of a phosphopeptide, f-gpyeei (5 m), in the presence of w4-[r5] and w-dodecanoyl-[r5] (10 m) against sk-ov-3 and ccrf-cem cell lines (1 h incubation). cells with no treatment were used as control. to evaluate whether the presence of peptides can enhance the pharmacological effect of a molecular cargo, the antiproliferative activity of doxorubicin (dox) was examined in the presence of dodecanoy-[r6] ([ r6]-c12) in mcf-7 cells in a time-dependent assay. as it is shown in figure 10, the antiproliferative potency of dox (5 m) was enhanced by 7%, 11%, and 14% in the presence of [r6]-c12 (5 m) when compared with that of the drug alone after 24, 48, and 72 h incubation, respectively. these results showed that the cell proliferation of mcf-7 cells was inhibited in a time-dependent manner suggesting a sustained drug release by peptide leading to a higher inhibitory effect. the presence of the peptide possibly improved the cellular uptake of dox and enhanced the antiproliferative activity. time-dependent antiproliferative activity of dox (5 m) in the presence and absence of [r6]-c12 (5 m). in conclusion, acylated cyclic polyarginine peptides were synthesized and examined as cpps and potential molecular transporters. acpps showed higher potency as molecular transporter compared to the corresponding linear counterpart and cyclic polyarginine without fatty acid. the mechanism of the peptide internalization was found to be energy-dependent endocytosis. cyclization and acylation reactions on the structure of the peptide enhanced the intracellular uptake of polyarginine peptides although they carry a short length of sequence. this intracellular delivery property of acpps can be optimized by modifying the length of fatty acid chain. to the best of our knowledge, this is the first report of cyclic fatty acylated polyarginine peptide as molecular transporter of a cell-impermeable phosphopeptide. this study provided insights about how a combination of the cyclic nature and acylation can improve the cell internalization of polyarginines. further investigations are undergoing to determine whether conjugation of cell-impermeable hydrophobic drugs to acylated cyclic polyarginines can be an efficient method for designing novel drug delivery systems.

many of the reported arginine-rich cell-penetrating peptides (cpps) for the enhanced delivery of drugs are linear peptides composed of more than seven arginine residues to retain the cell penetration properties. herein, we synthesized a class of nine polyarginine peptides containing 5 and 6 arginines, namely, r5 and r6. we further explored the effect of acylation with long chain fatty acids (i.e., octanoic acid, dodecanoic acid, and hexadecanoic acid) and cyclization on the cell penetrating properties of the peptides. the fluorescence-labeled acylated cyclic peptide dodecanoyl-[r5] and linear peptide dodecanoyl-(r5) showed approximately 13.7- and 10.2-fold higher cellular uptake than that of control 5,6-carboxyfluorescein, respectively. the mechanism of the peptide internalization into cells was found to be energy-dependent endocytosis. dodecanoyl-[r5] and dodecanoyl-[r6] enhanced the intracellular uptake of a fluorescence-labeled cell-impermeable negatively charged phosphopeptide (f-gpyeei) in human ovarian cancer cells (sk-ov-3) by 3.4-fold and 5.5-fold, respectively, as shown by flow cytometry. the cellular uptake of f-gpyeei in the presence of hexadecanoyl-[r5] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[r5] and dodecanoyl-[r5], respectively. dodecanoyl-[r5] enhanced the cellular uptake of the phosphopeptide by 1.42.5-fold higher than the corresponding linear peptide dodecanoyl-(r5) and those of representative cpps, such as hepta-arginine (cr7) and tat peptide. these results showed that a combination of acylation by long chain fatty acids and cyclization on short arginine-containing peptides can improve their cell-penetrating property, possibly through efficient interaction of rigid positively charged r and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids.

PMC4144761

pubmed-57

as of 2014, the worldwide prevalence of type 2 diabetes mellitus (t2 dm) was estimated to be 9% among adults aged 18 years with great impact on mortality, particularly in low- and middle-income countries (lmic) [1, 2]. moreover, globally, approximately 25% to 75% of diabetes cases remain undiagnosed [3, 4], until further complications, especially at the macro- and micro-vascular level, manifest clinically. in latin america, an important strategy to prevent or delay t2 dm complications is the early identification of those with undiagnosed diabetes; yet, universal screening for diabetes at the population level is not practical in resource-limited settings. the american diabetes association recommends the use of glucose test as t2 dm screening in people with overweight and obesity as well as in those with other risk factors. as a result, risk assessment scores have been developed to address this problem in a simple and inexpensive way. most of the available algorithms for diabetes screening have been developed in caucasian [79] and asian populations [1013] and very few in other ethnic groups [14, 15]. to date, one diabetes risk score has been developed and validated in latin america so far which was derived from one urban area in brazil, thus bearing limited generalizability to the wider region. furthermore, it is well established that before adopting existing risk scores as screening tools in different populations and ethnic groups, their performance needs to be evaluated, calibrated, or validated in local settings. as the american diabetes association, the peruvian ministry of health recommends diabetes screening in general population with fasting glucose in adults aged 40 to 70 years with risk factors. however, fasting glucose is not always available in primary care settings, especially in semiurban and rural areas. as a result, a major challenge to be overcome in many countries is the implementation of a simple, fast, and laboratory-free based screening method. consequently, we aimed to develop a simple laboratory-free risk score to identify people with undiagnosed diabetes and incident diabetes in peru, a latin american country that spans coastal, andean, and rainforest settings. in order to do so, this work benefited from two large-scale population-based surveys: the first one, representative at the national level, was used to develop the score, and the second one, a cohort study, was utilized for external validation. the national survey of nutritional and biochemical indicators for noncommunicable diseases (eninbsc in spanish), conducted by the peruvian national institute of health, was used to develop our predictive model. this was complemented with the cronicas cohort study, whose baseline and longitudinal information was used to validate the risk score. the eninbsc is a national population-based survey carried out in peru between august 2004 and april 2005, designed to estimate the prevalence of hypertension, type 2 diabetes mellitus, and other risk factors for noncommunicable diseases at the national and regional level. potential participants were those aged 20 years, habitual residents in the study area, and able to provide consent for their participation in the study. pregnant women and those currently breastfeeding were excluded from the study. as per design, the eninbsc sample was stratified according to peru's five major regions of the country: lima, rest of the coast, urban highlands, rural highlands, and jungle. in each stratum, cluster of blocks the cronicas cohort study is an ongoing cardiopulmonary project aimed to estimate the prevalence and incidence of hypertension, diabetes mellitus, and obesity in four different settings in peru that differ in terms of their urbanicity and altitude: pampas de san juan de miraflores, in the highly urbanized lima, puno in the altitude (3,825 meters above the sea level) contributing with rural and urban areas, and tumbes, a semiurban area in the northern coast of peru. the study started in september 2010 and a follow-up visit was completed in march 2014. a sex- and age-stratified sample was selected at random for each of the settings and all participants aged 35 years, full time residents in the study area, and able to consent, were enrolled. follow-up data used for this analysis was collected, on average, at 30 months after baseline. the first one lasted on average 40 minutes and was carried out to apply a face-to-face questionnaire regarding data about household characteristics, demographics, lifestyles behaviors, risk factors, and blood pressure measurements. the second visit lasted 30 minutes on average and was planned to have an appropriate period of fasting for blood sampling for glucose, total cholesterol, hdl-cholesterol, and the remaining anthropometric measures (height, weight, and waist circumference) using standard procedures. similarly, the procedures of the cronicas study has been published elsewhere. in brief, participants responded to a face-to-face questionnaire applied by trained community health workers. data collected comprised risk factors for cardiovascular disease based on a modified version of the who step approach questionnaire for surveillance of noncommunicable disease. a period of 8 to 12 hours of fasting was required for blood sampling to collect fasting glucose, total cholesterol, and hdl-cholesterol. height, weight, and waist circumference were also assessed, and blood pressure was measured in triplicate after five minutes of resting using an automatic monitor (omron hem-780) previously validated in adult's population. in both studies, diabetes was defined as any of the following conditions: fasting glucose 7.0 mmol/l (126 mg/dl) and/or self-report of physician diagnosis. fasting glucose was assessed by an enzymatic colorimetric method (glucose oxidase god-pap) in both studies. after excluding individuals without known diabetes, undiagnosed diabetes variables included in the analyses were built to guarantee similarities between both studies: sex; age (< 55 and 55 years); education (in years); self-reported smoking (current versus never/former smoker); alcohol use (user versus never user); self-reported diabetes in first-degree relatives (participant's parents and/or siblings); and levels of physical activity (low versus moderate/high levels, based on the transport-related domain of the ipaq). anthropometric measurements included in the analysis were body mass index (( bmi),<25, 2529.9, and 30 kg/m), waist circumference (< 90, 9099.9, and 100 cm), waist-to-height ratio (< 0.50, 0.500.59, 0.600.69, and 0.70), and hypertension (measured or previously diagnosed). a total of 4,206 participants were enrolled in the eninbsc, but only 2,472 were included in this analysis. reasons for exclusion were 1,524 because of age<35 years to make both databases comparable, 129 because of no data about fasting plasma glucose levels being available, and 81 because of known diagnosis of diabetes. in the cronicas study, 3,601 participants were enrolled at baseline but only 2,948 records were analyzed as 465 had no data about glucose levels, and 188 were excluded because of previous diagnosis of diabetes. in addition, only data from 2,577 participants was used in the longitudinal assessment of the risk score (comparison of baseline characteristics among those included and excluded from longitudinal analysis is shown in online supplement: e-table 1; see supplementary material available online at http://dx.doi.org/10.1155/2016/8790235). initially, population characteristics of both studies were tabulated using proportions in the case of categorical variables and means and standard deviation (sd) with numerical variables. then, the prevalence and 95% confidence intervals (95% ci) of total diabetes and undiagnosed diabetes were estimated in each study. the risk score was derived from data of the eninbsc survey taking into account the multistage sampling strategy of the study. each potential risk factor (i.e., sex, age, family history of diabetes, etc.) was assessed in bivariate models using logistic regression and undiagnosed diabetes as the dependent variable. then, risk factors with a p value<0.10 in the bivariate analysis were included in a multiple logistic regression model using stepwise backward elimination with a significance level of 5%. the hosmer-lemeshow goodness-of-fit test was used to assess how well the predicted prevalence matched the observed prevalence of undiagnosed diabetes (i.e., p values over 0.20 indicate that model fits well). as we sought for an easily applicable and implementable algorithm, the risk factors in the final model were each assigned a weighted score by rounding up all regression coefficients in the final model to the nearest integer as in a previous report. for the evaluation of the risk score, the area under the receiver operating characteristic (roc) curve, sensitivity, specificity, and positive and negative predictive values (ppv and npv) were calculated. the optimal cut-point was determined using the youden index, a single statistic that captures the performance of a diagnostic test (i.e., sensitivity+specificity 1). as one of the main aims of a nonlaboratory risk score is to identify people who warrant having a blood test (i.e., fasting glucose, glycated haemoglobin, etc.), the cut-point with the highest sensitivity was also estimated and described. we assessed the performance of our score using bootstrap techniques as well as carrying out an external validation using the cronicas cohort study. the resulting 1,000 prediction models were then assessed to estimate the bootstrap auc using the bias-corrected version of the confidence intervals. in addition, using baseline data from the cronicas cohort study, validation measures (auc, sensitivity, specificity, predictive values, and likelihood ratios) were estimated. to evaluate the performance of our algorithm, the peruvian risk score was compared to previously published models for undiagnosed diabetes including the brazilian risk score, the qingdao score, the indian risk score, the kuwaiti risk score, the patient self-assessment score, and the rotterdam risk score using the c-statistic. finally, using the follow-up data of the cronicas cohort study, the risk score was also evaluated to detect incident cases of t2 dm by excluding those with diabetes diagnosis at baseline. analyses were performed using stata 13.0 (statacorp, college station, tx, usa). the protocol and informed consent forms of the eninbsc study were reviewed and approved by the instituto nacional de salud and the centro nacional de alimentacin y nutricin, both part of the ministry of health in lima, peru. in the case of the cronicas cohort study, protocol and consent forms were reviewed and approved by the institutional review boards of the universidad peruana cayetano heredia and the ngo asociacin benfica prisma in lima, peru, and the johns hopkins university in baltimore, usa. overall, participants from the cronicas study were 5 years older, reported consuming lower levels of alcohol, and were less physically active than those from the eninbsc survey. the overall prevalence of diabetes was 5.1% (129/2538; 95% ci: 4.2%5.9%) in the eninbsc survey and 8.7% (272/3135; 95% ci: 7.7%9.7%) in the cronicas cohort study's baseline. after excluding those with known diabetes, undiagnosed diabetes was present in 2.0% (48/2457; 95% ci: 1.4%2.5%) in the eninbsc survey and in 2.9% (85/2948; 95% ci: 2.3%3.5%) in the cronicas cohort study. after stepwise backward logistic regression, age, diabetes in first-degree relatives, and waist circumference were independently associated with undiagnosed diabetes (table 2). the hosmer-lemeshow test showed that the final model fitted relatively well (p=0.21). the peruvian diabetes risk score was constructed based on the coefficients of that final regression model. the score gave an auc of 0.73 (95% ci: 0.650.78), and the optimal cut-point for undiagnosed diabetes using the youden index was 2 (figure 1). with this cut-point, about 34.8% of participants were categorized as at high risk of diabetes: sensitivity 69.6%, specificity 65.8%, and ppv and npv of 3.9% and 99.1%, respectively. with a cut-point 1, 69.8% of participants would be at high risk of diabetes with improved sensitivity (93.5%) but lower specificity (30.6%). table 3 shows the performance of the risk score for detecting undiagnosed diabetes at different cut-points. when bootstrap was used, the performance of our risk score was similar to the obtained in the development model (auc=0.72; 95% ci: 0.650.78). in addition, when the risk score was evaluated by applying the score to the cronicas cohort study's population, the auc for undiagnosed diabetes was 0.68 (95% ci: 0.620.73). at the suggested cut-point of 2, 42% would be categorized as undiagnosed diabetes with sensitivity, specificity, ppv, and npv of 70.2%, 58.9%, 4.8%, and 98.5%, respectively (table 4). on the other hand, with a cut-point 1, 80% would be categorized as undiagnosed diabetes with sensitivity, specificity, ppv, and npv of 94.0%, 20.0%, 3.3%, and 99.1%, respectively. when previous published algorithms for undiagnosed diabetes were applied to the cronicas cohort study, the performance of the rotterdam score (p<0.001), indian score (p<0.001), and qingdao score (p<0.01) was poorer than our score; however, our algorithm performed similar to the other assessed models, such as the brazilian risk score (p=0.93), the kuwaiti score (p=0.26), and the patient self-assessment score (p=0.74), but having only three variables. the performance of this risk score was also assessed to predict incident cases of diabetes using the longitudinal data from the cronicas cohort study. one hundred twenty-one new cases of diabetes were found accounting for 6,207 person-years at risk, with an overall incidence of 1.95 (95% ci: 1.632.33) cases per 100 person-years of risk. the auc of the score was 0.66 (95% ci: 0.610.71). with a cut-point 2, 42.5% of participants were categorized as at high risk of developing diabetes: sensitivity, specificity, ppv, and npv were 69.4%, 58.9%, 7.8%, and 97.4%, whereas, for a cut-point 1, the respective values were 79.9%, 91.9%, 20.7%, 5.5%, and 98.1%. using a national population-based survey, a simple nonblood based risk score based on age, history of diabetes in first-degree relatives, and waist circumference was built and shown to perform moderately in detecting undiagnosed diabetes when externally validated. moreover, the performance of the score was almost similar for detecting incident cases of diabetes in the peruvian population. a relatively recent systematic literature search found 23 different blood-free prevalent diabetes risk scores: ten from europe, nine for asian populations, two from the united states, and two from middle east. in addition, and not included in the aforementioned review, only one risk score was developed in latin america using brazilian urban population. the same systematic review reported that auc for these predictive models was greater in the development studies (range: 0.65 to 0.88) than in the validation studies (range: 0.63 to 0.80), similar to our findings. another systematic review found that several noninvasive algorithms were created using variables such as age, gender, waist circumference and/or bmi, and family history of diabetes in the final model. as impracticality due to use of the algorithms was a common barrier to the uptake of risk scores by healthcare staff and individuals, our model, created with three of these more common variables, reached a moderate-to-high sensitivity depending on the used cut-point. moreover, two of these variables are easily evaluable during medical appointment or through individual's self-assessment, and only a measuring tape and no calculations are required to be implemented in clinical practice or at the population level. from a cross-sectional point of view, with a cut-point 2, from 1000 participants assessed by the peruvian diabetes risk score, a total of 420 would be classified as undiagnosed diabetes with the detection of 20 cases and only 6 will be missing. on the other hand, with a cut-point 1, from 1000 screened individuals, a total of 804 would be categorized as having undiagnosed diabetes with the detection of 27 cases and only 7 will be missing. thus, the reduction of the cut-point of the risk score would increase sensitivity but reducing the specificity and imposing the need of performing a confirmatory test (i.e., fasting glucose) to almost the double of individuals, with the benefit of having only 7 more people diagnosed. longitudinally, the same risk score would detect an important number of participants at risk of developing diabetes: 43% of screened individuals would be classified at high risk of diabetes, and of them, 8% would develop diabetes in the next 2.5 years. according to a previous study, 17 reports described a noninvasive model to predict the development of diabetes and included a median of six risk predictors, ranging from 2 to 11. although our score did not perform as good as other well-known longitudinal models in the literature such as the findrisc or the aric scores [35, 36], it only included three variables and was built using cross-sectional information. in addition, some variables used in the aforementioned studies are difficult to standardize within a country as peru, that is, food portions, physical activity, or sedentarism, limiting therefore its use on a wider scale and in a simple pragmatic fashion. our algorithm performed better than the rotterdam, the indian, and the qingdao risk scores in our population, which highlights the need of calibration and/or development of a specific score for different ethnic groups before its adoption. as there are ethnic differences in risk factors for diabetes and peru is considered a multiethnic country, it is necessary to create specific scores or recalibrate existing algorithms before applying in specific contexts. in addition, with only three variables included, the performance of our predictive model was similar to the other assessed scores included in the analyses. taken together, the score developed has the potential to augment, in a pragmatic manner, initial rapid screening for diabetes, especially at various nonspecialized primary healthcare services. our findings also demonstrate that approximately 35% of cases of t2 dm (39% in the eninbsc survey and 33% in the baseline of the cronicas cohort study) are not aware of their disease. results are similar to those reported in previous studies in our context and in similar settings in latin america. as the developed risk score is simple, it does not require a blood test or laboratory services, and it might be easily implemented in clinical practice. moreover, because our score asks for general information in the form of age and diabetes in first-degree relatives and is complemented by a simple anthropometric measure of waist, there is potential for the score to be self-administered. according to our results, any patient aged 55 years and above and having at least one first-degree relative with t2 dm has greater probability of having undiagnosed diabetes but also is at risk of developing diabetes in the future. in addition, a greater central obesity, that is, 100 cm or more, independent of the other terms of the score is alone a good predictor of diabetes as reported in previous studies. our algorithm included waist circumference instead of body mass index as other risk scores, providing a better indicator of accumulation of visceral fat and metabolic dysfunction in our context. recently, the peruvian ministry of health has published the guide of clinical practice for diagnosis, treatment and control of diabetes mellitus in primary care and only recommends screening in general population with plasma glucose among adults between 40 and 70 years with obesity or overweight as suggested by the american diabetes association. as in other lmic, plasma glucose is not always available in primary care, especially in semiurban and rural areas; therefore, a major challenge to be overcome in many countries is the implementation of a simple, fast, and laboratory-free based screening method. moreover, within the peruvian context, no risk score has been proposed as part of the aforementioned guide. thus, our algorithm might fill a gap to facilitate further specialized assessment of high risk individuals for diabetes, an approach that may be of utility to various other countries facing similar challenges. the strengths of this study include the use of a national population-based survey, including urban and rural areas across major geographical regions, to develop the peruvian diabetes risk score, as well as its validation using bootstrap but also an independent longitudinal cohort study. additionally, it is only based on three variables ensuring its simplicity to be used and implemented. however, the study has also some limitations. first, we have utilized fasting plasma glucose as the gold standard for diagnosing diabetes instead of an oral glucose tolerance test (ogtt). although the ogtt is more sensitive and specific than the fasting plasma glucose, more cases would have been detected with the overload of glucose; it is rarely performed as part of the routine clinical practice. second, the cronicas cohort study did not include information from the amazon rainforest as did the eninbsc survey. when a sensitivity analysis was performed excluding individuals from the jungle from eninbsc data, in addition, the score was created using a national survey to be applicable to the entire peruvian population. third, some variables were not assessed in our logistic regression model such as dietary intake or history of gestational diabetes as such data was not available. as a result, some caution should be made when our algorithm is compared to other risk scores. fourth, our model is based on the idea of risk stratification instead of individualisation; for instance, variables were categorized instead of being preserved as numerical. nevertheless, the performance of our score did not change when age and waist circumference were treated as numerical variables (data not shown). moreover, our idea was to develop a simple and easily applicable score instead of a complex algorithm for predicting undiagnosed and incident diabetes. finally, as other diabetes risk scores, the model warrants further scrutiny before it can be used in other populations. the peruvian diabetes risk score, built using age, self-reported diabetes in first-degree relatives, and waist circumference, proves to be a simple pragmatic screening tool for undiagnosed and incident cases of diabetes in peru. this experience in generating such simple, easy-to-use approaches for the identification of t2 dm can serve to inform other similar lmic efforts who are on early stages of diabetes prevention. this tool, due to its simplicity, can facilitate various initiatives oriented to introduce and scale up early preventative and management strategies on a wider scale.

objective. to develop and validate a risk score for detecting cases of undiagnosed diabetes in a resource-constrained country. methods. two population-based studies in peruvian population aged 35 years were used in the analysis: the eninbsc survey (n=2,472) and the cronicas cohort study (n=2,945). fasting plasma glucose 7.0 mmol/l was used to diagnose diabetes in both studies. coefficients for risk score were derived from the eninbsc data and then the performance was validated using both baseline and follow-up data of the cronicas cohort study. results. the prevalence of undiagnosed diabetes was 2.0% in the eninbsc survey and 2.9% in the cronicas cohort study. predictors of undiagnosed diabetes were age, diabetes in first-degree relatives, and waist circumference. score values ranged from 0 to 4, with an optimal cutoff 2 and had a moderate performance when applied in the cronicas baseline data (auc=0.68; 95% ci: 0.620.73; sensitivity 70%; specificity 59%). when predicting incident cases, the auc was 0.66 (95% ci: 0.610.71), with a sensitivity of 69% and specificity of 59%. conclusions. a simple nonblood based risk score based on age, diabetes in first-degree relatives, and waist circumference can be used as a simple screening tool for undiagnosed and incident cases of diabetes in peru.

PMC5027039

pubmed-58

atherosclerosis is a coronary heart disease (chd), and is the most common cause of death in the industrialized world. high blood pressure (bp), hypercholesterolemia, diabetes mellitus (dm) and smoking are considered as the major risk factors for the development of atherosclerosis. in addition, increasing age, male sex, family history and genetic factors predispose patients to chd.1 there is some data that have suggested the possible potential role of some infectious agents in the pathogenesis of atherosclerosis.26 the possibility of infectious agents may trigger the process of atherosclerosis by direct or indirect inflammatory effects, especially at younger ages.5 although different studies have been carried out to prove this hypothesis some infections may contribute in the pathogenesis of atherosclerosis; nothing has ever been conclusively proven. the possible role of some agents such as chlamydophila pneumoniae,6 hepatitis c. virus,7 human immunodeficiency virus,7 hepatitis b virus,8 cytomegalovirus,9 herpes virus,9 epstein-barr virus,10 mycobacterium tuberculosis,11 and helicobacter pylori,3 have been considered in the pathogenesis of atherosclerosis. among these pathogens, c. pneumoniae is important, because it could be effectively treated by antibiotics.12 c. pneumoniae is an obligatory intracellular organism, and is responsible for at least 10% of community-acquired pneumonias. this bacterium has also been associated with pharyngitis, bronchitis, otitis, influenza-like illness, sinusitis and myocarditis.1214 conflicting results have been reported about the relationship between c. pneumonia and atherosclerosis. west et al.15 apfalter et al.,16 and zibaeenezhad et al.17 reported no association between c. pneumoniae and atherosclerosis but others found significant association between c. pneumoniae and atherosclerosis.61819 the results of jha et al.,6 dabiri et al.18 and sessa et al.19 support the idea that c. pneumoniae may have a role in the development of atherosclerosis. this controversy about the role c. pneumoniae in the pathogenesis of atherosclerosis emphasizes the importance of more research works. the aim of this study was to demonstrate whether there is any difference between two group of patients with coronary atherosclerotic plaque and subjects without coronary atherosclerosis for the presence of c. pneumoniae dna by polymerase chain reaction (pcr) method. this case-control study was carried out on 60 formalin-fixed paraffin-embedded (ffpe) coronary artery biopsies in the molecular pathology laboratory, ghaem hospital, mashhad, iran in 2010. coronary artery tissues were selected from another study that was performed previously by our colleagues for determining prevalence of atherosclerotic plaques in autopsy cases with non-cardiac death in northeast iran. in this study, left coronary artery (lca), right coronary artery (rca) and left circumflex artery (lcx) had been evaluated grossly and microscopically. our materials included 30 coronary artery tissues with atherosclerotic plaques that were defined as study group, and 30 coronary artery tissues without atherosclerotic plaques that served as the control group. archived slides of the two groups were reviewed by two pathologists for confirmation of diagnosis, tissue adequacy and selection of the best paraffin blocks for dna extraction. for all subjects, the weight and height were determined, and the body mass index (bmi) was calculated. five to seven, 5-m-thick sectionswere cut from each ffpe specimen under sterile conditions, and then the dna was extracted by using proteinase k and non-heating dna extraction method.20 dna concentration was determined by using the thermo scientific nanodrop 2000 spectrophotometer, and specimens with low dna content (< 20 ng/l) were excluded from the study. pcr was performed for detecting c. pneumoniae dna by using the pcr kits (dna technology (jsc), pcr kit, moscow, russia). 10 l of pcr master mix and 0.5 l unit of taqpolymerase (hot start taq dna polymerase) were added into each paraffin sealed tube and were mixed, then 5 l (100 ng) of dna samples were added (except for positive and negative controls) and were spun at 1,000 rpm for 3-5 s. the tubes were placed into applied biosystems (abi) veriti thermal cycler and pcr was performed with the program of 180 s at 94c for the first step and then 45 cycles was run as follows: 50 s at 94c, 50 s at 64c, 50 s at 72c. amplified pcr products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light by gel documentation instrument. pcr was interpreted as positive when the dna band corresponds to the band of the positive control (254bp) in addition to the internal control band (ic:370 bp). results of pcr in control and atherosclerotic groups were analyzed with spss version 11.5 by a statistician. we used fisher's exact test for comparison of categorical variables and independent sample t-test for continuous variable in patients and control groups. the age range in the control group was 20-46 years with a mean (standard deviation) age of 26.0 (6.41) years. in the study group, it ranges from 18 to 50 years with a mean (sd) age of 32.0 (9.46) years and the t-test showed no significant difference between the ages of patients in the study and control groups (p=0.97). twenty-five patients (83.3%) were males and five patients (16.7%) were females. the male to female ratio was 5:1 and no significant difference was seen between study and control groups for gender (p=1.00) by fisher's exact test. the mean bmi of the patients in the study and control groups were 24.4 (3.77) and 23.9 (2.62) kg/m respectively [table 1]. the frequency of atherosclerotic plaques in the different vessels of heart of the patients in the study is as shown in table 2. as a result, the possibility of the appearance of advanced atherosclerotic plaques s in lca was higher than the two other vessels. concurrent occurrences of different stages of atherosclerosis were seen in our patients. only one vessel involvement with fibro-fatty morphology (mild) was seen in five patients (16.7%), one vessel with advanced plaque was observed in three patients (10.0%), seven patients (23.3%) had one vessel with fibro-fatty and one vessel with advanced plaque, two vessels with advanced plaque were noticed in six patients (20.0%), three vessels with advanced plaque were observed in five patients (16.7%) and four patients (13.3%) had two vessels with advanced plaque and one fibro-fatty morphology. comparisons between patients with coronary atherosclerosis and controls for weight, height and body mass index the frequency of atherosclerotic plaques in different vessels of the heart in the study group positive pcr result for c. pneumonia was seen in one (3.3%) sample among the 30 coronary artery tissues with atherosclerosis (advanced plaque), while all the control samples were negative. fisher's exact test showed no significant difference for detection of c. pneumoniae between samples of coronary artery with and without atherosclerotic plaque (p=1). ischemic heart disease could be observed in the absence of its major risk factors such as increasing age, male gender, hypertension, hyperlipidemia, smoking and dm. inflammatory cells are seen during all steps of atherogenesis, and some infectious agents may trigger this inflammation.113 although some studies showed that c. pneumoniae can increase the risk of atherosclerosis, 61819 but we did not find any significant difference in the frequency of c. pneumoniae dna isolation in the coronary arteries with and without atherosclerosis by pcr method. we believe that at least two important factors can account for this discrepancy in the results from previous studies: methodology and epidemiology. c pneumonia infection can be diagnosed by various methods such as serology with an increase in serum antibodies against the organism, dna detectionby pcr, immunocytochemistry, electron microscopyand c. pneumonia isolation by culture.14 in other studies, the most widely used method for identifying of c. pneumoniae infection was based on antibody detection. the frequency of antibody against c. pneumoniae starts to rise in children and is observed approximately in 50% of adolescents.1214 somesero-epidemiologic studies have shown a relationship between c. pneumoniae and atherosclerosis. saikku et al.,21 podsiady et al.22 and romano et al.23 detected a higher level of antibody against c. pneumoniae in chd compared to control group but zibaeenezhad et al.,17 romeo et al.,24 and ericson et al.,25 found that the presence of antibodies was unable to predict coronary artery events. we used pcr for detection of c. pneumonia; this assay appears to be more sensitive than cell culture.1426 our finding was in concordance with those of west et al.,15 jantos et al.27 and satpathy et al.28 similarly, reszka et al.,29 studied presence of c. pneumonia dna in aortic vessels, voorend et al.30 in cerebral vessels, and kwon et al.31 in carotid arteries and could not establish any significant difference in pcr results between the study and control groups. these findings suggest that serology could detect past as well as present infection, whereas pcr detects current c. pnuemoniae infection. considering the study of west et al.15 and satpathy et al.28 who showed a higher c. pnuemoniae igg antibody in patients with chd compared to control group but could not detect the organism by nested pcr assay in any specimen strengthens our hypothesis. we believe that the lack of standardized methods for the detection of c. pnuemoniae infection used in the aforementioned studies is probably one of the important reasons for the discrepancies in their results. c. pnuemoniae is a common human pathogen; the majority of infected subjects have few or no symptoms. the frequency of antibodies to c. pneumoniae is approximately 50% in the northern hemisphere.1432 exposure to c. pneumoniae is probably common in iran. moghaddam et al.33 reported 38% seropositivity for c. pneumonia antibodies (igg and igm) in healthy subjects by elisa method in tehran, iran. studies carried out in iran on the association between c. pneumonia infection, and atherosclerosis revealed conflicting results; while some studies showed the association between c. pneumonia infection, and atherosclerosis1834 others didnt demonstrate any significant associationbetween c. pneumonia and atherosclerosis.1735 the anatomical localization of vessels may also be a factor influencing the results from previous studies. jha et al.6 showed that c. pneumoniae may be associated with chd and coronary artery was more susceptible to c. pneumoniae as compared with carotid artery. bahrmand et al.25 and ericson et al.34 examined the relationship of c. pneumoniae infection to the severity of coronary atherosclerosis by pcr and serology respectively. they examined coronary artery samples with two different methods, and both revealed a high rate of reactivity to this bacterium in severe atherosclerosis and a much lower rate in mild atherosclerosis; therefore, they concluded that severity (stage) of atherosclerosis can be presumed as an important factor in diagnosis of c. pneumoniae infection. our study was performed on 30 archived ffpe coronary artery tissues of patients with atherosclerosis. this sample size may be too small for final decision; therefore, future study with a larger number of case groups and also using fresh specimens are helpful. in conclusion, we did not find any significant association between c. pneumoniae infection and coronary artery atherosclerosis by pcr method. further studies involving a greater number of patients and also using more specific methods for detecting living c. pneumonia organisms by culture can be helpful.

background: atherosclerosis is a coronary heart disease, andis the most common cause of death in the industrialized world. some studies suggested that atherosclerosis may be triggered by infectious agents, mostly chlamydophila pneumoniae. however, the role of c. pneumoniae in the pathogenesis of coronary atherosclerosis is still controversial. objectives:this study was performed to evaluate whether there is a significant association between coronary artery atherosclerosis and c. pneumoniae by the polymerase chain reaction (pcr) method. materials and methods: this case-control study was carried out on formalin-fixed paraffin-embedded tissue biopsies of the coronary arteries obtained from 30 patients with coronary atherosclerosis and 30 subjects without atherosclerosis living in northeast of iran. all subjects ' weight and height were determined, and the body mass index was calculated. we also reviewed the medical history and previous laboratory reports of patients. deoxyribonucleic acid (dna) was extracted, and c. pneumonia dna was amplified and detected using pcr assay. results:the age of the patients in the study group was from 18 to 50 years, and the male to female ratio was 5:1. only oneout of the 30 coronary tissue samples had positive pcr for c. pneumoniae (3.3%), while it was negative for patients in the control group. conclusions:this study showed that c. pneumoniae infection is not strongly associated with coronary artery atherosclerosis in northeast of iran.

PMC3644743

pubmed-59

confrontation with the consequences of diabetes causes a crisis in physical, mental, and spiritual dimensions. sometimes the spiritual crisis can be tremendous. since spiritual health coordinates different aspects of human life, this study aimed to identify the spiritual health of patients with defects caused by diabetes this was a qualitative-phenomenological and descriptive study and the participants were selected from rehabilitation centers in isfahan and valiasr hospital in zanjan. outcome of this phase of the study was 173 codes and 2 groups that included hindering factors in spiritual health and the promotion of the relation with god. the concepts that patients had experienced as hindering factors of the treatment process were disappointment and hopelessness, guilt, feeling distant from god, quitting obligatory acts and knowing god as cruel. the concepts that patients had experienced as contributing factors to the healing process were resorting to imams, god s ordering the disease as a reward, fear of god s punishment, believing in miracles, being closer to god, believing in the mercy of god, returning to religious practice, feeling of enjoying life and knowing that the disease is the atonement of sins. with regard to the importance of spiritual and religious care as one of the tasks of nurses, as the key members of health team, they should respect the patients beliefs and values in addition to considering their physical and mental conditions. although the mortality rate in diabetic patients due to ketoacidosis and infection is declining, deaths from diabetes complications including diabetic foot complications has increased dramatically. according to some reports, its rate in the first year after amputation is 1340 percent, in the third year, 3565 percent and in the fifth year it is 3980 percent, which figures are comparable to death rates in malignancies. in addition, the loss of job and the need for medical and nursing care, reduction in social and family interactions, and lifestyle changes are the major problems that influence the family and socioeconomic status of these patients. the absence of the feet and lower limbs can be extremely problematic for these patients and exerts too much energy for walking; therefore, amputation of higher limbs or development of ulcers may occur. ultimately, these patients with amputated legs need long-term hospitalization, rehabilitation, home care and social support. organ amputation due to diabetes causes crisis in the physical, mental, and spiritual life of the patients. in this case, the spiritual health is the unique element that can harmonize physical, mental and social aspects of the human life and is necessary for coping with the disease. as a matter of fact, when the spiritual health is seriously at risk, a person may experience emotional disturbances such as loneliness, depression and loss of meaning in life. the spirit of prayer is a deep human instinct, which is the center of humanity, and where the passion and knowledge to communicate with the world comes from. forms and statements of people who pray are different: speaking, listening, waiting and wailing. it exposes them to severe stress and they seek different approaches to cope and adapt to life. koenig believes that religion creates a positive attitude towards the world and makes the person powerful against unfortunate events in life such as diseases and helps to improve the life with motivation and energy. this increases tolerance and acceptance of situations that can not be changed. in many cases of emergency in which science is unable to help a person, this issue is of vital importance especially in serious cases. the increasing interest of medical scientists to the impact of praying in the treatment of other diseases can be indicative of treatment of some diseases in case of modern medicine failure. on the other hand, medical researchers also acknowledge the importance of medical procedures of traditional and complementary therapies including prayer to treat the diseases. even the western scientific community has recently published articles showing that doctors can not be indifferent to the religious beliefs of patients for treatment. this study was conducted to understand the spiritual experiences of the patients with organ amputation due to diabetes. the aim of phenomenological studies is to understand the phenomena of human experience through the analysis of participants verbal explanations of experiences. the study population consisted of patients with defects, caused by diabetes, who had referred to rehabilitation centers in esfahan and zanjan provinces. sampling was purposive and the inclusion criteria for participants were the injury caused by diabetes and the willingness to participate in the interview. there were 15 participants entered the study (8 men and 7 women at 5070 years of age). all interviews were fully recorded and all participants were given a code based on the interviews. in this study, the researcher tried to be cautious with the information collected to be free from any kind of bias and to avoid persons with poor memory and those who could not provide the correct information. in the interviews, the researcher attempted to withdraw his assumptions about the phenomenon under study and thus help to strengthen the data. they were asked that how has your life changed compared to before the disease? in response, they described their life experiences with this disability, and how their performance was changed due to the disease. during the interview, the patients focused on cases where the disease led to their functioning. when it was necessary to clarify the data in specific areas, the questions were asked in details. finally, participants were asked if they wanted to provide alternative explanations in relationship with their disease. the researcher wrote down the participants statements through repeated listening to in-depth interviews. after the formation of concepts and ideas from each interview, the next interview was conducted. the above process was repeated for each interview (second stage of colaizzi s method). after the all interviews, the concepts were formulated in particular subjects and were classified into categories (third stage) and eventually all ideas were combined so that a complete description of the phenomenon under the study was derived and presented (the fourth, fifth and sixth stages). finally, the concepts were returned to the participants and were analyzed (the seventh). in this study, the criteria for validity and reliability of data were lincoln and guba. in order to make the study believable, the participants and the research partners also reviewed all the data. to achieve reliability and objectivity of the research data, the account access was used. to increase the portability of the present study, the complete research project was introduced. there were 15 patients participating in this study, comprised of 7 women and 8 men. the age range of participants was between 50-75 years with mean age of 59.7 years. length of diabetes was between 530 years and the length of organ amputation caused by diabetes also varied from 2 weeks to 11 years with mean of 4.2 years. the education level comprised of uneducated to junior high school. regarding employment, 53.3 percent of the participants (8 people) lost their jobs and were unemployed due to organ amputation caused by diabetes. about 66.6 percent (10 people), a leg was amputated below the knee and 26.6 percent of the participants (4 people) both legs were amputated below the knee area. in one patient both legs below the knee and the middle finger were amputated due to diabetes. the concepts derived from patients experiences as factors interfering spiritual health in the treatment process were disappointment and hopelessness, guilt, feeling distant from god, quitting obligatory acts and considering god cruel. factors promoting the healing process were deemed by god, fear of god punishment, belief in miracles, being closer to god, believing god to be merciful, returning to religious practice, enjoying life and indicating the disease as the atonement of sins. most of the participants, encounteringdiabetes-related organ amputatio, said that in such circumstances, intellectual protections, religious sources, and having a strong relationship with a higher power could improve the quality of life, and support the individuals and reduce the severity of symptoms. one of the participants expressed his experiences in turning to practice religion as follows: i was deemed to cut my foot to know god, i am sure there is a wisdom behind this, i ve been weak in a prayer. i was wavering, but when my leg was cut, i celt much closer to god, i know god took my foot, but after this incident, i feel that god wanted to try me, before this, if my prayer was late, i did not become upset. but now, if i do not perform my prayer on time, i will get upset, i feel that i did not do an important job, i become worried with no reason (participant 1). another participant stated when my uncle came to visit me, he said the disease is the expiation of sins. whenever this is said, my heart becomes calm, but i did not commit a sin to deserve such a punishment. even, i had not bothered anything in my life. i do not remember, i wish, everything could be finished in this world (participant 3). since my foot (right foot) has been cut off, i rub it every day. as i can not bend i m afraid; it may give testimony against me in the doomsday and tells god that i had been overeating because our hands and legs have rights and we must consider this (participant 5). our tenant is a believer who always reads the quran. when she saw that i was confused, she said, let me read the koran for you to be calm. although i am illiterate and i do not understand anything from the quran, when i hear the peaceful verses of the quran, i forget my sorrows i participate in the middle of moharram mourning ceremony every year and in mourning ceremonies every thursday, but when my leg was cut, i did not go there anymore because i feel i m too far from god and as a result, these ceremonies can not help when the wounds of the foot were fine after surgery, at fast; i performed my prayer in sitting position, but i realized that i do not feel good in just sitting and praying, i just hurt myself (participant 15). the prayer and vow are likely to enhance the human s tolerance against diseases and problems communication with the source of existence and asking him to help would repair the physical and mental powersof man and relieves a lot of disorders and diseases. therefore, the appeal to the imams in patients with organ amputation due to diabetes is high. several studies have also shown that when the spiritual health is at a serious risk, prayers in acute cases may face difficulty. in these cases, praying for the patient can be an important spiritual intervention. one of the most useful sentences in the form of prayers could be the short ones, asking god for fulfillment of the needs, melting away the fears and giving hopes to the patient and ultimately remembering that god is able to meet the needs of the patients. if the patient is depressed, angry or upset or suffering from extreme pain, the expression of praying sentences is like the salt to the wound. nurses should pay close attention in the implementation of these provisions, and if the patients are upset, they should leave them alone. in this regard, a research by tracy et al. was conducted on two thousand nurses in critical care units in which 55% of nurses reported that patients and families were asking them to pray. another study which studied the effects of prayer on blood infection, randomly employed 1691 experimental and 1702 control patients. parameters studied included duration of fever in patients, duration of hospitalization and mortality in the hospital. the results of this study showed a significant difference between the duration of fever and length of patients stay that was lower in the intervention group compared to the control group. nowadays, some of the organizations that evaluate health care systems and are responsible for granting accreditation to them, suggest that the spiritual needs of the patients in health care should be also evaluated. finally, the importance of spiritual and religious care, which is today considered as one of nurses tasks, urges them as the key members of health teams to respect patients beliefs and values as well as their physical and mental aspects.

background: confrontation with the consequences of diabetes causes a crisis in physical, mental, and spiritual dimensions. sometimes the spiritual crisis can be tremendous. since spiritual health coordinates different aspects of human life, this study aimed to identify the spiritual health of patients with defects caused by diabetes. materials and methods: this was a qualitative-phenomenological and descriptive study and the participants were selected from rehabilitation centers in isfahan and valiasr hospital in zanjan. a purposive sample of 15 participants underwent deep interviews. colaizzi s method of analysis was used to analyze the data. findings:outcome of this phase of the study was 173 codes and 2 groups that included hindering factors in spiritual health and the promotion of the relation with god. the concepts that patients had experienced as hindering factors of the treatment process were disappointment and hopelessness, guilt, feeling distant from god, quitting obligatory acts and knowing god as cruel. the concepts that patients had experienced as contributing factors to the healing process were resorting to imams, god s ordering the disease as a reward, fear of god s punishment, believing in miracles, being closer to god, believing in the mercy of god, returning to religious practice, feeling of enjoying life and knowing that the disease is the atonement of sins. conclusions:with regard to the importance of spiritual and religious care as one of the tasks of nurses, as the key members of health team, they should respect the patients beliefs and values in addition to considering their physical and mental conditions.

PMC3696216

pubmed-60

vitamin d deficiency was once thought to exclusively affect bone metabolism, but now there is ample evidence of its role in many other conditions including metabolic syndrome, autoimmune diseases, and cancer. vitamin d receptors are recognized to be in numerous extraskeletal tissues, such as pancreas and muscle. a systematic review by pittas and colleagues reported that vitamin d may have a beneficial effect on the action of insulin, either directly or indirectly. several observational studies in adults, including the framingham heart study, have reported an inverse association between vitamin d status and insulin resistance. however, data from children and adolescents do not consistently report this inverse relationship [510]. even in studies reporting this association in adolescents and children, further, none of the studies in adolescents have studied the effect of puberty on this association. a fall in insulin sensitivity with compensatory increase in insulin secretion has been reported in puberty [11, 12]. high prevalence of cardiometabolic risk factors, including insulin resistance (64.8% of normal weight children had at least one cardiometabolic abnormality), even in healthy young children of normal weight, has been reported in indian children and adolescents. south-asian adolescents, including those with normal bmi, have a higher prevalence of insulin resistance when compared with age and bmi matched european adolescents [14, 15]. this ethnic difference in insulin sensitivity was attributed to the higher body fat percentage in south-asian children. an alternative hypothesis to explain the ethnic differences in insulin sensitivity is vitamin d deficiency. despite plenty of sunlight available throughout the year, a high prevalence of vitamin d deficiency has been reported in all age groups including toddlers, school children, pregnant women and their neonates, and adult males and females residing in rural and urban india. vitamin d deficiency has been reported in 8492% children in the age group of 1018. in view of high prevalence of both, insulin resistance and vitamin d deficiency in asian-indian children and adolescents, we sought to examine the relationship between vitamin d status and insulin resistance. we also sought to determine if puberty affected the relationship of vitamin d status and parameters of insulin resistance and sensitivity in asian-indian children and adolescents. the study was conducted as a prospective observational study after approval from the ethics committee of the all india institute of medical sciences (aiims), new delhi. subjects were selected from the children and adolescents attending the endocrinology outdoor services at aiims for evaluation of obesity. the inclusion criteria included children between the ages of 617 years with obesity as per international obesity taskforce (iotf) criteria. the exclusion criteria included subjects with diagnosed diabetes mellitus or taking metformin or any weight reducing drugs, subjects with any known systemic illness or endocrine or metabolic disorders, known to be associated with obesity, or subjects with symptoms to suggest hypothalamic obesity were excluded from the study. blood samples were collected in fasting state (minimum 9 hours fasting), followed by oral glucose tolerance test (anhydrous glucose=1.75 gm/kg bodyweight, maximum of 75 gram, dissolved in 250 ml of water, ingested over 5 minutes) with sample collection at 60 and 120 minutes after glucose ingestion. height was measured with holtain's stadiometer (holtain inc., crymych, pembs. waist circumference was measured (nonelastic measuring tape) at the end of normal expiration at the midpoint between the iliac crest and the lower edge of ribs in the midaxillary line. blood pressure (bp) was measured in the right upper limb in sitting position with a mercury sphygmomanometer after 5-minute rest with an appropriate size cuff. total body fat was measured with dual energy x-ray absorptiometry (dxa, hologic qdr 4500 with pediatric software, hologic inc., fat mass index (fmi, fat mass in kg/height in meter) and fat-free mass index (ffmi, lean body mass+ bmc)/(height in meter) were calculated. bone mineral area, content (bmc) and areal density (abmd) at the femoral neck, lumbar spine (l1-5), and forearm were measured, using a hologic qdr 4500a fan beam dxa machine. hologic spine phantom (hologic spine phantom number 21373) was scanned daily before subject evaluation. the measured phantom bone mineral density was stable throughout the study period at 0.9150.945 gm/cm. similarly, whole body phantom (hologic wb number 1252) was also scanned before subject evaluation and remained stable during study period. the in vivo precision error for adults was 0.62% for femoral neck abmd, 0.65% for lumbar spine abmd, and 0.77% for forearm abmd. however, in view of additional radiation exposure, the reproducibility of these scans was not assessed among children. pubertal stage was assessed by a single endocrinologist, based on breast stage and pubic hair development in girls and genitalia development in boys. b-mode ultrasonography was used to measure cimt (7.510 mhz probe, philips envisor ultrasound machine) using standard protocol. insulin resistance (ir) was calculated by computer-based model called homeostasis assessment model (homa-ir) utilizing fasting blood glucose and fasting serum insulin levels .the whole body insulin sensitivity index (wbisi) or matsuda index was calculated by the formula suggested by matsuda and defronzo .area under curve (auc) was calculated for blood glucose (0, 60, and 120 minutes) and serum insulin (0, 60, and 120 minutes) by using trapezoidal rule. insulin resistance (ir) was calculated by computer-based model called homeostasis assessment model (homa-ir) utilizing fasting blood glucose and fasting serum insulin levels. the whole body insulin sensitivity index (wbisi) or matsuda index was calculated by the formula suggested by matsuda and defronzo. area under curve (auc) was calculated for blood glucose (0, 60, and 120 minutes) and serum insulin (0, 60, and 120 minutes) by using trapezoidal rule. complete blood counts, liver function tests, renal function test, serum calcium (corrected calcium with serum albumin), phosphate, alkaline phosphatase, uric acid, and blood glucose were measured in all subjects with an automated chemistry analyzer (roche hitachi 912 chemistry analyzer, gmi inc. glycosylated hemoglobin (hba1c) was measured in whole blood using ion-exchange high performance liquid chromatography (bio-rad laboratories inc., ca, us). serum insulin was measured on an autoanalyzer (roche elecsys 2010) using electrochemiluminometric assay. serum total cholesterol (tc), triglyceride (tg), and hdl cholesterol (hdl-c) levels were estimated directly with automated analyzer, while ldl cholesterol (ldl-c) was estimated by using the friedewald equation. vitamin d deficiency was defined as a serum 25(oh)d levels less than 20 ng/ml. this was further subdivided to severe, moderate, and mild vitamin d deficiency if serum 25(oh)d levels were<5 ng/ml, 5<10 ng/ml, and 10<20 ng/ml respectively. statistical analysis was carried out using spss (spss version 11.5; spss inc., continuous variables not showing normal distribution were treated with appropriate log transformations before any parametric analysis. student's t-test was used to determine statistical difference between subjects with serum 25(oh)d<10 ng/ml or more. pearson's correlation was used to assess association between continuous variables and partial correlations were used to determine relationship after adjusting for relevant covariates. stepwise linear regression analysis was used with serum 25(oh)d as dependent variable in the model. values for serum 25(oh)d, serum insulin, homa-ir, and matsuda isi were not normally distributed; therefore, they were logarithmically transformed which yielded normal distribution. a total of 62 subjects participated in this cross-sectional study from june 2008 to may 2009. the mean age of subjects was 13.0 3 years (35 boys; 27 girls). assessment of pubertal status by tanner's method showed 19 subjects in stage 1, 11 in stage 2, 6 in stage 3, 3 in stage 4, and 23 subjects in puberty stage 5. no significant difference was observed between boys and girls except higher whr in boys than girls (0.93 0.05 versus 0.89 0.05; p=0.013). all study subjects were classified as vitamin d deficient (mean sd 8.5 4.2 ng/ml; maximum minimum: 3.919.2 ng/ml, median=6.9 ng/ml). severe vitamin d deficiency (< 5 ng/ml) was seen in 11 subjects (17.7%) while 30 subjects had serum 25(oh)d levels between 5<10 ng/ml (48.3%). serum 25(oh)d equal to or more than 10 but less than 20 ng/ml was present in 21 subjects (33.8%). pearson correlation coefficient analyses showed significant inverse relationship between serum 25(oh)d and total body fat percentage (r=0.31, p=0.01) and serum 25(oh)d and fmi (r=0.33, p=0.01, figure 1). partial correlation analysis showed persistence of significant correlation between serum 25(oh)d and total body fat percentage (r=0.37, p=0.005) and between serum 25(oh)d and fmi (r=0.36, p=0.006) even after adjustment for age, sex, pubertal stage, and bmi. although blood glucose and serum insulin levels showed inverse relationship trends with serum 25(oh)d, but these were not statistically significant (data not shown). similarly, no statistically significant correlation was seen between serum 25(oh)d and parameters of insulin sensitivity or resistance. fmi showed positive correlation with homa-ir (r=0.26; p=0.04), auc insulin (r=0.41; p=0.001), auc glucose (r=0.31; p=0.018), and negative correlation with wbisi (r=0.42; r=0.001). however, total body fat did not show any correlation with parameters of glucose and insulin parameters. on the basis of serum 25(oh)d, subjects were divided into two groups, group a with serum 25(oh)d level<10 ng/ml (n=41) and group b with serum 25(oh)d level 10 ng/ml (n=21) (an arbitrary cutoff to compare subjects with mild vitamin d deficiency with moderate and severe vitamin d deficiency, 25). no statistical significant difference was seen in blood glucose, serum insulin, and auc for both, glucose and insulin between group 1 and group 2. trends for higher insulin resistance (homa-ir) and lower insulin sensitivity (wbisi) were seen in subjects with lower serum 25(oh)d concentrations but not statistically significant. total body fat percentage and fmi were significantly higher in group 1 in comparison to group 2 (p=0.015 and 0.007, resp.). however, no significant difference was seen in waist and hip circumference and waist hip ratio. similarly, ffmi, lipid parameters, or thyroid profile were also not different between the two groups. group 1 (prepubertal group; tanner stage 1) had 19 subjects, group 2 (peripubertal group; tanner stage 2, 3, and 4) had 20, while group 3 (postpubertal group; tanner stage 5) had 23 subjects. no significant correlation was seen between vitamin d status and any other parameters including total body fat percentage, fmi and parameters of insulin resistance and sensitivity in the prepubertal (group 1) or peripubertal groups (group 2). partial correlations after adjusting for age, sex, and bmi were also nonsignificant for both groups (data not shown). in postpubertal subjects (group 3, n=23), a significant inverse correlation was seen between serum 25(oh)d and homa-ir (r=0.41, p=0.03, figure 2). this association between serum 25(oh)d and homa-ir persisted even after control for age, sex, and bmi (r=0.505, p=0.023). positive trends of association between serum 25(oh)d and wbisi were also seen, but this was not significant (r=0.29, p=0.17), even after adjusting for age, sex, and bmi (r=0.35, p=0.13). serum 25(oh)d showed significant inverse correlation with total body fat percentage (r=0.58, p=0.003) and fmi (r=0.49, p=0.016) which improved after adjusting for age sex and bmi (r=0.63, p=0.002 for total body fat percentage; r=0.582, p=0.007 for fmi). serum 25(oh)d also showed significant inverse relationship with fasting serum insulin levels (r=0.44, p=0.034) which improved after adjusting for age, sex, and bmi (r=0.53, p=0.016). when prepubertal subjects (group 1) were compared with the prepubescent subjects (group 2), no significant difference was seen in any of the parameters including blood glucose, serum insulin, homa-ir, and wbisi. similarly, when prepubertal subjects were compared with all other subjects (subjects in group 2 and 3 combined), significantly higher insulin sensitivity (wbisi, mean sd 6.07 5.7 versus 3.42 2.7, p<0.01) and lower insulin resistance (homa-ir, mean sd 3.68 2.7 versus 5.22 4.0, p=0.04) were seen in prepubertal subjects. this is the first study examining the relationship between serum 25(oh)d levels and parameters of insulin resistance in obese asian-indian children. our study confirms a significant inverse relationship between serum 25(oh)d levels and body fat indices as reported in other studies in children [6, 10]. we demonstrated trends for that higher serum 25(oh)d levels are associated with better insulin sensitivity and less insulin resistance though this correlation did not reach statistical significance. one of the main reasons for the absence of significance could be the universal presence of vitamin d deficiency (< 20 ng/ml) resulting in very narrow range of serum 25(oh)d. we also show, for the first time, that the association between serum 25(oh)d and insulin resistance and sensitivity is influenced by pubertal staging. when subjects were stratified by pubertal stage, only subjects in tanner stage 5 exhibited a significant correlation between serum 25(oh)d and homa-ir, while prepubertal subjects did not show statistically significant correlation. high prevalence of vitamin d deficiency has been reported across the age range in pediatric, adolescent, and adult in obese individuals [5, 6, 2631]. this may be just reflection of very high prevalence of vitamin d deficiency in asian-indians [16, 17]. however, high prevalence of vitamin d deficiency has been reported in obese children and adolescents from populations where vitamin d deficiency is not that prevalent [5, 6, 10]. reis et al., in a study of 3577 adolescents in the age group of 1219 years, found significantly lower serum 25(oh)d levels in adolescents with bmi>95th centile in comparison to subjects with bmi<95th centile (p<0.001). it has been postulated that increased sequestration of vitamin d in fat tissues, leading to decreased vitamin d bioavailability, low dietary vitamin d intake due to poor nutritional habits, and minimal sun exposure due to sedentary indoor lifestyle are important factors associated with high prevalence in the obese population [29, 32]. it has also been suggested that vitamin d deficiency is a cause of common obesity and can account for the secular trends in the prevalence of obesity and for individual differences in its onset and severity. the inverse relationship between serum 25(oh)d and insulin resistance in adults have been reported in most studies, though some have failed to find this association. in comparison, reports in children and adolescents have shown conflicting results. delvin et al., in a study of 1745 french-canadian children, reported modest but significant negative associations between serum 25(oh)d and homa-ir. each 10-nmol/l (4 ng/ml) increase in serum 25(oh)d was associated with lower glycemia and homa-ir. did not find significant correlation between serum 25(oh)d levels and insulin, homa-ir and homa-b %. in another study based on nhanes data of 3577 adolescents from 20012004 showed that serum 25(oh)d levels were inversely associated with plasma glucose concentration (p=0.01). lenders et al., in a study of 58 obese adolescents, did not found any correlation between insulin indices and serum 25(oh)d in both, adjusted and unadjusted models. in our study, we did not find a statistically significant association between serum 25(oh)d and parameters of insulin sensitivity and resistance. however, trends for higher blood glucose and serum insulin levels were seen in subjects with low serum 25(oh)d level. no significant difference was seen in insulin resistance and sensitivity between group 1 and group 2. this is in contrast to the observations made by ashraf et al. who reported in 51 african-american obese female adolescents that higher serum 25(oh)d concentrations were associated with significantly higher wbisi (p=0.018) but no difference in homa-ir. these differences may be due to marked difference in severity of obesity as mean bmi of study subjects was 43.3 9.9 in study by ashraf et al., while it was 29.3 9.8 kg/sqm in our study subjects. studies in adult population have shown that parameters of insulin sensitivity and insulin resistance as calculated by hyperinsulinemic euglycemic clamp study (m value) correlated better with serum 25(oh)d levels than indirect parameters like homa-ir. studies with direct measures of insulin sensitivity like clamp studies are required to further investigate these issues in pediatric and adolescent population. the phenomenon of pubertal insulin resistance has been well described in cross-sectional as well as longitudinal studies. a longitudinal study of insulin resistance in american children showed that during puberty, insulin sensitivity decreased by 50%, with a compensatory increase in plasma insulin which is independent of the changes in body fat. these changes are thought to be mediated by interaction of various hormones during puberty including increased growth hormone secretion. no studies have yet reported the relationship between serum 25(oh)d concentrations and insulin sensitivity and resistance in pediatric and adolescent population and the potential influence of puberty except one. lenders et al. reported no significant association between serum 25(oh)d and insulin indices, even after adjusting for tanner stage. however, our study, for the first time, shows that the relationship between serum 25(oh)d concentrations and insulin resistance is influenced by puberty. when subjects with all stages of puberty were analyzed, no significant correlation was found, but when subjects in tanner stage 5 were analyzed alone, significant inverse correlation was seen between serum 25(oh)d concentrations and homa-ir (r=0.41, p=0.03). this association persisted/improved after controlling for age, sex, and bmi (r=0.505, p=0.023) but disappeared when subjects were controlled for fmi (r= 0.27, p=0.24). most of the evidence of an association between adiposity and vitamin d status comes from adult studies [4, 33]. a recently published study of 382 healthy subjects aged 621 years with dxa scans showed that serum 25(oh)d concentrations were more likely to be lower in those with greater bmi z-scores and fm, but not ffm. when this association was adjusted for possible covariates in the model, it became nonsignificant.. showed that obese adolescents with and without vitamin d deficiency differed according to fm and fm percentage, but not lean mass and this relation of fm percentage to serum 25(oh)d remained significant after potential confounding variables were adjusted for. showed that vitamin d deficiency was associated with higher visceral adipose tissue in white and greater subcutaneous adipose tissue in black american children and adolescents. in agreement with these studies, our findings indicate that body fat indices (total body fat percentage and fmi) are inversely correlated with serum 25(oh)d level. this correlation persisted even after adjustment for possible covariates like age, sex, and bmi. the differences in study findings may be explained in part by differences in study design and underlying sample characteristics like bmi and variation in serum 25(oh)d concentrations. the major strengths of our study are the use of robust measures of insulin sensitivity and resistance based on ogtt rather than fasting sample and all our study subjects belonged to one ethnic group. we have used dxa for assessment of body fat as opposed to several studies which have used bmi as a surrogate for body fat. the limitations of our study included, small sample size, presence of vitamin d deficiency in all study subjects which gave us very narrow range of serum 25(oh)d concentrations thereby limiting comparisons. absence of pth measurement was another important limitation, since it has been implicated in pathogenesis of insulin resistance and metabolic syndrome. another important limitation was inability of dxa technique to differentiate between subcutaneous and visceral adipose tissue. in conclusion, our study demonstrates a significant inverse relationship in obese asian-indian children between body fat indices and serum 25(oh)d concentrations which persists even after adjustment of covariates. the association between serum 25(oh)d and parameters of insulin sensitivity and resistance is influenced by puberty as a significant association was found between these two variables in postpubertal subjects (tanner stage 5) only which remained significant even after adjustment for covariates. as association is not equal to causation, future longitudinal research studies are required in determining role of puberty on association between serum 25(oh)d and insulin resistance in both, obese as well as subjects with normal bmi .

to study the effect of puberty on the relationship between serum 25-hydroxyvitamin d (25(oh)d) and parameters of insulin kinetics in obese asian-indian children. material and methods. the study population included 62 obese asian-indian children and adolescents in the age group of 617 years. blood glucose, serum insulin, and serum 25(oh)d were measured. total body fat was measured by dual energy x-ray absorptiometry. indices of insulin resistance (homa-ir, auc for insulin) and sensitivity (wbisi) were calculated after oral glucose tolerance test. result. a total of 62 subjects (35 boys; mean age=13.0 3 years; bmi=29.3 4.8 kg/sq m; 19 subjects in tanner stage 1, 11 in stage 2, 6 in stage 3, 3 in stage 4, and 23 subjects in tanner stage 5) were studied. all study subjects were vitamin d deficient with a mean serum 25(oh)d of 8.5 4.2 ng/ml. no significant relationship was observed between serum 25(oh)d and parameters of insulin kinetics in prepubertal children. however, a significant inverse correlation was seen between serum 25(oh)d and homair (r=0.41, p=0.03) in postpubertal subjects. conclusion. the relationship between vitamin d status and parameters of insulin kinetics are affected by puberty.

PMC3432876

pubmed-61

a 21-year-old man (height=180 cm, weight=95 kg, body mass index=29.3 kg/m) presented to the outpatient department with a long history of frequent and severe snoring. on physical exam, after a diagnosis of obstructive sleep apnea was made, a bilateral tonsillectomy and uvulopalatopharyngoplasty was planned for treatment. prior to surgery, the patient had normal laboratory results with hemoglobin (hb) of 15.8 g/dl and no systemic or hematological diseases or history of bleeding disorders. on the day of the surgery, the patient received no premedication due to the history of sleep apnea. intraoperative monitors included electrocardiogram, non-invasive blood pressure (bp), pulse oximetry (spo2), and end-tidal co2. pre-anesthetic vital signs were bp 120/82 mmhg, hr 68 bpm, and spo2 96%. general anesthesia was induced with remifentanil 0.5 g/kg/min and propofol 180 mg iv at 10:10 am. at loss of eyelash reflex, cisatracurium 16 mg was administered, and the trachea was intubated after a short period of facemask ventilation. anesthesia was maintained with sevoflurane 2 vol% in 50% oxygen-enriched air and remifentanil 0.2 g/kg/min. the neuromuscular block was antagonized with glycopyrrolate 0.6 mg and pyridostigmine 15 mg iv without neuromuscular monitoring upon the completion of the surgical procedure at 11:25 am. postoperatively, a physical examination confirmed the presence of acute bleeding in the left inferior pole of the tonsillectomy site in the post anesthetic care unit. the surgeon requested an emergent re-operation under general anesthesia for the control of bleeding. a rsi for a second round of general anesthesia was performed with propofol 120 mg, midazolam 5 mg, and rocuronium 100 mg at 11:55 am. the surgeon confirmed active bleeding due to an aberrant arterial blood supply in the left inferior pole of the tonsillectomy site. after the completion of the surgical procedure, no twitch was shown after train-of-four (tof) stimulation (tof=0) with neuro-stim (model ns-3cc, houston, tx, usa) at 12:40 pm. the neuromuscular block was then antagonized with sugammadex 200 mg iv and the rocuronium-induced nmb was completely reversed within 2 min. the patient was transferred to the recovery room, and there were no specific findings on physical exam. a post-operative laboratory assessment showed hb of 14.6 g/dl (table 1). after 6 h and 20 min, the surgeon again requested emergent anesthesia and re-operation for control of bleeding. the surgeon attempted to control bleeding at the ambulatory otolaryngology clinic but was unable to do so. when the patient arrived at the operating room (or), vital signs were within normal limits and hb was found to be 13.2 g/dl. a third round of general anesthesia was induced with propofol 120 mg and rocuronium 40 mg by the on-call resident at 19:00 pm. the neuromuscular block was antagonized with sugammadex 200 mg iv and a tof=0 at 20:00 pm. the patient was again extubated successfully and transferred to the recovery room with no specific findings (table 1). after 2 h and 35 min, when the patient arrived at the or, vital signs were stable and hb was 11.6 g/dl. a fourth induction of general anesthesia was performed with propofol 120 mg and rocuronium 40 mg by the same resident at 22:35 pm. during tof stimulation, 1 twitch was seen, and an additional dose of rocuronium 10 mg was given due to bucking 20 min after the induction of anesthesia. at the end of the procedure, the neuromuscular block was antagonized with glycopyrrolate 0.4 mg and pyridostigmine 10 mg iv with a tof=1 at the end of the surgical procedure at 0:20 am. extubation was carried out after confirming the recovery of consciousness, regular respiration, and the absence of fade on tof monitoring. after extubation, the patient vomited a large amount of blood on the operating table (approximated to be more than 200 ml). however, the patient was alert and aware of his surroundings and had regular respirations (table 1). after 20 min, the surgeon requested a re-induction of general anesthesia for additional bleeding control. an rsi for a fifth round of general anesthesia was performed with propofol 120 mg and rocuronium 100 mg at 0:40 am. the neuromuscular block was antagonized with glycopyrrolate 0.4 mg and pyridostigmine 10 mg iv with a tof=1 after the surgical procedure was completed at 1:30 am. approximately 15 min passed after the nmb was antagonized as above, but no evidence of recovery from the rocuronium-induced nmb was seen. therefore, we injected a bolus of iv sugammadex 200 mg to reverse the residual nmb. once again, the nmb was completely reversed within 2 min, and tracheal extubation was performed in the or uneventfully (table 1). a decision by surgeon was made to perform angiography of the left external carotid artery with subsequent embolization of the bleeding vessel. embolization of the left ascending pharyngeal artery was performed with polyvinyl alcohol particle (350 m) and tornado embolization microcoil (cook, bloomington, usa) (3/2 mm) (fig. the patient's postoperative course was uneventful, and hb was found to be 10.5 g/dl. pth occurs in approximately 1 in 20 adults (5.1%), and more than half of patients who bleed are likely to require a procedure of some type to control their hemorrhage. aberrant arterial blood supply to the tonsillar region derived from the internal carotid artery or the carotid bulb may be present. to control bleeding, electrocauterization, packing of the pharynx, or angiographic embolization of the feeding artery may become necessary. the endovascular embolization due to refractory bleeding after tonsillectomy proved to be a valuable treatment method as an effective alternative to surgical intervention. it is a safe and permanent treatment option in this potentially life-threatening complication. in a case such as this, the aberrant arterial blood supply in the left inferior pole of tonsillectomy site caused continuous bleeding. the anesthesiologist has experienced difficulties, because an experienced surgeon has embarrassed due to repetitive bleeding. in the first induction of anesthesia, no problems were encountered with conventional anesthesia, although neuromuscular monitoring was not performed. the most common anesthetic used for rsi during the management of children with pth is succinylcholine. since the introduction of sugammadex, rsi with rocuronium followed by reversal with sugammadex allowed for earlier establishment of spontaneous ventilation than with succinylcholine, which has many adverse side events. during the second round of anesthesia, rocuronium 100 mg (1 mg/kg) was injected for rsi, followed by reversal with sugammadex 200 mg (2 mg/kg) at deep nmb (tof=0). during the operation, nmb monitoring was difficult due to problems attaching leads to the arms during the operation. tof stimulation was applied at the ulnar nerve using conventional instrumental monitoring rather than quantitative monitoring due to convenience. deep nmb was maintained for bleeding control in an attempt to not disturb the procedure. during nmb reversal, we found an absence of fade on tof monitoring just 2 min after sugammadex was administered. re-administration of 0.6 mg/kg rocuronium could then be given after sugammadex at the recommended waiting time of 6 h. during the third anesthesia induction, an on-call resident used rocuronium 40 mg for muscle relaxation during intubation followed by reversal with sugammadex 200 mg at deep nmb (tof=0). an absence of fade on tof monitoring was shown 2 min after sugammadex was given. for the fourth round of anesthesia, the same resident used rocuronium 40 mg for intubation 2 h and 35 min after the previous nmb reversal with sugammadex. however, we believe this decision was made without considering the recent sugammadex use. when rocuronium is used within a short time interval (less than 6 h) after sugammadex administration, the onset time of rocuronium may be prolonged and may also have an unpredictable duration of action. we suspect that the additional dose of rocuronium 10 mg due to bucking may be related to the short time interval between sugammadex and rocuronium administration. in situations such as these, the benzylisoquinoline class of medications are recommended instead of re-administering rocuronium within 6 h after sugammadex. when considering this case, cisatracurium for muscle relaxation and intubation may have been a better choice for the fourth round anesthesia. however, one drawback of cisatracurium is the slower onset time than rocuronium. reversal of the rocuronium-induced nmb with pyridostigmine at a moderate nmb (tof=1) was uneventful. after extubation, the patient vomited more than 200 ml of blood on the operating table; however, he was alert and showed regular respirations. postoperative nausea and vomiting (ponv) after an operation for pth is common due to blood swallowed during the procedure. therefore, patient awareness and complete reflexes are important, but aspiration must be avoided and monitored closely during the recovery period. however, ponv increases the risk of primary hemorrhage and unexpected postoperative hospital admissions. in this case the patient did not aspirate, but did have an increased risk of pth due to ponv. for the fifth round of anesthesia, rocuronium 100 mg (1 mg/kg) was injected for rsi after a time interval from sugammadex administration of 4 h and 40 min. it has been reported that re-administration of 1.2 mg/kg rocuronium could have been given after sugammadex 2 mg/kg without a waiting time. however, neuromuscular monitoring did not show the expected recovery, and the patient had residual paralysis after 15 min. it has been shown previously that the reversal of nmb with anticholinesterase drugs may not be complete after a large dose of a neuromuscular blocking agent. sugammadex resolved the residual nmb within 2 min, even after reversal with anticholinesterase agents, and is a safe alternative to reversal of nmb induced by steroidal non-depolarizing agents. in this case, deep rocuronium-induced nmb can be reversed successfully with sugammadex at doses of 2 mg/kg and in as little as 2 min. re-administration of rocuronium within a short time interval after sugammadex may result in unpredictable effects of the nmb agent. sugammadex made it possible to perform a rapid, complete reverse when the residual block was maintained by an incomplete reversal of anticholinesterase.

post-tonsillectomy hemorrhage (pth) is the most frequent complication of tonsillectomy, and occasionally results in a lethal outcome. a 21-year-old man (height 180 cm, weight 95 kg) was scheduled for a bilateral tonsillectomy and uvulopalatopharyngoplasty for treatment of obstructive sleep apnea. he required 5 rounds of general anesthesia due to recurrent pth. the anesthesiologist used sugammadex a total of 3 times to achieve the successful reversal of the deep neuromuscular blockade (nmb) induced by rocuronium. after sugammadex 2 mg/kg was administered, the nmb was reversed in 2 minutes each time. re-administration of rocuronium within a short time interval after sugammadex may result in unpredictable effects of neuromuscular blocking agents. sugammadex made it possible to perform a rapid, complete reverse when the residual block was maintained by an incomplete reversal of anticholinesterase.

PMC4121494

pubmed-62

infertility has been recognized as a public health issue worldwide (1) leading to an increasing need to the use of assisted reproductive technologies (art), including in vitro fertilization (ivf). after the first reported case of ivf in 1978(2) art enabled millions of people to have their own children in cases when pregnancy did not occur under natural circumstances. 700,000 cycles a year in the usa and europe together (3,4). despite of evolving intracytoplasmatic sperm injection (icsi) technique a success rate of 25% and 28% has been reported in 2005(7) and 2008(8), respectively. nowadays, this rate went up to 32% (9), which can not be considered as a significant development. earlier clinical protocols preferred multiple embryo transfer, but multiple gestations can result in the increased risk of preterm delivery (10-16). other studies report that multiple gestations also increased the risk of low birth weight cerebral palsy (17). in the us alone, preterm births resulting from multiple pregnancies during ivf cause a 1 billion usd extra cost to the social insurance (18). in order to exclude the discussed risk factors, it is imperative, however, that accurate and economical methods should be developed to ensure that the most viable euploid embryo is selected for transfer. ideally, such tests would be noninvasive, lessening the risks to the embryo and reducing costs and workload in the embryology laboratory (19). the biggest issue with pre-implantation viability assessment is that due to ethical reasons any assay should be completely non-invasive because no one can predict what kind of interference would be the unwanted result in the later embryonic development. the most apparent and routinely applied-way of the assessment of viability is the morphological evaluation of in vitro fertilized embryos using microscopy. there are several morphological features described which could be used for viability assessment purposes, these are dependent on the time spent after fertilization. right after fertilization in the 1-cell embryo the size and symmetry of the two pronuclei can be examined. the time of the first cell division is also a good predictor of later implantation potential, as zygotes that divide early tend to develop more frequently to the blastocyst stage. criteria as cleavage rate and blastomere shape and symmetry, an adequate trophectoderm layer (te) and an inner cell mass (icm) is a morphological marker of the later stages (5,20). not only can the morphology of the fertilized embryo be used for further prediction of implantation potential, but morphological defects of the retrieved oocyte as well. fertilization and pregnancy rate correlates with the grade of cumulus-oocyte complexes, and embryos originating from dysmorphic oocytes show a larger grade of pregnancy loss (21-23). the cleavage stages of morulae and blastocysts or the symmetry and patterns of cell division are also notable and frequently used aspects, and are often examined during the prediction of embryo viability (23). the biggest issue of morphological assessment is that it is still a highly subjective method (20). the reason is partly due to the fact that the final decision is made by a clinician, and not by an objective test result, and secondly it is does matter how important are the individual morphological features in the final conclusion (24-26). to overcome the different practice of laboratories worldwide in 2011 an international consensus (istanbul consensus) has been reached on embryo viability assessment (27). the selected morphological markers of respective stage embryos, the weighing of individual features and a scoring system has been set up. the limitations due to static time-point observation, is now solved with the use of time-lapse microscopy (28,29). time-lapse microscopy also enables the observation of dynamics of cytoplasmic movements and cytokinesis, reflecting the functionality of microtubule and actin cytoskeleton, which is critical for proper development. in our laboratory, we aimed to improve the success rate of implantation by adopting and further optimizing the istanbul consensus. this score has been called as the optimized criteria system (ocs). according to this scoring, 3-day old embryos were divided into two subgroups: the subgroup with low blastomere number (less than 7) and with high blastomere number (7 or more). symmetric position of blastomeres indicates the rate of symmetry of holoblastic cleavage along the embryo axis. it was classified as good (full symmetry); fair (light asymmetry); or poor (evident asymmetry). the percent values of fragmentation are based on the ratio of fragmented to total cell numbers. as a further modification to the istanbul consensus, embryos were considered as good if the fragmentation rate was<15% (instead of the original 10%). this shift from 10 to 15% was the result of our observation that a fairly high proportion of the embryos between 10-15% appeared to be viable. in summary the optimized criteria system (ocs) highlights 3 modified or new parameters: fragmentation (with a more permissive criterion of<15% in the good category); symmetry and the blastomere number. in addition, the blastomere size was evaluated according to the original istanbul consensus. a scoring-map was created to facilitate the evaluation (table 1) as far as the 5-day old embryos are concerned, we modified the original istanbul consensus for blastocysts by leaving out the hatched stage from the evaluation. the istanbul consensus for the 5-day old embryos has a shortcoming, i.e. it does not express the viability of embryos with a single category (good, fair, poor). we tried to overcome this by using a scoring map (table 2). in conclusion, we constructed a composite score for day-3, as well as day-5 old embryos, based on morphological parameters. as it is evident from the results, this composite score is sensitive to evaluate viability (figure 1) another possibility for non-invasive embryo viability assessment is the metabolomic examination of the culture medium surrounding the in vitro fertilized embryo. metabolomic, (proteomic) profiling of spent embryo culture (sec) offers an exceptional, non-invasive opportunity for the assessment of embryo viability (30,31). the metabolomic profiling (32,33) of early embryo development might mean the analysis of the total metabolome by following the changes of several selected compounds, metabolomic analysis using unidentified, but significantly differing metabolomic changes, or by the analysis of a limited population of nutrients or end products. the common feature in all three concepts is that they are concentrating on the metabolomic alterations caused by differently developing embryos in the culture medium. very simple idea is the monitoring of glucose consumption or pyruvate formation, since this would directly indicate the metabolism of the developing embryo and it is an obvious conclusion that a metabolically active embryo would have higher implantation potential. some authors reported that the identification of these parameters resulted in successful prediction of embryo implantation potential, but other research groups describe contradictory results (34, 35). the amino acid profile of culture media is also used in the prediction of implantation potential, though not exclusively as an independent parameter, rather in combination with morphological features (36). the detection of unidentified metabolomic changes using near infra-red (nir) or raman spectroscopy (37, 38) is a very interesting and challenging possibility. more complicated is the concept when unknown, new biomarker molecules of embryo viability are searched for, assuming that these biomarkers were secreted by the embryo. the difficulty of the concept is that only 4-8 cells are present in the culture medium; thus a very sensitive analytical tool is required. mass spectrometry (ms) has the potential of specific and sensitive quantification in a wide spectrum of molecular mass ranges and therefore suites well the needs of metabolomic or proteomic fingerprinting and quantification. in parallel to the spreading of mass spectrometry, proteomics is also an emerging field in the understanding of embryo development (39,40). the analysis of the embryonic secretome (41,42) provides information of the total transcriptome of the developing embryos. mass spectrometry can be used both in targeted and discovery analysis with accurate quantification of identified biomarkers after molecular identification by bottom-up or top-down proteomics using tandem or multiple ms (43-46). in a recent publication from our laboratory (47) using liquid chromatography coupled mass spectrometry (lc-ms), a fragment of the human haptoglobin molecule was identified in the culture medium. rather than analyzing the embryonic secretome, the aim this experiment was to use preexisting molecules present in the cell culture media as biomarkers. haptoglobin-which was detected in the culture medium-is not a product of the developing embryo; the polypeptide is a contaminant of the human serum albumin standard used to supplement the culture medium (47,48). during the first three days of embryo development the formation of a subunit (alfa-1) of the human haptoglobin molecule was observed. this subunit similar to the total haptoglobin molecule was detectable in the blank control medium samples as well. the differentiation of the viable and non-viable embryos was done using the observation that compared to blank controls the samples of embryos which later did not resulted in pregnancy contained the alpha-1 subunit in a much larger quantity than the samples of embryos which did (figure 2). clinical statistical analysis of the results revealed that the specificity of the diagnostic test was 64%, while the sensitivity was 100%. it is more informative that the positive predictive value of the assay was 51% and maybe more importantly the negative predictive value was 100%. receiver operating characteristic (roc) analysis provides tools to select possibly optimal models and to discard suboptimal ones. roc analysis is related in a direct and natural way to cost/benefit analysis of diagnostic decision-making. the roc curve of the morphological versus metabolomic approach in relation to the correct prediction of pregnancy outcome is illustrated in figure 3. it is obvious that our biochemical investigation method enables a selection of the embryos by sorting out the non-viable ones. the test selected with 100% potential the embryos, which did not lead to successful implantation at all. one of the areas of collaboration between clinicians, the clinical laboratory and the research laboratory at the university of pcs is related to the research of infertility. since the clinical background gives the beauty and the medical importance of laboratory research, it was of outstanding importance for us to receive the eflm-abbott diagnostics award for excellence in outcomes research in laboratory medicine (paris, 2015), the award given to the best published paper (47), as judged by an independent panel of experts, which demonstrates improved outcomes arising out of the application or improved utilization of an in-vitro diagnostics test.

human reproduction is a relatively inefficient process and therefore the number of infertile couples is high. assisted reproductive technologies (art) have facilitated the birth of over five million children worldwide. art, however, superimposes its own relative inefficiency on the preexisting inefficiency of normal reproduction. the efficiency (expressed as pregnancy rate) is generally not more than 30%. modern reproductive medicine is gradually moving from multiple embryo transfer to the transfer of a single embryo, mainly because of obvious and unwanted side effects of multiple embryo transfer (e.g. epidemic multiple pregnancies). this concept, however, requires a fast, professional selection of the most viable embryo during the first few days of art. thus the aim of a modern art is the safe transfer of a healthy, viable, single embryo. accurate and rapid methods of quantifying embryo viability are needed to reach this goal. methodological advances have the potential to make an important contribution, and there has been a drive to develop alternative non-invasive methods to better meet clinical needs. metabolic and genetic profiling of spent embryo culture (sec) media should offer an exceptional opportunity for the assessment of embryo viability. the current review focuses on the latest non-invasive diagnostic approaches for pre-implantation viability assessment of in vitro fertilized embryos.

PMC4975227

pubmed-63

leaf senescence is the last phase of plant development and a highly coordinated process regulated by a large number of senescence associated genes (sags) (12). leaf senescence can either be naturally induced during development stages, or stimulated by environmental factors including darkness, nutritional deficiency and various stresses (13). premature senescence is an important factor leading to the decrease of crop yield and quality, which becomes an increasing concern due to the global climate change in the recent years. many advances in the understanding of leaf senescence at the molecular level had been achieved through the identification and characterization of hundreds of sags and senescence-related mutants in arabidopsis thaliana, lycopersicon esculentum and nicotiana tabaccum (14). microarray expression profiling in arabidopsis revealed that more than 800 genes are up-regulated during the course of leaf senescence (5). among them, more than 200 transcription factors, including wrky, nac, mads, myb, bzip and bhlh family members, are involved in the regulation of leaf senescence, indicating that leaf senescence is governed by complex transcriptional regulatory networks. molecular and genetic studies of leaf senescence in recent years led to the accumulation of a large volume of scattered information related to sags. the construction of a leaf senescence related database with wide-spread collection and systematic annotation of sags may provide a useful resource and a good starting point for the further study of the molecular aspects of leaf senescence. some initial efforts to this end had been made; including the online website of plant senescence network (sennet) constructed by thomas and his colleagues (http://www.sidthomas.net/plant_senescence/) and the corresponding senwiki web pages (http://www.sidthomas.net/senwiki/). sennet brings together various community information including meetings, laboratories, websites and useful links related to plant senescence, while senwiki provides general information and knowledge of senescence with texts and images. however, a wide-ranging compilation and detailed annotation of known sags that would be of great help and demand to the systemic study of leaf senescence at the molecular level has yet to be done. in this regard, we have developed a leaf senescence database (lsd) (http://www.eplantsenescence.org/) by retrieving and integrating information from research papers and public databases. at present 1145 sags from 21 species were manually curated and categorized into several groups according to their function. users can browse the entries in the database to obtain information including literatures, mutants, phenotypes, expression profiles, mirna interactions, orthologs in other plants and cross links to protein domain and family databases. users can also search the database easily through the text search interface with locus names, keywords and author names, etc. we have implemented the blast tool kit for sequence similarity search against nucleotide or protein sequences of these sags in the lsd. a major feature of the lsd is the integration of a bioinformatics platform weblab we had developed previously (6), which allows users to perform extensive sequence analysis of the sags they are interested in. all the sag sequences are freely available for downloading. help information including user guides, a tutorials and faqs are also available online. we plan to identify putative sags from completely sequenced genomes and add them into the database in the near future, and improve the user interface based on user feedback, provide more help documents with case studies, add more links to other useful sites, and update the database in time with more leaf senescence related data available. we hope that lsd could be a useful resource for the leaf senescence research community, as well as a gateway for the collaborative project we are working with both domestic and international colleagues. we made an extensive literature survey and collected approximately 200 leaf senescence related papers published before october 2010 on major plant biology journals including plant cell, plant physiology, plant journal, new phytologist, journal of experimental botany, among others. a total of 1145 sags from 21 species including arabidopsis, rice and so on, were identified and manually verified based on genetic, genomic, proteomic, physiological and other experimental evidence. among them, 96 and 58 genes were supported by mutational investigation or transgenic over-expression study, respectively (table 1). table 1.number of sags and mutants in 21 speciesspeciescommon namesagsmutantstransgenicarabidopsis thalianathale cress9499035oryza sativarice10439medicago truncatulabarrel clover3100brassica napusrape1500lycopersicon esculentumtomato803nicotiana tabacumtobacco503brassica oleraceabroccoli400pisum sativumpea410glycine maxsoybean401sorghum bicolorsorghum400solanum tuberosumpotato303zea maysmaize300hordeum vulgarebarley300astragalus sinicuschinese milk vetch101chenopodium rubrumred goosefoot110festuca pratensis huds.fescue110ipomoea niljapanese morning glory101medicago sativaalfalfa101rosa hybridrose100triticum turgidumwheat101triticum aestivumwheat100total2111459658 number of sags and mutants in 21 species the information of 154 leaf senescence related mutants such as name, ecotype and mutagenesis method were also retrieved from literatures. expression profiling data were acquired from a classical and systemic research paper (5). in addition to manual curation, computational approaches were also employed to annotate these sags. we predicted the potential mirna targets for the sags using the rnahybrid method (7). the orthologs of each sag in other plants were retrieved from the online database orthomcl-db (8). finally, putative function domains of sags-encoding proteins were identified with interproscan (9). to meet the general requirement of data analysis, we integrated the sequence similarity search tool blast (10) and the sequence analysis platform weblab (6) into our leaf senescence database. users can either retrieve the sequence from lsd, or upload their own sequences to search homologs against different divisions (gene, mrna, cds and protein) in the lsd. weblab is a web-based bioinformatics platform developed by our center and publicly available worldwide (http://www.weblab.org.cn/) (6). a user space is provided to save input data and analysis results for registered users. users may retrieve sequence data and submit to weblab directly to perform extensive analysis for dna, mrna and protein sequences of the sags they are interested in. the lsd database enables users to retrieve and analyze sags through the browse or search page. users may browse the entries by clicking the buttons of species, mutants or phenotypes at the main page. a tree-like structure was designed for both species and phenotypes, and a table was created for mutants. currently, the major source of sags was from the two model organisms a. thaliana (949 entries) and oryza sativa (104 entries). the phenotypes of all sags were divided into the following groups: (i) natural senescence, (ii) dark induced senescence, (iii) nutrition deficiency induced senescence, (iv) stress induced senescence and (v) others. the text search interface allows users to make queries with three types of data: (i) locus name, genbank i d, alias, species and description of genes; (ii) name, type and ecotype of mutants; and (iii) title, author, journal and date of literature papers. figure 1 shows the annotation for a typical lsd entry ntl9, a member of the nac transcription factor family and a membrane-associated gene that mediates osmotic stress signaling in leaf senescence (11). general information such as locus name, alias, organism, taxonomy was retrieved from the literature. functional category, effect and evidence of senescence, as well as a brief description of this gene were manually annotated (figure 1a). expression profiles generated from microarray data can be found for most arabidopsis sags (figure 1c). we predicted potential mirna targets for some sags and added links to mirbase (12) for these mirnas (figure 2a). orthologs from other plants are listed with links to the orthomcl database (figure 2b). putative functional domains of proteins encoded by sags were identified and annotated using the interproscan program (9), and matches were displayed with cross links to several protein domain and family databases such as prosite and pfam (figure 2c). (a) basic information, (b) mutant information and (c) expression profile. figure 2.computational annotations for the arabidopsis nac transcription factor. (a) mirna targets, (b) ortholog groups and (c) cross links to other databases. a typical entry in lsd, the arabidopsis nac transcription factor. (a) basic information, (b) mutant information and (c) expression profile. (a) mirna targets, (b) ortholog groups and (c) cross links to other databases. in addition to sequence similarity search with the blast tool kit implanted in the database, users may also perform extensive analysis for sequence data retrieved from the lsd. for each entry, links to different sequence types (genomic, mrna, cds and protein) are provided. users can click these links to bring up the corresponding sequence and submit it to weblab for further analysis, such as predicting gene structures, making pairwise or multiple sequence alignment, generating sequence logos, constructing phylogenetic trees, finding sequence motifs, etc. we will update the database regularly with more leaf senescence related data available, and predict putative sags from completely sequenced plant genomes in the near future. we will improve the user interface with comments and suggestions from the user community and add more documents including case studies to help user to make thorough analysis of sags. we hope that lsd can be a platform not only for the domestic and international collaborators we are working with, but also for the research community of leaf senescence worldwide. national natural science foundation of china (31071160 to j.l., 30625003 and 30730011 to h.g.); ministry of science and technology of china (2009cb119101 to h.g.); ministry of education of china (ngi2008-108-3 to j.l., ed20060047 to h.g.). funding for open access charge: national laboratory of protein engineering and plant genetic engineering.

by broad literature survey, we have developed a leaf senescence database (lsd, http://www.eplantsenescence.org/) that contains a total of 1145 senescence associated genes (sags) from 21 species. these sags were retrieved based on genetic, genomic, proteomic, physiological or other experimental evidence, and were classified into different categories according to their functions in leaf senescence or morphological phenotypes when mutated. we made extensive annotations for these sags by both manual and computational approaches, and users can either browse or search the database to obtain information including literatures, mutants, phenotypes, expression profiles, mirna interactions, orthologs in other plants and cross links to other databases. we have also integrated a bioinformatics analysis platform weblab into lsd, which allows users to perform extensive sequence analysis of their interested sags. the sag sequences in lsd can also be downloaded readily for bulk analysis. we believe that the lsd contains the largest number of sags to date and represents the most comprehensive and informative plant senescence-related database, which would facilitate the systems biology research and comparative studies on plant aging.

PMC3013730

pubmed-64

female study participants were recruited from participants in a cross-sectional study assessing bmd in young women with type 1 diabetes and age-matched community control subjects (7). cases for the cross-sectional study (baseline) were recruited from a regional tertiary hospital pediatric diabetes center and from endocrinology practices in western new york. at the time of the initial study, the diabetes center followed over 600 patients aged 221 years with type 1 diabetes. female patients aged 1321 years in that clinical center were offered the opportunity to participate in the baseline study (n=138). recruitment of older women involved contacting former center patients (aged>21 years), patients from regional endocrinology practices, and affected relatives of subjects screened by our diabetes center for the diabetes prevention trial. the comparison group included young women recruited from the same region as the case subjects; most were classmates or acquaintances of the case subjects or were volunteers who learned of the study through flyers posted at the university and hospital. the baseline study enrolled 72 case subjects (diabetes duration>2 years) and 91 control subjects aged 1337 years who were at least 2 years postmenarchal (7). all type 1 diabetic case subjects and nondiabetic control subjects who participated in the baseline study were recruited 2 years later to assess change in bmd over time. contact was maintained with study participants between the baseline and follow-up exam via phone and mail. inclusion criteria for the follow-up study were participation in the baseline study, current negative pregnancy test, and signed informed consent. for individuals younger than 18 years of age, exclusion criteria for both the baseline and follow-up study included systemic illness affecting bmd (other than diabetes), other endocrine disorders (except autoimmune thyroiditis), and diagnosis of juvenile osteoporosis or other bone disease. the study was approved by the institutional review boards of the women and children's hospital of buffalo and the university at buffalo. at baseline, study participants completed questionnaires relating to personal and family health, lifestyle habits, dietary intake (food frequency questionnaire), and medication intake including use of calcium supplements. participants also reported prior fracture history, menstrual history, and demographic information. for participants with type 1 diabetes, information on disease duration, insulin dose schedule (injections versus continuous subcutaneous insulin infusion), and diabetes complications were reported. bmi was calculated as weight in kilograms divided by the square of height in meters. dexa (hologic qdr-4500a; hologic, waltham, ma) was used to measure bmd in the anterior/posterior (l14) spine, total hip, femoral neck, total forearm, and whole body. a single technician performed all baseline scans and 65% of the follow-up scans; two additional technicians performed the remainder of the follow-up scans (27% and 7%, respectively). coefficients of variation (cvs) were determined for measures of all sites for all technicians throughout the study and were all<1% throughout the study. the following biomarkers were measured in nontimed blood samples: serum osteocalcin, igf-1, igf binding protein (igfbp)-3 (by radioimmunoassay), and a1c (by high-performance liquid chromatography with bio-rad variant; bio-rad, richmond, ca). random urinary n-teleopeptide levels were measured by enzyme-linked immuno-absorbent assay. serum for hormonal assays was frozen at 80c and stored until sent for analysis in batches to esoterix laboratory (calabasas hills, ca). a1c (fresh plasma) was assayed in a sequential fashion at the women and children's hospital of buffalo laboratory (kaleida health). intra-assay cv was<3% for n-telopeptide, 6% for igf-1 and osteocalcin, and 13% for igfbp-3. interassay cv was<7% for n-telopeptide,<9.7% for igf-1, 13% for osteocalcin, and<17% for igfbp-3 (esoterix laboratories). data from study participants were analyzed in stratum based on age at initial enrollment (7). demographic, lifestyle, metabolic characteristics, and bmd were compared between diabetic case subjects and nondiabetic control subjects, stratified by age. comparisons were made for baseline differences, differences at follow-up, and percent change between the two time points. results were presented as both unadjusted comparisons and comparisons of adjusted means (adjusted for age, bmi, and oral contraceptive [oc] use). unadjusted differences for continuous variables were examined using student's t test and for categorical variables using the test. data analyses were performed using sas version 8 (sas, cary, nc). post hoc power analysis of follow-up total hip bmd (adjusted for age, bmi, and oc use) indicated that our dataset of 63 case subjects (n=37,<20 years of age) and 85 control subjects (n=36,<20 years of age) gave our study 80% power to detect a difference between case and control subjects with an of 0.05 and a target effect size of 0.45 in women<20 years of age and 0.63 in those>20 years of age. table 1 includes baseline and follow-up characteristics of women who participated in this follow-up study. for comparative purposes, the follow-up group included 63 women with type 1 diabetes (58.7%<20 years of age) and 85 control subjects without diabetes (42.4%<20 years of age) at ages 1539 years at enrollment. of the participants in the baseline study with diabetes, 87.5% (63 of 72) participated in this follow-up study, and 93.4% (85 of 91) of control subjects participated in both examinations. more subjects from the baseline cohort 20 years of age were lost to follow-up (13.8 vs. 3.94%, cohort<20 years of age), with the highest attrition being from individuals with diabetes 20 years of age (21.2%). participants in this follow-up study were predominantly non-hispanic caucasian (95%) and were similar in age at menarche and years since menarche. participants 20 years of age with diabetes used oc therapy longer than control subjects (p 0.05). although few participants were smokers, more women with diabetes reported current smoking; there were no smokers among the control subjects<20 years of age. at follow-up, women with diabetes, particularly the older women, continued to have higher bmi than control subjects. weight change (in percent) was not significantly different over the follow-up period. as reported previously, more patients in the older cohort were using continuous subcutaneous insulin infusion (p<0.01, data not shown). while a trend toward lower daily insulin requirement was present in the older cohort (0.86 0.42 vs. 0.75 0.22 units kg day), that difference was not statistically significant. subjects 20 years of age were more likely to have diabetes-related complications, although no statistical differences were found. table 2 presents bmd values at baseline and follow-up for each body site measured. unadjusted means and means adjusted for age, bmi, and oc use are shown stratified by age-group and diabetes diagnosis. in those<20 years of age, both case and control subjects had an increase in bmd over the 2-year interval. the percent increase in bmd from baseline to follow-up was not statistically different between younger subjects and control subjects. for all participants in the population 20 years of age, the bmd from baseline to follow-up remained stable. significant differences in bmd were identified at the total hip, femoral neck, and whole body in the case subjects 20 years of age compared with the control subjects. as in the previous report, no differences in bmd were seen between groups in the younger cohort. in the adjusted model (for age, bmi, and oc use), bmd values at the total hip (baseline, p=0.003; follow-up, p=0.007) and femoral neck (baseline and follow-up, p<0.001) were significantly lower in older women with diabetes compared with control subjects. after adjustment, the whole-body bmd was no longer significantly different between case and control subjects; however, both age-groups showed a trend toward lower whole-body bmd in the case subjects. further adjustment for dietary intake of calcium and vitamin d as well as physical activity did not alter the bmd results (data not shown). when the analysis was restricted to nonsmokers, the adjusted means changed very little. for the older cohort, however, the p values were attenuated for the total hip (p=0.017) and whole body (p=0.215). in nonsmokers>20 years of age, the percent change difference at the total hip became more pronounced between case and control subjects and reached statistical significance (p=0.008). overall, igf-1 levels were lower for all groups compared with baseline (table 3). this is consistent with a physiologic decrease in igf-1 in postteenage years (esoterix normative data 11). at the 2-year follow-up, however, the igf-1 level was significantly lower in the younger diabetic subjects than in the control subjects, although still within the normal range. igf-1 levels were not statistically different between diabetic participants and control subjects in the older cohort. within the older age-group, igfbp-3 levels were lower in participants with diabetes than in control subjects (p<0.05). igfbp-3 levels were comparable for control and diabetic subjects in the cohort<20 years of age. serum osteocalcin levels were lower at follow-up for all groups, even with increased bone mineral accrual in the group<20 years of age (table 3). there was no difference in osteocalcin or n-teleopeptide levels between case and control subjects<20 years of age (table 3). after adjusting for age and bmi, case subjects had significantly lower osteocalcin levels than control subjects in the population 20 years of age (p<0.04; table 3). in addition, the percent change in osteocalcin level from baseline to follow-up was significantly different in diabetic women 20 years of age compared with control subjects after adjustment (p<0.03). markers of bone formation did not correlate with bmd in our subjects. metabolic control, as measured by a1c, was poorer in the younger diabetic cohort, as we reported previously (7). overall metabolic control was stable within a given age stratum between the baseline and follow-up studies. no correlation was found between a1c and any of the bmd measurements (data not presented). when evaluating for associations between metabolic control and bmd measurements that were statistically lower in case than in control subjects at follow-up (table 2), the pearson correlation coefficient (r value) for a1c and bmd was between 0.121 (total hip) and 0.334 (femoral neck) (p>0.095, data not shown). as in our previous study, there was no association found between bmd and diabetes duration, baseline a1c, or years since menarche. while longitudinal studies have examined the effect of insulin therapy on bmd in patients with long-standing type 1 diabetes (12), few have reported on the natural history of metabolic bone disease in younger women. here we present bmd data for a well-characterized cohort of young women with type 1 diabetes at baseline and 2 years later. the baseline study demonstrated that the older cohort had significantly lower bmd at the femoral neck and lateral spine than age-matched control subjects (7). the data presented here demonstrate that case subjects in the older cohort had persistently lower bmd than age-matched healthy control subjects. in this cohort, bmd at the total hip and femoral neck remained lower than in control subjects after adjusting for age, bmi, and oc use, even with no significant decrease in bmd from baseline to follow-up (table 2). similar to our initial study, there was no difference in bmd between women with diabetes aged<20 years and control subjects at any of the sites measured. however, there was a trend toward lower bmd at the total hip and whole body in women with diabetes. this difference occurred in the face of bmd increases (percent change) at both the total hip and whole body in this population, suggesting that bone mineral accrual is altered in a subtle manner during late adolescence in women with type 1 diabetes. individuals with type 2 diabetes have higher than average bmd compared with control subjects (13), in part due to the mechanical load of obesity. in our study, women with type 1 diabetes had higher bmi than control subjects. however, bmd continued to be lower in older women with type 1 diabetes, even after adjusting for bmi; thus, overweight status does not confer protection against poor bone mineralization in these young women. although the younger cohort had poorer diabetes control than the older cohort, we were again unable to demonstrate an association between bmd measures and metabolic control, as measured by a1c. others have reported similar findings with respect to bmd in children and adolescents with diabetes (14). a1c measures short-term diabetes control; perhaps cumulative life-time glycemic control is a better indicator of osteoporotic risk. this hypothesis is supported by studies demonstrating that decreased lumbar spine and femoral neck bmd in adults with type 1 diabetes is associated with retinopathy, nephropathy, and peripheral neuropathy, all of which are long-term complications of poor metabolic control (1517). in addition, our data did not find a correlation between diabetes duration and bmd, further supporting a possible relation to lifetime diabetes control. the adverse effects of chronic hyperglycemia related to osteoblast function, osteoclast activity, and formation of advanced glycosylated end products that may impact bone quality as well as bone quantity (18) are difficult to measure in a systematic fashion. the role of insulin as a direct anabolic agent in bone metabolism is unclear (19). animal models of spontaneous and pharmacologically induced diabetes demonstrate that as insulinopenia develops, there is a suppression of osteoblast markers (20). it has been postulated that in addition to insulinopenia, relative igf-1 deficiency, whether systemic or local, contributes to low bmd in diabetes. igf-1 levels are lower in individuals with type 1 diabetes, and poor glycemic control negatively impacts igf-1 production by the liver. in adults with type 1 diabetes, igf-1 levels were lower in individuals with osteopenia at the femoral neck (1). additionally, in a study of 127 children (aged 620 years) with diabetes, low bone mineral content correlated with low igf-1 levels (9). in our study, igf-1 levels of case subjects were significantly lower than those of control subjects only for the younger cohort. however, igf-1 levels of subjects with diabetes were lower in the group 20 years of age compared with the group<20 years of age, and igfbp-3 levels in the older cohort were significantly lower compared with control subjects. given that no biochemical markers of bone metabolism correlated with bmd at the hip or femoral neck in our study, it could be hypothesized that a cumulative history of insulinopenia and low igf-1 levels are better correlates for low bone density. our data demonstrate significant differences in bmd at the femoral neck and total hip that are likely to be clinically significant and may explain why women with type 1 diabetes have an increased risk of hip fractures later in life (4,5). our data also indicate that alterations in bone mineral accrual occur within years of achievement of peak bone mass and that there is not a later period of catch-up bone mineralization. the most clinically relevant factor is whether a decrease in bmd correlates with fracture risk, which can not be determined in this study. besides bmd, multiple factors impact fracture risk including, but not limited to, age, nutrition status, smoking history, degree of frailty, and fracture history (21). the well-characterized population, the large percentage of women who participated in the follow-up study, and the standard procedures used in this longitudinal study all represent significant strengths. yet, we acknowledge certain limitations. the study population is small, limiting its ability to detect true differences that may still exist. study control subjects were community based and may not represent the general population of women. finally, the results presented should be interpreted keeping in mind that multiple comparisons were made. we are aware of the lack of unanimity of opinion regarding the statistical approach when adjusting the statistical testing for many comparisons (22). because many of our hypotheses are nested, and not independent, we feel that using a bonferroni approach is overly conservative. however, we have interpreted our results with caution and believe they are stronger when considered as a whole rather than as independent statistical tests. our findings demonstrate lower bmd in young women with type 1 diabetes compared with control subjects. although bone density testing is not routinely performed in young women, these data suggest that screening may be important in young women with type 1 diabetes. in addition, these women should be counseled regarding lifestyle interventions that may improve bone health, including adequate intake of calcium and vitamin d, and exercise.

objective individuals with type 1 diabetes have decreased bone mineral density (bmd), yet the natural history and pathogenesis of osteopenia are unclear. we have previously shown that women with type 1 diabetes (aged 1335 years) have lower bmd than community age-matched nondiabetic control subjects. we here report 2-year follow-up bmd data in this cohort to determine the natural history of bmd in young women with and without diabetes.research design and methods bmd was measured by dual-energy x-ray absorptiometry at baseline and 2 years later in 63 women with type 1 diabetes and in 85 age-matched community control subjects. a1c, igf-1, igf binding protein-3, serum osteocalcin, and urine n-teleopeptide were measured at follow-up. resultsafter adjusting for age, bmi, and oral contraceptive use, bmd at year 2 continued to be lower in women 20 years of age with type 1 diabetes compared with control subjects at the total hip, femoral neck, and whole body. lower bmd values were observed in cases<20 years of age compared with control subjects; however, the differences were not statistically significant. lower bmd did not correlate with diabetes control, growth factors, or metabolic bone markers. conclusionsthis study confirms our previous findings that young women with type 1 diabetes have lower bmd than control subjects and that these differences persist over time, particularly in women 20 years of age. persistence of low bmd as well as failure to accrue bone density after age 20 years may contribute to the increased incidence of osteoporotic hip fractures seen in postmenopausal women with type 1 diabetes.

PMC2518333

pubmed-65

familial adenomatous polyposis account for 1% of colorectal cancers, and provides a model of apc inactivation as an early genetic event for the approximately 80%85% of cancers that develop from sporadic polyps. colorectal cancers arising in patients with familial adenomatous polyposis can be largely prevented by polyp surveillance and prophylactic colectomy. total proctocolectomy with construction of a conventional ileostomy or ileoanal anastomosis with preparation of an ileal pouch, has various effects on the function of the terminal ileum and the intestinal bacterial flora. this may deteriorate cholesterol metabolism, as absorption of cholesterol in duodenum and jejunum requires micellar solubilization with bile acids, fatty acids, monoglycerides, and phospholipids. hypothetically, ileal-pouch-anal anastomosis and ileostomy patients might differ with regard to the presence of various fatty acids in feces and their relationship to other reflections of lipoprotein metabolism, but we found no previous study focusing upon this issue. dietary fatty acids are incorporated into blood and tissues, and the fatty acid composition in these tissues are often used as biomarkers of fat intake. furthermore, the fecal amount and composition of fatty acids reflect fat ingestion, intestinal fatty acid absorption, and the activity of colonic bacteria. although some of the discrepancies between studies may be due to the use of different methods to analyze fatty acids, differences in diet, or the fact that assessments have been performed in different body compartments, modifications in the metabolism of fatty acids have been suggested in cancer patients [5, 6]. it is not clear at what steps in the multistage carcinogenesis process a possible distorted fatty acid metabolism occurs. notably, if such alterations occur in the development of carcinogenesis, this may affect the biological functions of essential fatty acids and their derivates. very little is known about fatty acid metabolism in familial adenomatous polyposis, although chemoprevention affecting the fatty acid derivates and the cyclooxygenase enzymes is often administered to familial adenomatous polyposis patients. deregulation of the cyclooxygenase-2 pathway appears to affect tumorigenesis via a number of distinct mechanisms: promoting tumour maintenance and progression, encouraging metastatic spread, and perhaps even participating in tumour initiation. cyclooxygenase-1 and -2 are the rate limiting enzymes in the synthesis of prostaglandins and thromboxanes. arachidonic acid is the main substrate for these enzymes, leading to the synthesis of prostaglandins which have growth promoting effects. substituting arachidonic acid with omega-3 fatty acids has been shown to lead to the production of less potent prostaglandins. since cyclooxygenase-2 is a fatty acid metabolising enzyme, the relationships between cyclooxygenase-2 and fatty acid composition of different tissues is of interest. colectomized familial adenomatous polyposis patients had a deviant fatty acid profile with high levels of arachidonic acid and docosahexaenoic acid and low levels of linoleic acid and alpha-linolenic acid in serum phospholipids, which is in accordance with studies in patients with other types of cancers [5, 1013]. in a previous familial adenomatous polyposis study, comparable treatment effects of a cyclooxygenase-2 inhibitor were observed on the fatty acid composition in serum phospholipids and duodenal lesions, presumably and most importantly the nonbeneficial effects involving essential fatty acids. the results describe the content of fatty acids in feces, and relate this to the proportions of fatty acids and levels of cyclooxygenase mrna expression in duodenal biopsies, diet, and levels of serum lipoproteins. because the effects of ileostomy construction may differ from that of ileal-pouch-anal anastomosis because of scarcity of the bacterial flora and different surface area of the terminal ileum, we did separate analyses for these two groups. previous familial adenomatous polyposis studies have focused upon faecal sterols and bile acids [1517], but none has to the best of our knowledge analysed the content of fatty acids in feces, including very long chained fatty acids. data from the present study are taken from a randomized double-blind placebo-controlled intervention study with a cyclooxygenase-2 inhibitor [12, 14, 18]. the main aim was to compare the effect of rofecoxib treatment on duodenal lesions (data in preparation). the present study comprises data from baseline for only the patients operated with ileal-pouch-anal anastomosis and ileostomy (77% of total study group). all of them had been colectomized, and duodenal lesions had been verified by endoscopy and histology. rikshospitalet university hospital is a highly specialized university hospital with national responsibilities in the area of complicated treatments, such as follow-ups on the norwegian familial adenomatous polyposis patients. inclusion criteria were verified familial adenomatous polyposis, colectomy, 1870 years of age, and documented duodenal lesions graded as spigelman i, ii, or iii and the largest adenoma10 mm. exclusion criteria were indications for surgical treatment, suspected or documented intestinal obstruction or stenosis, patients unwilling or unable to adhere to protocol, known cardiac failure requiring medical treatment, and pregnancy. as for the adenomas sampled, they were all assessed histologically as mild/moderate dysplasia, all below 10 mm and all flat, located in the descending part of the duodenum. samples of feces were transferred to tubes and mixed to make a homogeneous mass of which 100200 ul were used for lipid extraction by adding 50 ul butanol, 100 l h2o, 1 ml methanol, and 1 ml chloroform. after 10 min under n2, the mixture was filtered and the filter washed with 1 ml chloroform +1 ml chloroform/methanol (2: 1)+0.8 ml 0.73% nacl. after mixing for 15 s, the tubes were centrifuged for 30 min at 1000 g. the upper phase was discarded and the lower phase transferred to new tubes and evaporated under n2 at 65c using a heat block. then 0.4 ml 0.5 n naoh in methanol was added and the solution kept for 7 min at 100c under nitrogen. after cooling to room temperature, 0.5 ml 12% bf3 was added and the solution kept for 10 min at 100c under nitrogen. after cooling to room temperature, 1 ml heptan was added under nitrogen, and mixing for 30 s, 1 ml of saturated nacl solution was added. after mixing, the solution was centrifuged for 10 min at 1000 g, and the upper phase transferred to new tubes. finally, the heptan was evaporated and 300 l hexan added before continuing with the gas chromatography procedure referred to earlier. dietary intake was assessed by a validated food frequency questionnaire, designed to cover as much of the total habitual diet as possible. questions were related to habitual frequency of consumption and the amount of foods eaten during the last year. they were asked to identify their habitual choices of edible fats by pointing at specially prepared pictures, in order to increase the validity of the estimate of dietary fat. each biopsy was put into a tube containing 600 l trizol and immediately mixed on a mixermill (retsch gmbh, germany). gene expression analyses were carried out on a 7900 ht real-time pcr machine from applied biosystems. based on the results from running a housekeeping gene test (taqman human endogenous control plate, applied biosystems) with rna from isolated human leucocytes (data not shown). the primers and probes were initially designed as three assays per gene and validated for efficiency and specificity. the primers and probes for the cox-1 assay were: forward primer: 5-cttccaggagctcgtagga-3, probe: 5-agaaggagatggcagcagagttggag-3, and reverse primer: 5-acgcatcaatgtctccatacaat-3. cox-2 forward primer: 5-tggaacatggaattacccagt-3, probe: 5-tgttgaatcattcaccaggcaaattgct-3, and reverse primer: 5-tcctaccaccagcaaccct-3. gus forward primer: 5-gaaaatatgtggttggagagctcatt-3, probe: 5-ccagcactctcgtcggtgactgttca-3, and reverse primer: 5-ccgagtgaagatccccttttta-3. tbp forward primer: 5-ctggaaaagttgtattaacaggtgc-3, probe: 5-agcagaaatttatgaagcatttgaaaacatctaccctatt-3, and reverse primer: 5-cattacgtcgtcttcctgaatc-3. taqman universal pcr master mix (applied biosystems) was added as reaction mix. the reaction conditions were initiated by a step of 2 min. at 50c and 10 min. at 95c, followed by 40 cycles of denaturation at 95c for 15 sec. and annealing at 60c for 1 min. a combination of cdna from several samples were made and diluted in order to make a dilution curve that was included on each plate. the average of the three values for each gene was divided by the average of the corresponding tbp values, generating a normalized value of the gene expression which is a unit less value used to compare the relative amount of mrna for each gene in the different samples. patients were informed by a physician, and in addition thoroughly written information was given to all patients. the protocol was explained to the subjects that had to give their consent before inclusion. was approved by the norwegian health authorities and the regional committee of medical ethics 20/06/2002 (reference: s-02127). nonparametrical statistical methods were chosen, as some of the variables were skewed and the number of observations limited. age and fecal content of palmitolic acid, linoleic acid, and arachidonic acid differed between the ileostomy and the ileal-pouch-anal anastomosis patients (table 1). the content of linoleic acid in feces correlated negatively to the proportion of eicosaenoic acid in duodenal biopsies (r=0.6, p<.05). the content of arachidonic acid in feces was correlated negatively to the proportions of eicosapentaenoic acid (r=0.7, p=.02) and docosahexaenoic acid (r=0.7, p=.008) in duodenal biopsies. total cholesterol were negatively correlated to the content of palmitic acid (r=0.7, p=.03), palmitoleic acid (r=0.7, p=.02), stearic acid (r=0.7, p=.02), oleic acid (r=0.7, p=.02), linoleic acid (r=0.8, p=.002), and monounsaturated fatty acids (r=0.7, p=.02) in feces. the levels of triglycerides was negatively correlated to the content of linoleic acid (r=0.8, p=.003) and eicosaenoic acid (r=0.7, p=.03) in feces. no significant correlations were found between the content of different fatty acids in feces and the fatty acid dietary intake (data not shown). with the exception of the palmitoleic acid/palmitic acid ratio and levels of cyclooxygenase-2 expression in duodenal biopsies (r=0.8, p=.003), no relationships were found between fatty acids in feces and the levels of cyclooxygenase mrna expression (figure 1). significant correlations were found between the content of oleic acid in feces and the proportion of myristic acid (r=0.8, p<.001), oleic acid (r=0.5, p<.05), and eicosaenoic acid (r=0.5, p=.03) in duodenal biopsies. moreover, the content of linoleic acid in feces correlated significantly to the proportion of myristic acid (r=0.8, p<.001) in duodenal biopsies, whereas the content of alpha-linolenic acid in feces correlated inversely to eicosaenoic acid (r=0.6, p<the content of palmitic acid in feces was positively correlated to levels of ldl-cholesterol (r=0.05, p=.05). no significant correlations were found between the content of different fatty acids in feces and the levels of cyclooxygenase mrna expression in duodenal biopsies (data not shown). several different fatty acids in feces correlated positively to dietary intake of fatty acids, most predominantly monounsaturated fatty acids (table 2). as expected, we found a difference between ileal-pouch-anal anastomosis and ileostomy patients concerning associations between fecal fatty acid composition and other variables involved in lipoprotein metabolism. possibly, our results relate to metabolic differences caused by the different intestinal reconstructions, but the data are not suitable to explain the findings. we found no significant relationships between the levels of cyclooxygenase mrna expression in duodenal biopsies and the content of fatty acids in feces, except for the estimates of the content of delta-9-desaturase (stearoyl-coa desaturase, scd) activity, namely the ratio of palmitolic acid/palmitic acid [19, 20] in the ileostomy group. stearoyl-coa desaturase is the central lipogenic enzyme catalyzing in vivo reactions in the synthesis of monounsaturated fatty acids, particularly oleic acid and palmitoleic acid, which are the major monounsaturated fatty acids of membrane phospholipids, triglycerides, wax esters, and cholesteryl esters. both delta-9-desaurase and cyclooxygenase-2 are ppar alpha regulated, although this possible link is only a speculation. a study in healthy subjects showed that human fecal water contains components can affect both the cyclooxygenase-2 protein level and enzymatic activity. one recent study claims that it is possible to detect cyclooxygenase-2 mrna in feces of colorectal cancer patients irrespective of clinical stage. notably, increased cyclooxygenase-2 expression in duodenal compared with colonic tissues in familial adenomatous polyposis was recently reported. the significant negative correlation between content of arachidonic acid in the feces and amounts of omega-3 fatty acids in duodenal biopsies which was found in the ileostomy group is interesting, since substitution of arachidonic acid with omega-3 fatty acids has been shown to lead to the production of less potent prostaglandins. a previous familial adenomatous polyposis study established a positive correlation between the reduction in tissue prostanoid levels and clinical response, as measured by the reduction in size and number of adenomas, when patients were treated with sulindac. however, this correlation only became consistently significant when prostanoids were assayed after tissue homogenates were first incubated with arachidonic acid. we may suspect transformation of linoleic acid to arachidonic acid in feces, but data are limited as with regard to feces from colectomized familial adenomatous polyposis subjects. however, the dietary assessment showed that the intake of such foods was normal among these familial adenomatous polyposis patients. dietary controlled intervention studies on ileostomy subjects have shown that dietary manipulation with fat and fiber may modify cholesterol absorption and sterol excretion. in the ileostomy group, levels of total cholesterol were negatively correlated to the content of palmitic acid, palmitolic acid, stearic acid, oleic acid, linoleic acid, and monounsaturated fatty acids (< 18 c atoms) in feces, which is interesting since the present levels of total cholesterol are lower than those among a healthy reference group. in the ileal-pouch-anal anastomosis group separately, the content of monounsaturated fatty acid in feces was positively correlated to dietary intake of monounsaturated fatty acids in particular. moreover, positive correlations were found between the content of oleic acid in feces and the proportion of myristic acid, oleic acid, and eicosaenoic acid in duodenal biopsies. the precise role of monounsaturated fatty acids synthesis in cell proliferation and programmed cell death remains unknown. the strong correlation of high levels of monounsaturated fatty acids and neoplastic phenotype may suggest that the regulation of stearoyl-coa desaturase must play a role in cancer development. this was an observational study, and not a metabolic balance study that was designed to investigate the metabolism of fatty acids in familial adenomatous polyposis patients. although some studies have found that the absorption rate of fatty acids are unaffected by age [26, 27], we can not exclude the possibility that some of the observed differences between the ileal-pouch-anal anastomosis and ileostomy patients might be due to age, rather than type of surgery, since lipoprotein clearance has been shown to be altered by age, and that might affect the relationship between dietary fatty acids and the fatty acid profiles found in tissues. moreover, no conclusions can be drawn from the present study because this is an observational study.. however, the study addresses the need to learn more about the dietary and lifestyle habits of this subset of at-risk patients. the present study includes a number of patients close to the maximum possible number of eligible norwegian familial adenomatous polyposis patients. we may, however, suspect that the low sample size and use of a food frequency questionnaire, may have weakened any associations between the content of fatty acids in feces and dietary intake. notably, these data do not contradict that the nutrient intake among these patients should at least meet the recommendations for healthy subjects. nevertheless, we suggest that these data are compelling enough to suggest that future familial adenomatous polyposis studies should investigate overall fatty acid metabolism, molecular mechanisms relevant to fatty acid metabolism, inflammation, and angiogenesis, in addition to nutrition requirements. in conclusion, we found a difference between ileal-pouch-anal anastomosis and ileostomy patients concerning associations between fecal fatty acid composition and other variables involved in lipoprotein metabolism. we may suggest that fatty acid content in feces is related to dietary intake, serum lipids, and fatty acid composition in duodenal biopsies, even in colectomized familial adenomatous polyposis patients. this observational study may represent hypothesis-generating suggestions for future familial adenomatous polyposis studies .

a few familial adenomatous polyposis studies have focused upon faecal sterols and bile acids but none has analysed the fecal content of fatty acids. we report here findings of an observational study on 29 colectomized familial adenomatous polyposis patients that describe the fecal content of fatty acids, and relate this to the proportions of fatty acids and levels of cyclooxygenase mrna expression in duodenal biopsies, levels of serum lipoproteins, and diet. in the ileostomy group separately (n=12), the fecal content of arachidonic acid was correlated negatively to the proportions of eicosapentaenoic acid and docosahexaenoic acid in duodenal biopsies. total serum-cholesterol was negatively correlated to the fecal content of saturates and monounsaturates. the fecal palmitoleic acid/palmitic acid ratio was positively correlated to the levels of cyclooxygease-2 expression in duodenal biopsies.in the ileal-pouch-anal anastomosis group separately (n=17), significant correlations were found between the fecal contents of oleic acid, linoleic acid, and alpha-linolenic acid, and the proportions of myristic acid, oleic acid and eicosaenoic acid in duodenal biopsies. dietary monounsaturates were positively correlated to different fecal fatty acids. future studies should focus on molecular mechanisms relevant to fatty acid metabolism, inflammation, and angiogenesis, in addition to nutrition.

PMC2967835

pubmed-66

local anesthesia and pain control is one of the most important elements of dentistry, and particularly prosthetic dentistry. according to the american dental association, fear of pain is the most important factor preventing patients from visiting their dentists. different kinds of fear related to previous clinical experience affect patients ' attitudes to local anesthesia or dentist20. although local anesthesia techniques do not provide pain-free treatment, this pain is generally tolerable. pain can result from the mechanical trauma of needle introduction into the site of injection, or from the sudden distension of the tissues, resulting from a rapid discharge of the contents of the syringe. pain can also be caused by the stimulation of the first few drops of the local anaesthetic3,14. contrary to prevalent ideas, needle penetration of tissue is not the primary reason for discomfort. volume and pressure of the local anesthetic being injected causes more distress and/or pain. administering local anesthetic injection may not only provoke anxiety in patients, but also in the dentist29. despite that, administering local anesthesia by injection for pain relief is still the most common method used in dentistry. however, there are several ways to alleviate pain before dental procedures begin, or the often painful nature of the injection in local anesthesia. transcutaneous electrical nerve stimulation (tens)24, use of intraoral lidocaine patch11 and computerized local anesthesia7 (the wand) are newly developed methods to alleviate the pain and anxiety of dental patients, as alternatives to conventional local anesthesia. the wand (milestone scientific, livingston, nj) is a computer-controlled local anesthesia delivery system that drives local anesthetic from a conventional local anesthetic cartridge through plastic microtubing into a plastic handle to which a luer-lok needle is attached. the computer-controlled flow is initiated by exerting pressure on a foot pedal. the pump allows administration of local anesthetic at two slow but constant rates, and the computer compensates for variation in resistance to flow4. during needle insertion, continuous positive pressure delivers an anesthetic drip that precedes the needle. the combination of an anesthetic pathway and controlled flow rate results in a virtually imperceptible injection and rapid onset of profound anesthesia. the wand system offers several advantages over conventional syringes, including excellent tactile sensation afforded by a lightweight plastic handle and the ability to rotate the needle as it is introduced into tissues, producing a coring penetration that minimizes needle deflection. wand devices automatically deliver the anesthetic more slowly and precisely than a conventional syringe, thereby reducing patient discomfort5,15. anesthetic solution gently pumped into the soft tissue can be absorbed at the same rate by soft tissue in the wand system. the theory is that when the needle is advanced slowly, the drops of solution anesthetize the tissue ahead of the needle. all techniques of local anesthesia, such as maxillary and mandibular infiltration, mandibular block22, intraligamentary26, and anterior middle superior alveolar (amsa) injection8, and anterior superior alveolar (asa) injection25 can be performed with the wand system. conventionally, maxillary teeth have been anesthetized by administering an infiltration injection on the buccal or labial aspects of the target tooth. palatal injections have also been administered by plastic syringe for this objective, but can be painful5,7. the amsa derives its name from the injection's ability to anesthetize both the anterior and middle superior alveolar nerves57. the middle superior alveolar (msa) and asa nerves branch from the infraorbital nerve before they exit from the infraorbital foramen. the msa nerve is thought to innervate the maxillary premolars and plays some role in pulpal innervation of the mesiobuccal root of the first molar. the anterior, superior alveolar nerve supplies pulpal innervation to the central and lateral incisors and canines18. the plexus where the 2 nerves join is the target site for the amsa injection. the 10 maxillary teeth extending from the second premolar to contralateral second premolar, and the associated palatal tissue, can be anesthetised with bilateral amsa injection57. wand research is largely attributable to pediatric dentistry1,2,9, restorative dentistry7, endodontics14, periodontology17,26,27, maxillofacial surgery16, comparison of pain perception to the wand and a traditional syringe21,28, influence of anesthetic flow rate delivered by the wand on pain response to the palatal injection23. the hypothesis of the present study was that the wand anesthesia technique reduces the pain at different phases (needle insertion, delivery of local anesthetic and tooth preparation) versus conventional buccal anesthesia with conventional plastic syringe. therefore, the study compared pain levels at needle insertion, during delivery of local anesthetic solution and during tooth preparation, for the conventional syringe technique on the buccal or labial aspect on one side, and the amsa technique with wand local anesthesia application on the other side of the maxilla. a 5-point verbal pain rating scale (vrs)12,17 was used on 16 patients who were under prosthodontic treatment in the ondokuz mayis university clinic sixteen adult patients who had partially edentulous states of both left and right maxillary arch, and were to undergo fixed prosthodontic treatment, participated in this study. they ranged in age from 27 to 64, were predominantly women (12 women and 4 men), and were in a good health. patients were not taking any medication that would alter their pain perception, as determined by a written medical health form and oral questioning. allergy to lidocaine, mepivacaine, sulphides or articaine, history of significant medical problems, taking central nervous system depressants within 48 hours, pregnancy, or inability to give informed consent, were the exclusion criteria. the human subjects review committee of ondokuz mayis university approved this study (28.11.2005 dated, and 328 numbered), and written, informed consent was obtained from each patient. all patients had previously experienced a conventional syringe, no patients had previously received the wand injection, and all patients had at least one tooth absent both on the left and right maxillary arch. the amsa technique with the wand for half the maxillary arch and conventional plastic syringe anesthesia on mucobuccal fold (control group) for opposite maxillary arch were administered by the same operator. the order of anesthesia techniques was randomly selected, and anesthesia procedures and tooth preparations were completed in the same session (table 1). vrs (0 no pain, 1 minimal pain, 2 slight pain, 3 moderate pain and 4 severe pain). the pain level descriptions were as follows: no pain, minimal pain, slight pain, moderate pain and severe pain (0, 1, 2, 3, 4). patients were blindfolded with a commonly used sleeping mask so they would not distinguish which anesthetic delivery system was used. the wand produces audible beeps as the injection is administered. because the beeping tones can not be turned off with a switch, the sound were produced during both injection methods (the wand or conventional-control) so the patients were not aware of the method being used. immediately after needle insertion, delivery of local anesthetic and tooth preparation, patients were asked to remove mask and rate the pain for both conventional and wand injection techniques. no topical anesthetic was used because the wand manufacturer's instructions suggest that topical application is optional. this study was conducted according to a split- mouth design, with both injections given to all patients. for each patient, the amsa technique with single wand injection was used on one side for central incisors, lateral incisors, upper canine, first and second premolars, and the traditional plastic aspirating syringe injection was used on the mucobuccal fold of the related tooth on the opposite side. the amsa injection site is located at a point that bisects the maxillary first and second premolars, and midway between the crest of the free gingival margin and mid-palatine suture. the needle is orientated at a 45 degree angle with the bevel facing the palatal tissue, while rotating the wand needle. all injections were given by the same operator who had had two months experience using the wand at the department of prosthodontics, faculty of dentistry, ondokuz mayis university, turkey. the ampul form of articaine hydrochloride with adrenalin (hoechst marion roussel, deutschland, gmbh, germany) was injected with conventional plastic syringe and 27gauge dental needle. the cartridge form of articaine hydrochloride with adrenaline (hoechst ag, frankfurt, germany) was administered with the computer-controlled anesthesia device (the wand, milestone scientific, livingston, nj, usa), with 27gauge handpiece and needle (milestone scientific). approximately 0.9 to 1.0 ml local anesthetic was used both for amsa with the wand on one side and control group for each tooth on the other side. groups were labelled as follows: group (c), pain perception scores at needle insertion for the conventional technique; group (c), pain perception scores for local anesthetic delivery by the conventional technique; group (c), pain perception scores at tooth preparation for the conventional technique; group (w), pain perception scores at needle insertion by the wand technique; group (w), pain perception scores for local anesthetic delivery for the wand technique; and group (w), pain perception scores at tooth preparation for the wand technique. power analysis were performed to detect adequate sample size using statistical software (pass 2008, ncss, kaysville, ut, usa). since our variable was an ordinal level, the mann-whitney u test was used to determine the number of times a score from the conventional method is ranked than a score from the wand techniques at 1% significance level. statistical tests were performed with the statistical software (spss version 12.0; spss inc, chicago, il, usa). differences in pain rating were analyzed at needle insertion, delivery of local anesthetic and during tooth preparation for conventional versus the wand techniques. sixteen adult patients aged from 27 to 64 (12 women and 4 men) participated in this study. median scores, minimum and maximum pain rating values after needle insertion, and delivery of anesthetic and tooth preparation time for both conventional and wand techniques, are shown in table 3. the wand technique registered a highly significantly lower pain level during the needle insertion phase (z=- 4.064, p<0.01), with the wand technique having a lower pain level compared to the conventional. during the anesthetic solution delivery phase, the pain level for the wand technique was again highly significantly lower than for the conventional technique (z=- 3.897, p<0.01). however, there was no difference between the wand and conventional techniques for pain level during tooth preparation (z=- 0.671, p>0.05). the research hypothesis stated that the wand anesthesia technique reduces the pain at different phases (needle insertion, delivery of local anesthetic and tooth preparation) versus conventional buccal anesthesia with conventional plastic syringe. the wand anesthesia technique reduces the pain compared the conventional anesthesia for needle insertion and anesthetic solution delivery phases (p<0.01), with the exception of tooth preparation phase (p>0.05). the majority of literature on the computer-controlled injection system (the wand) has dealt with the pain of injection with the computer-assisted injection system, compared to injection using a conventional syringe1,2,8l0,l6,2l,27,28. in general, the results have been favorable1,8,9,16,23,27 for the computer-assisted injection system, with only 2 studies showing no difference2,28, and 1 study showing higher pain ratings10. palatal injections are generally considered the most painful injections13; wahl, et al.30 showed that palatal injections caused significantly more pain than other intraoral injections. the authors of18,30 speculated that palatal injection pain might be mainly the result of pressure. the traditional route for administration of anesthesia to the maxillary teeth is supraperiosteal infiltration delivered in the mucobuccal fold near the apex of the tooth to be anesthetized. although effective in achieving pulpal anesthesia of the neurovascular bundle by diffusion of anesthetic through the porous maxilla, this approach routinely anesthetizes the surrounding tissues (lips, surface of the face), and also affects muscle activity. the patient's natural repose, as well as active facial expressions are temporarily affected, including an distortion of the smile. the patient also experiences the annoying sensation of facial numbness, and the smile line is temporarily invalidated as an esthetic reference for restorative and prosthetic dentistry. an alternative to maxillary infiltration is the infraorbital nerve block injection which, if properly performed, achieves anesthesia of the anterior superior alveolar branch of the trigeminal nerve. this injection site, however, is also close to muscles of expression, and anesthesia of cutaneous fibers of the infraorbital nerve results in a corresponding loss of sensation in the face and the upper lip29. the palatal approach amsa nerve block can be attempted with a traditional manual syringe, but the precise pressure and volume ratios of the wand are virtually impossible to reproduce5,6. the clinician can anesthetize several teeth in the maxillary arch, extending from the mesiobuccal root of the first molar to the central incisor, by means of a single palatal infiltration. in addition to profound pulpal anesthesia, palatal soft tissue anesthesia is realized, which also extends from the maxillary first molar to the central incisor and the associated palatal tissues, without numbness to the lips and face, and without interference with the muscles of facial expression for at least 60 min16,25. during needle insertion with a coring motion, the drops of solution anesthetize the tissue ahead of the needle when the needle is advanced slowly7,15. the combination of an anesthetic pathway and controlled flow rate results in virtually imperceptible needle insertion and injection, and rapid onset of profound anesthesia28. the same efficacy of anesthesia was achieved with conventional buccal anesthesia and the palatal approach amsa technique with the wand device25. the current study was in agreement for tooth preparation, but contradictory for needle insertion and delivery of local anesthetic. numbness of the lips and face was not observed in the current study for the palatal approach when using the amsa technique with the wand. another study using the vrs (verbal rating scale) compared computer-controlled and conventional local anesthesia delivery systems for performing scaling and root planing on patients with moderate periodontal disease; amsa injections with the wand were considered less painful than the conventional syringe17. two further benefits of the amsa injection technique are that it also eliminates the potential for an intravascular injection because no major vessels are encountered in this region of the palate5, and the palatal bone is porous enough to permit the anesthetic solution to diffuse through the tissues and anesthetize both the anterior and middle branches of the superior alveolar nerve when the injection is deliberately slow and steady. for this reason, the successful deposition of the anesthetic solution through the fibrous tissue is said to be accomplished more easily with the computer-controlled delivery device that regulates the pressure and volume ratio of solution delivered5,7. the wand system offers several advantages over conventional syringes, including excellent tactile sensation afforded by the lightweight plastic handle and the ability to rotate the needle as it is introduced into tissues, producing a coring penetration that minimizes needle deflection. presumably, the slow rate of anesthetic flow reduces patient discomfort compared with palatal injections administered with a traditional syringe4. decreasing the total amount of anesthetic and vasoconstrictor necessary for maxillary anesthesia, shorthening the total anesthesia time, and diminishing patient-operator anxiety however, the wand's beeping sounds make the injection seem longer and may make the patient feel uncomfortable. other negative aspects of the wand are frequent breakage of the anesthetic cartridge on activation of the unit, and a separate instrument is required for removing the cartridge from the unit10. additionally, 1.4 ml anesthetic solution can be delivered in about 1 min in rapid injection mode, and in 4 min 45 s in slow injection mode9. fukayama, et al.8 examined the efficacy of the amsa anesthesia technique using the wand system to assist with the electrical pulp test. electrical pulp stimulation indicated that the lateral incisors, canines, and first and second premolars were more anesthetized than the central incisors and first molars. they also stated that anesthetic applied via amsa with the wand has a slow onset and further studies are required to evaluate its effectiveness for this reason. however, amsa injection using the wand method seems to both preclude severe injection pain and be very effective for pulpal anesthesia of lateral incisors, canines, and premolars. pain scores were higher for amsa technique in the central tooth than in other teeth (p<0.05) in another related study16. in the present study, in two cases of amsa using the wand, pain was at the minimum level (vrs score 1) during tooth preparation of central incisors. the reason for this pain rating could be that the central incisors were innervated from the opposite arch, or alternatively, fukayama, et al.8 and lee, et al.16 stated that the effectiveness of the wand is lower in the central incisors and first molar teeth than lateral incisors, canines, and premolars. friedman and hochman57 stated that careful needle insertion and slow anesthetic delivery could reduce the sensation of needle insertion. primosch and brooks23 showed that slow anesthetic delivery produces significantly lower pain scores than fast anesthetic delivery, and the wand decreases the pain for both slow or fast anesthetic delivery rate, but contrary to the manufacturer's claims, it did not eliminate pain elicited by palatal injections in some patients. in the present study, a highly significant (p<0.01) difference for pain rating was obtained between conventional buccal and amsa using the wand injection for needle insertion and anesthetic solution delivery. however, contrary to the manufacturer's claims, pain was not eliminated at slow anesthetic flow rate in this study there have been limited prosthodontic or restorative studies for amsa using wand injection. in one study, amsa was bilaterally applied with the wand and 6 anterior maxillary teeth were prepared. incisal reduction of these teeth was achieved with reference to the lip, which is not affected by amsa. bilaterally, the amsa technique using the wand was repeated for tooth sensitivity during the cementation procedure5,6. as in previous studies, amsa using the wand was also of benefit for lip guidance in the present study. another benefit of palatal amsa injection using the wand is that it reduces the number of injections, and also the amount of anesthetic solution compared to conventional buccal infiltration anesthesia that applies multiple injections to each tooth. in addition, more teeth can be anesthetized with a single injection, without numbness of lips and face, compared to the conventional technique. the amsa technique administered using the wand in a prosthodontic treatment reduced pain highly significantly more than conventional buccal anesthesia, both in inserting the needle and in the of delivery of local anesthetic. when comparing the anesthesia efficiency of amsa using the wand injection and conventional buccal infiltration techniques, no statistical difference was found. although the slow anesthetic flow rate proved superior in pain control, the pain score was not zero for the wand, contrary to the manufacturer's claim. computer-controlled anesthesia using the wand appears advantageous for restorative procedures because more teeth are anesthetized with one palatal injection, and without numbness of lips and face, in contrast to multiple conventional buccal anesthetic injections for each tooth.

objective: the objective of this study was to compare the pain levels on opposite sides of the maxilla at needle insertion during delivery of local anesthetic solution and tooth preparation for both conventional and anterior middle superior alveolar (amsa) technique with the wand computer-controlled local anesthesia application. material and methods: pain scores of 16 patients were evaluated with a 5-point verbal rating scale (vrs) and data were analyzed nonparametrically. pain differences at needle insertion, during delivery of local anesthetic, and at tooth preparation, for conventional versus the wand technique, were analyzed using the mann-whitney u test (p=0.01). results: the wand technique had a lower pain level compared to conventional injection for needle insertion (p<0.01). in the anesthetic delivery phase, pain level for the wand technique was lower (p<0.01). however, there was no difference between the wand and conventional technique for pain level during tooth preparation (p>0.05). conclusions;the amsa technique using the wand is recommended for prosthodontic treatment because it reduces pain during needle insertion and during delivery of local anaesthetic. however, these two techniques have the same pain levels for tooth preparation.

PMC4327666

pubmed-67

chronic obstructive pulmonary disease (copd) is a progressive lung disease which is characterized by airway obstruction and lung parenchyma destruction. patients may experience dyspnoea or shortness of breath, a persistent cough with sputum production, decreased exercise tolerance and decreased quality of life [1, 2]. globally, copd is the fourth most common cause of death and is predicted by the world health organisation to be a major health concern in the coming decade. in australia, 1.2 million people are estimated to have moderate-to-severe copd (gold stages ii iv). however, the true number is potentially much higher as copd is commonly underdiagnosed [4, 5]. the total cost of living with copd (including nonmonetary costs) is estimated to be $83 000 aud per person each year [6, 7]. as the severity of copd greatly impacts the cost of care, providing early diagnosis and effective management of the individual is an essential component in reducing its economic impact [5, 810]. pr is a multidisciplinary approach which incorporates self-management education with exercise training, psychosocial and nutritional support [11, 12]. it is a fundamental part of managing people with copd and has been shown to be effective in improving exercise tolerance, reducing dyspnoea, improving, quality of life and reducing the severity of acute exacerbations [1, 1117]. although pr has been shown to be effective in managing symptoms and reducing hospital admissions, poor participation and adherence are a current problem among outpatient programs. factors include difficulty travelling to the program's location [1922], inconvenience of hospital attendance, or difficulty accessing programs. australia is one of the least densely populated nations (2.7 people per square kilometre) with up to 11.8% of the population residing in outer regional or remote locations. conducted a study in kyabram (rural victoria) to assess the efficacy of their pr program and noted that patients who lived outside the town centre were more likely to withdraw from pr as the program was conducted within the town hospital. this finding is also supported by de angelis and colleagues who found that transport, travel distance, and related costs were the main reason for nonattendance of a cardiac rehabilitation program. difficulty in accessing outpatient programs has led to the development of home-based programs. it is well established that home-based pr programs are safe and effective [13, 2931] and are recommended by many international guidelines and reviews [2, 11, 12, 17]. given the effectiveness of independent home-based pr programs one could ask what additional benefits supervised remote-based programs would provide. the main advantage of supervised remote-based programs is the ability to minimise anxiety in both patient and therapist. this benefit has been shown in remote-based cardiac exercise programs where the use of transtelephonic exercise monitoring (tem) has been used to relieve patients ' and trainers ' anxiety during exercise [3234]. it is anticipated that a person with copd could exercise to a higher intensity without the risk of undetected adverse events (e.g., exercise-induced oxygen desaturation). telerehabilitation is an emerging technology used to facilitate the delivery of rehabilitation services at a distance by combining telecommunication and electronic transmission of health information. it has the potential to provide equitable access for people living in rural or remote areas where access to health service is limited. for telerehabilitation to be successful, however, there must be accurate assessment and outcome measurement tools available to therapists [37, 38]. in pr, the pulse oximeter is a noninvasive device which continuously monitors a person's peripheral blood oxygen saturation (spo2) and heart rate (hr). it is used clinically to measure a person's exercise intensity and warn of exercise-induced desaturation. the concept of remote monitoring of physiological data including spo2 and hr for exercise rehabilitation purposes has received attention in the literature. as early as the mid-1980s, physicians were successful at remotely monitoring a patient's electrocardiogram (ecg) through the analogue telephone network during a home-exercise program [40, 41]. however, the majority of electronic healthcare literature on pr has focused on telemonitoring systems [4246] or consultations services [36, 47]. there has been little research using e-health to deliver real-time, live supervision of patients for treatment purposes. this study aimed to develop and verify the use of a remote pulse oximetry system on healthy individuals. specifically, it aimed to establish the technical feasibility of transmitting pulse oximetry data remotely, to determine the validity of remote measurements when compared to conventional face to face measurements, and to evaluate the participants ' perception on the usability of the technology. it was hypothesised that remote pulse oximetry would not only be technically feasible but would also be valid when compared to conventional units and would be easy for participants to use. a favourable result will enable the development of remote pulmonary rehabilitation programs for people living in rural or remote areas. this study was reviewed and granted ethical clearance from the appropriate medical research ethics committee at the university of queensland. thirty-seven healthy individuals were recruited to participate in a single remote cardiopulmonary exercise session. the following exclusion criteria were applied: cardiovascular, respiratory, metabolic, or orthopaedic conditions that would prevent participation in an exercise program, chronic infectious diseases, pregnancy, and mental or physical impairment. the exercise session was conducted using the ehab telerehabilitation system (v2.0, uniquest, brisbane, australia) which is a computer-based videoconferencing and remote consultation system. two ehab devices were used, one with the participant and the other with the therapist. the participant and therapist were located in separate rooms. the ehab device with the participant was configured to receive data from a bluetooth pulse oximeter module (onyx ii 9560, nonin medical inc., plymouth, mn) (figure 1). this information was transmitted by the participant device at a frequency of one hertz via an encrypted data channel to an offsite server via an active server page (asp) web service. the data was subsequently stored in a mysql database on the server. a therapist device retrieved this data via an asp web service to produce a graph of the participant's heart rate and spo2 recordings in near real time. for this study the ehab telerehabilitation system utilised a local wi-fi network connection; however, the connection speed was throttled to 128 kbit/s to ensure compatibility with the 3g/hsdpa mobile network available in remote areas of australia. after written consent was obtained, participants were taken to a room with the ehab device, remote pulse oximeter module, headset, and treadmill. they were asked to remove any nail polish from their index finger on the nondominant hand or jewellery which may disrupt accurate recordings. participants were given an information sheet with instructions on how to affix the pulse oximeter module to the index finger of their nondominant hand and how to establish a connection with a therapist in another room via the ehab device. once a videoconference was established, the therapist guided the participant through an exercise session. the modified borg score (mbs) was used to assess the participant's exercise intensity. as per current copd exercise protocols the session began with a warm-up phase (mbs 2/10) for 5 minutes before gradually progressing to a moderate/somewhat severe intensity (mbs 4-5/10). the last 5 minutes consisted of a cool-down phase (mbs 2/10). if any adverse signs were observed, the exercise session would be ceased immediately. interrupted sessions due to technical difficulties were noted, and those participants were asked to repeat the exercise session at a later date. at the conclusion of the exercise session, participants completed a questionnaire about their experience with the remote pulse oximeter module and ehab device by marking on a 100 mm visual analogue scale (vas) and providing any open-ended comments. during each session, the participant ehab device logged heart rate (hr) and spo2 values from the pulse oximeter and time stamped the data. this data was saved into a database stored locally on the computer (dataset #1). the participant device also transmitted the data to the remote therapist device where another time stamp was applied to enable the calculation of transmission latency. this data was saved into a database stored locally on the therapist device (dataset #2). the mean data transmission rate was determined by measuring the bandwidth requirement (kbit/s). the satisfaction questionnaire rated the comfort, quality, and usability of the ehab device and remote pulse oximetry module. the validity of remote pulse oximetry was assessed by comparing the remote measurements (therapist data) with conventional face-to-face measurements (participant data). these were percentage agreement, mean absolute difference (mad), and the bland and altman's limits of agreement method [49, 50]. percentage agreement was calculated as the proportion of data successfully transferred between participant and therapist. the limits of agreement method enabled the quantification of the agreement between the two data sets in terms of the clinical significance of the transmission error. specifically, it gave the upper and lower bounds which show the 95% confidence interval for the difference between the remote and conventional measurements (i.e., mean 1.96 sd). when applying the limits of agreement method comparing the two datasets, the average of 10 second intervals of data was used. in terms of clinical significance, the minimum clinically important difference (mcid) was used as a threshold value to represent meaningful and worthwhile change. remote measurements were classified as valid if the measurement error was lower than the mcid. the mcid for hr, however, varies depending on the individual's exercise tolerance. for the purpose of this study, the manufacturer's device accuracy of 3 beats per minute (bpm) was used as the mcid, which was a conservative estimate. data was analysed using statistical package for the social sciences (v17.0 spss inc., chicago, il). a p value of<0.05 was considered to be significant, where applicable. thirty-seven healthy individuals (14 males and 23 females) completed a total of 15 exercise hours. participants had a mean age of 30 years and standard deviation of 15 years (range: 18 to 62). there were 34 successfully completed sessions on first attempt confirming the feasibility of the method, while three had to be repeated due to technical difficulties with the bluetooth or wifi connection. the mean (sd) transmission latency of the pulse oximetry data (not the videoconference) between the computers was 2.94 (0.59) seconds. the mean (sd) bandwidth usage for data transmitted was 6.5 (1.0) kbit/s. of the remaining data, 9.0% was found to be omitted from the therapist device, while 11% was found to consist of duplicated numbers. the mad and limits of agreement between participant and therapist data are shown in table 1. overall, participants rated their experience with the remote pulse oximeter and ehab device highly. the mean response to each of the eight questionnaire items is shown in figure 2. positive written comments included the following: i would be comfortable using [this device] at home (n=4) and [the ehab was] simple and easy to use negative comments included worried [remote pulse oximeter] could dislodge during vigorous exercise (n=5), remote pulse oximeter was uncomfortable (n=4), and audio or video quality was jumpy (n=3). however, inconvenience, distance, or availability of pr programs can lead to lack of participation and poor compliance. this study demonstrated the feasibility of a remote pulse oximeter for use during a pr program. remote measurements of spo2 and hr were valid when compared to conventional face-to-face measurements. these findings provide confidence for the development of a remote pr program. in our study, there was an unexpectedly high mean rate of data omission (9.0%) and duplication (11%) between participant and therapist data. although these error rates appear comparatively high, they do not necessarily indicate a failure in the data transmission. rather, the logging of data points by the participant device appears to not be perfectly synchronized with data retrieval from the therapist device. this may be a function of the variable delays that can be present with web service calls which were used in this study to transmit data. asynchrony in web service responses may have resulted in either repetition of data or omission of single values. moreover, a visual inspection of the data confirms a random distribution of error throughout the data file. despite this asynchrony, the mad analysis revealed a small difference (spo2 0.04%; hr 0.21 bpm) in comparison to the mcid of 4% for spo2 and 3 bpm for hr. similarly, the limits of agreement demonstrated a small difference in comparison to the mcid for spo2 and hr. the upper and lower bounds for spo2 were 0.67 to 0.67% and for hr were 2.90 to 2.89 bpm. both are within the mcid, and this provides further evidence for the validity of remote measurements. unlike mad, which analyses the actual difference, the limits of agreement give a conservative estimation of difference through a 95% confidence interval. clinically, it means 95% of the time of the difference due to measurement error will be within the relevant limits for spo2 and hr. the mad and limits of agreement values show that remote measurements are sufficiently accurate and valid for real-time monitoring purposes. overall, as indicated by questionnaire responses, participants found the remote pulse oximeter easy to use (mean vas 89/100) and felt confident that they would be able to use it at home (mean vas 87/100). this is similar to literature which has found the ehab device user friendly [5557]. however, some participants (n=4) stated that the remote pulse oximeter was uncomfortable. unlike a conventional pulse oximeter, the remote module has the sensor, display and battery unit all incorporated on the fingertip (see figure 1). this creates additional weight compared to a conventional sensor, and it could be a contributing factor to the feeling of instability experience by these participants. future studies should compare the comfort with other remote pulse oximeters that have the display and battery unit located at the wrist. furthermore, this needs to be investigated in the copd population as they may be more accustomed to pulse oximetry than healthy adults. five participants commented that they were worried the remote pulse oximeter could dislodge during vigorous exercise. this could be a result of the weight of the device and high intensity of exercise, where most participants needed to jog or run in order to reach an mbs of 5/10. problems with video and audio quality have been identified as key issues in telehealth [5860]. during testing, there were occasional disruptions in audio and video transmissions during the exercise session, and on three occasions, the videoconference unexpectedly exited due to connectivity issues on the wifi network, requiring the call to be reestablished. the low frame rate is likely due to the low bandwidth available (128 kbit/s) on the network connection. however, it should be noted that the remote pulse oximeter only uses a very small bandwidth (6.5 kbit/s) for data transmission and, therefore, is unlikely to have attributed to the reduced video/audio quality. there were several limitations of this study (1) our sample size was small (n=37) and consisted primarily of young, healthy university students (mean age 30 years). our healthy participants did not show any clinical changes (e.g., exercise-induced desaturation), and this may have affected the magnitude of error in mad and limits of agreement. furthermore, the young population may be more confident to use or learn about new technology, and this may have therefore influenced the result of the satisfaction questionnaire. (2) this study was conducted within the university clinic. although the participant and therapist were in separate rooms, they were in close proximity to each other, with the therapist on call if any difficulties occurred. whist the ehab system was bandwidth throttled to ensure compatibility with the 3g/hsdpa mobile network, other factors such as mobile network coverage, signal strength, and speed were not assessed, and this may have affected the performance and the latency of transmission. future studies should include a trial of the remote pulse oximetry system in the home environment with a copd population on the 3g/hsdpa mobile telephone network. a large sample size is required to more rigorously assess the validity and reliability of data transmissions. furthermore, it has been shown that patients demonstrate more positive views of telecare encounters than their healthcare providers. a therapist questionnaire is therefore required to assess their satisfaction and confidence in safely conducting a remote exercise session. our pilot study demonstrated that remote pulse oximetry was feasible, and the data transmission method produced valid data at the remote end when compared with conventional face-to-face measures. although this study was performed on healthy individuals, participants found the ehab device easy to operate and were confident it could be used at home. further studies are needed to address the technical feasibility and validity in a home environment and the therapist's confidence in conducting a safe exercise program.

pulmonary rehabilitation is an effective treatment for people with chronic obstructive pulmonary disease. however, access to these services is limited especially in rural and remote areas. telerehabilitation has the potential to deliver pulmonary rehabilitation programs to these communities. the aim of this study was threefold: to establish the technical feasibility of transmitting real-time pulse oximetry data, determine the validity of remote measurements compared to conventional face-to-face measures, and evaluate the participants ' perception of the usability of the technology. thirty-seven healthy individuals participated in a single remote pulmonary rehabilitation exercise session, conducted using the ehab telerehabilitation system. validity was assessed by comparing the participant's oxygen saturation and heart rate with the data set received at the therapist's remote location. there was an 80% exact agreement between participant and therapist data sets. the mean absolute difference and bland and altman's limits of agreement fell within the minimum clinically important difference for both oxygen saturation and heart rate values. participants found the system easy to use and felt confident that they would be able to use it at home. remote measurement of pulse oximetry data for a pulmonary rehabilitation exercise session was feasible and valid when compared to conventional face-to-face methods.

PMC3462379

pubmed-68

acute mountain sickness (ams) occurs during exposure to high altitude (ha) and is a clinical syndrome characterised by headache, insomnia, malaise, and gastrointestinal symptoms. it is common, developing in 1030% at 25003000 meters and in up to 60% of those ascending to around 4500 meters. it causes significant morbidity and is a challenging clinical condition in remote environments. a biochemical marker of ams, particularly one available as a point-of-care test (poc), could have widespread clinical utility. the pathophysiology of ams is not clearly understood but involves alterations in fluid balance, endothelial function, vascular permeability, inflammation, and oxidative stress. the renal response to ha is an important factor in acclimatization, and ha exposure leads to renal arteriole constriction and relative hypoxia [3, 4]. despite the relative renal hypoxia, ngal (neutrophil gelatinase-associated lipocalin) is a 25 kda peptide, part of the lipocalin family of small soluble proteins. it is produced in a number of human tissues, notably the distal nephron but also in the lung ngal rises rapidly in the nephron in response to a renal insult and an ngal 150 ng/ml following acute kidney injury (aki) is predictive of acute renal failure (arf) well before creatinine has risen. ngal is also an acute-phase protein, has a role in inflammation [8, 9], and is upregulated in the lung during inflammation [5, 10, 11]. ngal is also known to rise in conditions associated with oxidative stress [12, 13], and oxidative stress has been implicated in ams [14, 15]. we therefore hypothesised that ngal would increase at ha secondary to these various stimuli and that the magnitude of any increase might relate to the presence of ams. we therefore studied a combined cohort of trekkers from 2 expeditions to ha. in order to clarify the relative contribution of ams, hypoxia or exercise to ngal levels, we also studied a cohort pre- and postexercise at near sea level, and a further cohort exposed to acute normobaric hypoxia. the potential role of inflammation in stimulating ngal was assessed by the measurement of highly sensitive c-reactive protein (hscrp) in a subset of participants. all study protocols were approved by the ministry of defence research ethics committee, whitehall, uk, and satisfied the requirements of the declaration of helsinki. in all studies informed, thirty-two subjects participating in a defence medical services (dms) trekking expedition (trek 1) in the khumbu region of nepal were studied. blood samples were taken from the antecubital fossa at 3 study altitudes: on day 2 at 3400 m, day 6 at 4270 m, and day 10 at 5150 m (following ascent to everest base camp at 5364 m). all samples in this study were collected immediately following a day trekking (posttrek) to the study altitude. twenty subjects from a further dms expedition (trek 2) to nepal were also studied. blood samples were again taken at 3 study altitudes: on day 2 (3400 m), day 6 (4270 m), and day 10 (5150 m) (following ascent to kala patthar (kp), 5643 m). samples in this study were again collected immediately following a day trekking (posttrek). additional samples were taken at rest in kathmandu (kat) at 1300 m and at rest the next morning at the 3 study altitudes. as serving members of the military, all subjects were able to fulfil the fitness criteria of their relevant service. this broadly includes an age-adjusted ability to run 1.5 miles in under approximately 11 minutes and to perform an age-adjusted number of sits-ups and push-ups within two blocks of 2 minutes. fourteen subjects underwent a 3-hour exposure to normobaric hypoxia (fio2 11.6%, equivalent to 4800 m altitude) in a hypoxic chamber. this exposure included a 5-minute step test (step height of 25 cm, 1 complete step every 2 seconds) at 95 minutes. a group of 22 subjects had ngal assayed at rest and after exercise at sl in the uk following ascent from sea level to 1085 m over 6 hours (an equivalent gain in altitude and duration of exercise similar to that experienced on a trekking day in nepal). two subjects from trek 2 were part of the sl exercise group, but data collection occurred several months apart. ngal was analysed in the field on a biosite triage point of care monitor (alere ltd, stockport, uk) using a triage ngal test kit. the triage ngal test is a point-of-care, fluorescence-based immunoassay used which gives a rapid (15 minutes) quantitative measurement of ngal in a range from 60 to 1,300 ng/ml. oxygen saturation (digitally on warm hands at rest) was measured using a nellcor np-20 pulse oximeter (covidian, ma, usa) during trek 1+2 and in the hypoxic chamber study at the same time as blood samples were taken. during trek 1+2, twice-daily ams scores were assessed using the lake louise score (lls) questionnaire. the lls allocates a score of 0 to 3 (symptom not present to severe) for symptoms of ams (headache, gastrointestinal symptoms, fatigue/weakness, dizzy/light-headedness, and difficulty sleeping). a score of 3 or more in the presence of headache is consistent with ams, a score of 6 or more with severe ams. the commercially available, highly sensitive, immunoturbidimetric assay (roche diagnostics) was used to measure crp in trek 2 at the same time points as ngal. mg/l and a between-run coefficient of variation between 2.5 and 5.7%. for statistical calculations, the software package spss 14.0 was used. for subjects with a ngal below the limit of detection of the assay (60 ng/ml), a value of 60 ng/ml was assigned for the purposes of statistical analysis. all data were tested for gaussian distribution using the kolmogorov-smirnov test and shapiro wilks statistic. for the analysis of dependent variables that were normally distributed, changes were tested by student's paired t-test. for independent variables that were normally distributed, a within-subjects anova was performed to investigate any serial changes in ngal with ascent at rest and post-trek. a two-way mixed anova with either resting or after trek ngal at each study altitude as the within-subjects factor and the presence of ams (according to the ll score at multiple altitudes) as the between-subjects factor if the mauchly sphericity test was significant, then p values were expressed after multiplication by the greenhouse-geisser epsilon. correlation analyses for normally distributed data were performed by calculating the pearson coefficient of correlation. a p value<0.05 (two-sided) was considered significant. as the ascent profile and route were closely matched in trek 1 and trek 2, data were combined and analysed as a whole. taking medication (acetazolamide, dexamethasone) had no apparent effect on ngal values, and therefore these subjects (n=11) were not excluded from the analysis. demographic data for the field study (trek 1+2), the controls, and the hypoxic chamber study are shown in table 1. in the 22 subjects ascending to 1085 m in the uk, there was no significant (p=0.084) rise in ngal following exercise: resting sl ngal was 64 11 (ng/ml, mean sd, range 60104) and postexercise ngal was 71 14 (ng/ml, mean sd, range 60100). of the 52 subjects, spo2 (%, mean sem) dropped from 97 2 at kat (1300 m) to 84 5 and 79 7 at 4270 and 5150 m, respectively (p<0.001). there was a moderate inverse correlation between ngal and spo2 at 5150 m (r=0.477, p=0.001) (figure 1) with a weaker inverse correlation between ngal and spo2 at 4270 m (r=0.340, p=0.019). within the subjects, anova demonstrated a significant change in ngal with ascent both at rest (p=0.007) and after trek (p=0.001) (figure 2). spo2 (%, mean sem) dropped from 99 0.4 at baseline to a nadir of 79 5 (p<0.001). despite an equivalent drop in spo2 to that seen in trek 1+2, ngal (ng/ml, mean sd, range) showed no change between baseline and 180 minutes: 63 26 (2980) versus 67 25 (2784), p=0.538. in trek 2, serum creatinine {mol/l, mean sem, (range), (p value versus baseline at kat)} was 78 2 (6395) at baseline; at 3400, 4270 and 5150 m it was 87 3 (72120) (p=0.001); 84 2 (72104) (p<0.001); and 94 5 (76142) (p<0.001). according to their ll scores at the highest study altitude (5150 m), there were 23 subjects with no ams, 16 subjects with mild ams, and 7 subjects with severe ams. there was a significant difference between ngal depending on the presence or absence of ams at 5150 m (figure 3) with higher values in those with ams and severe ams. a two-way mixed anova revealed a significant change (p=0.003) in resting ngal with ascent and an interaction with ams at 4270 m (p=0.017) and 4910 m (p=0.002 for change in ngal, p=0.027 for interaction with ams). hscrp was (mean sem, range): 1.6 0.4 (0.337.53) at baseline; at 3400 m, 4270 m, and 5150 m post-trek: 7 2.9 (0.7847.93) (p=0.002 versus baseline); 25.7 8.1 (0.58104) (p<0.001 versus baseline); 9 3.2 (0.5644.62) (p=0.003 versus baseline). at 3400 m, 4270 m, and 5150 m at rest: 6.2 2.9 (1.8725.1) (p=0.001 versus baseline); 21.6 5.7 (0.4983.9) (p<0.001 versus baseline); and 5.8 2.1 (0.5426.59) (p=0.012 versus baseline). ngal at 5150 m, after trek was moderately correlated with hscrp at 5100 m after exercise (rho 0.526, p=0.036). this is the first report to describe an association between ngal and both the presence and severity of ams at ha. the significant novel findings are that ngal rises in response to sustained hypobaric hypoxia but not acute normobaric hypoxia or near sl exercise and that this rise is related to ams at 5150 m. the rise in ngal following trekking (by day 2 at 3400 m) was to the levels normally associated with the subsequent development of arf (> 150 ng/ml), but this did not occur. although creatinine rose significantly with altitude, the rise was very modest, and we suspect that a combination of factors other than a simple renal insult is responsible for the increase in ngal at ha. our data suggest an inverse correlation between spo2 and ngal at 5150 m (and to a lesser extent at 4270 m). although no such correlation was found at 3400 m, this may still suggest that prolonged renal hypoxia could be a significant drive to ngal release. in addition to renal hypoxia, we suspect that other factors may also contribute to the rise in ngal at ha. the significantly greater ngal in those with severe or mild ams versus those without at 5150 indeed, ngal is an acute-phase protein with a role in inflammation [8, 10, 11]. exercise stimulates an immune response, and hypoxia is also known to cause a response in immune and endothelial cells with inflammatory markers such as hscrp increasing with ha [1922]. consistent with this, we saw a significantly higher hscrp at all altitudes compared to baseline. limited data have suggested hscrp may be associated with ams but we did not demonstrate any evidence to support this. there was a weak correlation between hscrp and ngal at 5150 m but this can not explain the rise in ngal as a whole. ngal also rises with oxidative stress [9, 12] which is increased by exercise, and ha-induced oxidative stress has been implicated in ams. as such, it is interesting to note that we found a higher ngal following trekking and in those with ams at the highest altitude. in an attempt to clarify the relative influence of exercise and hypoxia on ngal, we measured ngal before and after exercise of a similar duration (6 hrs) and similar incremental altitude (1085 m) as that experienced daily in nepal and also in a hypoxic chamber. in neither scenario did ngal rise. this may reflect inadequate duration or severity of stimulus but may also reflect that the ngal response is not due to exercise or hypoxia alone but is multifactorial involving hypoxia, oxidative stress, an inflammatory response, and other, as yet unidentified, stimuli. we also acknowledge limitations such as a lack of serum markers of oxidative stress and a lack of resting ngal data in trek 1. in addition, we did not measure ngal at sl before departure to nepal, although the ngal recorded as a baseline at kat (1300 m) (68 ng/ml) was no different to those recorded at sl in the uk (63 and 64 ng/ml). we also acknowledge the fact that although we measured creatinine in trek 2, we did not continue to monitor it after the cessation of trekking. as a consequence of creatinine rising more slowly in response to a renal insult than ngal, in conclusion, there are several interesting and novel findings that are worthy of further exploration. ngal rises in response to prolonged hypobaric hypoxia; marked increases in ngal may occur without concomitant arf and the degree of ngal rise at ha is associated with the presence or absence of ams. the fact that ngal does not appear to rise secondary to acute normobaric hypoxia or exercise in isolation suggests that the rise at ha and relation with ams may have common pathways, perhaps related to prolonged hypoxia and an inflammatory response. with the huge and increasing popularity of recreational sports undertaken at both moderate and high altitude assessment of ngal takes a matter of minutes using poc testing, and its use in identifying ams requires further evaluation.

acute mountain sickness (ams) is a common clinical challenge at high altitude (ha). a point-of-care biochemical marker for ams could have widespread utility. neutrophil gelatinase-associated lipocalin (ngal) rises in response to renal injury, inflammation and oxidative stress. we investigated whether ngal rises with ha and if this rise was related to ams, hypoxia or exercise. ngal was assayed in a cohort (n=22) undertaking 6 hours exercise at near sea-level (sl); a cohort (n=14) during 3 hours of normobaric hypoxia (fio2 11.6%) and on two trekking expeditions (n=52) to over 5000 m. ngal did not change with exercise at sl or following normobaric hypoxia. during the trekking expeditions ngal levels (ng/ml, mean sd, range) rose significantly (p<0.001) from 68 14 (60102) at 1300 m to 183 107 (65519); 143 66 (60315) and 150 71 (60357) at 3400 m, 4270 m and 5150 m respectively. at 5150 m there was a significant difference in ngal between those with severe ams (n=7), mild ams (n=16) or no ams (n=23): 201 34 versus 171 19 versus 124 12 respectively (p=0.009 for severe versus no ams; p=0.026 for mild versus no ams). in summary, ngal rises in response to prolonged hypobaric hypoxia and demonstrates a relationship to the presence and severity of ams.

PMC3817649

pubmed-69

when deposited on various substrate surfaces, rod-like, -conjugated, small organic molecules are well-known for their tendency to form highly anisotropic crystal shapes, which are frequently called fibers or needles. whereas the epitaxial growth has been studied on various substrates, in particular anisotropic surfaces seem favorable to conserve the highly anistropic morphology and optical properties, for example, polarized emission or adsorption provided by a parallel molecular orientation obtained by self-assembly. consequently, cu(110), tio2(110) and muscovite mica(001) are frequently chosen as a proper fundament to study the epitaxial growth of rod-like small molecules. in this paper, the epitaxial growth of 2,2:6,2-ternaphthalene (nnn) on muscovite mica(001) is reported. as indicated in figure 1a, the molecule is built from three naphthalene units, which are linked together by c the coexistence of needle-like structures and island-like crystallites is verified. structural analysis reveals two different crystal orientations. whereas island-like structures are built up by upright standing molecules orientated with a (001) contact plane relative to the muscovite mica substrate (see figure 1b), needles consist of nnn molecules with a (111) lying orientation (see figure 1c). both crystal configurations provide a well-defined azimuthal alignment, which is discussed based on force field simulations and a recently reported growth model. the azimuthal alignment of island like structures is explained by ledge directed (b) a side view of nnn molecules packed in the observed crystal structure. each unit cell houses two nnn molecules. molecules are approximately standing on the s (001) contact plane, which is indicated in blue. (c) a side view along the long molecular axis visualizing the edge-on/flat-on herringbone stacking of nnn. the blue area represents the orientation of the b (111) contact plane where molecules are aligned in almost in lying configuration. this all-aromatic compound could be obtained in high yield by coupling 2 equiv of 2-naphthaleneboronic acid (1) with 1 equiv of 2,6-dibromonaphthalene (2), as described in the supporting information. the final product, 2,2:6,2-ternaphthalene (nnn), was obtained as a colorless product, which appears to be highly insoluble in common solvents and could only be recrystallized from 1,2,4-trichlorobenzene (colorless platelets). the material was checked with gas chromatography and mass spectroscopy and found to be>99% pure before thermal sublimation. all samples have been fabricated on muscovite mica(001) substrates (spi, structure probe, inc.). muscovite mica is a representative of sheet silicate minerals and provides a layered structure of aluminum silicate sheets weakly bound by layers of potassium ions. each layer is characterized by a high symmetry direction identified by parallel aligned surface grooves. between the individual sheets, the high symmetry direction alternates by 120 leading to a periodic stacking sequence along direction. immediately after cleaving, the mica substrates were transferred to the hot wall epitaxy (hwe) chamber. the hwe technique was applied for the deposition of the organic material, which allows us to perform the growth process close to thermodynamic equilibrium, and in further consequence relatively high vapor pressure of the organic deposit in the substrate region can be achieved. therefore, the requirements concerning vacuum conditions are reduced compared with, for example, molecular beam epitaxy. the source material nnn was purified twice by thermal sublimation before filling it into the quartz tube of the hwe reactor. muscovite mica substrates were transferred into the deposition chamber via a load lock and subsequently preheated at the deposition temperature (80 c) for 30 min to ensure a stable temperature during the whole deposition process. the deposition was performed thereafter under a base pressure of 9 10 mbar. optical microscope images have been acquired by a nikon labophot 2a microscope in combination with a nikon type 115 digital camera. scanning force microscopy (sfm) studies of the deposited organic films were performed using a digital instruments dimension 3100 in the tapping mode. the 10 10 m images have been acquired at scan speeds of 46 m/s using sic tips (masch, hq: nsc15/al bs) exhibiting a cone angle of 40. nominal values for resonance frequency and tip radius are 325 khz and 10 nm, respectively. x-ray diffraction (xrd) measurements were carried out on a philips xpert x-ray diffractometer using cr k radiation (= 2.29) and a secondary graphite monochromator. please note that the monochromator is transparent for, /2, /3, etc., so despite the weak intensity of the bremsspektrum, it can give clear bragg peaks due to the scatting on the single crystalline mica substrate. specular scans were performed in bragg brentano configuration by varying the z-component of the scattering vector q. consequently it is possible to detect lattice planes that are parallel to the sample surface. pole figures were acquired by measuring at a constant length of q and only varying its direction. the unit cell parameters of nnn, which were used for analysis, are defined by a= 8.148 0.005, b=5.978 0.005, c=19.45 0.2, and =94.6 0.2 describing a monoclinic lattice (p21/a). the unit houses two nnn molecules in planar configuration. the van der waals (vdw) interaction between the organic molecule and the dielectric substrate is modeled by lennard-jones-type potentials. corresponding parameters are taken from the universal force field implemented in a matlab program. the molecules and substrates are assumed to be rigid where the internal structure of an isolated nnn molecules is determined from the crystal structure. simulations were performed for the adsorption of a single nnn molecule as well as a crystal stack. by assuming that a single nnn molecule prefers to lie flat on the surface, the energy minimization procedure is simplified in the following way: we consider only four molecular degrees of freedom, the x-, y-, and z-positions of the molecular center of mass and the angle. the angle defines the azimuthal molecular alignment and is probed by rotating the nnn molecule around the z-axis (surface normal). we perform a grid-based optimization to search for the best molecular adsorption geometry using a grid of 81 81 points for the lateral position. the adsorption angle was tested with a step size of =1. the surface structure of the substrate has been assumed to be the same as in the bulk where the corrugation is about 0.2. the substrate surface is assumed to be terminated by the tetrahedral layer of muscovite mica. for the simulation of the 7 2 nnn stack, the molecular packing has been deduced from the crystal structure of a (111) orientated nnn crystallite. due to the presence of flat and edge-on molecules, the adsorption distance has been optimized, yielding a distance of 1.6 of the lowest h atom of edge-on nnn molecules to the substrate surface. because energetic minima are significantly narrow for the molecular stack, an angular resolution of =0.5 has been chosen for the calculations. in a first step, nnn was deposited by hot wall epitaxy (hwe) on muscovite mica(001). whereas the substrate temperature was kept constant at 80 c, the deposition time was continuously increased. scanning force microscopy was chosen to study the sample morphology versus deposition time, and obtained images (10 10 m) are depicted in figure 2. as the maximum height scale z0 significantly changes with increasing deposition time, the corresponding values are indicated for each sample. the morphology of all samples is dominated by the presence of several micrometer long needle like structures that are aligned along multiple orientations. as exemplified by the cross-section (1), these fibers reach height levels up to 200 nm and are characterized by similar dimensions in width. a more detailed analysis is provided in figure 2a, plotting the mean needle height versus growth time. as indicated by the solid line, the growth rate can be approximated by a linear fit, yielding a slope of 17.8 1.2 nmmin. interestingly, the fibers surface coverage is approximately constant for the whole sample series yielding a value of 12.5%. the sfm analysis reveals that flat islands start to nucleate at the side walls of the fibers and continuously fill the substrate surface between the fibers with increasing deposition time. the latter observation is underlined by analyzing the islands surface coverage, which is depicted in figure 2b as a function of growth time. the solid line, which represents a guide for the eye, indicates an asymptotic approach to 85% of the surface area. again, a representative sample position has been chosen to deduce a cross-section (2), which is presented in the bottom right of figure 2. as reported for other rod-like molecules, a steplike morphology with height levels in the range of 1.82 nm the obtained value corresponds to approximately one monolayer of upright standing nnn molecules. contrarily, the step size of the boundary, defined by the islands and the substrate surface, is significantly larger reaching values in the range of 15 nm. these steps are further characterized by straight extensions, which suggest the formation of well-defined crystal facets. scanning force microscopy (sfm) images showing the sample morphology versus deposition time. all samples are dominated by needle-like structures. with increasing growth time, island-like structures start to nucleate at the needle side walls covering continuously the substrate surface between the fibers. exemplary cross sections for both morphologies are indicated in the bottom part of the figure. as indicated in part a, the height of the fiber like structures linearly increases with growth time reaching values of some 100 nm. contrarily, the surface coverage by needles stays approximately constant (part b). step heights in the center part of the islands approximately correspond to a monolayer of upright standing molecules. in a next step, x-ray diffraction (xrd) has been chosen to study the structural properties of the organic crystallites. in order to obtain sufficient diffraction intensity, a sample with 216 nm high nnn fibers has been selected. figure 3a reports the acquired specular xrd diffraction pattern, which is dominated by a series of {00n} diffraction peaks. these peaks are characteristic for island-shaped crystal morphologies, built up by approximately standing nnn molecules, and are consequently abbreviated by s-orientations (q001=0.324). arrows in the upper part of figure 3a indicate the positions of (00.2n) diffraction peaks stemming from the muscovite mica(001) substrate. additionally, a diffraction peak arises at qz=1.36, which correlates with (111) orientated nnn crystallites, abbreviated as b orientation. the orientation is characteristic for a nearly flat lying molecular configuration and thus explains the presence of needle-like crystallites as revealed by sfm analysis. a more detailed analysis is reported in the supporting information. in order to analyze the azimuthal alignment of the nnn crystallites relative to the muscovite mica(001) substrate, xrd-pf measurements have been performed and are reported in figure 3b. for a profound analysis, xrd-pf have been acquired with a maximum sensitivity to diffraction intensities stemming from the scattering at {202} and {201} netplanes and are depicted in the bottom leftand right part of figure 3b. the diffraction intensities show a distinct azimuthal distribution, which underlines a well-defined epitaxial relationship to the muscovite mica substrate. the obtained symmetry of the diffraction intensities further underlines the presence of an terminated muscovite mica surface, which is characterized by a mirror plane along the [110]mica orientation. the orientation of the substrate has been determined from diffraction patterns stemming from muscovite mica (001) and are indicated by black solid circles. xrd-pf patterns of the organic crystallites can be constructed by mirror operation from the top hemisphere, sketched by a gray shaded sector. moreover, the xrd-pf patterns reveal a 2-fold rotational symmetry, which can be understood by an approximately equivalent adsorption energy for 180 turned organic crystallites. consequently, discrimination between both crystal alignments has been omitted and simulated 2-fold symmetric diffraction spots are labeled identically. based on these geometrical considerations, only the diffraction spots of a single quadrant have to be analyzed and labeled. (a) specular x-ray diffraction (xrd) spectrum of nnn on muscovite mica(001). scanning force microscopy images revealed a needle height of 216 nm for the chosen sample. the spectrum is dominated by a series of s (001) diffractions peaks, which are representative for island-like morphologies. (b) xrd pole figure (xrd-pf) analysis of {202} and {201} diffraction peaks, providing information about the azimuthal crystal orientation. as indicated by the simulated pole distribution (bottom), all diffraction spots can be explained by the presence of three differently aligned (001) crystallites, labeled as s13 (red). the mirror symmetry plane of the muscovite mica (001) surface is indicated by a horizontal line, which explains the presence of mirrored s13*crystallites (blue) with a (001) contact plane. moreover, xrd-pf reveal a well-defined azimuthal orientation of b1 (111) crystallites (red circles). again mirror symmetric crystallites b1*(111) are indicated by blue symbols. diffraction intensities from the muscovite mica substrate are indicated by black solid filled circles. diffraction intensities that are characteristic for s-orientated crystallites are located at =63 (74) in the left (right) xrd-pf, and their azimuthal distribution can be explained by the presence of three crystal orientations labeled as s13. consistently, both xrd-pf measurements hint the strongest diffraction intensities originating from s1 crystallites. diffraction spots, which can be attributed to b*(111)/b (111) crystallites are expected to appear at 51 for both diffraction geometries and are marked by blue/red filled circles. as each quadrant reveals the presence of one diffraction spot, the azimuthal alignment of b orientated fibers again, mirror symmetric crystals are labeled as b1*and are characterized by a (111) contact plane. based on the simulated xrd-pf diffraction peaks, the long needle axis (lna) and long molecular axis (lma) orientations of b orientated fibers have been deduced and are presented in figure 4 by solid filled arrows. whereas the lna coincides with the [110] orientation, the lma can be approximated by the alignment of [101] relative to the muscovite mica substrate. mirror symmetry of the muscovite mica substrate leads to the generation of two energetically equivalent crystallites. fibers that are built up by b1*(111) orientated crystallites (blue) are aligned with their lna (lma) 59.5 (49) relative to the muscovite mica substrate s [110]mica crystallographic orientation. contrarily, their mirror symmetric twins (b1) can be constructed by flipping the b1*crystallites upside down (red arrows), azimuthally aligned with their lna (lma) 59.5 (49) relative to [110]mica. in order to verify the lna alignment, which has been constructed based on xrd-pf measurements, optical microscopy has been chosen, and an image of a representative sample area is depicted in figure 4. the sample morphology is dominated by fibers that are aligned in a v-shaped, herringbone fashion. as indicated by large red and blue arrows, which represent the expected b1 and b1*lna orientations deduced by xrd-pf analysis nevertheless, additional needle orientations, which are present in minor fraction, can be observed and are marked by small red (b2) and blue (b1 *) arrows, respectively. in order to gain better statistics, a microscopy image of a larger sample area has been chosen to perform a fast fourier transformation (fft) and the obtained pattern is depicted beside. the fft is dominated by two stripes, which are characterized by an enclosing angle that perfectly reflects the lna orientation of b1 and b*1 crystallites (indicated by red and blue arrows). long needle axis (lna) (left) and long molecular axis (lma) (right) orientations of b1 (red solid filled arrows) and mirror symmetric b1*(blue solid filled arrows) crystallites, deduced by x-ray pole figure (xrd-pf) measurements. in the outer ring of the lna polar plot, additionally the [110] crystallographic orientations of s13 (red) and it is visualized that the [110] crystallographic direction of s1 crystallites provides the same azimuthal orientation as the lna of b1 fibers. below, the obtained lna orientations are verified by the observed sample morphology using optical microscopy. beside b1 and b1*fibers additionally minor fractions of approximately horizontally aligned crystallites are observed (marked by small red and blue arrows). by using fast fourier transformation (fft) (depicted beside), the dominant fraction of b1 and b1*crystal orientations is further underlined. in a next step in particular, the azimuthal alignment of their [110] directions has been deduced and is depicted in the outer ring of figure 4 (lna). because s1 and s1*crystallites represent the major fraction, they are indicated by large arrows approximately aligned 60 relative to [110]mica. the azimuthal orientation perfectly coincides with the lna of b1 and b1*crystallites, which already suggests an epitaxial relationship of both crystal types. in order to analyze the latter observation in more detail, the sfm image, which is shown in figure 5, is dominated by two approximately vertically aligned fibers. between fibers, the presence of an s orientated island can be observed, which is terminated in the bottom part of the image by a sharp l-shaped boundary. the observed boundary shape perfectly correlates with the expected angle between the and [110] crystallographic orientations of an (001) orientated crystallite. the generation of the observed crystal shape can be understood by the formation of (11n) and (11n) side facets, which represent low index planes for n=1, 0, or 1. an extracted cross-section of the observed fiber is presented in the left part of figure 5. the observed fiber is terminated in the right part by a flat plane, which is aligned parallel to the substrate at a height of approximately 100 nm. contrarily, the left side of the fiber shows a constantly decreasing height level. the slope of the side facet approximately correlates with a 25 nm decrease in height along 100 nm of the needle width (14). the lna of b*type crystallites is defined by their [110] orientation and consequently all crystallographic planes (11n) are aligned parallel to it. because the angular tilt of 13.8 between the low index planes (110) and (111) perfectly correlates with the observed sfm analysis, the theoretically expected cross-section of a b1*fiber has been modeled and is depicted below the experimental data. although, the observed steep height decrease at the fiber side walls is below the resolution limit of the sfm, a termination of the fibers by (001) and (001) facets can be assumed. scanning force microscopy (sfm) image showing vertically aligned fibers and s orientated islands in between after the deposition of 60 min nnn at 80 c substrate temperature. the island s boundaries correlate with the geometrical alignment of and [110] orientations. the extracted fiber cross-section (top, left) can be explained by the formation of (111), (110) and (001) facets. based on the observed crystal shapes, their crystallographic orientations relative to each other has been deduced and is visualized by a 3d model below. the epitaxial relationship between fibers and islands is consistent with the structural analysis and can be explained by a nucleation of nnn molecules at the fiber side walls, also called ledge directed epitaxy. based on the latter analysis, a three-dimensional model of both crystal types has been generated and is depicted in the bottom part of figure 5. as indicated by the xrd-pf analysis, which is presented in figure 4 (lna), b1*and s1*crystallites shared the same azimuth for their crystallographic [110] orientations. in that way, the tilt angle of standing nnn molecules within the s-type crystallite approximately correlates with the tilt angle of the fiber (001) low energy plane. analogous observations were demonstrated for 6 t fibers and are explained by the nucleation of islands at the sidewalls of already existing needles. moreover, the latter picture is perfectly consistent with the sfm analysis presented in figure 2, which reports a continuously increasing island coverage for longer deposition times. such epitaxial alignment based on a geometrical fit between nucleating crystallites and already existing topographic features on the substrate is called ledge directed epitaxy. based on the latter analysis together with xrd-pf data presented in figure 4 (lna), it can be concluded that the minor fraction of s23 crystallites has most likely nucleated at b-type fibers, which are orientated approximately 30 tilted relative to [110]mica of the muscovite mica substrate. this conclusion is further consistent with the microscopy image, presented in figure 4, revealing the presence of a minor fraction of such fibers (below the detection limit of xrd-pf measurements), which are subsequently labeled by b2 and b2*. in order to understand the observed growth behavior of b1 and b2 fibers on muscovite mica(001), the left part of figure 6 depicts a planar nnn molecule in gas phase. analogous to 6 t, a planar molecule is characterized by a mirror plane h, which is aligned in the plane of the naphthalene rings, and a 2-fold rotational axis, which is aligned normal to h., it should be assumed that single nnn molecules tend to adsorb lying flat on the muscovite mica substrate in order to maximize their contact area. in that way, h is orientated parallel to the substrate, and the molecule becomes chiral when adsorbed on an arbitrary surface. molecules that are intrinsically achiral but obtain a form of 2d chirality when adsorbed on a substrate surface are also called prochiral. analogous to 6 t, two mirror symmetric nnn enantiomers (sketched as red and blue molecules) can adsorb on the muscovite mica surface, which can not be brought into congruence by translation and rotation. taking a top view of the molecular stacking at the contact plane of b-type crystallites, which is depicted in the right panel of figure 6, reveals that (111) orientated fibers are built up by an alternating assembly of only one enantiomer (red) and edge-on nnn molecules. contrarily, their twin crystallites b*(111) are built up by the mirrored molecular configuration (blue) only. the latter observation further explains the consistent choice of a red and blue color code for molecules and crystallites. beside a real space model for b1 crystallites, which is deduced by xrd-pf analysis, the right part of figure 6 further includes the proposed geometry of b2 fibers. based on a growth model that has been deduced for 6 t fibers, it is assumed that two needle orientations, for example, b1 and b2*can originate from one molecular adsorption site. the existence of these two lna orientations is explained by a mirror symmetric molecular stacking during crystal nucleation. interestingly, epitaxially grown 6 t on muscovite mica showed a comparable fraction of both stacking types, which explains the observation of four lna orientations. the latter phenomenon is further explained by force field simulations for both crystal types, yielding a similar adsorption energy with a deviation of some millielectronvolts/molecule. contrarily, xrd-pf analysis revealed that nnn fibers are dominantly present only in one configuration, which reduces the observed lna orientations to two (see the fft in figure 4). consequently, it can be stated that both crystal types seem to significantly differ concerning their adsorption energy, which should be investigated and underlined by the discussion of force field simulations within the next paragraphs. molecular symmetry of a planar nnn in gas phase and when adsorbed flat on an arbitrary surface. due to the presence of a mirror symmetry plane h parallel to the naphthalene rings and a 2-fold rotational axis, aligned normal to it, the nnn molecule in gas phase can be described by the c2h point group. contrarily, when adsorbed flat on a substrate surface, nnn can form two mirror symmetric enantiomers (sketched by red and blue molecules), which follow c2 symmetry. the right panel depicts a real space model of the discussed crystal orientations (top view). the molecular alignment of nnn within the surface unit cell has been deduced from its bulk structure, oriented with a (111)/(111) contact plane for b/b*crystallites. the orientation of the long molecular axis (lma) or long needle axis (lna) is indicated by blue or red arrows. taking a closer look at the molecular stacking at the contact plane of b-type crystallites reveals that (111) orientated crystals are alternately assembled by red enantiomers and edge-on nnn molecules. contrarily, their mirror symmetric twins b*only consist of blue molecular configurations. the real space image of b1 (b1 *) and b2*(b2) the adsorption energy as a function of the long molecular axis (lma) orientation () is depicted in the top panel in terms of a polar plot. red and blue arrows indicate the molecular orientation deduced by experiments. at the indicated positions, force field simulations reveal a broad maximum for the corresponding enantiomer. contrarily, the adsorption site seems less favorable for the mirror symmetric molecule (e 20 mev). a real space model that sketches the lateral position for the molecular adsorption angle at =57 simulations reveal a preferred alignment of the terminating naphthalene units in the surface corrugations of the muscovite mica substrate (indicated by horizontal lines). below, an analogous analysis has been done for a 7 2 molecular stack representative for the contact plane of a b (111) crystallite. simulations reveal a strongly pronounced adsorption maximum at the experimentally observed adsorption angle (red arrow) and a significantly different adsorption energy for a b*crystal with opposite stacking sequence (blue arrow, e 300 mev/molecule). a real space model of the optimized adsorption position is depicted in the right panel. by comparing the alignment of the nnn molecules with the muscovite mica unit cell, an approximately periodic alignment can be recognized along mica/[110]mica for an/terminated surface. in a first step, the optimal adsorption energy of a single nnn molecule has been deduced based on force-field simulations by selecting the most favorable adsorption site for each angle. the angle characterizes the azimuthal alignment of the lma relative to mirror symmetry plane of the muscovite mica substrate surface. the molecules are assumed to adsorb flat on the substrate and the adsorption energy, ead, is defined as the difference between the energies of the isolated subsystems and the energy of the combined system. therefore, maxima in the ead versus curves evidence the favorable adsorption geometries. to increase the readability, ead curves are presented in figure 7 in terms of a polar plot, where ead=e0 ead. the parameter e0 represents the adsorption energy at the least favorable angle, yielding a value of e0=2.49 ev for the isolated molecules. because nnn molecules can adsorb either in their left- or right-handed configuration, simulations have been performed for both enantiomers and are color coded by red- and blue-filled curves. simulations yield, for both molecular configurations, multiple adsorption maxima, which are located for the blue marked enantiomer at max, blue=42, 57, 102, 161, and 178. due to 2-fold rotational symmetry of the nnn molecule, identical values are obtained for ead(+180). moreover, optimized adsorption positions for the red molecular type are found at max, red=max, blue due to mirror symmetry of the substrate surface. experimentally obtained adsorption angles are further indicated by a blue (red) arrow at =49 (131). both adsorption angles correlate with the broadest maxima obtained by simulations and are importantly consistent with the simulations of the corresponding enantiomer. the fact that beside experimentally observed adsorption geometries force field calculations also yield additional maxima is attributed to the usage of empirical potentials, which in some cases may yield the wrong energetic ordering of competing structure solutions. nevertheless, it has to be underlined that simulations indicate a significant less favorable adsorption site for the mirror symmetric molecular configuration (20 mev). in general, adsorption energies for both molecular configurations significantly differ, which in further consequence leads to a nonequal distribution or even breakup of both enantiomers depending on the adsorption angle. contrarily, simulations as well as experimental data that are reported for 6 t indicate a significantly lower energetic splitting between both molecular configurations, which may result from a higher symmetry of the molecule. the latter statement can be understood by the fact that thiophene molecules with an odd ring number, for example, quinquethiophene or septithiophene, are characterized by a mirror symmetry plane when adsorbed on a surface and consequently do not show a prochiral character. contrarily, the asymmetric alignment of the c c bond between two naphthalene units of nnn inevitably leads to a prochiral behavior when adsorbed on a substrate surface and consequently plays an essential role concerning the energetic separation of both enantiomers at a defined adsorption angle. the experimentally confirmed adsorption position of nnn molecules is further depicted in the right part of figure 7. for both molecules, an adsorption angle=analogous to calculations for p-6p and 6 t, nnn molecules tend to align their rings in the surface corrugations, which are indicated by vertical solid lines. in order to study the adsorption energetics of a b-type crystallite, a 7 2 molecular stack has been deduced from a (111) orientated crystallite. analogous to the force field simulations of an isolated molecule, the adsorption energy ead for the stack has been probed depending on the molecular orientation and the adsorption energy at the least favorable adsorption angle is given by e0=0.41 ev/molecule. because the curve calculated for the a b*(111) stack follows the same behavior as discussed for a single molecule (mirror symmetric), only the results for a b contact plane are depicted in order to increase readability. interestingly, simulations reveal that not only the number of energetically favorable adsorption sites decreases but also the angular acceptance, which becomes visible by well pronounced peaks. simulations further indicate the presence of two adsorption maxima, which are located at =12 and 48. again, the experimentally obtained adsorption angle for b1 crystallites is indicated by a red arrow and underlines a nearly perfect agreement. moreover, it can be recognized that the adsorption energy for a b2 crystal (at+48) becomes even more unfavorable than that for a single molecule, which is manifested by a much lower value of ead in the range of some 100 mev/molecule. consequently, force field simulations not only reflect the experimental observations but also explain the dominant fraction of b1 crystallites by a preferred nucleation of their stacking sequence in contrast to b2 crystallites. the observed behavior can be even better understood by analyzing the real space model of the simulated adsorption position at =48. besides the molecular alignment, also the surface unit cells of the muscovite mica and b crystal have been indicated. obviously, the unit vector [110] of the nnn crystal stack, which also defines its lna, tends to align parallel to one surface unit vector of the muscovite mica crystal, which is defined by the mica, ([ 110]mica,) orientation for an () terminated surface. the epitaxial growth of ternaphtalene (nnn) on muscovite mica(001) has been investigated by combining structural (xrd-pf) and morphological (sfm) methods. consistently, both methods reveal the formation of s (001) orientated nnn island-like structures which have nucleated at the sidewalls of b (111) orientated fibers. it is demonstrated that the latter nnn crystal types tend to align along two dominant directions, which leads to the formation of a v-shaped sample fiber morphology. because the tilt angle of nnn molecules within s-orientated crystallites correlates with the tilt angle of the fiber side facets, the island nucleation is explained by ledge directed epitaxy. based on this growth model, it can be understood that both crystal types provide a well-defined azimuthal orientation relative to the muscovite mica substrate. by use of force field simulations, the growth of the fibers is further analyzed. the epitaxial growth of sexithiophene (6 t) on muscovite mica showed the formation of four well-defined fiber orientations, which can be explained by mirror symmetry of the muscovite mica substrate and two differently stacked 6 t crystallites, which can nucleate at a molecular adsorption position. contrarily, experimental investigations indicate that nnn crystallites tend to stack in a single configuration, which explains the dominant formation of only two fiber orientations. based on force field simulations, the latter observation is further investigated and explained by significantly different adsorption energies of both crystal types. it is further demonstrated that the observed behavior results from an interplay of the molecular adsorption and lattice match.

the morphology and structure of 2,2:6,2-ternaphthalene (nnn) deposited on muscovite mica(001) substrates was investigated by scanning force microscopy (sfm) and specular x-ray diffraction measurements. consistently, both methods reveal the coexistence of needle-like structures with a {111} contact plane and {001} orientated island-like crystallites, which are built up by almost upright standing nnn molecules. both orientations are characterized by a well-defined azimuthal alignment relative to the substrate surface, which is analyzed by x-ray diffraction pole figure (xrd-pf) measurements. based on xrd-pf and sfm analysis, the azimuthal alignment of {001} orientated crystallites is explained by ledge-directed epitaxy along the fibers sidewalls. these fibers are found to orient along two dominant directions, which is verified and explained by a doubling of the energetically preferred molecular adsorption site by mirror symmetry of the substrate surface. the experimental findings are confirmed by force-field simulations and are discussed based on a recently reported growth model.

PMC3919176

pubmed-70

a-38-year-old female patient consulted to our emergency outpatient clinic with complaints of fever, sore throat, coughing, shivering, chills, joint, and muscle pain, headache, chest pain continuing for nearly 2 weeks and she was hospitalized to investigate the etiology of fever. physical examination findings were as follows: bp 130/80 mm hg, body temperature, 39.8 c; hr 120/min; moderately well general physical status with full cooperation, markedly hyperemic pharynx, and tonsils, herpetic rashes around her lips, bilaterally diminished respiratory sounds heard over basal segments and tachycardia. her neck rigidity was evaluated however the kernig, and brudzinsky signs could not be elicited. laboratory test results were as follows: hb 11 g/dl (12.117.2), mcv 80.8 fl (82.299), wbc 22.10 k/ul (410), neu 90.5% (3773%), esr 92 mm/s (020), procalcitonin 0.8776 ng/ml (< 0.1), total protein 5.66 g/dl (6.48.3), albumin 2.9 g/dl (3.55), inr 1.15 (11.5), ferritin 2889 ng/ml (14150). glucose, creatinine, ast, alt, alp, na, k, complete urinalysis, ana, rf, and ena profile were unremarkable. on thoracal imagings, bilateral hilar lymph nodes of which the largest being 2 cm in diameter, bilateral pleural effusion, and atelectasic areas in the parenchyma surrounding the pleural effusion were observed (figure 1, 2). on abdominal tomograms, no bacterial growth was detected in urine, throat, csf, and blood cultures. lumbar punction performed because of suspect neck rigidity did not reveal any abnormal cells in csf. cephtriaxone at daily parenteral doses of 2 g). during follow-up period, rashes, and swelling on her right, and left wrists were detected, so she was consulted to rheumatology clinic, and naproxen sodium therapy was started with the indication of suspect aosd. on physical examination dullness over traube s space however her hyperfebrile state persisted, echocardiograms obtained for the differential diagnosis of infective endocarditis. posteroanterior chest radiogram costodiaphragmatic sinuses are not distinctly visualized, high-lying diaphragms, and an enlarged mediastinum were observed. biochemical analysis of the fluid drained by thoracenthesis of the patient whose computed tomograms demonstrated bilateral pleural effusion revealed the presence of 2 gm/dl albumin, 3.34 g/dl protein, 483 u/l ldh (concurrent serum albumin 2.9 gm/dl, total protein 5.99 g/dl, ldh 393 u/l). antibiotherapy of the patient with pleural effusion (exudate) was discontinued, and methylprednisolone treatment at daily doses of 16 mg was initiated. after initiation of steroid therapy, she maintained an afebrile state, her joint manifestations regressed, and her complaints decreased during her follow-up period. the patient whose pleural effusion completely regressed with steroid therapy discharged to be further followed up by rheumatology outpatient clinic (figure 3). adult onset still s disease is a disease whose diagnosis can be made by excluding infectious, malign, autoimmune, and autoinflammatory diseases, and in consideration of its characteristic clinical, and laboratory findings. for definitive diagnosis, yamaguchi criteria can be used. its major criteria consists of higher body temperature at 39c lasting for at least one week, marked nonpruritic macular/maculopapular salmon-colored rashes on the torso or extremities persisting for at least 2 weeks, leukocytosis (> 10.000/ml), and neutrophilia (> 80%). minor criteria include sore throat, lymphadenopathy, hepatomegaly or splenomegaly, abnormal hepatic function tests (increases in especially ast, alt, and ldh levels), and ana, and rf negativities. for the establishment of diagnosis, 5 diagnostic criteria (including at least 2 major criteria) should be met. in our patient, during approximately 4 weeks up to initiation of steroid therapy, fever exceeding 39c every day, symptoms of arthralgia, marked arthritis of both wrists, leukocytosis, neutrophilia, sore throat, and lymphadenopathies fulfilled the required diagnostic criteria, and pleural exudate seen during the clinical course of the disease was assessed to be associated with the disease. in the adult onset still s disease pleural effusion, transient pulmonary infiltration, and pericarditis can be seen in 3040% of the patients. affected individuals may complain of mild episodes of coughing, pleuritic chest pain, and shortness of breath. even in some patients it may progress to acute respiratory distress syndrome (ards) [6, 7]. rarely, myocarditis, arrhytmias, heart failure, and cardiac tamponade can be seen. myocarditis, and ards are more frequently seen especially in patients with mas. in the differential diagnosis, infectious diseases (ebv, cmv, hbv, rubella, parvovirus, coxsackie, hiv, subacute bacterial endocarditis, meningococcemia, tuberculosis, syphilis); vasculites (takayasu arteritis, polyarteritis nodosa, serum disease, thrombotic thrombocytopenic purpura); malign diseases (leukemia, lymphoma), connective tissue diseases (ra, sle, mixed connective tissue disease), granulomatous diseases (sarcoidosis, crohn disease); autoinflammatory diseases (kikuchi disease, sweet syndrome), and drug hypersensitivity reactions should be considered. in our patient serological tests, cultures, and imaging modalities were used for differential diagnosis which led to the establishment of the diagnosis of aosd. higher ferritin levels are among the most important laboratory findings of aosd, however markedly lower glycosylated ferritin levels are detected (< 20%). increased ferritin level is an indicator of the disease activity, and also a serologic marker of treatment response. in our patient ferritin levels may increase as a response of hepatocytes to cytokines as il-18, and il-1b which may be thought to play a role in the pathogenesis of aosd. as the first-line therapy nsaids (especially endomethacin), and corticosteroids are recommended. in cases of severe organ involvement pulsatile high doses of steroids for few days can be used, than daily dose is dropped down to 1 mg/kg. in cases of recurrence or with the intention to decrease steroid dose, methotrexate at a weekly dose of 7.525 mg can be given. hydroxychloroquine, sulphasalazine, azothioprine, cyclosporine can be used separately or in combination with methotrexate. in recent years, effectiveness of biological agents (infliximab, etanercept, anakinra, and tocilizumab), and ivig therapies have been reported in the treatment patients with serious organ involvement, and mas. still many characteristic features of aosd are not known, and a standard follow-up or a treatment protocol of the disease is not available. in patients who met diagnostic criteria, pleural effusion which may be seen in the clinical course of the disease should be presumably associated with the disease. besides regression of pleural effusion can be expected with the treatment of underlying desease, additional locally invasive diagnostic, and/or therapeutic interventions should be avoided.

adult onset still s disease is a rare systemic inflammatory disorder. at the onset of the disease sore throat, pharyngitis; which does not respond to antibiotics, one or two times peaking febrile episodes, marked salmon-colored rash on the trunk and extremities, arthralgia, arthritis, myalgia, fatigue, loss of appetite with nausea and weight loss; hepatosplenomegaly and lymphadenopathy can be seen. among laboratory examinations levels of ferritin and other acute phase reactants distinctly rise, and neutrophilic leukocytosis; ana and rf negativity are detected. pleural and pericardial effusions, transient pulmonary infiltration, and rarely myocarditis can be seen during the course of the disease. here we report a patient who was examined for fever of unknown origin and diagnosed with adult onset still s disease which is a rare etiology of pleural effusion.

PMC5175095

pubmed-71

dual-polymerizing resin cements have been extensively used for placement of indirect restorations and posts. the dual-polymerizing materials were developed to compensate for the lack of polymerization in the absence of light and to represent a combination of auto- and light-polymerizing components. in some clinical situations, such as in dark zones at the apical region and during the cementation of indirect restorations, the severe light attenuation results in low degree of conversion (dc), which can compromise the mechanical properties and consequently the longevity of the indirect restorations.14 modifications in the viscosity of the resin cements allow their use in different clinical situations. the option for low viscosity versions offer some benefits, such as minor thickness of the pellicle that was formed following the restoration placement. the lowest film thickness generates smaller polymerization shrinkage, reducing the possibility of gaps formation and premature marginal leakage.5,6 the difference in the cement formulations that change the viscosity is related to the proportion between resin matrix and filler particle content.7 high volume fraction of fillers can increase the viscosity and the elastic modulus and strength of composites.7,8 low viscosity or flowable resins and resin cements present lower filler loading than regular restorative materials. most direct dental restorative composites use bisphenol-a-diglycidylether dimethacrylate (bis-gma), which is considered a very viscous monomer, and when mixed with higher filler loadings, it becomes a nearly solid mass and unusable product. vinyl groups (e.g., ethylene glycol dimethacrylate) are added as a thinner or diluent monomer for uncured pastes, which are considered another approach to change the viscosity of resin-based materials. the filler loading and the viscosity of composites may interfere in the monomer conversion, since they could restrict the mobility of monomers and the propagation of polymerization reaction.7,911 the restorative resin-based materials must reach a high degree of monomer conversion in order to present better clinical performance and longevity and also to reduce the early failures. for the dual-polymerizing resin cements, the self-cure mode should ensure the high level of conversion, especially in the cervical proximal areas, the root canal and in the internal and deep areas of the cavity preparations.1214 several methodologies have been used to analyze the dc of resin-based materials; however, most of them use fourier transform infrared spectroscopy (ftir). although many researchers have evaluated the polymerization effectiveness to determine the physical and mechanical properties of dental materials,9,15,16 little information is known about the influence of different viscosities of dual-cured resin cements on their physical properties, such as the degree of conversion in situations of self-and dual-polymerization. thus, the purpose of this study was to measure the dc of two commercially dual-cured resin cements in different viscosities (low and high) when they were light-activated or when the materials were allowed to self-cure solely, after 5 minutes and 24 hours from the mixture of pastes (base and catalyst). the hypothesis tested was that curing mode, evaluation time and viscosity would affect the dc of the resin cements. two commercial dual-cure resin cements in high and low viscosities were evaluated: variolink ii (ivoclar vivadent, schaan, liechtenstein) and nexus 2 (kerr corp., orange, ca, usa). the resin cements consist of two paste components that were equally dispensed and mixed together according to manufacturers instructions. after mixing, resin cements (n=5) were applied to the horizontal attenuated total reflectance znse crystal at 45 (fourier transform infrared spectrometer, ft-ir spectrometer 520, nicolet instrument corp, madson, wi, usa) at room temperature. adhesive tape (3 m, sumar, sp, brazil) was placed on the znse crystal surface to act as a spacer, ensuring standard thickness for all specimens (100120 mm). a mylar strip (quimidrol com. ltda, joinville, sc, brazil) was placed over the cement and was pressed flat to spread the material on the crystal surface. the spectrum of unpolymerized material was obtained and the cements were either light-cured (dual-polymerizing mode) or allowed to self-cure only (autopolymerizing mode). each specimen was left on the crystal surface and further spectra were obtained 5 minutes and 24 hours after post mixing. for light-cured groups, a halogen light curing unit (xl 3000, 3 m espe, st. paul, mn, usa) was used during 40 seconds (600 mw/cm). the light intensity was periodically checked with a radiometer (curing radiometer, model 100, kerr corp., orange, ca, usa). spectra were obtained between 4000 cm and 750 cm at a resolution of 4 cm. monomer conversion was calculated (%) according to the changes in the ratio between the absorbance peaks corresponding to the aliphatic (cc) (1638 cm) and aromatic (1608 cm) carbon double bonds prior to and 5 minutes and 24 hours after polymerization initiation for both curing modes.10 the aromatic peak is used as an internal reference because the intensity does not change during the polymerization reaction.9,11 the effect of curing mode, viscosity and time intervals on the dc were evaluated for each material. three-way repeated measure analysis of variance (anova) (curing mode, viscosities and evaluation time) was performed and tukey s post-hoc test was used to detect pair wise differences among experimental groups. the data was analyzed using the statistical program sas 9.1 (sas institute, cary, nc, usa) and all statistical testing was performed at a pre-set alpha of 0.05 (p<.05). the statistical analysis of the data showed significant differences in curing mode, viscosity and evaluation time factors for both resin cements (p=.01). there was significant interaction between factors: the curing mode x evaluation time (p=.01). other double-interactions and the triple-interaction (curing mode x viscosity x evaluation time) were not significant (p>.05). the dc of resin cements was higher when the materials were light-cured and when the low-viscosity versions were used than when self-curing mode and the high-viscosity resin cements were tested, respectively (p<.05). the results of this study showed that the curing mode (auto- and dual-polymerization), the viscosity (low or high) and the evaluation time (5 minutes or 24 hours) influenced the dc of resin cements. thus, the research hypothesis stating that these factors would result in significant changes in the dc was accepted. as the dc is dependent on the monomeric composition and the concentration and type of the photoinitiator in each product,9,17 the statistical analysis comparing the dc between resin cements the main factors under study (curing mode, viscosities and time intervals) produced similar effects for the resin cements, which presented different formulations regarding the resin matrix composition and type and concentration of filler particles. the variations on the proportion of components of resin matrix and filler particles that produced the low and the high versions caused no difference in behavior between the two cements results.7 a high monomer conversion with cross-linked polymeric network formation is essential for the durability of the resin-based restorations.911 studies have shown the importance of a high dc of resinous materials in order to reach better mechanical properties, since the presence of a high amount of residual methacrylate monomers compromises the hardness, the abrasion and the fracture resistance. moreover, the low dc can increase the sorption and the solubility, interfering in the color stability and mass loss of the resin cement.2,4,9,14,1720 the resin cements showed significant difference between the evaluation times, 5 minutes and 24 hours from the polymerization initiation, independently of viscosity or curing mode. thus, the polymerization reaction for both resin cements continued after 5 minutes of the mixing of the base and catalyst pastes. the continuation of the chemical part of the polymerization reaction (benzoyl peroxide+tertiary amines) was primarily responsible for the increase of the dc after 24 hours. additionally, the percentage of dc increasing after 24 hours was higher for the self-cure mode than the dual cure mode, which ranged from 38.3 to 42.4% and from 26.6 to 28.4%, respectively. the continuation reaction 24 hours after polymerization initiation resulted in a reduction of absorbance peaks that corresponded to the aliphatic carbon double bonds10 and the dual polymerization increased dc, which corroborates with several studies that compared the polymerization modes for resin-based materials.2,11,21,22 for both resin cements used in this study, the direct light-polymerization provided dc that was higher and ranged from 10 to 30% higher than those observed for the self-curing mode. also, it was higher at 5 minutes (24 to 30%) than for 24 hours (10 to 12%) from the polymerization initiation. the auto-polymerization may be insufficient to result in an adequate dc for the long lasting restorations. clinically, the low viscosity version ensured higher dc, and 24 hours elapsed time after setting of restoration is appropriate for the final appointment, since the higher dc could better support the final oclusal adjustments, finishing, and polishing procedures. the direct light-polymerization can be considered a limitation of this study, since the light is applied directly to the resin cement layer only at the periphery of restorations. on the other hand, the inner part of indirect restorations is reached by an attenuated light and resin cement may depend only on self-cure activation. regarding the differences in the dc between high and low viscosities, higher mean values were found for low viscosity versions of both resin cements. low viscosity materials can be due to lower filler loading and/or higher addition low viscosity diluents monomers. the low viscosity monomer allows better mobility and distribution of free radicals inside the resin material, which can increase the polymerization reaction and the monomer conversion.9 the increase of dc promoted by the low viscosity versions in percentage, following the polymerization mode ranged from 0.9 to 3.1% for the dual-polymerizing groups and ranged from 1.5 to 3.9% for the auto-polymerizing mode, which was the highest percentage among the groups from nexus 2 resin cement. for the variolink ii, dual-polymerizing groups ranged from 1.4 to 2.8%, while the auto-polymerizing mode ranged from 1.7 to 2.4%. the direct light-activation and the low viscosity version were important to provide higher dc for the dual-polymerizing resin cements. twenty-four hours after seating the indirect restoration is appropriate for final oclusal adjustments, finishing, and polishing procedures, which are capable of generating stress to the resin cement layer.

objective: the purpose of this study was to evaluate the effects of the viscosities and curing modes on the degree of conversion (dc) of two resin cements. methods:eight experimental groups were evaluated (n=5), according to the dual-cured resin cements (nexus 2/variolink ii), viscosity (low and high) and evaluation time (5 minutes and 24 hours). the resin cements were applied to surface of a horizontal attenuated-total-reflectance unit and were polymerized either with self-cure (sc) or light exposure (xl3000/3 m espe) for 40 seconds. infrared spectra were obtained after 5 minutes and 24 hours (nicolet 520 ft-ir/thermo scientific inc.). dc was calculated according to changes in aliphatic-to-aromatic peak ratios pre- and post-curing. data (%) were analyzed by 3-way repeated measure anova (curing mode, viscosity and time interval) and tukey s post-hoc test (p<.05). results: the dual-polymerizing mode provided higher dc than auto-polymerization. the dc mean values increased for both resin cements after 24 hours. the low-viscosity resin cements from light-activated or self-cured groups exhibited higher dc than high viscosity version. conclusion:the dc of resin cements was higher for the low viscosity version, following the light-polymerization and when were tested after 24 hours.

PMC3571514

pubmed-72

pleura is divided into a parietal layer which lines the inner aspect of the chest wall and a visceral layer which covers the interlobar fissures. fluid collection within the pleural cavity can be assessed with clinical and radiological means. when pleural effusion is detected, the characteristics of the fluid (exudate or transudate) must be revealed using thoracocentesis. pneumonias and congestive heart failure are the most frequently encountered causes of transudative and exudative effusions, respectively. accumulating knowledge about cytokines demonstrated their important roles in the pathogenesis of most of the infectious diseases. interleukins (ils) mediate the reactions of the our organism against foreign antigens and harmful agents. the ils act through autocrine or paracrine rather than endocrine pathways. in various infectious diseases significant alterations in il concentrations of blood, as well as of body fluids, occur. although il levels in serum and various body fluids have been studied in various diseases, very few researches involving il levels in pleural effusions of children have been conducted [3, 4]. it is already acknowledged that established criteria used for the differentiation between exudative and transudative pleural effusions in adults are not valid for children and further studies are required to determine diagnostic criteria for children. the aim of this study is to compare il levels in exudative and transudative pleural fluids by measuring il levels in pleural effusions developed owing to various etiological factors in childhood. we aimed to examine whether the changes in pleural fluid protein, glucose, and lactate dehydrogenase (ldh) parameters, as well as in il-1, il-2, il-6, and il-8 levels, were significant in differential diagnosis of childhood pleural effusions. the study group consisted of 26 patients admitted in the department of pediatrics, medical faculty, firat university with a diagnosis of pleural effusions. thoracal ultrasonograms were obtained from patients thought to have pleural effusions in consideration of medical history, physical examination, and chest x-rays. the presence and the amount of pleural fluids were determined and then thoracocentesis was performed. all samples were obtained by thoracentesis at the diagnosis time (table 1, figure 1). the first samples were centrifuged immediately for the measurement of il concentration and the supernatant layers were stored at 70c until testing for cytokines. the second samples were inoculated in aerobic, anaerobic, and lwenstein-jensen media and stained with gram, giemsa, and ziehl-nielsen dyes. the third samples were analysed for protein, glucose, and ldh. as indicators of infection, peripheral leukocyte counts (wbc), erythrocyte sedimentation rate (esr), c-reactive protein (crp) measurements, blood culture, and purified protein derivate (ppd) tests were performed. pleural effusions were categorized as exudates and transudates according to the clinical and laboratory findings. the cutoff values for the differentiation between the pleural transudates and exudates were determined as follows: protein, 3 g/dl; ldh, 200 iu/l; pleural fluid/serum protein ratio, 0.5; and pleural fluid/serum ldh ratio, 0.6. pleural effusions with values above these cutoffs were classified as exudates and those below as transudates. empyematous effusions were characterised by the presence of gram-positive bacteria in pleural fluid smears, attendant pneumonia with or without bacterial growth in the cultures of pleural fluid samples, and typical glucose (< 40 mg/dl) and/or protein (> 5 g/dl) and/or ldh (> 1000 the samples of pleural fluids with higher glucose concentrations (> 40 mg/dl) whose gram-stained smears did not reveal any specific characteristics or bacterial growth were considered as parapneumonic effusions. the patients whose pleural fluid smears revealed afb which could be cultured in lwenstein-jensen media and also responded favorably to the antituberculostatic treatment were acknowledged as cases with tuberculous pleurisy [5, 6, 7]. pleural fluids preserved at 70c were thawed and il-1, il-2, il-6, and il-8 levels in the samples were measured using enzyme-linked immunosorbent assay (elisa) and il-1, il-2, il-6, and il-8 bender medsystems commercial kits. the clinical and laboratory characteristics of the patients were given as means (standard deviation (sd)). statistical evaluations between different groups were done with kruskal-wallis variance analysis and post-hoc tukey-scheffe tests using spss 10.0 software programs. in our study group pleural effusions were of exudative (n=27, 75%) and transudative (n=9, 25%) type. the ages of the study subjects ranged between 9 months and 14 years. twelve parapneumonic (44%), 12 empyematous (44%), and 3 (12%) tuberculous pleural effusions were detected in the exudate group (table 1). a statistically significant difference was found between crp values of the exudate (140.2 92.4 mg/dl) and the transudate (9.6 3.2 mg/dl) groups (p<.001) (figure 2). protein levels in pleural fluids were measured as 5.1 1.2 g/dl and 1.6 0.5 g/dl in the exudate and the transudate groups, respectively (p<.001). glucose levels in pleural fluids were measured as 53.3 36.6 mg/dl in the exudate group and 83.8 14.7 mg/dl in the transudate group (p<.001) (figure 3). il-1 levels of pleural fluids in the exudate and the transudate groups were detected as 304.1 127.7 pg/ml and 70.1 23.4 pg/ml, respectively (p<.001). il-1 levels were demonstrated as 210 43.5 pg/ml in the parapneumonic effusion group, 430.3 69.9 pg/ml in the empyematous effusion group, and 175.3 36.3 pg/ml in the tuberculous effusion groups. statistically significantly higher values were found in the empyematous group when compared with the parapneumonic and tuberculous pleural effusion groups (p<.001). il-2 concentrations of pleural fluids were ascertained to be at undetectable levels in 12 (44.4%) and 176.6 173.6 pg/ml in 15 (55.6%) patients in the exudate group. statistically significant difference was uncovered between the exudate and the transudate groups (p<.001). statistically significant differences were not revealed between the empyematous group and the other two groups (p>.05). statistically significantly higher values were recognized in the tuberculous pleural effusion group rather than the parapneumonic effusion group (p<.01). il-6 levels were detected to be 18589.5 3631.8 pg/ml in the exudate group and 65.6 16.0 pg/ml in the transudate group (p<.001). the corresponding values were 15295.0 22678.1 pg/ml in the parapneumonic effusion group, 20518.0 1410.9 pg/ml in the tuberculous pleural effusion group. in the empyematous group, statistically significantly higher values were obtained when compared with those of the parapneumonic effusion group, while the corresponding values detected in the tuberculous pleural effusion group were also significantly higher than those found in the parapneumonic effusion group (p<.001). pg/ml in the exudate group and 108.8 92.0 pg/ml in the transudate group (p<.001). the concentrations of il-8 were 1884.5 366.7 in the parapneumonic effusion group, 4789.8 1013.2 pg/ml in the empyematous effusion group and 919.0 9454.5 pg/ml in the tuberculous pleural effusion group. the values were statistically significantly higher in the empyematous group when compared with the other two groups (p<.001) (figure 4). pleural effusion can be seen due to the intrusion of an infectious agent or an irritating foreign substance inside the pleural cavity or due a direct access of harmful materials or neoplastic cells into the pleural cavity via hematogenous route. it can be observed following pleural trauma or in association with asbestosis related pleural diseases. pleural effusions are known to develop secondary to trauma, cardiac, renal, collagenous diseases and malignancies besides pneumonia [9, 10, 11]. from 61% to 80% of the cases with pleural effusions empyema is also an important cause of mortality in the developing countries. in the remaining 20%39% of the cases, empyema develops secondary to trauma, malignancies, and renal diseases [5, 12, 13, 14, 15]. in our study group many studies have been conducted concerning the levels of il-1, il-2, il-6, and il-8 in body fluids in various infections. although many studies investigating the alterations in levels of il according to the exudative or transudative nature of pleural effusions in adults have been performed, similar studies pertaining to children are relatively few [16, 17, 18]. for that reason many studies have analysed il-1 levels in patients with meningitis, sepsis, urinary tract infections, and empyema. variable results have been obtained dependent on the presence of different etiological agents [17, 18]. in patients with bacterial and tuberculous meningitis, il-1 levels of cerebrospinal fluids were found to be significantly higher than those with aseptic meningitis and in the control group. umblical plasma levels of il-1 of infected newborns with early-onset sepsis were found to be higher than those of the control group. il-1 levels in synovial fluids were detected to be higher than those in the pediatric cases with suppurative arthritis due to other causes. silva-mejias et al have found comparatively higher il-1 levels in the empyema group. similar to our findings, alexandrakis et al revealed that il-1 levels in pleural effusions were higher in the exudate group compared with those of the transudate group. naito et al ascertained that il-1 levels in pleural effusions were higher than those found in bacterial infections due to other causes. in accordance with the literature in our study, il-1 levels were nearly 4.5 times higher in the exudate group. il-2 levels in pleural effusions due to malignancies were reported to be lower than those with tuberculous pleural effusions. shimokata et al stated that il-2 levels were at undetectable levels in 25% of tuberculous pleural effusions and 75% of cancerous pleural effusions. in our study il-2 levels in pleural fluids were at undetectably lower levels in 44% of the patients in the exudate group while the il-2 levels of the remaining 56% were assessed as 176.6 33.4 pg/ml. however all the patients in the transudate group demonstrated undetectable il-2 levels. in our study a statistically significant difference was not present between the empyema group and the other two groups. however a statistically significant difference was detected between the parapneumonic and the parapneumonic pleural effusion groups. various studies have been performed concerning the levels of il-6 which is the mediator and the regulator of inflammatory responses in miscellaneous inflammatory processes. blood il-6 levels were found to be higher in children with mycoplasma pneumoniae infections who stayed febrile for more than 3 days compared with those having shorter febrile episodes. they were also statistically significantly higher in pediatric cases with urinary tract infections when compared with the control groups. il-6 levels of cerebrospinal fluid in the patients with bacterial meningitis were reported to be significantly higher than the aseptic meningitis and in the control groups. levels above 100 pg/ml were measured in pediatric cases with viral (14%) but especially with bacterial meningitis (53%). xirouchaki et al and yokoyama et al reported the levels of il-6 in pleural fluids to be significantly higher in the exudate group rather than the transudate group. they also found il-6 concentrations significantly higher in the tuberculous group rather than the parapneumonic effusion group. in our study not only the levels of il-6 were different between the groups with parapneumonic and empyematous effusions, but at the same time statistically significant differences were found between the empyematous and the parapneumonic or tuberculous pleural effusion groups. alexandrakis et al measured il-6 levels in serum and pleural fluids and ascertained that serum il-6 levels did not differ significantly between the exudate and the transudate groups. however pleural fluid il-6 levels were detected to be meaningfully higher in the exudate rather than the transudate group. a definite proportion between the serum and pleural fluid il-6 concentrations could not be assessed. however we found il-6 levels in the pleural fluid to be 285 times higher than those detected in the exudate group. il-8 is the mediator and the regulator of chemotaxis of leukocytes in inflammatory processes. in adults il-8 levels were found to be higher in cases with infectious pleural effusions compared with the patients with noninfectious effusions [3, 26, 27, 28, 29, 30, 31, 32]. ceyhan et al reported higher levels in empyematous/parapneumonic effusion groups rather than the tuberculous group. however antony et al detected higher levels in cases with parapneumonic effusions unlike those found in the group with tuberculous effusions. il-8 concentrations in the empyematous group were reported to be higher than those measured in the parapneumonic group. in accordance with this study, in our study the highest levels of il-8 were detected in the empyematous, parapneumonic, and tuberculous pleural effusions in decreasing order. dlugovitzky et al discovered statistically significantly higher il-8 values in the tuberculous pleural effusion group in contrast with findings in the parapneumonic pleural effusion group. ashitani et al and broaddus et al found higher levels in the empyema group compared with the other groups. miller and idell have detected significantly higher levels in the exudate group rather than the transudate group. in this study, similar to the findings of other investigations, il-8 levels in the exudate group were reported to be 28 times higher than those found in the transudate group. this study have shown that for the differentiation between the exudative and transudative pleural effusions, in addition to the parametres such as protein, glucose and ldh, pleural fluid il-1, il-2, il-6, and il-8 levels could be used. etiological factors can be differentiated by determining pleural fluid il levels. taking these levels into consideration accordingly, a definitive diagnosis, a successful treatment and reduction in mortality can be achieved .

the aim is to examine whether the changes in pleural fluid interleukin (il)-1, il-2, il-6, and il-8 levels were significant in differential diagnosis of childhood pleural effusions. il-1, il-2, il-6, and il-8 levels in pleural fluids of all 36 patients were measured. the levels of il-1, il-2, il-6, and il-8 in pleural fluids were statistically significantly higher in the transudate group compared with those of the exudate group. the levels of il-1, il-6, and il-8 were also found to be statistically significantly higher in the empyema group compared with both the parapneumonic and the tuberculous pleural effusion groups. the levels of il-2 and il-6 were detected to be statistically significantly higher in the tuberculous pleural effusion group in comparison with those of the parapneumonic effusion group. the results showed that pleural fluids il-1, il-2, il-6, and il-8 could be used in pleural fluids exudate and transudate distinction.

PMC1513055

pubmed-73

literature analysis showed few articles about this complication, but no publication has described the management of open patella fracture around total knee arthroplasty. we report a unique case of an open patellar fracture above a total knee arthroplasty, sustained by a 56-year-old female patient. despite the poor outcome of operative management in patellar periprosthetic fracture, this approach should be considered for acute and post traumatic fractures in young patients with a good remaining bone stock. the demographic changes with a high rate of aging and active population, as well as the improved life expectancy during the last decades have led to an increase in the number of total knee arthroplasties (tka) [1, 2, 3, 4], and consequently a raise of post-operative complications. among these complications, periprosthetic fracture after tka is the most frequent cause of post-operative pain, and revision surgery. they may occur in the supracondylar region of the femur, followed by the patella, then the proximal tibia [1, 2, 3, 4, 5, 6, 7]. this entity can be very challenging to manage, with unpredictable and poor outcome, especially for displaced fracture or in cases with implant loosening [1, 2, 3, 4, 5, 8]. in this article, we report a unique case of an open periprosthetic patellar fracture that was managed with operative treatment. it also involves a literature analysis with description of the risk factors and treatment options of this uncommon entity. a 56-year-old female underwent a cemented postero-stabilized condylar tka with fixed tibial plateau through an antero-medial approach, in our department. she had severe osteo-arthritis of the left knee with a major stiffness (irreducible flexum at 25). initial (1) and post operative(2) knee radiographs before, and after the arthroplasty. three weeks later, the patient tripped and fell off with all her standing height striking the anterior aspect of the left knee with hyper flexion. she was taken to the emergency room with left knee pain and a large skin wound. physical examination showed a large, deep and longitudinal wound measuring 15 cm in length due to a total breakdown of the suture line (the antero-medial approach performed during the tka) (fig. 3, 4). fig 3. a clinical picture showing the open patellar fracture a clinical picture showing the open patellar fracture. lateral knee radiograph showing the patellar fracture above a total knee arthroplasty, without any signs of implants loosening. lateral knee radiograph showing the patellar fracture above a total knee arthroplasty, without any signs ofimplants loosening. the knee radiographs showed a short oblique fracture at the inferior pole of the patella without any loosening of the implants or dislocation of the knee: this fracture is classified as type i according to ortigueraet d.j. she urgently received intravenous antibiotic (augmentin, gentamicin), as well as tetanus vaccination. her fracture was temporarily stabilized in a long-leg plaster splint before she was transported to the operating room. she underwent wound irrigation, debridement of non-viable soft tissue from the wound, open reduction and fixation with a figure-eight cerclage wiring, without any revision of the implants. the wound was closed without any skin tension, after placing an intra-articular drain. complementary immobilization with a removable knee pad at full extension, for six weeks was prescribed. post operative knee radiographs post operative knee radiographs she received intra venous antibiotics: augmentin for 2 weeks associated with gentamicin during the first five days, as well as subcutaneous heparin. she was discharged from the hospital after she was instructed to remain non-weight bearing with a follow-up in 2 weeks. at 6 months follow-up, physical examination did not reveal any signs of local or deep infection. her knee was pain free and she was able to walk without crutches, but she had a mild stiffness: the knee range of motion was 0/60. no antero-posterior or frontal laxities were noted. clinical pictures at last follow-up showing a clean and dry wound (7), and knee range of motion (8, 9) clinical pictures at last follow-up showing a clean and dry wound (7), and knee range of motion (8, 9). the patient achieved union of the fracture: knee radiographs showed a healed patellar fracture, with hardware in place. since the late 1960s, total knee arthroplasty has been the most effective treatment for knee osteoarthritis, providing good functional outcomes and improvement in the patient s quality of life. constant advances in the joint replacement surgery, and especially the total knee arthroplasties have led to an increase in the number of complications following this type of surgery, such as periprosthetic fractures. [1, 2, 3, 4] this entity is very challenging to treat even for the most skilled and experienced surgeons [7, 8, 10]. the femur is the most frequent fracture site, followed by the patella and the tibia [1, 2, 3, 4, 5, 6, 7]. patella fracture after tka can occur after trauma, or result from a stress fracture due to patella osteonecrosis, which is the most common cause of this type of fracture [1, 2, 3, 4, 5, 8, 10, 11]. chalidis et al (2007) have reported in a systematic review of a 752 periprosthetic patella fracture that in 88.3% of the cases, the fracture was not associated with a traumatic event, asymptomatic, and discovered during routine follow-up. many risk factors of these stress fractures have been described in the previous literature [1, 2, 3, 4, 5, 7, 8, 9, 10, 11] including: poor blood supply for patella after tka especially for antero-medial approach, and in case of lateral release or unwarranted fat pad removal: knee replacement surgery is likely to cause disruption of the vascular peri- patellar network especially the medial and the superior lateral genicular arteries.mal-alignment of the femoral and tibial implantspatella resurfacing arthroplastiesan excessive (thickness<15 mm) or asymmetric bone resection during patella resurfacingosteonecrosis due to bone cement polymerization: the heat of polymerization of the polymethylmethacrylate may exceed the coagulation temperature, causing thermal necrosis of the bonethe use of large central fixation peg designs for patellar implant, instead of small peripheral peg design.revision surgeryosteoporosis and bone losshyperflexion of the kneerhumatod arthritis poor blood supply for patella after tka especially for antero-medial approach, and in case of lateral release or unwarranted fat pad removal: knee replacement surgery is likely to cause disruption of the vascular peri- patellar network especially the medial and the superior lateral genicular arteries. mal-alignment of the femoral and tibial implants patella resurfacing arthroplasties an excessive (thickness<15 mm) or asymmetric bone resection during patella resurfacing osteonecrosis due to bone cement polymerization: the heat of polymerization of the polymethylmethacrylate may exceed the coagulation temperature, causing thermal necrosis of the bone the use of large central fixation peg designs for patellar implant, instead of small peripheral peg design. osteoporosis and bone loss hyperflexion of the knee open patellar fracture above a total knee arthroplasty is a very uncommon injury, and challenging to treat. to our knowledge, reported a unique case of patellar fracture associated with open knee dislocation after a total knee arthroplasty. it was treated with open reduction and internal fixation of the patella with band tension wiring. greco et al, reported an open tibial shaft fracture below an ipsilateral tka, managed with intramedullary nailing. some factors should be considered to succeed in the management of this type of fracture such as displacement of the fracture, function of the extensor mechanism, stability of the patellar implant, remaining bone stock [1, 2, 3, 4, 5, 8, 9, 10, 11, 13]. it includes immobilization by a brace locked in full extension or a cylinder cast in case of non-operative treatment; open reduction and internal fixation, implant revision in case of loosening, partial or total patellectomy, repair of the quadriceps tendon or patellar ligament in case of surgical treatment [4, 5, 7, 8]. the periprosthetic patellar fractures are usually managed by a non-operative treatment especially for type i and ii, because of the high infectious risk of open reduction and internal fixation [13, 14], and consequently a high risk of arthroplasty failure. literature analysis showed that conservative or non-operative treatment was more successful for the management of non-displaced patella fracture with stable implant and an intact extension mechanism. ortiguera and berry reported good results in 37 out of 38 peri-prosthetic patellar fractures managed with conservative treatment. dennis reported a complication rate of 39% of operative treatment (in a series of thirty one patients who sustained a peri- prosthetic patellar fracture). chalidis s systematic review has shown that open reduction and internal fixation failed in 92% of the cases. in this particular case, we were forced to opt for an internal fixation with a cerclage wiring after intensive wound irrigation, debridement and soft tissue repair. although no conclusion can be established from a single case report, we think that the successful result in our case can be explained by the relative young age of the patient (56 years old), the good bone stock, the acute and post-traumatic characteristics of the fracture, the early and adequate management necessary to avoid infectious complication. the surgical management may reduce the ability of patella to heal because of the evacuation of peri-fractural hematoma as well as the supplementary disruption of the vascular network around the patella after a knee replacement surgery. nevertheless, the high failure rate of the operative treatment in the patellar peri-prosthetic fracture can be explained by: the high proportion of stress, atraumatic fracture (88%) due to osteonecrosis which suggests a poor remaining bone stock.the stress fracture, unlike the acute and post traumatic ones, has a higher risk of non-union.the poor outcome of partial or complete patellectomy, which is a type of operative treatment.in our point of view, the post traumatic fractures in young patients are more likely to heal than stress fractures in the elderly. the high proportion of stress, atraumatic fracture (88%) due to osteonecrosis which suggests a poor remaining bone stock. the stress fracture, unlike the acute and post traumatic ones, has a higher risk of non-union. the poor outcome of partial or complete patellectomy, which is a type of operative treatment. in our point of view, the post traumatic fractures in young patients are more likely to heal than stress fractures in the elderly. peri-prosthetic patella fracture is a very uncommon entity, and challenging to treat, especially the open ones, because of the high risk of infectious complications. although several previous studies report a high rate of failure and unpredictable results of open reduction for periprosthetic patellar fracture, this approach (especially the internal fixation with a strong and dynamic construct) should be considered in young patients with a good remaining bone stock, who sustained an open or a displaced fracture after a high energy trauma. orthopaedic surgeons should consider open reduction and internal fixation as a good management alternative for periprosthetic patellar fracture, in case of a post traumatic and acute fracture in young patients with a good bone stock.

introduction: periprosthetic patellar fracture after total knee arthroplasty (tka) is a rare complication. their management can be very challenging with unpredictable results. literature analysis showed few articles about this complication, but no publication has described the management of open patella fracture around total knee arthroplasty. case presentation: we report a unique case of an open patellar fracture above a total knee arthroplasty, sustained by a 56-year-old female patient. conclusion:despite the poor outcome of operative management in patellar periprosthetic fracture, this approach should be considered for acute and post traumatic fractures in young patients with a good remaining bone stock.

PMC5040584

pubmed-74

hypertension is a common chronic medical condition affecting people in pakistan and the rest of the world. it is an important modifiable risk factor for cardiovascular morbidity and mortality, particularly for stroke (accounting for 51% of all stroke deaths worldwide), ischemic heart disease (45% of all deaths), chronic kidney disease, congestive heart failure, aortic aneurysm, and peripheral arterial disease. prevalence of hypertension (systolic blood pressure>140 mm hg or diastolic blood pressure>90 mm hg, or on antihypertensive medications) in pakistan has increased from 17% in 1980 to 35% in 2008 in adults aged 18 years and older. the increasing prevalence of hypertension together with a deficient control makes this one of the frequent conditions that require urgent medical attention. the prevalence of uncontrolled hypertension varies around the world, with the lowest prevalence in rural india (3.4% in men and 6.8% in women) and the highest prevalence in poland (68.9% in men and 72.5% in women). however, the control of hypertension was 23% from a community based data in urban population from karachi, pakistan. uncontrolled hypertension can progress to hypertensive crisis defined as a systolic blood pressure>180 mm hg or a diastolic blood pressure>120 mm hg. hypertensive crisis can be further classified as a hypertensive urgency or hypertensive emergency depending on end-organ involvement including cardiac, renal, and neurologic injury. hypertensive urgency refers to severe hypertension without evidence of new or worsening end-organ injury while hypertensive emergency refers to a severe hypertension that is associated with new or progressive end-organ damage. hypertensive crises (76% urgencies, 24% emergencies) represented more than one-fourth of all medical urgencies/emergencies. hypertensive urgencies frequently present with headache (22%), epistaxis (17%), and psychomotor agitation (10%) and hypertensive emergencies frequently present with chest pain (27%), dyspnea (22%), and neurological deficit (21%). the reason for uncontrolled hypertension in pakistan is high due to lack of awareness, knowledge, adherence, and attitudes of pakistani patients with hypertension. however there is no data on patients with hypertensive crisis from tertiary care hospitals in pakistan. additionally the number of patients who complicate towards stroke, myocardial infarction, and chronic kidney disease is also not known. hence, it is essential to have figures on prevalence and clinical presentation from our setup. therefore, we conducted this study to determine the prevalence of hypertensive crisis, its management, and its outcome in patients presenting to a tertiary care center in karachi. this was a retrospective study conducted at the aga khan university, karachi, pakistan. the aga khan university hospital (akuh) has 563 beds in operation and provides services to over 50,000 hospitalized patients and to over 600,000 outpatients annually. ethical approval from the ethics review committee of the aga khan university (1985-med-erc-11) was taken for conduct of the study. adult inpatients (> 18 yrs) presenting to the er who were known hypertensive and had uncontrolled hypertension were included. controlled blood pressure was defined as systolic blood pressure (sbp)<140 mm hg or diastolic blood pressure (dbp)<90 mm hg. uncontrolled hypertension was defined as sbp>140 mm hg and dbp>90 mm hg in both diabetic and nondiabetic patients who were either aware of their problem or under pharmacological treatment. the sample was drawn using computerized medical record system international classification of diseases-9-coordination and maintenance (icd-9-cm) at health information management system in the hospital. patients admitted with primary diagnosis of hypertension crisis, uncontrolled hypertension, hypertensive emergency, and hypertensive urgency were selected through the icd-9-cm (i-10: essential (primary) hypertension, i-11: hypertensive heart disease, i-12: hypertensive renal disease, i-13: hypertensive heart and renal disease, and i-15: secondary hypertension). patients whose medical records did not contain minimal clinical information to allow case classification (hypertensive urgency or emergency) were excluded from the study. data over a period of 5 years, from year 2005 till year 2010, was used. a sample of 1336 consecutive patients fulfilling the inclusion criterion was selected. all patients gave a general consent on admission; however informed consent was not taken as data was extracted later through icd-9-cm. data on demographics, comorbid conditions, clinical symptoms, blood pressure readings at subsequent time intervals, length of stay, and antihypertensive drug therapy was recorded by trained data collectors. a history of physician-diagnosed diabetes mellitus (dm), chronic kidney disease (ckd), ischemic heart disease (ihd), and stroke was noted from the patient's medical record file. dm was defined as fasting plasma glucose 126 mg/dl at a prior visit. ckd was defined as rise in serum creatinine of>1.2 mg/dl for 3 months. blood pressure readings, at different time intervals, were recorded from vital sheets for nursing services. hypertensive crisis was defined as a systolic blood pressure>180 mm hg or a diastolic blood pressure>120 mm hg. management of patient was assessed by recording the list of medication from the computer generated pharmacy sheet attached inside the medical record file. antihypertensive treatment was divided into two types: medication given per oral and medications given intravenously. i values where available were recorded using the patient profile viewer, an online hospital database software. mean length of stay was recorded by calculating the time interval between the patient's admission date/time and discharge date/time from er/ward. various complications like myocardial infarction, stroke, aortic dissection, acute renal failure, and pulmonary edema developed during the hospital stay were recorded using the discharge summary notes filled by the primary consultant. myocardial infarction was diagnosed when blood levels of sensitive and specific biomarkers such as cardiac troponin or ckmb are increased in the clinical setting of acute myocardial ischemia with electrocardiographic changes. according to who definition, stroke was defined as a rapidly developing clinical sign of focal (at times global) disturbance of cerebral function, lasting more than 24 hours or leading to death with no apparent cause other than that of vascular origin. aortic dissection was referred as the condition when a separation has occurred in aortic wall intima as diagnosed on ct scan, causing blood flow into a new false channel composed of the inner and outer layers of the media. acute renal failure was diagnosed when the plasma urea nitrogen (pun) or serum creatinine did not stabilize within 72 hours. acute pulmonary edema was defined as alveolar or interstitial edema verified by chest x-ray and/or with o2 saturation<90% on room air prior to treatment accompanied by severe respiratory distress, with crackles over the lungs and orthopnea. a minimum sample size of 237 patients was required to estimate a proportion of 19% of patients with hypertensive crises presenting to er, with bound on error of 5% and alpha of 5%. mean and standard deviation were used for qualitative variables and frequency and percentage for qualitative variables. chi-square test was used to compare categorical variables and student's t-test was used to compare quantitative variables. a total of 73,063 hypertensive patients presented to the er between years 2005 and 2010 out of which a sample of 1336 (1.8%) patients was taken for this study. the prevalence (%) of uncontrolled hypertension was 28.9% (387). prevalence of hypertensive crisis overall was 16.3% (218) and among those with uncontrolled hypertension is 56.3% (218). mean age (sd) of patients presenting to the er was 56.7 (14.7) and 175% (45.2) of patients were male. overall, dyslipidemia was the most common comorbidity in patients presenting with uncontrolled hypertension to the er with the prevalence of 43.2% (167) followed by diabetes mellitus, 36.9% (143), and ischemic heart disease, 21.4% (83), and 13.9% (54) of them were smokers. the baseline characteristics of patients overall, with hypertensive crisis and without hypertensive crisis, are given in table 1. headache was the most common presenting symptom, 35.7% (138), followed by dyspnea, 32.6% (126), chest pain, 21.4% (83), dizziness, 21.2% (82), vomiting, 17.3% (67), epistaxis, 5.2% (20), and neurologic deficit, 3.6% (14). on comparison of patients with hypertensive crisis with those with no hypertensive crisis the clinical symptoms that were statistically significant were as follows: headache was present in 42.2% (92) versus 27.2% (46) p value=0.002 and chest pain was present in 17.4% (38) versus 26.6% (45) p value=0.02. the mean (sd) systolic blood pressure (sbp) recorded in patients with hypertensive crisis versus no hypertensive crisis in er was 202 (17.971) and 158 (13.387) (p value 0.001). the mean (sd) diastolic blood pressure in patients with hypertensive crisis versus no hypertensive crisis in er was 108 (17.429) mm hg and 87 (14.984) mm hg, respectively, (p value 0.001). the trend of blood pressure recorded in the er is shown in figures 1 and 2. antihypertensive medications used in management of patients with hypertensive crisis. calcium channel blocker was the most widely used oral antihypertensive medication in the er, 35.4% (137). intravenous (iv) nitrate was the most commonly administered iv medication in er, 22.7% (88). the mean (sd) drop in sbp in patients with hypertensive crisis who received intravenous medications versus oral medications was 53.1 (29) mm hg and 43 (27), respectively (p value=0.01). the mean drop in dbp in patients with hypertensive crisis who received intravenous versus perioral medications was 25.8 (19) and mm hg and 17.8 (22) mm hg, respectively (p value=0.006). comparison of blood pressure trends in patients with and without hypertensive crisis on intravenous or per oral medication is shown in table 2. the maximum drop in systolic blood pressure and diastolic blood pressure was achieved by sodium nitroprusside: 80 (15) mm hg and 37.5 (7.77) mm hg. types of intravenous medications and drop in sbp and dbp are shown in table 3. the total length of stay (in days) for patients with hypertensive crisis was 2.46 (0.164) whereas the total length of stay (in days) for patients without hypertensive crisis was 2.20 (0.158). overall prevalence (%) of complications was 47.7% (104) in patients with hypertensive crisis. acute renal failure was the most common complication with the prevalence (%) of 41.3% (43) followed by myocardial infarction 28.8% (30) and pulmonary edema 18.26% (19). stroke accounted for 6.5% (12) of complications. on comparison of patients with hypertensive crisis and patients without it, myocardial infarction occurred in 7.2% (15) versus 9% (15), stroke occurred in 2.4% (5) versus 4.2% (7), and acute kidney injury occurred in 11.5% (24) versus 11.4% (19) patients, respectively. on further categorization of patients with hypertensive crisis who received intravenous antihypertensive in er versus not receiving intravenous medication, there was no significant difference in the complications in both groups. we have shown in this study on patients presenting to er with hypertensive crisis that the prevalence of hypertensive crisis is high (56.3%). mean length of stay was longer in patients with hypertensive crisis and acute renal failure was the most common complication in these patients. the incidence of hypertensive crisis was 47.22% in a study conducted in bosnia and 16% in a study conducted in brazil [22, 23]. the reasons for high prevalence of patients with hypertensive crisis in our setup are multiple. lack of knowledge about control of hypertension and poor compliance to antihypertensive medications is a major issue in pakistan. also, lack of proper health infrastructure in public sector leading to inability of the poor population to access healthcare adds onto the severity of hypertension in these patients. in previous studies [25, 26], the most frequent clinical sign of patients presenting to the er was headache (22% and 42%) and dizziness was reported among 30% of emergency department patients. this study showed (36%) headache as the most common clinical sign followed by dizziness (21%) which supports the previous findings about the characteristics of hypertensive crisis patients. complications of hypertensive crisis reported in previous studies conducted in bahrain and italy were acute coronary syndrome (32%), left ventricular heart failure (38%), and stroke (29.3%) and cerebral infarction (24%), pulmonary edema (23%), and hypertensive encephalopathy (16%). however acute renal failure, myocardial infarction, and pulmonary edema are the most common complications in our setup. the reason for high prevalence of acute renal failure in our setup may be the higher number of patients with hypertensive emergency. the most common intravenous medication used to control hypertensive crisis in this study was nitrates. recommendation for use of intravenous nicardipine was reported by a study conducted in 2005 whereas sodium nitroprusside, because of its direct vasodilator effect and immediate onset of action, was recommended for hypertensive emergency in a subsequent study in 2006. we observed in this study that sodium nitroprusside was the most potent intravenous antihypertensive in dropping blood pressure. however it was not the most commonly used drug in this study. in a similar study, intravenous labetalol was reported as the most frequently used antihypertensive medication for emergency department patients presenting with hypertensive urgency. this indicates that the use of intravenous nitrate as the most common antihypertensive for management of hypertensive crisis (apart from use in patients with cardiac cause) is not completely in accordance with the recommendations. however in some studies, oral labetalol, beta blockers, diuretics, ace inhibitors, and calcium channel blockers have been recommended. calcium channel blockers have also been found to be the most common antihypertensives used for management of hypertensive in this population and have been found to have good control rates. calcium channel is now recommended as a first line antihypertensive in the recent nice guidelines. the reduction in systolic and diastolic blood pressure reported in our study shows a smooth decline in order to avoid risks of potential side effects of a much rapid decline. it is recommended that the initial reduction in mean arterial pressure (map) in case of hypertensive emergency should not be more than 20%25% below the pretreatment blood pressure or that map be reduced within the first 3060 minutes to 110115 mm hg. this can be comparable to our study which reported an overall decrease of blood pressure of about 52 mm hg in sbp from intravenous medication from the time of admission until 2 hours after admission in er. a rapid decline in blood pressure is associated with acute deterioration in renal function, ischemic, cardiac, or cerebral events, and occasional retinal artery occlusion and acute blindness. the goal is to reduce bp by 1015% over a period of 3060 minutes, with the exception of the patient that presents with aortic dissection or acute intracranial bleed. however treatment should be individualized to each patient based on the type and extent of end-organ damage, degree of bp elevation, and the specific side effects that each medication could have on a patient's preexisting comorbidities. the strength of this study is that it is the first study from this region to report figures on prevalence of hypertensive crisis, as well as prevalence of complications as a result of hypertensive crisis among patients. moreover, this study also reports management of these patients in terms of the treatment received in the er and into the ward and their mean length of stay. however there are limitations in this study; firstly it has limited external validity as the sample is not representative for an entire population. it represents population visiting a single-tertiary care hospital and, hence, it is not representative of the entire population of pakistan. secondly, compliance issues with the medication regimen have not been taken into consideration; hence, the medications administered to the patients may be misjudged. thirdly, some patients may also have gotten discharged against medical advice; hence, the optimal length of stay for these patients may not have been achieved. fourth, this retrospective study can not strongly give a cause and effect association and we have not reported the hypertensive urgencies and emergencies separately. we did not report data separately on hypertensive emergency and urgency and retinopathy could not be examined and reported in all patients. the prevalence of hypertensive crisis is high in our subjects. per oral calcium channel blocker and intravenous nitrate are the most commonly administered medications in the er and ward.

objectives. hypertension, if uncontrolled, can lead to hypertensive crisis. we aim to determine the prevalence of hypertensive crisis, its management, and outcome in patients presenting to a tertiary care center in karachi. methods. this was a cross-sectional study conducted at the aga khan university, karachi, pakistan. adult inpatients (> 18 yrs) presenting to the er who were known hypertensive and had uncontrolled hypertension were included. results. out of 1336 patients, 28.6% (387) had uncontrolled hypertension. the prevalence of hypertensive crisis among uncontrolled hypertensive was 56.3% (218). per oral calcium channel blocker; 35.4% (137) and intravenous nitrate; 22.7% (88) were the most commonly administered medication in the er. the mean (sd) drop in sbp in patients with hypertensive crisis on intravenous treatment was 53.1 (29) mm hg and on per oral treatment was 43 (27) mm hg. the maximum mean (sd) drop in blood pressure was seen by intravenous sodium nitroprusside; 80 (51) mm hg in sbp. acute renal failure was the most common complication with a prevalence of 11.5% (24). conclusion. the prevalence of hypertensive crisis is high. per oral calcium channel blocker and intravenous nitrate are the most commonly administered medications in our setup.

PMC4590913

pubmed-75

a basic set of proteins and mrnas are differentially expressed among cell types, temporally and spatially, generating a vast assortment of cell phenotypes and/or activation states within a single tissue. outlining this protein/mrna portrait is thus crucial for understanding not only the uniqueness characterizing cells, but especially their distinguished functions. this becomes of major relevance when the balance between cell-intrinsic properties and identity cues received and provided by each cell to its neighboring cells then shapes the cell-to-cell cross talk during physiopathological conditions. in the cns, neuroinflammation is the typical consequence of the interchange among different cell types, particularly neurons, astrocytes, oligodendrocytes and microglia, of a variety of cues as neurotransmitters, cytokines, chemokines, toxic metabolites that condition the final protein/mrna profiles of cells, their activation states and functional outcomes [2, 3]. since neuroinflammation accompanies a large variety of neurodegenerative diseases, there is increasing interest in determining how the different cell phenotypes and cellular interconnectivity might contribute to reduce inflammation and reverse neurodegeneration. microglia actively participate to the context-dependent, neuroprotective/neurotoxic molecular network that is triggered during neuroinflammation. among the molecular cues having a key role in this process, extracellular nucleotides are major responsible for intercellular communication and propagation of inflammatory stimuli [57]. this occurs by specific binding to various receptor subtypes, termed ionotropic p2x and metabotropic p2y, which are simultaneously localized on several different cell phenotypes. among these, the p2y12 receptor subtype belonging to the gi class of g protein-coupled receptors is activated by adp. two transcript variants apparently encoding the same protein isoform have been identified so far for p2ry12 gene, but the determinants for cell specificity of p2y12 protein expression are still unknown. p2y12 is found on the surface mainly, but not exclusively, of blood platelets, where it acts as blood clotting regulator and target for the treatment of thromboembolisms [9, 10]. in the nervous system, the tissue- and cellular-selective expression of p2y12 exhibits a pattern throughout white and gray matter consistent with astrocyte expression, although it has not been found colocalization between p2y12 and gfap-positive astrocytes in rat brain cortex and nucleus accumbens, despite the abundant presence of p2y12 mrna. moreover, we previously established in vivo the expression of p2y12 in oligodendrocytes and myelin sheaths of rat cerebral cortex, subcortical areas, and periventricular white matter. this localization is confirmed throughout the corticospinal tract, therefore suggesting high conserved tissue-homogeneity and phenotype-specificity, and a hypothesized role in myelination [1215]. p2y12 is finally observed in brain and spinal cord resident microglia, where it affects activation, chemotaxis [1619] and neuropathic pain, but it is not observed, for example, in peripheral macrophages in spleen [18, 20]. p2y12 expression in primary microglia is variable with postnatal development and shows sexually dimorphic behavior. through the use of all the available p2y12-immunoreactive antibodies recognizing the c-terminus or the second intracellular loop of the receptor, the aim of the present work is to provide comparative evidence about p2y12 cell specificity in microglia versus oligodendrocytes particularly from the healthy and diseased cns under neuroinflammatory conditions as those sustained during amyotrophic lateral sclerosis (als) and multiple sclerosis (ms). adult b6.cg-tg (sod1-g93a)1gur/j mice expressing high copy number of mutant human sod1 with a gly93ala substitution (sod1-g93a) were originally obtained from jackson laboratories (bar harbor, me, usa) and housed in our indoor animal facility as described in apolloni and collaborators ,. the animals were euthanized, according to the guidelines for preclinical testing and colony management. also neonatal wistar and adult lewis rats (from charles river laboratories, lc, italy) were housed in our indoor animal facility. animal procedures were performed according to european guidelines for the use of animals in research (86/609/cee) and the requirements of italian laws (d.l. ethical procedures were approved by animal welfare office, department of public health and veterinary, nutrition and food safety, general management of animal care and veterinary drugs of the italian ministry of health. all efforts were made to minimize animal suffering and number of animals necessary to produce reliable results. microglia primary cultures prepared from mouse cortex as previously described were about 98% pure, as verified by immunofluorescence with cd11b (for microglia), glial fibrillary acidic protein (gfap, for astrocytes), neuronal nuclei (neun, for neurons), and smi94 (for oligodendrocytes). purified cultures of oligodendrocytes were prepared from forebrain of postnatal day 1-2 wistar rats, according to minor modifications from a previously described method. after removing the meninges, cortices were minced, digested, and dissociated by passage through 70 m nylon cell strainer (bd biosciences, europe). cells were plated in dulbecco's modified eagle's medium (dmem) supplemented with 20% heat-inactivated fetal bovine serum (fbs), 4 mm glutamine, 1 mm sodium pyruvate, 50 u/ml penicillin, 50 g/ml streptomycin, and 100 g/ml gentamicin in t75 poly-d-lysine-coated flasks, at about 10 million cells/flask. the cultures were maintained at 37c in a 5% co2 and 95% air atmosphere for 14 days. mixed glial cultures were then shaken at 200 rpm at 37c for 1 h and 98% pure microglia collected from the supernatant of each flask, as verified by immunofluorescence with gfap, neun, neural/glial antigen 2 (ng2, for oligodendroglial precursor cells), myelin basic protein (mbp, for mature oligodendrocytes), and cd11b. after further shaking at 200 rpm and 37c for 1518 h, the detached cell suspension was finally incubated for 1 h at 37c for differential adhesion of contaminating cells. the non-adherent cells were filtered through 40 m nylon cell strainer (bd biosciences), spun for 10 min at 100 g, resuspended in oligodendroglial precursor cells medium (basal chemically defined medium: dmem, 4 mm l-glutamine, 1 mm sodium pyruvate, 0.1% bovine serum albumin (bsa), 50 g ml apo-transferrin, 5 g ml insulin, 30 nm sodium selenite, 10 nm d-biotin, and 10 nm hydrocortisone) containing 10 ng ml platelet derived growth factor-aa (pdgf-aa) and 10 ng ml basic fibroblast growth factor (bfgf), and seeded at the density of 1 10 cells/cm into poly d, l-ornithine-coated plates. the cells were then induced to differentiate into mature oligodendrocytes when the basal chemically defined medium was added with 15 nm triiodothyronine, 10 ng ml ciliary neurotrophic factor (cntf) and 0,05 mg 10 ml n-acetyl-l-cysteine (nac), for 47 days. a 98% pure population of oligodendrocytes was thus obtained, as verified by immunofluorescence with ng2, mbp, gfap, neun, and cd11b antibodies. organotypic cerebellar slice cultures were prepared with modifications from previously published protocols [26, 27]. briefly, cerebella were excised from neonatal wistar rat (810 days old), cut on a mc ilwain tissue chopper (400 m) and parasagittal slices separated into cold hank's balanced salt solution (hbss). two to three slices were plated on each millicell cm culture inserts (millipore, italy) and kept in organotypic maintenance medium (50% basal medium eagle-bme, 25% hbss, 25% heat-inactivated horse serum, supplemented with 5 mg/ml glucose, 1 mm glutamine, 50 u/ml penicillin, and 50 g/ml streptomycin) at 37c in a 5% co2 and 95% air atmosphere. the medium was changed every 2 days and double immunofluorescence was performed in free floating after 710 days in vitro. organotypic cultures were washed three times with pbs, fixed with 4% paraformaldehyde for 1 h, rinsed, and blocked for 1 h in phosphate buffered saline (pbs)/10% normal donkey serum (nds)/0.4% triton x-100. primary antibodies were incubated for 24 h in pbs/2% nds/0.4% triton x-100 (table 1). the secondary antibodies used for double labelling are cy3-conjugated donkey anti-rabbit igg (1: 100, jackson immunoresearch, europe) or alexa fluor 488-affinipure donkey anti-mouse igg (1: 200, jackson immunoresearch). after rinsing, sections were mounted, covered with fluoromount medium (sigma-aldrich, milan, italy) and a coverslip, and analyzed by confocal microscopy. microglia and oligodendrocytes were washed three times with pbs, fixed with 4% paraformaldehyde for 10 min (oligodendrocytes) or 20 min (microglia), washed, permeabilized with 0.050.1% triton x-100 for 10 min, rinsed, and blocked for 30 min in pbs/1% bsa. microglia were stained for about 3 h at 37c in 1% pbs/bsa with 5 g/ml cy2-phalloidin (sigma-aldrich), in combination with primary antibodies against p2y12 receptor, as reported in table 1. the secondary antibodies used for double labelling are cy3-conjugated donkey anti-rabbit igg (1: 100, jackson immunoresearch) or alexa fluor 488-affinipure donkey anti-mouse igg (1: 200, jackson immunoresearch). cells were extensively washed and stained with the nucleic acid blue dye, hoechst 33342 (1: 1000). after rinsing, cells were covered with fluoromount medium (sigma-aldrich) and a coverslip and analyzed by confocal microscopy. animals were anaesthetized by intraperitoneal injection of chloral hydrate (500 mg/kg) and transcardially perfused with pbs followed by 4% paraformaldehyde at ph 7.4. tissue samples (mice spinal cord and rat brain) were then postfixed for 1-2 days, and cryoprotected in 30% sucrose in pbs at 4c. mice spinal cords (l3l5) were cut at 30 m thickness with a frozen microtome. sections were mounted on slide glasses and allowed to air-dry (1-2 h). a rectangle was then drawn around the sections with a pap pen (sigma-aldrich). rat brains were cut at 40 m thickness using a cryostat microtome and sections were processed in free-floating. double immunofluorescence analysis was performed after blocking in pbs containing 10% nds and 0.3% triton x-100 for 1 h at room temperature. sections were incubated with different combinations of primary antibodies (table 1), in pbs, 0.3% triton x-100 and 2% nds, for 2448 h at 4c. finally, sections were washed with pbs and incubated with appropriate fluorescent-conjugated secondary antibodies for 3 h at room temperature. after rinsing, sections were covered with fluoromount medium (sigma-aldrich) and a coverslip and analyzed by confocal microscopy. tissues supplied by uk multiple sclerosis tissue bank at imperial college, london, were collected postmortem with fully informed consent from both donors and close relatives. procedures for retrieval, processing, and storage have gained ethical approval from all appropriate committees. the brain tissues analyzed were from 7 neuropathologically confirmed cases of secondary progressive ms (spms). analysis was performed also on samples from patients who died due to nonneurological diseases (healthy). cerebral hemispheres were fixed with 4% paraformaldehyde for about two weeks, coronally sliced, and blocked. individual blocks were cryoprotected in 30% sucrose for one week and frozen by immersion in isopentane precooled on a bed of dry ice. cryostat sections (3040 m thick) were either stained with luxol fast blue and cresyl fast violet (kluver-barrera staining), in order to detect white matter lesions and their cellularity, or subjected to immunohistochemistry for mbp, in order to distinguish grey matter lesions. sections were processed in free-floating for double immunofluorescence studies, as described in amadio and collaborators. immunofluorescence was analyzed by means of a confocal laser scanning microscope (zeiss, lsm700, germany) equipped with four laser lines: 405, 488, 561, and 639 nm. the brightness and contrast of the digital images were adjusted using the zen software (zeiss). human p2y12 complete cdna was obtained by reverse transcription with enhanced avian rt-pcr kit (sigma-aldrich) from total human brain rna (life technologies, paisley, uk). the obtained cdna was then cloned into the xhoi and xbai sites of the eukaryotic expression vector cs2+mt to provide n-terminal 6x c-myc epitope-tagged mammalian expression plasmids, which has been validated by dna sequencing. oligos used for amplification were as follows: forward 5gcctcgagatgcaagccgtcgacaacctc3 and reverse 5gctctagagtttacattggagtctcttc designed on human p2y12 mrna (nm_022788.3). human embryonic kidney 293 cells (hek 293) and sloan-kettering neuroblastoma sh-sy5y clone (sh-sy5y) cells were grown in dmem supplemented with 10% fbs, 100 unit/ml penicillin, and 100 g/ml streptomycin at 37c in atmosphere containing 5% co2. one day before transfection, hek293 or sh-sy5y cells were plated and transfection of p2y12-cs2+mt or cs2+mt empty vector was performed with lipofectamine 2000 (life technologies), according to manufacturer instructions. primary rat microglial and oligodendrocyte cells were lysed with trizol (life technologies) and total rna was extracted following the manufacturer's instructions. after dnase treatment (qiagen, hilden, germany), equal amount of total rna (1 g) was subjected to retrotranscription by high capacity rna-to-cdna kit (life technologies) and 50 ng of each cdna were amplified with rat p2y12 specific primers (f: 5-gattgataaccattgacc-3; r: 5-ggtgagaatcatgttagg-3). amplification products (10 l of 20) were electrophoresed on 2% agarose gel containing ethidium bromide (1 g/ml, sigma aldrich), photographed under uv light using kodak image station 440cf, with molecular imaging software 4.0.1. in order to isolate total protein extracts, microglia and oligodendrocytes were harvested with ice-cold ripa buffer (pbs, 1% nonidet p-40, 0.5% sodium deoxycholate, and 0.1% sds), added with protease inhibitor cocktail (sigma aldrich). lysates were kept for 30 min on ice and then centrifuged for 10 min at 14,000 rpm, at 4c. snap-frozen blocks from cases ms114, ms125, and ms163 supplied by uk multiple sclerosis tissue bank at imperial college in london were homogenized in ripa buffer, added with protease inhibitor cocktail, incubated on ice for 1 h, and centrifuged at 14,000 rpm for 10 min at 4c. rat brain and mouse brain and spinal cord were homogenized and sonicated in ripa buffer, added with protease inhibitor cocktail, kept for 1 h on ice, and centrifuged at 4c for 10 min at 13,000 rpm. supernatants were collected and analyzed for protein content by bradford colorimetric assay (biorad, milan, italy). analysis of protein components was performed by polyacrylamide gel (sds-page) separation (biorad) and transfer onto nitrocellulose membranes (amersham biosciences, cologno monzese, it). after saturation, blots were probed overnight at 4c, with the specified antibody (table 1), and finally incubated for 1 h with hrp-conjugated secondary antibodies and detected on x-ray film (aurogene, rome, italy), using ecl advance western blotting detection kit (amersham biosciences). p2y12 protein was detected with molecular mass comprised between 40 kda (as predicted by amino acid sequence) and 49-50 kda (as predicted by the manufacturer data sheet). analysis was performed with the statistical software package medcalc (medcalc software, mariakerke, belgium). in order to provide wide ranging comparative analysis of p2y12 expression particularly in microglia and oligodendrocytes under neuroinflammatory conditions, we performed immunofluorescence and confocal analysis of receptor expression in primary cortical and organotypic cerebellar cultures, in tissue slices from rat striatum and cerebellum, in spinal cord sections from symptomatic and end stage sod1-g93a als mouse model, finally in autoptic cortical tissue from progressive ms donors. p2y12 receptor is formed by two transcript variants that give rise to identical proteins with 342 amino acids, a secondary structure constituted by seven hydrophobic transmembrane domains connected by three extracellular and three intracellular loops, with four extracellular cysteine residues most likely contributing to the nucleotide binding site (figure 1(a)). the commercially available p2y12 antibodies that we mostly used in our work are raised against the second intracellular loop, and precisely amino acids 125142 (here named intra1, intra2, and intra fl; see red circle in figure 1(a)), and against the c-terminus, here named c-ter (see green oval in figure 1(a)) (see also table 1). in order to validate the use of these different antibodies for p2y12 receptor, we compared them on recombinant p2y12 receptor protein obtained by cloning the complete cdna of the human receptor into the eukaryotic expression vector cs2+mt, to provide the expression of a n-terminal c-myc tagged fusion protein. after transfection into sh-sy5y and hek293 cell lines, we analyzed total protein extracts by sds-page and western blotting using c-ter, intra1, and intra2 antibodies. although with different intensities, all the antibodies recognize the myc-p2y12 protein band at the predicted molecular mass of 50 kda. these results confirm the specificity of the used antibodies toward denatured recombinant p2y12 receptor (figure 1(b)). in order to verify the expression of p2y12 mrna in rat primary oligodendrocytes (ol) and microglia (rmg), rt-pcr was performed using specific primers designed on receptor sequence. as shown in figure 1(d), rt-pcr on both rmg and ol reveals the presence of the predicted p2y12 mrna band. next, we validated the antibodies with protein extracts from dissociated primary cultures or tissues from different species. p2y12 protein is specifically recognized in extracts from human cerebral cortex snap frozen tissue, rat and mouse brain, mouse primary microglia (mmg) and ol (figure 1(c)). no signals are obtained when the immunoreactions are performed in the presence of p2y12 neutralizing peptides, when available from manufacturer (data not shown). in order to establish cell specificity of p2y12 receptor expression, we performed comparative immunofluorescence and confocal analysis in primary dissociated cortical microglia and oligodendrocytes (figures 2(a) and 2(b)), as well as organotypic cerebellar cultures (figure 2(c)). p2y12 is strongly recognized by both c-terminus- and second intracellular loop-recognizing antibodies (red, c-ter, upper left inset; intra fl, upper right inset; intra1, lower right inset and intra2, lower left inset), specifically distinguishing the very heterogeneous morphological features of mouse primary microglia, as shown by double fluorescence and confocal analysis performed with phalloidin (green), a marker for filamentous actin (figure 2(a)). fan-like cells (insets), elongated rod-like cell bodies with short and tiny branches, asymmetrical hairy cells with miniature processes or lamellipodia are simultaneously observed (c-ter phalloidin hoechst, merged). likewise, all the p2y12 antibodies (intra2, intra1, c-ter, and intra fl) recognize the multibranched morphology of rat mature oligodendrocytes (figure 2(b), ol, insets, red) in primary cultures, as confirmed by double immunofluorescence and confocal analysis with mbp antibody (ol, intra1-mbp, yellow merged image). in addition, both intra2 and c-ter antibodies distinguish the ng2-positive rat oligodendrocyte precursor cells (opc, merged insets). p2y12 immunoreactivity is confirmed in the ex-vivo system of organotypic rat cerebellar cultures (figure 2(c)). however, differently from mouse microglia and rat oligodendrocyte primary cultures, the second intracellular loop- (red, intra1) and c-terminus-recognizing (red, c-ter) antibodies surprisingly immunoreact with different cell phenotypes when the integrity and cytoarchitecture of the tissue is preserved, as in organotypic cultures. while intra1 antibody (red) exclusively labels mbp-positive fibers (green) and highlights myelin structures, c-ter antibody does not immunoreact with myelinated fibers, being present on cells likely resembling microglia (insets), as also confirmed by colocalization with microglial marker cd11b (data not shown). results similar to those found with intra1 were also obtained with intra2 antibodies, lots an01/02/04 (data not shown). we next verified if the cell type-specific presence of p2y12 receptor observed in organotypic culture, either in myelin structures or in microglia, respectively, by the use of the second intracellular loop-(intra1) or the c-terminus-recognizing (c-ter) antibody, is also confirmed in rat cerebellar and striatal slice tissues (figure 3). all the antibodies raised against the second intracellular loop (red, intra1, intra2, intra fl) identify the abundant presence of p2y12 receptor only on myelinated fibers from both cerebellum (figures 3(a), 3(b), and 3(c)) and striatum (figures 3(d), 3(e), and 3(f)), as confirmed by colocalization of signals obtained with intra fl and mbp antibodies in cerebellum (+ mbp, inset c2, merged, yellow) and striatum (data not shown); colocalization of signals obtained with intra1 and intra2 with mbp antibodies in cerebellum and striatum (data not shown); immunoreactivity of intra1, intra2, and intra fl antibodies for structures identical to those observed in cerebellum (data not shown) and striatum with mbp antibody (see for instance mbp, inset e1, green); identification by second intracellular loop p2y12 antibodies of structures totally different from both bergmann glia and astrocytes recognized by specific gfap antibody in cerebellum (inset b1, green), and from microglia identified by specific cd11b antibody in cerebellum (+ cd11b, inset c1, merged, green) and striatum (+ cd11b, inset f1, merged, green). of notice, all the 2nd intracellular loop antibodies are able to describe the specific cytoarchitecture of both cerebellum, where radiant and sparse fibers clearly characterize the lobuli, and of striatum, where white matter is instead organized in distinct bundles. as in organotypic cultures, the c-terminus antibody (red, c-ter) instead recognizes only microglia in rat cerebellum (figures 3(g), 3(h), and 3(i), and insets g1, h1, i1), striatum (figures 3(j), 3(k), and 3(l)) and cerebral cortex slices (data not shown), with a signal more uniformly distributed throughout the whole tissue. by comparing the cd11b and c-ter immunolabelling in the striatum, we furthermore observe that the mutual intensity of signals is cell-selective within the microglia population, with some cells exclusively positive for c-ter (arrow), and others instead showing different grades of cd11b-c-ter colocalization (see for instance orange, yellow and greenish cells in the merged field). regrettably, double immunofluorescence with c-ter and iba1 microglia marker is not practicable, since both antibodies are raised in the same species. however, no colocalization of signals is ever shown between mbp (green) and c-ter antibodies (+ mbp, inset j1, merged). in order to verify if p2y12 recognized by c-ter antibody exclusively in microglia from rat brain slices could be used as specific microglia marker during neuroinflammation, we validated its use in a typical neuroinflammatory disease such as als and for the first time characterized the presence of p2y12 receptor in sod1-g93a mouse model (figure 4). by immunofluorescence and confocal analysis on lumbar spinal cord sections (l3l5) of wild-type (wt) mice, we first compared the immunoreactive signals obtained with c-ter and specific microglia markers cd11b (red, recognizing ramified microglia) and cd68 (red, recognizing roundish activated microglia). in wt mice, c-ter (green) is abundantly and strongly immunoreacting with the microglia population and colocalizing with the majority of cd11b-positive cells, in both dorsal (dh) and ventral (vh) horns of spinal cord (figure 4(a), left panel, merged yellow signal). all cd11b-positive cells share immunoreactivity for c-ter, as proved by the absence of red cd11b signal. conversely, not all c-ter-positive cells immunoreact also with cd11b antibody, as proved by the presence of some green c-ter signals. in addition, we never observe colocalization with the rare activated cd68-positive (red) microglia cells present in wt healthy tissue (figure 4(a), right panel, merged and inset). to test if c-ter antibody can further recognize microglia activation during the progression of als, we next examined sod1-g93a spinal cord sections at two different stages of the disease, that is, 20 weeks, a phase when the disease accelerates, and end stage, that is, 23 weeks, when the animals reach the score of 1 accordingly to a behavioral score system. at both stages, sod1-g93a mice show a significant increase in cd68 immunostaining not only when compared to wt mice (figure 4(a)), but also in vh with respect to dh, and this is even more evident at end stage with respect to 20 weeks (red, figure 4(b)). remarkably, the immunoreactive signal of c-ter (green, figure 4(b)) decreases during disease progression and the effect is pronounced especially in vh, where motor neuron loss, tissue damage, and microglia activation are known to be increased. particularly at 20 weeks, c-ter-expressing microglia are still ramified in dh, where few cd68-positive cells are present, but start to disappear in vh, where instead cd68-positive cells are increased. at end stage, c-ter staining decreases in dh with respect to 20 weeks and disappears almost completely in vh, concomitantly with a robust increase in cd68 staining. a parallel decrease in c-ter and increase of cd68 immunoreactivities is also confirmed by western blot analysis performed on sod1-g93a lumbar spinal cord homogenates at end stage (figure 4(c)). as in rat organotypic cerebellar cultures (figure 2(b)) and rat cerebellar, striatal, cortical, or mouse spinal cord tissue slices (figures 3 and 4), a similar pattern of p2y12 receptor expression is shown in human frontal cortex autoptic tissue by using c-ter antibody that highlights only microglia uniformly distributed throughout the entire healthy tissue (red), but not mbp-positive myelinated structures (green), as detected by the absence of overlapping immunofluorescent signals (figures 5(a), 5(b), and 5(c), merged and insets a1, c1). on the contrary, intra1 (red, figures 5(d), 5(e), and 5(f)), intra2, and intra fl (data not shown) antibodies recognize exclusively myelinated fibers in tissue from healthy (insets d1, e1, and f1) and progressive ms donors (figures 5(d), 5(e), and 5(f)), as it is found with rat tissue (figures 2 and 3). moreover, we confirm a decrease of p2y12 receptor signal in proximity to the demyelinating lesion, as detected by loss of mbp-positive fibers (see asterisks). no colocalization of signals is shown with mhc ii-positive microglia (+ mhc ii, inset d3, merged). importantly, c-ter, intra1 (red, insets a-f2) and intra2, and intra fl (data not shown) antibodies all recognize the presence of p2y12 receptor in integrin ii/3-positive platelets (green) contained in the blood vessels of the analyzed tissues. as observed in als mouse spinal cord where p2y12 receptor detected by c-ter antibody is shown to temporally and regionally decrease in microglia as a function of increasing inflammatory damage (figure 4), we notice that microglia gradually lose immunoreactivity for c-ter antibody (figure 6) in proximity to the demyelinating active cortical lesions of ms expressing augmented positivity for mhc ii [3335]. moreover, by comparing mhc ii and c-ter signals, we recognize four areas (a d) inside and around the lesion, where microglia express different amount of these proteins. in zone a, at the edge of the lesion, we observe a predominance of c-ter immunoreactivity (red) compared to that of mhc ii (green), as depicted in the merged field by a major occurrence of red signal. in zone b, closer to the lesion, we notice a prevalence of active mhc ii-positive green cells, but still the presence of few red and yellow signals. c and d, apparently in the core or outside the lesion, respectively, where microglia express either almost exclusively mhc ii protein (c) or p2y12 receptor on ramified microglia (d). the interchange among different cell types of molecular cues that condition the cell specificity and the protein profile of each cell characterizes the morphological and functional heterogeneity in particular of microglia within various cns regions, developmental stages [37, 38] and, even more, states of activation during pathological conditions. in the case, for instance, of als, the release of signals from motor neurons apparently denotes one of the earliest phase of the disease, with microglia behaving as an m2 phenotype producing neuroprotective factors to repair motor neurons and preventing them against further injury. as disease rises, motor neurons start releasing alarm signals that in turn convert microglia from beneficial m2 to cytotoxic m1 phenotype, with consequent release of proinflammatory cytokines. for instance, m1 markers are abundantly expressed in normal appearing white matter and throughout active demyelinating ms lesions by activated microglia and macrophages, although in human active ms lesions microglia show an intermediate activation status. in addition, m2 microglia appear fundamental to guide oligodendrocyte remyelination in mice, and a switch from m1- to m2-dominant response occurs in microglia and peripherally derived macrophages when remyelination starts. only therapeutic procedures that both down-regulate the harmful responses and up-regulate the beneficial responses may hopefully slow pathological progression and provide meaningful hope for treatment. at the same time, the identification of clear markers involved in the m2/m1 microglia transition becomes mandatory for presymptomatic diagnosis, monitoring of disease progression, and efficacy of therapies. under this perspective, and consistently with previous findings establishing the role of purinergic receptors in the pathogenesis of both als and ms [14, 43], our present work serves this aim, by highlighting the gradual loss of p2y12 immunoreactivity as an early marker of neuroinflammation and microglia metamorphosis. we have indeed demonstrated here that p2y12 receptor protein identified in primary cultures of both microglia (figure 2(a)) [18, 44] and oligodendrocytes (figure 2(b)) by different but p2y12-selective antibodies can be instead recognized in branched microglia exclusively by the use of c-ter antibody (figures 36). this occurs only when the integrity and cytoarchitecture of the tissue is typically preserved in the presence of the least experimental manipulations, that is, in organotypic cultures (figure 2(c)) and tissues slices for instance from rat striatum and cerebellum (figure 3), mouse spinal cord (figure 4), and human cerebral cortex (figures 5 and 6). a similar difference in primary cultured cell versus tissue distribution of a protein was previously demonstrated with large-conductance calcium-activated potassium channel expression, in vascular endothelium. a first implication emerging from these results is that a reliable evidence about selective p2y12 expression in cells of healthy or neuroinflammatory states is genuine only when cell connectivity and tissue architecture are fully preserved. we have indeed shown this, by proving that the antibodies used for p2y12, recognizing either the c-terminus or the second intracellular loop of the receptor (figure 1 and table 1) and immunolabelling, respectively, microglia or myelinated fibers in the cns, are all still able to immunoreact for instance with platelets (figure 5), where the receptor was originally described to be present and to have a role in the processes of activation, aggregation [4649], primary hemostasis, and arterial thrombosis [5055]. a possible explanation for the microglia versus oligodendrocyte selectivity of the p2y12 antibodies might be that gi-coupling, and/or quaternary structure, post-transcriptional modifications, and subcellular localization of p2y12 that remain strictly preserved in platelets, are instead divergent in microglia with respect to oligodendrocytes/myelinated fibers. in this case, a cell-specific network of p2y12 oligomeric interactions and/or a distinct subcellular partitioning might simply mask the recognition sites of the different antibodies on p2y12 protein in different cell types. to support this hypothesis, we know that in platelets p2y12 indeed resides in subcellular lipid raft structures and its partitioning out from rafts causes for instance inactivation and that also the presence of another purinergic receptor, the ionotropic p2x3, in lipid rafts has cell-specific properties shared in cerebellar granule neurons and total brain tissue but not in neuroblastoma cells and dorsal root ganglia and that the specific antagonist clopidogrel inhibits p2y12 by breaking down the homoligomeric complex to single monomers and finally, that hetero-oligomerization of p2y12 is demonstrated with p2y1, p2y2, p2y13, and with adenosine a1, a2a receptors in different cellular backgrounds. all these features might very well explain also why it has not been found colocalization between p2y12 and gfap-positive astrocytes in rat brain cortex and nucleus accumbens, despite the abundant presence of p2y12 mrna and, moreover, why p2y12 is specifically observed in brain and spinal cord resident microglia but is not observed, for example, in peripheral macrophages in spleen [18, 20]. a second implication that emerges from our results is that the morphological metamorphosis that microglia undergo under neuroinflammatory conditions as those triggered during als and ms, can be remarkably highlighted by the progressive reduction of p2y12 immunostaining obtained with c-ter antibody that reacts, also in this case, exclusively with multibranched microglia still present in the tissue (figures 4 and 6). this closely reflects the expression of p2y12 that is robust in the resting/surveillant branched state but dramatically decreased after morphological transition and activation of microglia. our observations are also in line with the central role played by p2y12 in branched microglia membrane ruffling and inspection of the environment. on the other hand, they depict a morphological/functional state of microglia that only partially overlaps with cd11b and mhc ii immunoreactivities, which are furthermore known to increase during activation but instead highly contrasts with cd68 immunostaining that is totally absent in ramified microglia. in parallel to our results, these last antibodies actually accentuate the progressive transition of microglia from a lesser ramified shape to a significantly more activated amoeboid phenotype (figures 4, 6, and 7), thus suggesting the dual use of c-ter and cd68 antibodies as markers, respectively, for branched resting/surveillant versus roundish/activated microglia. a reduction of c-ter-p2y12 immunostaining that is concomitant to an increase, for instance, of mhc ii/cd11b/cd68 immunoreactivity, could thus become a feasible approach to detect an increasing neuroinflammatory condition. indeed, p2y12 is considered an essential component and primary site at which nucleotides such as adp act to promote directional microglia movement or chemotaxis at early stages of cns injury. in particular, microglia from mice lacking p2y12 exhibit normal baseline motility but are unable to polarize and to elicit directional branch extension and migration toward nucleotides in vitro, or sites of cortical damage in vivo. these notions are consistent with our results that fail to describe c-ter-p2y12 immunoreactivity on roundish phagocytizing, or polarized migratory microglia. however, we still do not know if reduction/absence of p2y12 c-ter-immunolabelling on activated microglia might be a cause or a consequence of morphological/functional transition or might simply reflect a cell-selective hindrance and lack of access to the immunogenic sites by the antibody. anyhow, we can assert that the distinctive recognition of multibranched microglia renders the c-ter antibody a novel and useful tool to discriminate among microglia morphological states, thus making p2y12 receptor a selective and early marker for the ramified phase. in parallel to this, we have also proven that all the several antibodies raised against the second intracellular loop of p2y12 (intra1, intra2, and intra fl) can likely be employed as markers for the presence of ms lesions. although with different intensities, they not only recognize the receptor specifically on myelinated fibers of organotypic cultures (figure 2(c)), tissues slices from rat striatum or cerebellum (figure 3) and human cerebral cortex, but also furthermore highlight the reduction of p2y12 signal that occurs for instance in ms tissue (figure 5) in correlation to the extent of demyelination found in all types of grey matter cortical plaques (i iii) and subcortical white matter. in brief, we have shown here that the presence of p2y12 receptor can be simultaneously identified by the c-terminus and the second intracellular loop antibodies. when this occurs, a condition of intact myelinated fibers and branched/surveillant microglia is represented at once that perhaps signifies a for instance in ms, a decrease in the second intracellular loop immunoreactions accompanied by an increase of c-terminus immunoreactivity will possibly depict the loss of myelin and replacement by ramified microglia that often occur in an inactive plaque. on the contrary, a decrease in c-terminus immunolabelling in the abundant presence of second intracellular loop-positive myelinated fibers would indicate an early active plaque where m1/m2 microglia reactivity starts to take place. by comparative analysis of all the available p2y12-immunoreactive antibodies recognizing the c-terminus or the second intracellular loop of the receptor, we have established here that, under experimental conditions of well-preserved cytoarchitecture and tissue integrity, p2y12 receptor expressed by both ramified microglia or oligodendrocytes/myelinated fibers might serve a dual function as specific marker, respectively, of branched/surveillant microglia as well as demyelinating lesions. we believe that p2y12 identification and modulation might potentially acquire an important predictive value under neuroinflammatory conditions, as those found in als and ms. p2y12 is likely to deserve a key role in the verge of a neuroinflammatory breakdown.

in the cns, neuroinflammation occurring during pathologies as amyotrophic lateral sclerosis (als) and multiple sclerosis (ms) is the consequence of an intricate interplay orchestrated by various cell phenotypes. among the molecular cues having a role in this process, extracellular nucleotides are responsible for intercellular communication and propagation of inflammatory stimuli. this occurs by binding to several receptor subtypes, defined p2x/p2y, which are widespread in different tissues and simultaneously localized on multiple cells. for instance, the metabotropic p2y12 subtype is found in the cns on microglia, affecting activation and chemotaxis, on oligodendrocytes, possessing a hypothesized role in myelination, and on astrocytes. by comparative analysis, we have established here that p2y12 receptor immunolabelled by antibodies against c-terminus or second intracellular loop, is, respectively, distributed and modulated under neuroinflammatory conditions on ramified microglia or myelinated fibers, in primary organotypic cerebellar cultures, tissue slices from rat striatum and cerebellum, spinal cord sections from symptomatic/end stage sod1-g93a als mice, and finally autoptic cortical tissue from progressive ms donors. we suggest that modulation of p2y12 expression might play a dual role as analytic marker of branched/surveillant microglia and demyelinating lesions, thus potentially acquiring a predictive value under neuroinflammatory conditions as those found in als and ms.

PMC4142314

pubmed-76

acute kidney injury (aki) requiring dialysis is a serious complication in critically ill patients, bringing increased morbidity, mortality, and costs of care [14]. aki requiring dialysis is usually considered the most severe form of kidney injury, and these patients have been conventionally regarded as a relatively homogenous group of patients, either when describing epidemiological information or while conducting clinical trials [5, 6]. however, studies examining interventions in dialysis patients (e.g., dialysis modality or frequency have not demonstrated unequivocal survival benefits [79]. it is well recognized that small changes in creatinine (mild-to-moderate aki) independently predict mortality [10, 11]; we also recently reported that patients with aki requiring dialysis represent a wider spectrum of severity of kidney injury, contrary to the prevalent notion. thus, it can be hypothesized that the degree of elevation of creatinine prior to initiating dialysis may discriminate risk-adjusted mortality, similar to the observations in nondialysis requiring aki. the acute kidney injury network (akin) has issued standard definitions of aki; currently, in these criteria, aki requiring dialysis is classified as stage iii (or severe) aki. the consensus panel also proposed that the examination of natural history of severe aki in icu to be one of the high-priority research areas, with a goal that new information may improve our ability in conducting prospective trials of intervention aki [14, 15]. to date, there are limited studies that have examined the course or progression of aki after icu admission until the point of dialysis initiation. whether the degree of severity of kidney injury prior to dialysis requirement independently influences mortality risk in aki is not known. in order to facilitate risk stratification that may be useful for the development of prospective studies, which are geared towards early diagnosis or intervention in severe aki, we characterized the spectrum of severity of kidney injury in a cohort of icu patients with aki who required dialysis. we specifically tested the association of creatinine elevation during icu stay, starting with icu admission and prior to dialysis initiation, with the risk of hospital mortality. we also examined the impact of severity of illness upon icu admission on the mortality risk in these patients. we examined a cohort derived from the veterans affairs (va) inpatient evaluation center (ipec); a national quality improvement program which reports risk-adjusted mortality, length of stay, and adherence to evidence-based practices in all va icus, by collecting data electronically from va computer databases. an analytic dataset was formed from the ipec retrospective cohort including all patients (n, 617,927) admitted to all 191 va icus in the usa between october 2001 and september 2006. we excluded patients with (i) less than three creatinine measurements during the icu stay (n, 204,963), (ii) readmissions to the icu (n, 50,874), (iii) transferred to other hospitals at discharge (n, 14,219), (iv) transplant recipients (n, 317), and (v) those with chronic renal failure defined as prior dialysis (n, 3,862 patients), or international classification of disease-9th clinical modification (icd-9-cm) codes for end-stage renal disease (esrd; 1,388 patients 585.6), or with a calculated glomerular filtration rate (gfr)<15 ml/min/1.73 m (16,571) (gfr estimated by four variable modified diet in renal disease equation); 296 additional patients who required dialysis in icu but had icd-9 codes for advanced ckd/stage v ckd were also excluded (based on random q/a, these codes identified esrd status in those who required dialysis). thus, 324,999 patients were available for analysis. the analyses were reviewed and approved by the university of cincinnati institutional review board and va r&d committees. briefly, from each hospital database, a customized program identifies patients whose hospitalization included an icu stay, and extracts administrative data from the index hospitalization that includes icd-9-cm codes representing the reason for admission to the icu, length of stay, and all procedure codes. measured values of 11 laboratory tests are extracted to estimate severity of illness from a period of time 7 days prior to icu admission through hospital discharge. mortality is defined at death during hospitalization and is validated using the vital status file at the va national database (austin, tx). aki was present if there was an increment of serum creatinine by>0.3 mg/dl (or 1.5 times increase) during the icu stay relative to the creatinine on icu admission. for this analysis, we specifically focused on examining characteristics and outcomes of those aki patients who progressed to new requirement of dialysis (aki-d) during icu stay (part of stage iii aki according to the akin classification system). dialysis requirement in icu included both intermittent and continuous dialysis, as prescribed by the clinician, and the modality can not be differentiated based on va electronic records that were available. we categorized aki-d into four groups based on the change in creatinine level after icu admission but prior to initiation of dialysis: group i had an increase in creatinine ranging from 0.3 mg/dl to<2-fold increase, group ii an increase in creatinine 2 times baseline but <3 times, group iii 3-fold increase in creatinine, and group iv included those patients with new dialysis requirement in icu but who experienced a creatinine elevation of<0.3 mg/dl. the purpose of this grouping was to determine an equivalent stage of aki that a patient may experience prior to initiating dialysis based on the standard definitions of aki put forth by the akin. some patients may have had creatinine values available after dialysis initiation, but these values were not considered for analysis. the study accounts for differences in patient characteristics using a previously validated logistic regression model that predicts mortality risk for each patient from independent predictors (age, thirty-one comorbid disease groups (by icd-9-cm codes), eighty-four mutually exclusive admission diagnoses determined from the icd-9-cm codes denoting the reason for admission to the icu, source of admission (emergency room, outpatient clinics, ward, hospital, operating room, or nursing home), and the worst value of each of the 11 laboratory tests measured within 24 hours of icu admission (sodium, urea, creatinine, bilirubin, albumin, glucose, hematocrit, white blood cell count, pao2, paco2, and ph). diagnosis and comorbid diseases were included in this model as binary variables, most laboratory values and age were included as cubic splines, and paco2 and ph were treated as a categorical interaction term. the model has been validated across multiple centers with excellent calibration and discrimination (c-statistic=0.88; brier's=0.06), and comparable with traditional methods of severity of illness assessment (e.g., apache). from the logistic regression model, a standardized mortality rate for groups can be determined (smr=observed/predicted mortality; where predicted mortality is estimated by the mortality model described above). a more detailed account of development and validation of these variables and risk adjustment methods has been published earlier [16, 19, 20]. univariate comparison of diagnosis categories, comorbid diseases, and their relationship with laboratory variables was tested by chi-square test and kruskal-wallis test. a two-step logistic regression model determined the independent contribution of patient groups (based on creatinine elevation) to hospital mortality. the first step predicted each individual patient's hospital mortality using the validated method described above. independent predictors in the second step included the predicted mortality (determined from the first step) and the patient group based on creatinine elevation before dialysis. based on the predicted mortality, as determined by the mortality model described above, we also estimated the standardized mortality rate (observed/expected mortality) for each of the patient groups under consideration. risk estimates were expressed as odds ratios (or) and 95% confidence intervals (95% ci). overall, 21.9% of patients developed aki (71,090/324,999) of whom 2,744 patients experienced severe aki requiring dialysis. 561/2744 (20.4%) patients were admitted to icu from the hospital floors, whereas 2,183/2,744 (79.6%) were either direct admission or admissions from emergency rooms. the median time for aki-d patients to meet the criteria for aki from the time of admission creatinine measurement was 23.4 hours (interquartile range, 12.2, 46.6). the median time to initiation of dialysis in icu after icu admission was 96.0 hours (interquartile range, 33.6, 231.6); median time to dialysis initiation from icu admission was 72 hours in group i, 145.1 hours in group ii, 216.0 hours in group iii, and 14.6 hours in group 4. when examined from the time when patients reached their peak creatinine value to the time of dialysis initiation, the median time was 43.0, 39.5, and 67.4 hours in groups i, ii, and iii respectively. figure 1 shows the percentage of patients initiating dialysis by days after icu admission. the proportion of patients in each group is shown in figure 2; of the 2,744 patients who required new dialysis during icu stay, only about a third of patients (31.5%) experienced greater than 3 times increase in serum creatinine prior to dialysis initiation, and a small proportion of patients (11.2%) required dialysis with little or no elevation in serum creatinine (likely for fluid-electrolyte/acid-base disturbances). the baseline comorbid and laboratory characteristics of patients who developed aki requiring dialysis, and their univariate comparison across groups i through iv are shown in table 1. group iii patients (those with>3 times increase in creatinine prior to dialysis) had the lowest levels of creatinine or bun on icu admission. in contrast patients in other groups, who sustained only milder or no elevation in creatinine before dialysis initiation, had significantly higher creatinine and bun levels on icu admission. table 2 shows the frequency of admission diagnosis categories across four subgroups of patients with aki requiring dialysis. the frequencies of patients admitted with primary icu admission diagnoses of kidney disorders or electrolyte and metabolic disorders significantly varied across four groups and were 24% and 21%, respectively, in group iv (< 0.3 mg/dl increase in creatinine before dialysis) compared with only 1.9% and 2.1% in group iii (> 3 times increase in creatinine before dialysis). this suggests that majority of patients in group iv may have been dialyzed for indications other than kidney injury (e.g., for electrolyte or metabolic disturbances). in contrast, cardiothoracic surgery admissions occurred at a higher frequency (26%) among group iii patients (> 3 times increase in creatinine), suggesting that these patients are more likely to have normal renal function on icu admission and hence sustain>3 times increment in creatinine prior to dialysis requirement. the degree of creatinine elevation prior to dialysis initiation was associated with increase in odds of death. table 3 shows odds ratios of mortality across all four groups, with group i as reference group. dialysis patients in group ii had greater odds of death (odds ratio (or), 1.76, 95% confidence intervals (95% ci), 1.40, 2.22)), those in group iii were more than twice likely to die (or 2.20, 95% ci, 1.792.71), whereas patients in group iv were less likely to die compared to group i (or 0.39, 95% ci, 0.29, 0.52). the smr (table 3) confirmed a graded association between the degree of creatinine elevation prior to dialysis initiation and mortality risk. the relationship between creatinine elevation and risk of death remained qualitatively similar even after stratifying patients by level of renal function on icu admission (stratified at creatinine level of 1.2 mg/dl), but the sample size was relatively small in these subgroups (data not shown). figure 3 shows smr associated with creatinine elevation stratified by severity of illness on icu admission. dialysis patients in groups ii and iii had a significantly higher smr than those patients in groups i and iv, when predicted mortality was<10% or 1030%; smr was not significantly different across all four groups in patients admitted with>30% predicted mortality on icu admission. the present study advances our understanding of natural history of progression of acute kidney injury during icu stay, in those patients who require dialysis. the study finds that, in patients with aki requiring dialysis, the risk of mortality is independently associated with the degree of severity of kidney injury sustained during icu stay prior to dialysis initiation and that severity of illness further influences this relationship. several epidemiological studies have confirmed that icu patients developing aki requiring dialysis experience a high risk of mortality across different clinical settings, including cardiovascular surgery, or in other medical or surgical icus [12, 2123]. it is also observed that the severity of kidney injury, determined by the degree of creatinine elevation, in non-dialysis requiring aki is associated with a graded increase in mortality risk [2, 10]. the present study is one of the first reports to show that in patients who developed aki requiring dialysis, the degree of kidney injury prior to dialysis initiation determines mortality risk, after accounting other major predictors of hospital mortality. it does so by examining a large, diverse, multicenter cohort that includes all icu settings, with an ability to perform electronic data extraction starting (including serial creatinine measurements) from the time of icu admission until the point of dialysis initiation in aki. the results indicate that majority of the aki-d patients sustain mild-to-moderate degrees of creatinine elevation (equivalent of stage i or stage ii aki) prior to dialysis, whereas less than a third of patients require dialysis after sustaining>3 times increase in serum creatinine during icu stay (or equivalent of stage iii aki). this confirms prior multicenter observations that aki requiring dialysis in icu usually represents a group of patients that sustain acute kidney injury on an underlying renal dysfunction. there were 11% of patients who required dialysis with<0.3 mg/dl increase relative to icu admission. although the specific indications for dialysis could not be ascertained by electronic data extraction, however, based on the distribution of comorbid conditions and admission diagnoses, it can be suggested that the indications for dialysis in this subgroup may have been for volume overload or electrolyte/metabolic abnormalities rather than the degree of kidney injury. these observations are consistent with our prior experience in cardiac surgery settings, where 1015% of cases were dialyzed for reasons related to volume overload or electrolyte disorders and not azotemia/creatinine elevation. in terms of mortality, however, this group was associated with the lowest risk of death among those who required dialysis consistent with the study hypothesis. prior large multicenter observations provide only a limited insight into the effects of severity kidney injury prior to dialysis initiation on patient outcome. somewhat indirect evidence comes from the studies that have reported the impact of baseline chronic kidney disease (ckd) in patients with aki requiring dialysis. for example, a third of patients in a multicenter us cohort (picard cohort; 618 aki cases, 60% of whom required dialysis) had stage iv or worse ckd prior to aki. by multivariate analysis, baseline ckd status conferred 43% lower adjusted odds of in-hospital mortality (or, 0.57, 95% ci, 0.390.84) in aki. similar observations have been reported in a medicare administrative sample that examined outcomes in aki. unadjusted mortality rate in patients that were coded for aki requiring dialysis on an underlying ckd was 22% compared to 30% among those with aki requiring dialysis but without underlying ckd [2426]. our study may offer one of the explanations to the seemingly paradoxical observation that ckd status in aki may confer a protective effect: that is, patients with impaired renal function on icu admission are likely to require dialysis after sustaining milder degrees of kidney injury than those admitted with relatively better renal function and thus experience a lower risk of mortality. severity of illness on icu admission further modified the relationship between the degree of creatinine elevation and risk-adjusted mortality in aki require dialysis. the degree of kidney injury sustained prior to dialysis did not discriminate mortality risk in those patients with very high severity of illness (> 30% predicted mortality) on icu admission. in contrast, in patients with low-to-moderate severity of illness, it is the degree of injury that determines excess mortality risk rather than dialysis status alone. first, the data indicates that in half of the patients, dialysis was initiated within 96 hours after icu admission, with ~25% of then requiring it in the first 48 hours. thus, in order to target these patients for early interventions, we need to develop better predictive models that are based on information derived within 24 hours of icu admission; similarly, the development of novel biomarkers that may predict dialysis requirement would need to discriminate acute kidney injury from underlying renal dysfunction at the time of icu admission. second, it indicates that the efficacy of therapeutic measures in aki requiring dialysis can not be equitably compared without accounting for both severity of illness on admission and degree of kidney injury sustained before dialysis initiation. for example, given the same predicted mortality or level of renal function on admission, patients in group i or iv could not be optimally compared with group iii in an intervention trial. finally, it also raises the question that whether early dialysis, as indicated by initiation of therapy after a relatively lower magnitude of rise in creatinine, may offer a survival benefit. the va patient population is predominantly represented by male gender and white race and hence may limit in terms of generalizability of findings to specific demographic subgroups. by way of retrospective design, and lack of standardized criteria to initiate dialysis, the data can not account for differences in dialysis practices across different centers. we, however, examine a multicenter, diverse cohort of icu patients that records patient level information that is available for automated extraction by computerized methods. our cohort derivation excluded patients with advanced renal dysfunction on icu admission, and based on the present findings it can be expected that a significant proportion of these patients may have required new dialysis in icu. we still chose to use this criterion to minimize the likelihood of misclassifying patients with dialysis dependent renal failure prior to icu admission but who were not appropriately coded for it. additionally, we considered the degree of creatinine elevation relative to admission creatinine, which may not reflect a true baseline assessment of renal function; this still allowed us to assess the relationship between magnitude of creatinine elevation and hospital mortality. another limitation is that we did not have information on modality or frequency of dialysis and hence could account for those variables in the multivariate analyses. the present study relies on the va icu mortality model instead of other traditional methods of measures of severity of illness assessment such as apache criteria. the rationale to do so is because the va icu mortality model is derived from an automated electronic data extraction method, which has been validated across multiple va sites with excellent predictive accuracy and calibration (c-statistic=0.88) and is comparable with other methods. in summary, within a cohort of patients who develop aki requiring dialysis during icu, the mortality risk is independently associated with the degree of creatinine elevation prior to dialysis initiation. the mortality risk is lowest in those who experience minimal or no elevation in creatinine and may represent less severe acute illness in patients with ckd, or easily reversible fluid-electrolyte or acid-base disturbances. the study also indicates that aki requiring dialysis represents a heterogeneous group of patients, and directly confirms earlier observations that only minority of these cases sustain severe kidney injury prior to dialysis initiation. we interpret the data to suggest that prospective studies aimed at examining therapeutic interventions in aki requiring dialysis (e.g., timing of dialysis initiation) would need to consider both the degree of kidney injury sustained prior to dialysis initiation and overall severity of illness, prior to performing equitable comparisons.

in a multicenter observational cohort of patients-admitted to intensive care units (icu), we assessed whether creatinine elevation prior to dialysis initiation in acute kidney injury (aki-d) further discriminates risk-adjusted mortality. aki-d was categorized into four groups (grp) based on creatinine elevation after icu admission but before dialysis initiation: grp i>0.3 mg/dl to<2-fold increase, grp ii 2 times but <3 times increase, grp iii 3-fold increase in creatinine, and grp iv none or<0.3 mg/dl increase. standardized mortality rates (smr) were calculated by using a validated risk-adjusted mortality model and expressed with 95% confidence intervals (ci). 2,744 patients developed aki-d during icu stay; 36.7%, 20.9%, 31.2%, and 11.2% belonged to groups i, ii, iii, and iv, respectively. smr showed a graded increase in grp i, ii, and iii (1.40 (95% ci, 1.291.42), 1.84 (1.662.04), and 2.25 (2.072.45)) and was 0.98 (0.781.20) in grp iv. in icu patients with aki-d, degree of creatinine elevation prior to dialysis initiation is independently associated with hospital mortality. it is the lowest in those experiencing minor or no elevations in creatinine and may represent reversible fluid-electrolyte disturbances.

PMC3556436

pubmed-77

many previous studies have revealed that parents, especially mothers, of children with developmental disabilities such as intellectual disabilities (ids), developmental delay, and physical and sensory handicaps are more likely to show signs of psychological distress or depressive symptoms and to exhibit lower well-being than parents of typically developing children [15]. it is generally accepted that caring for a child who has a developmental disability may involve significant and prolonged periods of time and energy. since the majority of this increased daily care-giving burden for these children is carried by their parents, they are supposed to have an increased risk for high levels of stress, and thus some cases may be linked to depression [7, 8]. recently, the mental health needs of adults with i d have also been taken up for discussion. studies have revealed that psychiatric disorders are more prevalent in people with i d compared within the general population. specialist psychiatric services for people with i d are available in some countries such as the uk and the usa; however, the provision of high-quality psychiatric services remains a major concern in many countries.. evidently, the current psychiatric services in japan are not adequate to meet the complex mental health needs of people with i d appropriately, sensitively, and effectively. failure to provide services that meet the needs of these individuals may lead to problems such as high-level caregiver stress and inappropriate therapy. the paucity of trained psychiatrists and other allied professionals is a major barrier to the development of this subspecialty in many countries. in japan, high-quality psychiatric services for the parents of children with i d are assumed to be not well established. so far, the major portion of resources has been directed to the treatment of and services connected with the children, and not enough attention has been paid to the actual mental condition and needs of the parents. to our knowledge, there have been no reports on a nationwide scale on the mental health and distress of parents having children with developmental disabilities in japan. since the understanding of the parents ' mental health may be essential for providing more appropriate support, we aimed to obtain a clear perspective on the situation facing the parents through this facility-based nationwide study. the current survey included 1,102 institutions and consultation centers for children with developmental disabilities distributing in all prefectures in japan, which were listed in the national register of agencies, organizations, and institutions related to intellectual disabilities and the directory of support centers for individuals with developmental disabilities. in japan, these institutions and centers play a key role in social support networks. professional workers in these facilities come into contact with children as their clients, and also with the children's guardians, mainly with the mothers, on a daily basis. the questionnaire was written in japanese and was divided into two sections with a total of 47 multiple choice questions. the first section sought information about the characteristics of the facility, such as staff adequacy, the scale of the facility, the health promotion services provided, the ages of their clients, and the clients ' main disabilities, namely, i d, pervasive developmental disorder (pdd), profound intellectual and multiple disabilities (pimd), physical disabilities, and others. the second section sought information about the clients ' parents, such as signs of mental health distress and their needs, from the professional caregivers ' point of view. also measures taken by the professional caregivers when encountering distressed parents were asked in detail. questionnaires were mailed in october 2008, with an explanatory letter to the person in charge of each institution or consultation center. when agreement was met to participate in this study, one or more service providers in each facility completed the questionnaire based on all sources of information available. the questionnaire was to be returned unsigned, using the stamped, self-addressed envelope enclosed. for ethical considerations, the information disclosed in the questionnaire was in a form which made it impossible to identify the facility and clients this study was approved and carried out under the supervision of the japan league on developmental disabilities (jidd). the statistical analysis including the chi-squared test was performed with the aid of the statistical package for the social sciences 15.0 (spss japan, inc.). a two-sided p value of 0.05 or less was considered to be statistically significant. answers to open-ended questions a total of 460 out of 1,102 facilities replied within four weeks, the response rate being 41.7%. according to the clients ' main disabilities, each facility was classified into the following categories: i d, pdd, pimd, physical disability group, and others (table 1). the last group was small in number and diverse in character and also included responders who provided incomplete information; thus, data from this group were omitted from analysis hereafter. also in table 1, the number of clients (mean persons per facility) as an index of facility scale, their mean age, and proportion of male gender are shown. their average age (sd) was 44.7 (9.5) years old for the i d group, 53.0 (7.6) for the pdd group, 48.3 (8.4) for the pimd group, and 55.3 (4.9) for the physical disability group. 373 out of the 415 facilities (about 90%) experienced cases with difficulties when communicating with the clients ' parents. the percentage was high regardless of the children's disabilities, being concretely highest in the pdd group (96.9%) and lowest in the physical disability group (86.7%). the proportion of such parents, out of the total, was reported to be almost none (5% or less) or few (less than one fourth of the clients) in most of the facilities (table 2). it should be noted that in the i d and pdd groups, about 15% of the facilities responded that about half or more of the parents were difficult to communicate with. in all groups, the characteristics of the i d and pdd groups were similar to each other, while the pimd and physical disability groups resembled each other. in other words, in the i d and pdd groups, slightly more difficulties were experienced with the fathers compared to in the pimd and physical disability groups, where they experienced more difficulties with other family members such as the grandparents and siblings compared to the i d and pdd groups. the primary reason for these difficulties was thought to be the parent's personal condition, such as poor mental health (table 2). this was especially true for the i d and pdd groups [(df=9)=41.9, p<0.001]. in the pimd and physical disability groups, the parent's poor socio-economic status and heavy care-giving burden, reflecting the child's level of disability, were considered to be major factors in approximately one-third of the cases. signs of depression or a depressive state were the most common psychiatric disturbances seen in the parents. among the four groups, the pdd group most commonly experienced cases which showed signs of depression or a depressive state in parents. the proportion of such parents was approximately 70% of the total [(df=12)=65.1, p<0.001]. well-treated cases were only 5% out of the total; thus, most of these conditions were considered to be not medically well treated. for further analysis, we selected 159 cases where symptoms of depression or a depressive state were reported in a descriptive manner. according to these reports, we asked about the time of the onset, that is, whether it was prior to the birth of the child, when the parent first noticed the child's disability, or after the child had been fully medically diagnosed. apart from the answer unknown which accounted for approximately half of the cases, prior to the birth of the child was relatively common in the pdd group (22.1%), whereas after the child had been fully medically diagnosed was relatively common in the pimd (23.5%) and physical disability groups (28.6%). when a parent suffered from signs of psychiatric disturbances, more than half of the children experienced maltreatment. physical neglect was most common in all groups, accounting for about 50% of the cases. in comparison with the other groups, in the pdd group, the child was likely to be emotionally disturbed and confined indoors (32.6%), in the pimd group, the child was mostly confined indoors (33.3%), and in the physical disability group, medical care neglect made the utilization of medical and welfare support impossible (50%) [(df=12)=27.6, p=0.006]. the leading measures were gathering and disclosure of necessary information, discussion among service providers within the facility about ways to deal with the parent, protection of the child by providing a safe environment, making approaches to other family members for cooperation, intensive collaboration with other organizations such as institutions and consultation centers for better community support, and medical support for the parent provided by specialists such as physicians and psychologists. many factors were reported to affect the mental health of parents. the presence or absence of support from other family members (i.e., a spouse) and the existence (or not) of a medical condition in the parent were major factors (table 5). practical use (or not) of social support networks, whether the child's level of disability was severe or not, and parent participation in face-to-face family support groups or not were also thought to be influential factors. in all groups, regardless of the child's disability, more direct welfare support for the child was considered to be helpful and thus requested to be improved, in order to meet the mental health needs of the parent. besides this, in the physical disability group, family support groups, in the pimd group, support (including financial support) for other family members, and in the i d and pdd groups, support for other family members (mainly siblings and the other parent) and medical treatment of the parent's psychiatric disturbance were also emphasized. to fulfill the demanded future role of their facility, respondents expressed the importance of more collaboration with other institutions and consultation centers and the necessity for more special training courses targeting support professionals and recognized the urgent demand for more specialists especially psychologists who can support the mental health needs of both the children and their parents. in the present study, we surveyed institutions and consultation centers for children with developmental disabilities in japan, which play a central role in the social support networks. this facility-based nationwide survey revealed the situation facing the parents of children with i d and other developmental disabilities from the professional caregivers ' point of view. almost all of the facilities experienced difficulties in keeping good relations with the clients ' parents, regardless of the children's disabilities, and the majority of service providers noted that the proportion of such parents, out of the total, was roughly between 5 and 25%, and still increasing. the primary reason for these difficulties was thought to be the parent's condition, such as their mental health and background. also, signs of depression or a depressive state were assumed to be the most common psychiatric disturbance seen. these results suggested that thorough consideration of the parents ' mental health needs, especially prevention of depressive conditions and support for parents suffering from them, is urgent-demanded task for facilities. i d, attention-deficit/hyperactivity disorder (ad/hd), and pdd are common developmental disorders, and the latter two are noted to have substantial genetic components. it has been reported that a lifetime prevalence of major mood disorders is higher in parents of children with autism and that the onset for the majority was prior to the birth of their child with autism. family history studies of autism consistently uncovered a large subgroup with a high incidence of major mood disorders among family members, suggesting the two entities are related clinically and genetically [19, 20]. the findings of the present survey must be interpreted allowing for the limitations of the use of unconfirmed data reported by professional caregivers ' who come into contact with children as their clients, and their parents on a day-to-day basis. evidently, the service providers ' perspective on parental mental health was consistent with previous reports from western countries on major mood disorders and pdd. in other words, facilities where the clients were diagnosed as having pdd experienced significantly more cases of parents with signs of a depressive state compared to other facilities, and the onset of depression was considered to be prior to the birth of the child in many of the cases. although these professional caregivers were well trained and well experienced, only, one-third were physicians, nurses or psychologists. on account of the limitations of these data, we emphasize confirming the diagnoses of psychiatric disturbances through direct contact with the parents by specialists such as psychiatrists is necessary in future studies, for accurate understanding of the overall problem. in japan, there is no official system to check and document the parents ' condition including their mental health status. parents are not accustomed to being asked about their own mental health, so there is a tendency to avoid discussion on matters concerning themselves. this was the major reason why we asked the professional caregivers about the parents ' condition, not the parents directly, in this study. but still we judged it to be worthwhile at this moment to summarize the professional caregivers ' impression on this issue, since the case in japan has never been described in detail despite of its importance. because the majority of burden for daily care of handicapped children is typically carried by mothers, particular concern is raised for their adaptation. in the past studies, the psychosocial outcomes in mothers were said to be better predicted by psychosocial factors such as more active social life and family resources. recent research focusing on parenting stress illustrated the value of the participation of fathers also. greater marital quality predicted lower parenting stress for both mothers and fathers, while greater social support predicted increased parenting efficacy for fathers. although the relationship between child characteristics and parental well-being has not reached an invariable agreement, the severity of a child's disability, intellectual functioning, and so forth are generally considered to be risk factors. since the burden for daily care is mainly carried by the mother, similarly in japan, service providers tend to be in contact with the mother more frequently than other family members. this may be partially the reason why service providers experienced difficulties in communicating with mostly the mothers in this study. the presence of support from other family members, such as a spouse, is considered to be a major influential factor for the mental health of parents in japan also. practical use of social support networks such as participation in face-to-face family support groups, more financial support for the household, more direct welfare support for the child, and medical treatment of the parent's psychiatric disturbance were reported to be helpful and thus requested to be improved, in order to meet the mental health needs of the parent. we would like to emphasize the fact that although the importance of medical treatment for the parents ' psychiatric disturbances was pointed out, it was also realized that these conditions were presently not being medically well treated. this shows that current psychiatric services in japan are not adequate to meet the complex mental health needs of the parents appropriately. to resolve this challenging issue, more intensive collaboration between other child-care facilities and organizations, aiming at the mental health needs of the parents, is demanded. the paucity of trained psychiatrists and other allied professionals also needs to be addressed, and in order to fulfill this, sufficient discussion and research on the establishment of an effective training system for specialists is warranted. providing direct advice for the parents on the utilization of social support networks is a reliable measure that can be immediately taken. training more professionals who can properly deal with the parents ' mental health needs is an urgent matter that must be tackled. overall, the present study is the first nationwide survey that examined the professional caregivers ' perspective on the mental health of parents of children with developmental disabilities, and it underscores the importance of understanding and supporting the parents ' mental health needs, which leads to more appropriate support for children with developmental disabilities.

parents of children with intellectual disabilities and/or physical disabilities are supposed to have an increased risk for parenting stress and psychological distress. we as professional caregivers sometimes experience difficulties in keeping good relations or communicating with the parents. professional workers in 460 institutions and consultation centers throughout japan answered a questionnaire on their clinical experiences. about 90% of the facilities experienced distressed parents, and the parents ' condition such as mental health seemed to influence this. signs of a depressive state were the most common psychiatric disturbances detected, and it was notable in the pervasive developmental disorder group. more welfare support, presence of support groups, support from other family members, and medical treatment of the parents ' problems were considered to be helpful and thus requested to be improved. training more professionals who can properly deal with the parents ' mental health needs is an urgent matter that must be tackled.

PMC3265212

pubmed-78

in may 2001, 24 cases of s. typhimurium dt160 salmonellosis were reported in the auckland region compared with an average of four sporadic cases each month with this serotype in the previous 7 months. raw and undercooked egg consumption was commonly reported by the first 10 case-patients interviewed. a case-control study and environmental investigation were undertaken to identify the vehicle of infection and source of the outbreak. recognizing the potential for a widely dispersed foodborne outbreak, we expanded the investigation throughout new zealand. we defined a case as diarrhea (3 loose stools in a 24-hour period) or vomiting after april 28, 2001, with a stool specimen positive for s. typhimurium dt160. patients were excluded if they had a history of contact with another person with culture-confirmed s. typhimurium dt160 infection, or if they had a history of recent overseas travel. each case was matched with two controls found from randomly drawn telephone numbers, matching for neighborhood and age (< 1, 14, 514,>14 years). the questionnaire covered symptoms (patients only) and contact with other symptomatic persons, bird or animal contact, and food consumption in the 3-day period before onset of illness (cases and controls). parents or guardians were interviewed on behalf of children ages<12 years. stepwise conditional logistic regression analyses were performed, also using sas, to identify the combination of variables that best explained the differences between case-participants and controls. samples from the drinking water supply of case-patients with a history of recent consumption of nonreticulated water were collected and tested for coliforms and s. enterica by using standard methods (6). brands of eggs eaten raw within the incubation period were sampled at random from retail displays at the case-patients purchase site. eggshell surfaces and contents were tested with standard methods (7). broken or cracked eggs were excluded from analysis. salmonella isolates were serotyped by using the kauffman-white scheme (8) and s. typhimurium isolates were phage typed by using the laboratory of enteric pathogens method (9). from may to august 2001, a total of 170 case-patients meeting the case definition were identified. of these, 119 (70%) agreed to participate and were enrolled in the study, along with 235 matched controls. the median age of case-patients was 8 years (range 4 months to 90 years), and 57% were female. the most frequently reported symptoms were diarrhea (97%), abdominal pain (77%), excessive tiredness (67%), and fever (66%). the median duration of illness was 7 days (range 144 days); 17 (15%) patients were hospitalized, and none died. case-patients and controls did not differ significantly according to age, sex, immunosuppressive therapy, treatment to reduce gastric acidity, or use of antibiotics. all s. typhimurium dt160 isolates were sensitive to ampicillin, cephalothin, chloramphenicol, ciprofloxacin, co-trimoxazole, gentamicin, streptomycin, sulfonamides, tetracycline, and trimethoprim. four represented different levels of contact with other persons with gastrointestinal illness (i.e., within 28 days of illness onset; within 3 days of onset; within the household; or outside the household). direct handling of dead wild birds, consumption of fast food, and consumption of food at a large gathering, such as at a party or large barbecue, were also significantly associated with illness. after stepwise regression, contact with a person with gastrointestinal illness in the 28 days before onset of illness in the case-patient (adjusted odds ratio [or] 2.8; 95% confidence interval [ci] 1.4 to 5.4), exposure to dead wild birds (adjusted or 10.5; 95% ci 2.3 to 47.5), and consumption of fast food (adjusted or 1.7; 95% ci 1.0 to 2.9) had independent significant associations with illness. dt, definitive type; d&v, diarrhea and vomiting; or, odds ratio; ci, confidence interval. twelve case-patients throughout new zealand indicated that they had drunk water from nonreticulated and untreated water sources. seven patients used roof-collected rainwater, and one used rainwater plus water from a dam. four of the five patients who had eaten raw eggs could identify the retail brand and outlet of purchase. these four patients had purchased six different brands of eggs from seven different retail outlets. samples for two brands were positive for s. thompson, both from shell surface washings. epidemiologic investigation of an outbreak of s. typhimurium dt160 infection in new from may to august 2001 found that contact with dead wild birds, contact with other persons with gastrointestinal illness, and consumption of fast food were all significantly associated with illness. in addition, s. typhimurium dt160 was found in roof-collected rainwater drunk by five patients. s. typhimurium dt160 had been previously identified as the cause of large numbers of sparrow deaths in new zealand in 2000, and analysis by pulsed-field gel electrophoresis (using the method described by barrett et al. and restriction enzyme xbai) demonstrated that bird and human isolates in 2000 were indistinguishable (4). in our study, information was not collected on exposure to environments contaminated by wild bird feces, such as parks and play areas, a fact that may have underestimated the avian contribution to human illness. s. typhimurium dt160 has previously been recognized as a bird pathogen in canada (11) and in england (12). before its emergence in new zealand, the human s. typhimurium dt160 infection had only been reported in the context of a 1979 institutional outbreak in the united kingdom, linked to food contamination by sparrow droppings (13). consumption of undisinfected water has previously been identified as a risk factor for salmonellosis linked to bird transmission (14). this risk factor was not confirmed by our case-control study, despite the finding of s. typhimurium dt160 in roof-collected rainwater. this discrepancy is probably because case-patients and controls were matched by neighborhood, and types of water sources are usually consistent within neighborhoods. the association of illness with contact with another person with gastrointestinal illness is likely underestimated because secondary salmonellosis cases were excluded. consumption of fast food was associated with illness; however, no single type of food outlet or food was identified. case-patients were equally likely to have eaten food from chain fast-food restaurants as from family-owned fast-food outlets. consumption of fast food may have occurred in environments contaminated by bird feces, or the foods themselves may have been contaminated, either during production or by infected foodhandlers (15). asymptomatic salmonella carriers would not have been excluded from selection as controls, potentially reducing the magnitude of observed associations. recall may have been influenced by delays between exposure and interview, although participants were asked to refer to a memory aid (personal diary or calendar). the investigation successfully excluded a single common source exposure for this outbreak and instead suggested that multiple exposures contribute to s. typhimurium dt160 infections in new zealand. strategies for addressing these exposures include routine treatment of roof-collected rainwater, hygienic disposal of dead birds, and promotion of hand-hygiene protocols and sick foodhandler policies in fast-food outlets. the source of this incursion of s. typhimurium dt160 into new zealand remains unknown: bird isolates have been exclusively from nonmigratory birds, s. typhimurium dt160 has not been identified in neighboring countries in the pacific basin, and early case-patients did not have a history of overseas travel.

an outbreak of human salmonella enterica serotype typhimurium dt160 infection in new zealand was investigated from may to august 2001. handling of dead wild birds, contact with persons with diarrheal illness, and consumption of fast food were associated with infection. contaminated roof-collected rainwater was also detected.

PMC2957965

pubmed-79

putting a halt to the rising trend in overweight prevalence has high priority in public health policy all over the developed world. prevention, preferably starting at an early age, is considered as the key strategy to achieve this goal, and considerable effort is put into the design of preventive measures. whereas we know in general terms that overweight is the result of an unbalance between energy intake and energy expenditure, development of effective preventive measures requires a much more specific understanding. specific dietary habits and (in)activities associated with overweight risk in specific age groups need to be identified and their quantitative contribution to the overweight prevalence in the population needs to be assessed. in addition, we need to know whether the importance of specific behaviors for the development of overweight differs between sub groups in the population. it is well established that children from families with a low socio-economic status and children with overweight parents are at increased risk to develop overweight [39]. it is therefore particularly important to assess the role of behavioral risk factors specifically in these children, since they are the priority target groups for interventions to prevent and reduce overweight. the aim of this study was to identify specific behavioral risk factors for overweight in young children and to assess their quantitative contribution to the prevalence of overweight in the general population and in high risk sub groups. in a large population-based birth cohort, we prospectively investigated associations between dietary habits, screen time and physical activity when the children were 5 and 7 years old and overweight at the age of 8 years. we also assessed the role of these behavioral factors separately in high risk sub groups: children of mothers with overweight and children of mothers with low education. we estimated the reduction of overweight prevalence that could be achieved if the prevalence of risk factors would be reduced. the study population consisted of children who participated in the dutch prevention and incidence of asthma and mite allergy (piama) birth cohort study. the baseline study population consisted of 4146 pregnant women, recruited from the general population in 1996-1997. postal questionnaires, including questions on the child's lifestyle and health, were sent to the parents during pregnancy, at the child's ages of 3 and 12 months, and yearly thereafter up to the age of 8 years. at the age of 8 years, the children were invited for a medical examination which included measurement of weight and height. the study protocol was approved by the medical ethics committees of the participating institutes, and all parents gave written informed consent. of the baseline population of 4146 pregnant women, 183 (5%) were lost to follow-up before any data on the child had been collected. parental completed questionnaires from age 3 months to 8 years were available for 3934, 3817, 3694, 3563, 3518, 3473, 3373, and 3269 children, respectively. when the medical examination of 8-year-old children was planned, 3668 children (93% of 3963) were still in the study, 3522 children were invited and 2214 participated (63% of those invited) and had their weight and height measured. 146 of the 3668 children were not invited for the medical examination, although they were still participating in the study. in the majority of cases the reason for this was that their families had moved and were now living too far away from the study centers where the medical examination was conducted. complete data on overweight and risk factors were available for 1871 children (47% of the 3963 children in the study at birth). during the medical examination of the 8-year-old children, weight was measured to 0.1 kg and height to 0.1 cm by trained research staff using calibrated measuring equipment. from the weight and height measurements bmi (body mass index: weight (kg)/height (m)) was calculated. overweight and obesity were defined according to age and gender-specific international standards that use cutoff points equivalent to the 25 kg/m and 30 kg/m cut-offs that are commonly used for adults. the term overweight is used for the total group of children who were overweight, including those who were obese. for physical activity, 3 indicators were selected: time spent walking or cycling to school (3 categories), membership of a sports club (yes/no), and time spent playing outside (3 categories). screen time (4 categories) included time spent watching television, video's, or at the computer. for dietary intake, 3 indicators were used: fast food consumption, snack consumption, and soft drink consumption. data on these exposures were obtained from the questionnaires administered at the ages of 5 and 7 years. these questionnaires contained a food frequency questionnaire with 40 different items and 5 answering categories per item (ranging from never in the last month to on 6-7 days per week), which was used to construct the dietary exposure variables. for fast food consumption a score was constructed based on the consumption frequencies of chips/french fries and of foods like hamburgers and hot dogs. snack consumption was based on the reported consumption frequencies of 7 different foods/food groups including pie/cake, muffins, candy bars, sweets, chocolate, biscuits, and crisps. soft drink consumption was based on the reported frequencies of consumption of 4 types of soft drinks. each of these variables was based on a number of different items, which differed with respect to their energy content. to be able to add up the different items, total weekly energy intake from each item was calculated based on the reported frequencies, assuming average portion sizes and average energy content per portion. data on maternal education and maternal weight and height were obtained from the questionnaire completed when the child was 1 year old. maternal bmi was calculated from reported weight and height and overweight and was defined as a bmi 25 kg/m. the associations between the exposure variables and overweight at the age of 8 years were analysed by logistic regression, adjusted for gender and birth weight. exposure variables were constructed using the data collected at both the age of 5 and at the age of 7 years in order to obtain a more stable estimate of exposure during the years preceding the measurement of weight and height. walking/cycling to school, playing outside and screen time were recorded in categories, numbered 1, 2, and 3 for walking/cycling to school, 1, 2, and 3 for playing outside, and 1, 2, 3, and 4 for screen time (see table 1). the data collected at the ages of 5 and 7 years, were combined by taking the mean of the numbers of the categories reported at the ages of 5 and 7 years respectively. this resulted in ordinal variables with 5 categories for walking/cycling to school (ranging from never at ages 5 and 7 to>1/2 an hour per day at both ages), 5 categories for playing outside (ranging from<1 x per week at ages 5 and 7 to>3 x per week at both ages) and 7 categories for screen time (ranging from<1/2 an hour per day at ages 5 and 7 to>2 hours per day at both ages). after having checked for nonlinearity fast food, snack and soft drink consumption at age 57 (the mean of the consumption scores at age 5 and at age 7), were included in the regression analyses as linear variables, but are shown in categories (tertiles) in table 1. overweight (including obesity) was used as the outcome measure in the regression analyses. the number of obese children was too small for analyses with fully adjusted regression models, and obesity was therefore not used as outcome measure in these analyses. in previous studies in the piama cohort, breast feeding was investigated in relation to overweight risk and was found to be associated with reduced overweight risk [12, 13]. although breastfeeding is not the subject of this study, results on breast feeding are shown in the tables for completeness. in additional analyses, the associations of physical activity and diet with overweight were also assessed separately for the exposures at age 5 and at age 7. thirteen percent of all the data potentially in the study (3963 participants 20 variables=79260) were missing and 2092 children (53%) had a missing value on at least 1 of the variables. if data are not missing completely at random (mcar), complete case analysis may lead to biased results [14, 15]. in our dataset several variables (including maternal education and breast feeding) were associated with the probability of a subject having one or more missing values. missing data were multiple times imputed, using the multivariate imputation by chained equations (mice) procedure [16, 17], that runs under the statistical program r version 2.5.0. for the multiple imputation we used all the data that were used in the analyses, as well as data on the children's weight and height that were measured and reported by the parents in the yearly questionnaires.. maternal education and maternal overweight could be confounders of the associations studied but could also be factors in causal pathways. in addition, the analyses were repeated, stratified for maternal education, maternal overweight and gender. differences between the associations observed in the different sub groups were tested by inclusion of interaction terms in the regression models. for the behavioral risk factors that were found to be statistically significantly associated with overweight, adjusted population attributable risk percentages (par%) were calculated, using the mantel-haenszel approach, as described by benichou. table 1 shows characteristics of the study population and the prevalence of overweight and obesity in different sub groups. children with incomplete data on either risk factors or on measured weight and height at 8 years were compared to the children with complete data, with respect to a number of characteristics assessed during the first year of the study (data not shown). children with incomplete data had, compared to children with complete data, a relatively high prevalence of low maternal education (27.5% versus 19.4%) and of no breast feeding (19.7% versus 15.9%). as expected, in the imputed data the prevalence of these characteristics was somewhat higher (23.6% and 17.9%, see table 1) than in the children with complete data. the prevalence of maternal overweight was similar in children with incomplete data and in children with complete data (24.1% and 26.1%, resp.). the prevalence of overweight in the children was almost the same in the imputed data and in the complete cases (13.8% and 14.4%, resp.) and the prevalence of obesity was the same in both datasets (2.7%). table 2 shows the associations between diet, physical activity, screen time and overweight. in the analyses adjusted for gender and birth weight only (model 1), fast food consumption and screen time, sports club membership, active transport to school, playing outside, snack consumption, and soft drink consumption were not associated with overweight. when all risk factors (including breast feeding) were included in the same regression model (model 2), the associations with overweight became somewhat weaker for most factors, indicating some association between the individual risk factors. the association of fast food consumption with overweight lost statistical significance in this analysis whereas the association with screen time remained statistically significant. surprisingly, an inverse association between snack consumption and overweight was observed. in additional analyses, the analyses shown in table 2 were repeated with the behavioral risk factors measured at age 5 and (separately) with those factors measured at age 7 instead of the variables combining the exposures measured at age 5 and age 7. for the physical activity indicators and for screen time, results of these analyses were similar to those shown in table 2, showing associations between screen time and overweight (aor (95% ci) at age 5: 1.35 (1.191.53) and aor (95% ci) at age 7: 1.22 (1.081.39)) but not between the physical activity indicators and overweight. snack consumption at age 5 was not associated with overweight at the age of 8 years (aor (95% ci): 0.87 (0.661.14)), but there was a significant inverse association between snack consumption at age 7 and overweight at the age of 8 years (aor (95% ci) 0.70 (0.540.91)). low maternal education and especially maternal overweight were strong predictors of childhood overweight (see table 2, model 1). the association between low maternal education and overweight weakened substantially in the model including the behavioral risk factors (an almost 25% change in the regression coefficient from 0.50 to 0.38), indicating that these specific factors explain at least part of the excess overweight prevalence in children of mothers with low education (see table 2, model 3). the association between maternal overweight and overweight in the child weakened only marginally in the model including behavioral risk factors (5% change in regression coefficient from 1.07 to 1.02), indicating that the association between maternal overweight and overweight of the child is only to a limited extent mediated by the behavioral factors studied (see table 2, model 4). the analyses were repeated after stratification for low maternal education, maternal overweight and gender, respectively. in the sub groups with low maternal education and with maternal overweight children of mothers with a low level education had a higher prevalence of high fast food and snack intakes, of>2 hours screen time per day and of not being member of a sport club than children of more highly educated mothers (see table 3). children of overweight mothers had a higher prevalence of high fast food intake and of>2 hours screen time per day (at the age of 5, but not at 7 years) than children of normal weight mothers (see table 3). girls had a slightly higher prevalence of overweight and slightly less screen time than boys (data not shown). the results suggest that the associations between screen time and overweight were weaker in children of mothers with overweight or low education than in children of mothers with a normal weight or higher level of education (table 4). interactions between the exposure variables and low maternal education and maternal overweight were not statistically significant, however. for boys and girls these associations were very similar (data not shown). of the behavioral risk factors studied, only screen time was statistically significantly associated with overweight. based on the prevalence and adjusted odds ratios for screen time>1 hr per day (see table 5), we estimated how much screen time contributed to the total prevalence of overweight. table 5 shows the adjusted population attributable risk percentage (par%) for a screen time of more than 1 hour per day at age 5 and/or age 7 as compared to less than 1 hour at both ages. of the total overweight prevalence in the low risk sub groups an estimated 17% could be attributed to screen time of>1 hour per day as compared to 10% in the high risk sub groups. this means that, if all children would reduce their screen time to less than 1 hour per day, the following reductions in overweight prevalence could be achieved: from 18.3% to 16.4% in children of mothers with low education; from 12.4% to 10.3% in children of mothers with higher education from 25.0% to 22.4% in children of overweight mothers and from 10.0% to 8.3% in children of normal weight mothers. all analyses were conducted in a data set containing only complete cases as well as in the imputed data. the odds ratios (95% ci) for screen time, for example, were 1.47 (1.241.75) and 1.41 (1.171.70) in model 1 and model 2, respectively, in the complete case analysis, compared to 1.45 (1.261.66) and 1.39 (1.211.61) in the analysis in the imputed data set. of 8 different potential behavioral risk factors, only screen time showed consistent and significant associations with overweight. in high risk sub groups of the population, screen time was higher than in low risk sub groups, but the associations between screen time and overweight were weaker (although not significantly weaker) in the high risk sub groups. important strengths of the study were the prospective design, the large study population, the repeated measurements (at ages 5 and 7) of different behavioral risk factors and the availability of measured (rather than parental reported) data on weight and height.however, a number of limitations have to be considered. thirteen percent of our data were missing and 53% of the study population had a missing value on 1 or more of the variables used in the analyses. we used multiple imputation to deal with missing data in our study in order to avoid bias due to selective missing of data and to make more efficient use of the available data. results of the complete case analysis and the analyses in the imputed data sets were similar. information on the behavioral risk factors was obtained from parental completed questionnaires and we therefore need to address the possibility of misreporting. the questions asked ranged from simple matter-of-fact items (like sports club membership yes/no) to items that are more difficult to observe and recall accurately (like the consumption frequency of a range of different food items). we expect that recalling or reporting problems will not have substantially influenced the data on sports club membership or transport to school and that any misreporting on these items will not have been systematically associated with overweight development. the data on foods and drinks consumption may well have been influenced by reporting bias. many of the foods included in the dietary variables have a bad reputation in relation to overweight, and selective underreporting by parents of children who were developing overweight may have occurred. another problem with respect to the diet variables is that data were available on the consumption frequencies of food items, but not on portion sizes. estimates of the intakes of fast food, snacks and soft drinks and the ranking of children according to their intakes were therefore based on reported consumption frequencies only, and the possibility of misclassification of food intake can not be excluded. membership of a sports club and active transportation to school were not associated with overweight in this population. the reason that we did not observe the hypothesized associations might be that in young children the duration and intensity of these activities are usually too low to have a sizeable impact on total activity. if this would be confirmed in further studies, it may have potentially important implications for the allocation of budgets for the prevention of overweight in this age group. in older children and adolescents active transport to school and sports club membership we found some evidence for an inverse relation between playing outside and overweight, but the associations were weak and not statistically significant. children who played outside 1 x per week seemed to have a higher overweight risk, but such children were a small minority in the study population. screen time was consistently and significantly associated with overweight and the association was largely independent of other lifestyle factors. a dose-response relationship was observed when it was included as a categorical variable in the regression analysis. our results thus suggest that, among the indicators of active and sedentary behavior that we studied, screen time is the most important risk factor for overweight in the age group studied. this result is in agreement with the conclusion of a recent study in which the evidence was summarized for six different strategies to prevent or treat pediatric overweight. our findings on screen time and overweight risk are also in line with the results of a comprehensive meta-analysis on the subject, which observed a statistically significant relationship between tv viewing and body fatness among children and youth. we estimated that a reduction in overweight prevalence of up to 2 percentage points could be achieved if all children would reduce their screen time to less than 1 hour per day. maximum achievable reduction of prevalence (marp) of 1.5 percentage points that was estimated for watching television>1 hr/day in a study on 5-6 year old german children. consumption of soft drinks and fast food at ages 57 was not associated with overweight whereas an unexpected inverse association between snack consumption and overweight was observed. snack consumption at the age of 5 years was not associated with overweight at age 8, but snack consumption at age 7 was inversely associated with overweight at age 8. as indicated previously, these results may have been influenced by selective underreporting of snack consumption by parents of children who were becoming overweight. besides selective underreporting, reverse causation may also have played a role, in the sense that parents of children who were becoming overweight really have reduced their children's consumption of specific foods and drinks. the results on fast food, snack and soft drink intakes seem to indicate that in particular the items included in our snack, variable are the kind of foods that come to parents ' minds when they think of trying to limit excessive weight gain in their child. the selective underreporting and reverse causation that are likely to be present in our dietary data, make it impossible to judge from our results how diet influences the development of overweight. we hypothesize that these mechanisms might play a role also in other epidemiological studies on diet and overweight. whereas, on theoretical grounds, it is hard to imagine that food intake is unrelated to weight status, observational studies have generally not produced consistent evidence for the expected associations [20, 2327]. our results do not provide an answer to the question whether consumption of soft drinks, fast food, and snacks causes overweight but seem to indicate that parents may see reduction of snack consumption as a method to counteract the development of overweight. further studies into the strategies that parents themselves use to influence their children's weight gain might provide insights that could be useful for the design of preventive strategies. low maternal education and maternal overweight were found to be strongly associated with overweight in the child. the association between low maternal education and overweight weakened substantially when the behavioral risk factors were included in the model, indicating that these factors explain part of the association. the behavioral risk factors had little influence on the association between maternal overweight and overweight in the child, indicating that other factors, possibly genetic factors, play a more important role in this association. we repeated the regression analyses in sub groups stratified by maternal weight status and maternal education. whereas in the high risk sub groups, screen time was higher than in low risk sub groups, the associations between screen time and overweight were weaker (although not statistically significantly) in these groups, and the population attributable risk percentage was lower than in the low risk sub groups. as the children of mothers with overweight and/or low education are the children at highest risk to develop overweight, this observation is potentially important for the development of preventive strategies and needs to be clarified in future observational as well as intervention studies. reduction of screen time should be part of interventions to prevent or reduce overweight in young children and could result in a reduction of overweight prevalence in the order of 2 percentage points in both high and low risk sub groups. our results also suggest however that interventions aimed to promote sports club membership, active transport to school, and playing outside may have little impact on the prevalence of overweight in this age group. the possibility that, due to reverse causation, associations between food intake and overweight can not be assessed in observational studies, needs further study.

objective. to prospectively identify behavioral risk factors for childhood overweight and to assess their relevance in high risk sub groups (children of mothers with overweight or low education). methods. in the piama birth cohort (n=3963), questionnaire data were obtained at ages 5 and 7 on screen time, walking or cycling to school, playing outside, sports club membership, fast food consumption, snack consumption and soft drink consumption. weight and height were measured at age 8 years. results. screen time, but none of the other hypothesized behavioral factors, was associated with overweight (aor 1.4 (ci: 1.21.6)). the adjusted population attributable risk fraction for screen time>1 hr/day was 10% in the high risk and 17% in the low risk sub groups. conclusion. reduction of screen time to<1 hr/day could result in a reduction of overweight prevalence in the order of 2 percentage points in both high and low risks sub groups.

PMC2915806

pubmed-80

the protection of water supplies and water distribution infrastructure such as reservoirs is of great concern for those defending against bioterrorism. the us military is concerned because there exist multiple biological warfare agents (bwas) that could be intentionally introduced into the water supply which could have serious implications for military operations as well as civilians located within a theater of operations. to protect potable water supplies, the department of defense (dod) seeks to develop a rapid field test that would determine if field supplies of water are safe to drink by the warfighter during a military operation. the us armed services have fielded biological defense systems that are designed to protect against a large scale attack via bwas dispersed through the air. these defensive systems utilize high-efficiency aerosol collectors that impinge the sampled air into a liquid buffer which is tested for bwas using antibody-based handheld assays (hhas). civilian first responders use similar versions of these antibody-based hhas for testing suspicious powder samples suspected to contain bwas. depending on which manufacturer of hha is being tested, the technology can be low cost, requires no electrical power, and requires minimal to no sample preparation to test a sample. it was an hha panel manufactured by the dod that first identified bacillus anthracis in the powder that spilled across a desk in senator daschle's office in the hart senate office building in 2001. three years later, a dod hha was again used to identify ricin toxin in the mail room of the dirksen senate office building. these instances demonstrate that, when hha technologies are used appropriately by an experienced operator trained to understand what the results mean, they can be a useful tool. additionally, hhas have proven fast and effective in the laboratory for the detection of francisella tularensis and biotoxins such as staphylococcal enterotoxin b (seb) and microcystins. the dod has initiated an effort called the joint chemical biological and radiological agent water monitor (jcbrawm) program to develop a system which will allow rapid field testing of drinking water supplies used by military personnel. the effectiveness of hhas in detecting biological agents in the air and in powders has resulted in a desire to test these assays to gauge their effectiveness to protect drinking water supplies used by us warfighters. in the present study, hhas from multiple manufacturers were evaluated for their effectiveness at detecting two specific targets, seb and ricin, in multiple water matrices. the hhas were not modified in any way to optimize for the detection of toxins within water supplies despite the fact that they were initially developed to test for concentrated agent collected from high-efficiency aerosol collectors and impinged into a phosphate buffered saline (pbs) solution rather than to test unbuffered water supplies. the ability of the hhas to detect seb and ricin in four different water matrices, representative of waters used by different branches of the armed forces, was evaluated. the four different water matrices were formulated tap water, formulated tap water with 1 mg/l free available chlorine (fac), water treated by reverse osmosis (ro) with 1 mg/l fac, and ro water with 2 mg/l total residual bromine. suggested health effect levels as used by the us military for ricin and seb are 455 ng/ml and 4.55 ng/ml, respectively. these minimal health effect levels were derived using the tri service standards for field water and based on an average consumption of 10 liters per day for seven days for a 70 kg service member. therefore, detection of ricin and seb in water at or below the suggested health effect level is desired. both singleplex (one-line) and multiplex (two-line and two-dot) hha models produced by two manufacturers were evaluated in the present study for the detection of ricin toxin and seb [5, 6]. active seb (toxin technologies, sarasota, fla, usa) and active ricin whole toxin (vector laboratories, burlington, on, canada) were obtained from the dod's critical reagents program (crp) for use in the present study. briefly, approximately 500 ml of astm type i deionized water was added to a one-liter volumetric flask to which each stock solution was added to achieve the desired final concentration and the total volume was brought to one liter with deionized water. after 1520 minutes, the ph was assessed and adjusted if needed (with minimal volume change) to 7.67.8. once prepared, synthetic tap water was stored at 4c with a shelf life of one week. briefly, one gram of calcium hypochlorite was added to twenty milliliters of astm type i deionized water and shaken vigorously for one to two minutes. briefly, approximately five microliters of the chlorine stock solution (supernatant, not solids) was added to 100 ml of the formulated tap water or ro water, which was prepared by running local tap water through a commercial ro unit. the fac was measured using a hach dr 2500 spectrophotometer per manufacturer's instructions, and the fac level was adjusted as needed to achieve the desired fac level of 1 chlorinated water was considered stable for only one day and therefore made fresh daily just prior to use. briefly, 25 ml of bromine resin was transferred to a 500 ml, 0.20 m filter unit (corning life science, lowell, mass, usa). total residual bromine in the collected water was measured using a hach dr 2500 spectrophotometer per manufacturer's instructions and adjusted as needed to achieve the desired final concentration of 2 mg/l. each water type was prepared fresh daily as described above and spiked separately with each target at desired concentrations. after spiking, 100 l of sample was loaded onto the sample well of the hha. the hha was allowed to develop for 15 minutes prior to reading the result by eye. each challenge concentration was tested a minimum of two independent times with a minimum of three handheld assays per concentration. limits of detection (lod) were determined for each hha type in each water matrix and defined as the minimum concentration of toxin that could be detected by eye. samples for circular dichroism (cd) were prepared by spiking ro water containing 1 mg/l fac with seb to a final concentration of 2 g/ml. control samples received no chlorine. after 24 hours, sodium thiosulfate (0.005% w/v final concentration; fisher scientific catalog number s446) was added to all samples to quench any remaining chlorine prior to analysis by cd. cd experiments employed a jasco model j-810 spectropolarimeter (jasco analytical instruments, easton, md, usa) equipped with a ptc-423s peltier thermoelectric temperature control system. cd spectra were obtained for seb at 25c from 260 to 190 nm with a data pitch of 0.5 nm, a band width of 1 nm, a response time of 1 sec, and a scanning speed of 10 nm/min. the cd was carried out on 2 g/ml samples in 1 cm quartz cuvettes and required long-time signal averaging. the concentration of seb was verified for a representative stock solution by uv absorption at 277 nm using an e for seb of 14. for each sample, the blank-subtracted cd curve for 100 averaged scans was converted to molar ellipticity using a molecular weight of 28,366 daltons and fit with the secondary structure estimation application in jasco's spectra manager (version 1.53.01) software to obtain the fraction of helix, beta sheet, turn, and random coil secondary structure. the percentages reported here were obtained using the reed reference set for the secondary structure calculations. samples for sds-page were prepared by spiking ro water containing 1 mg/l fac with seb to a final concentration of 2 g/ml. control samples received no chlorine. after one minute, one hour, and 24 hours, sodium thiosulfate (0.005% w/v final concentration) was added to all samples to quench any remaining chlorine prior to analysis by sds-page. reducing gel eletrophoresis was carried out using the mini-protean 2 cell and 1020% tris-hcl pager gold precast gels from lonza (rockland, me, usa). fifty nanograms of seb samples and 1: 20 diluted mark12 unstained standard (invitrogen) were prepared in sds sample buffer, followed by heating at 95c for 5 minutes prior to loading. running buffer was prepared by dilution from 10x tris/glycine/sds buffer (biorad). following electrophoresis, the gel was fixed with 50% methanol and 7% acetic acid (both v/v) for a total of 30 minutes. the rapid staining protocol with sypro ruby protein stain (invitrogen) was used for the detection of seb. the gel was washed with a solution of 10% methanol and 7% acetic acid (both v/v) for 30 minutes and imaged and documented with uv light using syngene (frederick, md, usa). all hhas were evaluated for their performance in formulated tap water, formulated tap water with chlorine, ro water with chlorine, and ro water with bromine for the detection of ricin and seb. each water type was spiked with multiple concentrations of each toxin and loaded onto hhas for testing until the lod was determined for each hha model. it was determined that ricin could be detected down to approximately 1 ng/ml for most hha models in both formulated tap water and formulated tap with chlorine, while exhibiting only slightly higher lods in ro with chlorine for most models. however, in ro with bromine, the lod for ricin increased to 3,000 ng/ml for all models tested. for seb, lods in formulated tap water were low, ranging from 0.9 ng/ml to 12.5 ng/ml. however, in both chlorinated and brominated water, lods increased to 2,500 ng/ml for singleplex hhas and even higher to 5,000 or 10,000 ng/ml for multiplex models. in all cases detection below the suggested health effect level was desired; however, seb was not detected below the suggested health effect level in chlorinated and brominated water and ricin was not detected below that level in brominated water. due to the increased detection limits observed in chlorinated and brominated water, cd spectroscopy, which assesses the three-dimensional fold of a protein, was performed on samples of untreated seb and seb exposed to chlorine in an effort to evaluate the basis for increased lods (figure 1). for each sample, the individual cd scans were compared prior to averaging to ascertain the structural stability of the sample. untreated seb at 2 g/ml was determined to contain 14.3% helix, 34.50% sheet, 17.6% turn, and 33.6% random coil (rmsd<0.1%). the cd spectrum of seb treated with 1 mg/l fac was stable over the time of experimentation. the curve-fitting calculation for chlorine-treated seb resulted in 25.9% helix, 0.0% sheet, 0.0% turn, and 74.1 random coil (rmsd<0.1%). sds-page was also performed to ascertain the overall effect of chemical reaction with chlorine on the molecular weight of seb. for the treated and untreated seb, 1.5 g of seb was loaded per well. as shown in figure 2, for untreated seb, the seb was visible on the gel as a band with a molecular weight of approximately 28 kda for all time points tested. for chlorine-treated seb, no band was visible at the same molecular weight as untreated seb for any of the time points tested. ricin and seb are considered potential biological threat agents as both can cause illness either by inhalation or ingestion. seb could potentially be used to attack low-volume water supplies; however, due to the fact that ricin is less toxic by oral ingestion, it may be a less likely threat to drinking water. however, detection of both agents in water is still a concern and technologies capable of detection in water must be investigated. performance of hhas for air and surfaces is well known; however, the use of hhas for detection of agents in water is less recognized. therefore, the application of hhas for detection in water was assessed in this study. in formulated tap water, detection of seb and ricin was achieved at or below the suggested health effect levels for each model of hha in almost every case. however, the presence of disinfectants proved problematic with regard to detecting either seb or ricin using all models of hhas. the increased lods observed when disinfectants were present are believed to be due to the degradation of the target or toxins by chlorine and bromine. therefore, it is likely that protein toxins would also be affected by these strong oxidants. in fact, sodium hypochlorite is known to completely inactivate seb and ricin toxin [11, 12]. therefore, when exposed to chlorine or bromine, protein toxins could become damaged or denatured causing antibodies present in an antibody-based assay, such as hhas, to no longer bind to the altered antigen as effectively. this decrease in binding efficiency could yield higher lods for the target, such as those observed in the present evaluation. to address this issue, cd spectroscopy and sds-page the cd results for untreated seb were similar to those obtained by singh et al. for seb at 0.5-0.6 mg/ml in 10 mm sodium phosphate. the cd results for chlorine-treated seb indicate a significant change in three-dimensional fold resulting in less well-defined structure for the chlorine-treated seb relative to the untreated seb. based on curve-fitting calculations, the percentage of random coil more than doubled upon treatment with chlorine. the secondary structure percentages obtained from the cd curve-fitting routine may well represent an average of multiple seb-based species in solution, which would be consistent with the sds-page results. the absence of bands for the chlorine-treated seb could occur if the seb was cleaved into smaller peptides that diffuse out of the gel or if multiple higher molecular weight species are formed, with no single species being concentrated enough to be visualized on the gel. in either of these cases, chiral components would be present in the sample and would lead to a measureable cd spectrum. the cd and page results together suggest a major change in seb structure that may result in multiple seb-based antigens, the majority of which are less easily recognized and not bound as effectively as untreated seb by the antibody present on the membrane of the assay. the decreased binding capacity for the seb-based antigen(s) would account for the increased detection limits seen for seb in chlorinated water. these results support the idea that increased lods in disinfected water are caused by disruption in antigen-antibody binding due to degradation of the antigen. in addition to the detection studies reported herein, currently studies are being performed to address whether both ricin and seb remain active, or toxic, after exposure to chlorine and bromine. however, regardless of whether activity remains, the ability to detect the presence of each toxin in disinfected water supplies is important. presence of these agents could indicate a potential compromise or attack on the water supply, thereby revealing security risks as well as potential health risks. additionally, it was noted in the present study that chlorine did not impact detection levels of ricin as significantly as detection levels of seb under the test conditions employed. additional exposure time may be required in order for ricin detection to be affected in the same manner as that observed for seb in chlorinated water. this could be due to a difference in protein structure or stability in chlorinated water between the two toxins. further tests allowing for prolonged incubation of ricin in chlorinated water prior to detection could address this issue.

development of a rapid field test is needed capable of determining if field supplies of water are safe to drink by the warfighter during a military operation. the present study sought to assess the effectiveness of handheld assays (hhas) in detecting ricin and staphylococcal enterotoxin b (seb) in water. performance of hhas was evaluated in formulated tap water with and without chlorine, reverse osmosis water (ro) with chlorine, and ro with bromine. each matrix was prepared, spiked with ricin or seb at multiple concentrations, and then loaded onto hhas. hhas were allowed to develop and then read visually. limits of detection (lod) were determined for all hhas in each water type. both ricin and seb were detected by hhas in formulated tap water at or below the suggested health effect levels of 455 ng/ml and 4.55 ng/ml, respectively. however, in brominated or chlorinated waters, lods for seb increased to approximately 2,500 ng/ml. lods for ricin increased in chlorinated water, but still remained below the suggested health effect level. in brominated water, the lod for ricin increased to approximately 2,500 ng/ml. in conclusion, the hhas tested were less effective at detecting ricin and seb in disinfected water, as currently configured.

PMC3139884

pubmed-81

with the goal of discovering the genes that contribute to the risk of common diseases, numerous susceptibility loci have been identified using linkage analysis. despite replication of many of these linkages in a second sample, some exceptions include tbc1d1, identified as the gene responsible for the obesity linkage on chromosome 4p15-14, and hoxb13, identified as the gene responsible for the prostate cancer linkage on chromosome 17q21-22. however, for many other common disease linkage signals, the underlying causal genes and variants await discovery. the expectation that a single gene accounts for a linkage peak may contribute to the difficulty in identifying causal genes. on the contrary attributed a triglyceride linkage on chromosome 7q36 to variants in five genes and christians et al. found that fine-mapping caused a single quantitative trait locus (qtl) for body size in mice to resolve first into three qtls, then one of those to split into two. furthermore, the clustering of causal genes may have stymied the multi-group effort to identify type 2 diabetes (t2d) genes on chromosome1q; two strong associations present in populations of european ancestry failed replication and confirmation in other ethnic groups. these examples suggest that abandoned linkage findings might yet reveal susceptibility genes if reappraised while considering the possibility of multiple causal genes. as for other common diseases, the challenge is even greater in african american (aa) populations where both prevalence and genetic diversity are higher, and pathophysiology may differ. genome wide case-control association studies (gwass) have identified single nucleotide polymorphisms (snps) with small effects on t2d risk, but few of the associated snps, identified primarily in european ancestry populations, are replicated in aas and few snps have been identified in aa populations. in general, the widespread rejection of family studies in favor of gwass has failed to produce the promised prognostic and diagnostic variants for t2d. as a resource for the discovery of genes related to t2d and its complications, the american diabetes association established the genetics of niddm (gennid) study. from 1993 to 2003 we have used this resource to increase understanding of the genetics of t2d by applying linkage and family-based association analysis to the african american (aa) subset of the gennid sample. genome-wide linkage analysis using genotypes on 5,914 snps identified chromosomal regions that potentially harbor risk genes for t2d and age of t2d diagnosis (aod). the strongest signal for t2d occurred on chromosome 2 at 95121 megabases (mb), with weaker support for aod in the same region and for t2d at 6895 mb. both t2d and aod also showed linkage on chromosome 13 at 1930 mb, but linkage was limited to t2d on chromosome 7 at 5079 mb and to aod on chromosome 18 at 3165 mb. other analyses inferred pleiotropy with triglyceride in the chromosome 2p region and pleiotropy with obesity in the chromosome 13 region. herein we present linkage and association analyses on the aa subset of the gennid sample using genotypes on 9,203 fine-mapping snps added to the genome scan snps used previously. our first goal was to more precisely localize the t2d susceptibility genes in each of the five chromosomal regions identified in the genome scan (chromosome 2 at 6895 and 95121 mb, chromosome 7 at 5079 mb, chromosome 13 at 1930 mb, and chromosome 18 at 3165 mb). our second goal was to test for association of the gene-based fine-mapping snps with t2d and aod in order to identify genes related to t2d in aas. the gennid study ascertained families through sibling pairs each with a t2d diagnosis. during phase 1, extended family members were also studied; one site ascertained aas. during phase 2, data collection beyond the sibling pair was limited to parents, or, if parents were unavailable, unaffected siblings; five sites ascertained aas. during phase 3, only affected sibling pairs and trios were studied; 5 additional sites ascertained aas. in total, 1,496 aa members of 580 pedigrees were studied at 10 sites. data cleaning and re-evaluation of t2d diagnoses by current criteria reduced the analysis sample to 1,344 members of 524 pedigrees; for this study, we selected an informative subset of 1,077 members of 415 pedigrees. t2d diagnoses, originally following national diabetes data group criteria, were re-evaluated using current criteria prior to all analyses; 84 affected and 13 unaffected sample members were re-assigned as unknown. body mass index (bmi) was computed from height and weight obtained from physical examination. we selected a subset of the sample for fine-mapping by excluding unaffected individuals below age 53, the age by which onset had occurred in 75% of cases, and then any individual, either affected or unaffected, who consequently had no family members in the sample. this sample subset maintained significant lod scores in each of the linkage regions and produced power over 80% to detect association. power to produce a p-value of 0.00001 was estimated from simulation of 1000 replicates of a snp with minor allele frequency 0.4 and heterozygous effect a 10% increase in penetrance at age 50 for t2d or 3 year increase in aod. for fine-map genotyping, we used snagger (http://snagger.sourceforge.net/) to select tagsnps within genes in the linkage regions on chromosomes 2, 7, 13, and 18. using hapmap phase 1 and 2 (http://hapmap.ncbi.nlm.nih.gov), variants in the region were tagged at linkage disequilibrium (ld) r 0.7. each snp selected had minor allele frequency>0.1 and illumina design score>0.4; snp pairs had minimum spacing of 60 base pairs (bp). the genetic map locations of the snps in centimorgans (cm) were obtained from the rutgers combined linkage-physical map of the human genome (http://compgen.rutgers.edu/maps); locations of snps not mapped directly were estimated using physical positions from ncbi dbsnp build 123 in a smoothing calculation (conway institute bioinformatics service; http://integrin.ucd.ie). genotyping errors were identified using pedcheck and merlin; we zeroed 4978 genotypes of 710 snps, 74 identified by pedcheck and 4904 by merlin. genotypes for 24 snps, available on less than 95% of the sample following data cleaning, were retained after confirmation of minimal effects on the results. multi-point identity by descent (ibd) probabilities were computed at each cm using merlin, treating as haplotypes snp sets with pairwise ld r>0.7. we identified 279, 175, 133, and 259 snp sets on chromosomes 2, 7, 13, and 18, respectively. likelihood analysis, as implemented in jpap, was used for univariate linkage analysis of unadjusted t2d (ut2d), t2d adjusted for bmi (bt2d), and aod and for bivariate association analysis of ut2d and aod. aod was adjusted for gender and modeled as a normal density with mean, standard deviation, and gender effect as parameters. t2d risk was modeled to account for aod in affected pedigree members, while allowing for censored observations with age, gender, and bmi (for bt2d only) effects and penetrance as parameters. for each trait, additional parameters included heritability, a quantitative trait locus (qtl) effect in linkage analysis, and an additive snp effect in association analysis. we applied variance components linkage analysis using the univariate model for each trait in conjunction with the ibd probabilities. at each cm across the 5 regions, all parameters were estimated for aod while only heritability and qtl effect were estimated for ut2d and bt2d with all other parameters for those traits fixed at estimates obtained upon maximizing the likelihood while correcting the likelihood for the ascertainment of each pedigree through an affected sib pair. the lod score at each cm was computed as the common logarithm of the ratio of the maximized likelihoods with the qtl effect estimated to the maximized likelihood with the qtl effect set to zero. we assessed each fine-mapping snp for association with ut2d and aod by coding the snp genotype as an additive covariate and testing its effect on both traits in a bivariate model. p values were determined using a 2 degree of freedom statistic computed as twice the natural logarithm of the ratio of the maximized likelihood with covariate effects estimated for both t2d and aod to the maximized likelihood with both effects set equal to zero. we controlled for multiple testing separately within each of the 5 linkage regions by specifying a false discovery rate of 0.05 accounting for 1291, 1780, 1819, 1336, and 2977 fine-mapping snps for chromosomes 2p, 2q, 7, 13, and 18, respectively. we tested for an independent effect of each secondary associated snp, conditional on the snp within the same gene that attained the highest significance, by comparing the maximized likelihood estimating t2d and aod covariate effects for both snps simultaneously to the maximized likelihood estimating t2d and aod covariate effects for the most significant snp alone. p<0.05 after bonferroni correction for the number of secondary snps in the gene supported the independence of the secondary snp. we genotyped 9,203 snps within five regions encompassing a total of 133 cm and 128 mb (table 1) and merged them with 244 (36 duplicates) genome scan snps. spacing between snps averaged 0.0145 cm (13,910 bp) and ranged from 0.0 to 5.3 cm and from 26 bp to 6 mb. to comply with the sample size limitations of the cidr genotyping platform, we selected a subset of the original linkage sample to be informative for both linkage and association. the genotyped subset included 1077 members of 415 pedigrees that ranged from 2 to 11 members (1 to 8 were genotyped). in this subset, aod and bmi differed little from the complete sample, but the unaffected sample members, selected for a minimum age of 53 years, were older (table 2). we previously identified 5 linkage peaks within four chromosomal regions from autosomal scans of t2d and aod; the chromosome 2 region contained 2 peaks separated by 30 mb and the centromere. using updated genetic map positions for the genome scan snps, we repeated the linkage analyses on the smaller sample: the genome scan lod scores for all 5 peaks (table 3) generally agreed with our published lod scores for bt2d and aod and for ut2d. upon adding the fine-mapping snps, linkage evidence remained in all 5 regions, but strengthened only on chromosome 2 (table 3). on chromosome 18, only aod supported linkage, although ut2d provided weak support (lod=2.62) upon elimination of sample members with bmi>45. the most remarkable effect of fine-mapping was the splitting of the lod score curves into multiple peaks in all of the regions except on chromosome 18. additional evidence that multiple susceptibility genes contribute to the linkage signals derived from the identification of associated snps that reside in multiple genes within each of the 5 regions, including on chromosome 18 (table 4). the number of associated snps ranged from 4 on chromosome 13 to 17 on chromosome 2q; the number of associated genes ranged from 2 on chromosome 13 to 9 on chromosome 7. although 20 of the 27 associated genes were identified through a single snp, 5 genes were identified through 2 or 3 snps and arhgap25 and dpp10 were identified by 8 and 10, respectively. however, six associated snp pairs exceeded ld r 0.7: arhgap25 (rs6714065 and rs7605681), ctnna2 (rs968820 and rs1368915), dpp10 (rs843417 and rs1823267, rs10204212 and rs13432035, rs4848376 and rs11694256), and hip1 (rs1179625 and rs1179622). nevertheless, conditional association analysis supported the independence of two or more snps in arhgap25, polr1b, dpp10, and mtus2 (supplementary table s1). the fine-mapping of five chromosomal regions allowed us not only to confirm our genome scan linkages, but also to infer the presence of multiple t2d susceptibility genes in each of the regions. two types of evidence supported the presence of multiple susceptibility genes underlying the linkage peaks. first, except chromosome 18, each lod score peak split into at least two peaks when fine-mapping snps were added to the linkage analysis. second, in every region including chromosome 18, two or more potential susceptibility genes were identified through the association with t2d and aod of fine-mapping snps residing in those genes. in fact, martin et al. attributed many failures to identify causal variants to the incorrect assumption that a single or a limited number of variants are responsible for a linkage signal. as evidence, they identified variants in five genes that fully accounted for a linkage peak for plasma triglyceride level. if small effects are typical of common disease susceptibility genes, their detection through linkage analysis may be limited to locations where the genes cluster. however, fine-mapping using next generation sequencing may provide sufficient power necessary to detect even isolated genes. although chromosome 18 failed to show t2d linkage and chromosome 7 failed to show aod linkage, we nevertheless expect that the susceptibility genes residing in all regions both increase t2d risk and decrease aod. variation between regions in the information available undoubtedly affects the relative strength of the corresponding lod scores for t2d or aod. as evidence, the elimination of t2d cases with bmi>45 kg/m revealed a weak t2d linkage on chromosome 18 and fine-mapping revealed aod linkages on chromosomes 2p and 2q. the lod scores are generally lower for aod than for t2d as expected since aod reflects onset age imprecisely and the familial correlation of aod may partially result from temporal clustering of t2d diagnoses among relatives. nevertheless, aod proved sufficiently accurate to produce linkage evidence in 4 of the chromosomal regions. few of the associated genes have previously been reported to associate with t2d or related traits. the exceptions include grb10 with t2d, nedd4l with diabetic nephropathy, and lipg with lipid metabolism. novel candidates reported herein include a cytokine (il36b) and genes involved in lipid metabolism (acoxl) and cell-cell and cell-matrix adhesions (magi2, cldn4, ctnna2). interestingly, the candidates also included genes involved in williams-beuren syndrome (wbscr28, wbscr17, cldn4), whose sufferers have a high prevalence of diabetes and pre-diabetes. dpp10 is related to dpp4, whose inhibitors are oral anti-hyperglycemics used in t2d therapy, and dpp6, which contributes to a triglyceride linkage, however, unlike dpp4, ddp10 lacks serine protease/dipeptidyl peptidase activity. support for the functionality of these genes derived from evidence that their expression levels are affected by our associated snps or proxies with high ld. we tested 14 snps in the 5 genes for which expression levels from transformed lymphocytes were measured on a subset of 160 sample members; nominal significance was obtained for one: p=0.0148 for an effect of rs7579103 on arhgap2 expression levels. in other populations and various tissues, expression levels of arhgap2, aff3, polr1b, hip1, lipg, and nedd4l showed evidence of an effect of an associated snp or its proxy (p<1105). for further insight into the nature of the associations, we haplotyped sets of 1530 fine-mapping snps, each encompassing two or more of the associated snps reported in table 4. for arhgap25, two haplotypes associated with t2d: the first extended across 4 of the associated snps and contained the risk alleles for all 4; the second extended across 6 associated snps and contained the risk alleles for all 6, but differed from the first haplotype for alleles at other snps. we propose that each haplotype harbors a distinct causal variant, despite sharing associations with the same risk alleles, demonstrating the complexity of disease associations. the advantages include the opportunity to exploit information on local ancestry to localize disease susceptibility genes. as with any such analysis, confirmation of these findings awaits replication in another sample. in summary, linkage and association analysis using genotypes on an additional 9,203 fine-mapping snps added to five chromosomal regions confirmed each linkage and identified potential susceptibility genes in each of the regions.

we previously localized type 2 diabetes (t2d) susceptibility genes to five chromosomal regions through a genome-wide linkage scan of t2d and age of diagnosis (aod) in the african american subset of the gennid sample. to follow up these findings, we repeated the linkage and association analysis using genotypes on an additional 9,203 fine-mapping snps selected to tag genes under the linkage peaks. in each of the five regions, we confirmed linkage and inferred the presence two or more susceptibility genes. the evidence of multiple susceptibility genes comprised: 1) multiple linkage peaks in four of the five regions; 2) association of t2d and aod with snps within 2 or more genes in every region. the associated genes included 3 previously reported to associate with t2d or related traits (grb10, nedd4l, lipg), and 24 novel candidate genes including genes in lipid metabolism (acoxl) and cell-cell and cell-matrix adhesion (magi2, cldn4, ctnna2).

PMC3692593

pubmed-82

the circadian system organizes the different biological functions in 24 h, such as sleep/activity, temperature, heart rate, glucose level, cortisol production, and oxidative stress. in mammals, this system is organized by a central clock localized in the suprachiasmatic nucleus of the hypothalamus (scn) and in a series of peripheral oscillators such as the liver, lung, adrenal gland, fibroblast cells, and others tissues [2, 4]. the peripheral oscillators are synchronized every day via nervous or humoral signals, and the most important humoral signal is the melatonin hormone, secreted by the pineal gland during the dark hours, and its impairment is associated with different disorders such as insomnia, cardiovascular disease, and cancer. the molecular clock is organized by transcriptional/translational feedback loop of clock genes named clock, bmal1, per13, and cry1-2. at the molecular level, the complex clock-bmal1 stimulates the expression of negative regulators per13 and cry1-2, and their protein inhibits the effect of the heterodimer clock/bmal1 [6, 7]. moreover, the molecular clock has different modulators which give fine tuning of output signals such as rev-erb, a negative regulator of bmal-1 expression [79], sirt1, a regulator of clock-mediated acetylase activity [10, 11], and pgc1, a stimulator of bmal1 expression [12, 13] (see figure 1). this system provides an output signal to genes such as hexokinase, dbp [1517], vegf, steroidogenic enzymes star, 3-hsd, and wee-1, giving a circadian oscillation of physiological functions such as metabolism, angiogenesis, cortisol production [2, 19], and cellular proliferation. the molecular clock can be modified by environmental changes and our modern lifestyle, resulting in physiological alteration and risk of pathologies; for example, during the monkey's pregnancy, the light exposition during night hours induces a lower body temperature and absence of circadian rhythm of temperature in the newborn. in humans, different reports showed that the alterations of the circadian system increased the risks of cancer [2123], preeclampsia, diabetes [3, 25], and mood disorder. curiously, a large quantity of polymorphisms has been detected in clock genes, which can be influencing the development of diseases through different physiological systems. this review will primarily focus on the hypothesis which states that the variation of genetic components of the circadian system, similar to environmental changes, can affect several physiological systems and produce an elevation of the risk of developing a disease. in recent years, there has been a significant increase in available information from polymorphic variations and epigenetic modification over clock genes and their risk of diseases. the report of health from the united states (2014) showed about 10% of population have a poor health associated with pathologies such as obesity (35.5%), hypercholesterolemia (30%), diabetes (32%), and cancer (6.4%). curiously, despite its low frequency in population, cancer is the second cause of death in the united states. the above pathologies add the infertility, which affects about 10.9% of women in the united states and 17% of women in other developed countries. during short-stay in the hospital, mood disorder and psychosis are the principal causer of hospital stay during 2014 and represent the third cause of morbidity worldwide. this present research reports the last single nucleotide polymorphism (snp) associated with an elevated risk of developing principal pathologies worldwide such as mood disorder, infertility, cancer, metabolism and diabetes and addictions, and a description of usefulness for a more detailed study in other pathologies. in mammals, the circadian system is a supraphysiological system that regulates biological functions every 24 h such as glucose homeostasis, temperature, blood pressure, tone in the coronary artery, and heart rate. the bmal1/clock complex target genes are related to the circadian expression of metabolic pathways as was observed by hatanaka et al. through high resolution the author detected a circadian expression of (i) glucose metabolism-related genes such as glut2, por, pck1, and gys2 and (ii) cholesterol metabolism-related genes such as cyp2a4, cyp2a5, cyp4a14, cyp7a1, and cyp2c55; and considering the very important role these systems have in metabolism, any alteration of them can have negative health effects. today there are several metabolic problems affecting the population, such as obesity, hyperglycemia, dyslipidemia, and hypertension. the cooccurrence of three or more metabolic disorders, including obesity, is defined as metabolic syndrome, showing a prevalence in the united states during 2009-2010 of about 22% of adults (21.4626.15). the insulin resistance or type 2 diabetes is one of the most important metabolic diseases currently affecting about 366 million people, causing more than 3.8 million deaths, and showing a rise in the number of affected people as observed in the united states population during 19992010. several factors could be increasing the risk of death by diabetes, such as dyslipidemia, hypertension, and obesity, all of which are driving to an elevated risk of cardiovascular disease. a variant of this pathology can also develop during the pregnancy (gestational diabetes mellitus) and is characterized by glucose intolerance in the mother and adverse consequences for her offspring, such as an increase in blood pressure, body mass index (bmi), and body fat and a decrease in hdl levels. at the molecular level, both diseases have in common the impairment of signal transduction of insulin receptors such as pi3kinase/akt and ras/map kinases [42, 45, 46], leading to metabolic dysregulations such as inhibitions of glycogen synthesis, impaired translocation of glut4 to plasmatic membrane, or antilipolytic effects of insulin in white adipose tissue [39, 46]. meta-analyses from 448 articles reporting shift work and health consequence have detected a strong relationship between shift work, a potent chronodisruptor, and diabetes mellitus type 2 (odd ratio about 1.42 times higher than day workers). however, this is not the only chronodisruption which we are exposed too. in animal models exposed to light/dark patterns similar to shift work, the length of the working day and changes of feeding times induce a modification of the liver weight and plasmatic glucose and a modification of the circadian profile for glucose, insulin, and triglyceride. these findings add to the observation in the circadian disruption in mutant animals, showing alterations of cholesterol metabolism, abolition of the circadian production of glycerol, free fatty acids, and impaired expression of rate-limiting lipolytic enzymes such as lipase, all of which add to increased weight gain, adipocyte hypertrophy, high level of glucose, glucose intolerance, and hypersecretion of insulin [4952]. polymorphic variations of clock genes can be incrementing the risks of developing a disease similar to chronodisruption by environmental changes as it has been detected in human metabolic disorders (see table 1). for example, the genotyping of clock genes in 346 greek pregnant women and their risk of diabetes was performed, detecting that the polymorphisms of bmal1 rs7950226 and rs11022775 are associated with gestational diabetes mellitus (p=0.025, or=1.46 and p=4.455e 06, or=2.64, resp.), while the study of the haplotype analysis of rs7950226/rs11022775 showed a major frequency in women with gestational diabetes mellitus (p=0.0069, or=6.96). similarly, a study performed in subjects from the united kingdom and pakistan reported that cry1 and cry2 polymorphisms rs2292912 and rs12315175, respectively, are associated with diabetes (p=0.015 and 0.008, resp.). moreover, the variant rs12315175 for cry2 has a tendency to elevate the risk at about 5% compared to the variation of cry1 (or=1.05 and 0.95, resp.). also, the authors did not find associations between diabetes and bmal1 polymorphisms rs7950226 and rs11022775, as occurs in gestational diabetes mellitus. genotyping of 19,000 adults from northern sweden for variants rs8192440 for cry1 and rs11605924 for cry2 is associated with higher level of glucose concentrations at 2 hours (p=0.06 and p=0.005, resp.). another study genotyping 1304 individuals from 424 british families, containing at least one patient with diabetes type 2 (diabetes in families study collection), demonstrates the relation between bmal1 polymorphisms rs7950226 and rs11022775 and diabetes (p=0.002), reinforcing what was previously described. similar relations are observed for bmal2 polymorphism rs7958822 in obese men and women (or 2.2 and 2.7, resp.) and the deletion/insertion of 54 base pair sequences of five repeat alleles on per3 gene (rs57875989) [57, 58]. in contrast, the per2 polymorphism rs7602358 is associated with a protection from type 2 diabetes in the uk population, which suggests that not all polymorphisms are negative for health. at the level of dyslipidemia, an interesting correlation has been detected between small dense ldl level and polymorphism of clock genes. in individuals carrying the polymorphism associated with the clock gene (rs1801260), the genotype tt or tc showed a major level of small dense ldl, which leads to increased triglycerides and increased risk of cardiovascular and obesity diseases, similar to the metabolic syndrome. likewise, genotyping in the japanese population identified clock gene polymorphism rs1801260 associated with higher odds ratio (or; 1.5) of type 2 diabetes; in contrast, a multicenter study detected the clock polymorphism rs4580704 is associated with prevention of diabetes and cardiovascular disease. the haplotypes of rs10002541 and rs4864546 from clock (cg and tg variations) are associated with abdominal obesity in chinese population (or 0.74 and 1.70, resp.) and polymorphism rs4864546 is associated with low level of hdl/apolipoprotein a1 ratio in spain. moreover, clock polymorphisms rs12649507 and rs3749474 are associated with higher intake of polyunsaturated fatty acid and fat intake, which can modify the body mass index (bmi). this is an antecedent which, bearing in mind the ideas above, suggests the genetic components of the circadian system as a critical factor in the development of metabolic diseases. in humans, infertility affects about 9% of couples, and about one-third of infertility cases associated with idiopathic male infertility are multifactorial, with 50% due to genetic abnormalities. the circadian system is important during reproduction and development [89, 90]; for example, it is involved in the timing of the lh surge [90, 91], stimulation of ovulation [90, 92], and regulating the level of steroidogenic acute regulatory protein (star) expression and is critical for cholesterol translocation inside mitochondria and sperm count. the genetic factors of clock genes are implicated in the pathogenesis of infertility (see table 2), as it occurs in male partners of infertile couples. the genotyping of male partners detected a correlation between single nucleotide polymorphism of clock rs11932595, rs6811520, and 6850524, with infertility (p<0.05 and or ranged between 1.4 and 1.9). similarly, the analysis of bmal1 polymorphism rs4757144 in slovenian and serbian caucasian men showed a significant correlation with infertility (p=0.047), suggesting that clock genes clock and bmal1 contribute to successful fertilization. mood or affective disorders are important causes of morbidity, where we can highlight depressive pathologies and hypomaniac and maniac disorders as major diseases affecting the population [29, 66], and it can be highlighted that depressive pathologies are the third largest source of morbidity in the world. curiously, a potent correlation has been detected between mood disorders, sleep/activity, and the circadian system suggesting that the circadian system can be modulating the neuronal activity. during pregnancy, the prevalence of mental illnesses in women is about 8%, suggesting an imbalance between physiology of sleep and/or the circadian system. this can be observed in the onset of a mental disease as has been observed in the impaired circadian production of melatonin during the pregnancy of depressed women (approximately 34 weeks of gestation), which shows an advance onset time of melatonin production of about 40 minutes and a minor production during the dark hours [97, 98]. moreover, workers who do shift work (circadian disruption) showed decreased alertness, cognitive functions, mood, social and work activities, and health [26, 99], which are associated with an impaired circadian system via melatonin suppression, all of which leads to the appearance of a mood disorder. for this reason, we can say that the circadian system may contribute to the risk of developing a mood disorder and the genetic component of clock genes can be involved in the development of mood pathologies (see table 2). at the genetic component level of the circadian system, a study of 744 people who carry polymorphisms rs2291739 and rs11171856 from tim, a member of the clock gene family which interacts with per1-2 proteins [100, 101], showed that they have an elevated risk of developing mood disorders ranging between 19 and 23% (or 1.19 and 1.23, resp.). moreover, gene variants of positive regulator clock have also been associated with mood disorders. people who carry the variants rs1801260 and rs11932595 showed a major risk of developing a bipolar disorder ranging between 45 and 37%, respectively, in comparison to a patients without polymorphism (or 1.45 and 1.37, resp.). in the same study, it was also observed that the polymorphisms rs2291739 and rs11171856 from tim gene are associated with unipolar disease, with a risk ranging between 37 and 40% in comparison to a patient without polymorphism (or 1.37 and 1.40, resp.)./rs11022779g/rs1122780 t (haplotype) are associated with mood disorders and bipolar disease [29, 66]. it has also been seen that the bipolar pathologies are associated with other genetic variations of bmal1 gene (rs4757144, rs1982350, and rs1481892) and the negative regulator per3 (rs2859387). curiously, a study performed an indian families reported the bmal1 polymorphisms rs2279287 are associated with seasonal affective disorder. in addition, the gene variant of cry2 gene (rs4132063) and deletion/insertion of 54 base pair sequences on per3 gene (rs57875989) may also be contributing to mood disorders via increased risk of developing bipolar disease. for example, a study in south india showed the prevalence of five repeat homozygotes from rs57875989 is associated with bipolar disease (or 1.72) but not with schizophrenia. at the level of depressive pathologies, a study performed in china compared 485 subjects (control) to 105 patients suffering from a depression disorder. the genotyping for clock genes showed that the variants of cry1 (rs2287161) and cry2 (rs10838524) are correlated to the depression disease with an odds ratio of 1.75 (p=0.012), which suggests that a patient with the allele has 1.75 times more risk of developing depression. moreover the authors detected a single nucleotide polymorphism for tef gene, the variant rs738499, is associated with 2.22 times more risk of development of depression (odds ratio of 2.22. finally, in a study performed in young adults, the risk of excessive intake of alcohol is minor when the polymorphism of per2 gene rs56013859 is present, suggesting a possible protector role for allelic variation of clock genes in addictions. in fact, this suggests that some polymorphisms are not negative for our health and it is necessary to study them in greater depth. cellular proliferation is a critical event for the survival and restitution of tissues of all living things and the circadian system is capable of delivering temporary information to the cell cycle [102104]. however, an uncontrolled cellular proliferation and an excessive tissue growth are observed by an altered cellular cycle during cancer, a pathology that showed a higher incidence in patients exposed to an impaired circadian system such as a shiftwork [106, 107]. the cell cycle is a finely regulated process from a cell that is capable of generating multiple cells through a series of cell divisions, including four critical and successive steps named g1 phase (growth phase 1), s phase (synthesis), g2 (growth phase 2), and m phase (mitosis). during the cell cycle, cyclin-dependent kinases (cdk) are critical for the transition between different stages, for example, cdc2 plus cyclin b protein kinase form the complex cdk1 which regulates g2/m transition [108, 109]. during dna replication, there are a number of controls or checkpoints that are critical in the cell cycle when dna damage is detected: the activation of the rad protein, which induces the action of cds 1 proteins, is triggered, as well as chk1 which are involved in cell cycle arrest via action of wee-1 and mik-1 proteins. however, the efficiency of this process changes depending on the time of the day when the lesion occurred. a lesion in the liver, occurring during the last light hours, induced a massive entry to m phase compared to a lesion which occurs early in the morning, which shows that the hour of surgery and the circadian system are critical for liver regeneration. similarly, oral mucosa is a highly proliferative tissue and it showed a circadian expression of clock genes bmal1, per1, and cry1 and thymidylate synthase activity, critical for dna synthesis during phase s. curiously, the peak of per1 mrna precedes the peak of thymidylate synthase activity, which suggests that the circadian system modulates the cellular proliferation in mucosa. wee-1 gene regulates cellular proliferation, and its promoter has three conserved sequence cacgtg (e-box) critical for the circadian expression of wee-1. its protein is capable of inactivating the cdc2-cyclin b complex via phosphorylation which inhibits transition between g2/m. curiously, knock-down of bmal1 in carcinoma cells of the colon (c26 cells), fibroblast cells (l929 cell), and intestine epithelial cells (iecs) produces cellular proliferation in vitro and increments the size of tumor cells injected subcutaneously, via the inhibition of apoptosis and the reduction of the time transition between g2/m, reduction of p53 and wee-1 expression. moreover, the knock-out for clock genes bmal1 y per2 in mice previously exposed to gamma radiation caused in mice hyperplasic growth and development of lymphoma, hepatic carcinomas, ovary tumors, and osteosarcomas via reduction of p53 expression. a precedent that reinforces the idea that clock genes can be modulators of cellular cycle via wee-1 and p53 proteins. at the genetic component level, different studies reveal the importance of the polymorphic variant of clock genes (see table 3) and how the chrono-type modulates the risk of cancer, such as observed in breast cancer, in which the risk is more elevated in premenopausal women (or, 2.43 and 2.55; resp.). similarly, women showed evening or night preference such as shown in the case of a study performed on norwegian nurses working in night shift which revealed two polymorphisms associated with breast cancer. the risk is incremented when women spend more time working during night hours, but this risk is higher when women have the alleles for bmal1 rs2290035 (or 1.91), rs969485 (or 1.64), and rs3903529 (or 2.77) or variant rs3750420 (or 1.6) of roreb gene. similarly, an incremented risk is associated with breast cancer in women from france when they have the allele rs11932595 in clock gene (or=0.74) or in connecticut (usa) when they have the polymorphisms rs7698022 (or 1.34) and rs1048004 (or, 1.43). curiously, when norwegian nurses are exposed to shift work during four nights, they showed three times more risk (or 2.75) of developing breast cancer when they were carrying clock polymorphism rs11133373, which suggests that the disruption of endogenous circadian rhythm by polymorphism associated with clock genes increases the risk of cancer. moreover, clock gene expression is induced in breast tissue from patients with breast cancer; this expression is associated with hypomethylation of clock gene. the silencing of clock gene lowered when women carry polymorphisms associated with cancer such as rs10448004 and rs7698022, which suggests that clock gene is a critical protagonist of cancer development. at level of colorectal cancer, a strong correlation is detected between cancer and the genetic component of the circadian system. for example, a polymorphism in the clock gene (rs1801260) showed a major prevalence in a cancer patient (p<0.0001, or=1.78 for c-allele) compared to a control patient. similarly, a screening performed in south carolina, usa, showed that a variation by deletion/insertion of a 54 base pair sequence on per3 gene (rs57875989) is associated with a higher risk of colorectal adenoma formation with odds ratio within 2.15.1. moreover, a population study performed on the residents of king county, washington (usa), detected six different types of polymorphism, which are significantly associated with the risk and aggressiveness of prostate cancer. these genetics variants are rs1012477 for per3 (or 1.3), rs7602358 for per2 (or 1.24), and rs2289591 in per1 (or 1.7). similarly, a genotyping of a patient with prostate cancer showed a strong correlation between cry1 polymorphisms rs7297614, rs1921126, and rs12315175 and fatal prostate cancer (or mean within 1.52) and a study conducted in china showed that the cry2 variant rs1401417 and the deletion/insertion of 54 base pair sequences on per3 gene (rs57875989) were associated with a major risk of developing prostate cancer (or; 1.7 and 1.3, resp.). a study performed in brazilian patients with pulmonary cancer showed a strong correlation of per3 polymorphism rs228644 and the risk of cancer (or 1.99). moreover, the authors reported the ancestral haplotypes for per3 rs228729, rs228727, rs707467, rs228644, and rs10462020 are associated with a higher cancer frequency; similarly, a meta-analysis performed by literature search showed that the variant insertion/deletion of per3 rs57875989 is associated with an increase of cancer susceptibility in about 17% or 70%. in a similar way, a soft risk is associated with breast cancer in premenopausal women from india, which suggests there is a relation between per3 and cellular proliferation. the frequency analysis of 1,538 breast cancer cases and 1,605 controls in china for clock gene variants showed the strong associations between three snps in circadian clock genes and the risk of developing breast cancer. the variants for cry1 rs1056560 are correlated to cancer, which elevate the risk in about 11% (or 1.11); per2 rs934945 in about 15% (or 1.15); and clock rs3805151 in about 35% (or 1.35). in contrast, tim protection for breast cancer development is detected in patients that carry the c-allele of rs7302060 (or, 0.54). the g allele of rs2291738 and the c-allele of rs7302060 are associated with reduced risk of breast cancer among estrogen receptor () or progesterone receptor () positive breast cancer cases (or, 0.46 and 0.36, resp.). the exogenous expression of human clock in cell lines of colorectal carcinoma induces the cellular proliferation in about 28%. in contrast, knock-down of endogenous clock gene expression inhibits the cell proliferation in about 34%. moreover, exogenous clock inhibits the apoptosis (42% reduction) via inhibition on apoptosis associated proteins expression of bax and bid and the increase of phosphorylation of akt. in vivo experiments by xenograft transplant of colorectal carcinoma cell line transduced with clock increase the tumor volume and tumor weight in about 61% and a 91%, respectively. however, the pharmacological inhibition of cry in human breast cancer by treatment with pharmacological agent ks15 inhibits the proliferation and cell viability by stimulation of wee-1 expression and stimulates the activity of heterodimer complex bmal1:clock [112, 113]. curiously, two polymorphisms for clock gene cry2, rs11038689 and rs1401417, have a protective action over the mammary cancer, which suggests that not all polymorphisms associated with clock genes are negative for our health. non-hodgkin's lymphoma is characterized by lymphoproliferation and advanced clinical stages, invasion to other tissues, and death. the estimated deaths from non-hodgkin's lymphoma in the united states amounted to 19,790 during 2015. analysis of cry2 variants showed the polymorphisms rs11038689, rs7123390, and rs1401417 increase the risk of lymphoma (or, 2.34; 2.40 and 2.97, resp.). moreover, a minor association of clock gene variants and glioma is detected for per1 rs2585405, clock rs11133391, and cry1 rs12315175 (or, 1.16; 1.08 and 1.02, resp.). genotyping of han chinese patients diagnosed with primary hepatocellular carcinoma showed an association between single snps of per3 rs228669 and cry1 rs3809236 with odds ratio of 1.41 and 1.26, respectively. these precedents reinforce the idea that clock genes can be modulators of cellular cycle and that modulates the risk of cancer. the circadian system is a supraphysiological system which modulates different physiological systems, and any alteration of this can have a negative impact on human health. different chronodisruptors have been described in literature such as light/dark pattern and inhibition of melatonin production as occurs in shifts work. however, other factors can be contributing on a minor scale, such as mealtimes or a genetic component. the relevance of the genetic variation of clock genes and how it can interact with the environment is unknown. but it has been described that the genetic component in the population predisposes the development of different pathologies such as diabetes, dyslipidemias, obesity, mood disorders, and addiction, all of which suggest the importance of this system to our health. however, it is also necessary to say that the genetic component could be protecting our health such as in the case of polymorphisms associated with per2 and cry2 genes in diabetes, alcohol intake, and cancer. these findings will need to be implemented and evaluated at the genetic interaction level and also the way in which the environment factors trigger the expression of these pathologies will be examined. finally, prospective studies are necessary to assess the predictive potential of these markers and to implement early treatment with consequent cost reduction for the health system.

the circadian system is a supraphysiological system that modulates different biological functions such as metabolism, sleep-wake, cellular proliferation, and body temperature. different chronodisruptors have been identified, such as shift work, feeding time, long days, and stress. the environmental changes and our modern lifestyle can alter the circadian system and increase the risk of developing pathologies such as cancer, preeclampsia, diabetes, and mood disorder. this system is organized by transcriptional/tranductional feedback loops of clock genes clock, bmal1, per13, and cry1-2. how molecular components of the clock are able to influence the development of diseases and their risk relation with genetic components of polymorphism of clock genes is unknown. this research describes different genetic variations in the population and how these are associated with risk of cancer, metabolic diseases such as diabetes, obesity, and dyslipidemias, and also mood disorders such as depression, bipolar disease, excessive alcohol intake, and infertility. finally, these findings will need to be implemented and evaluated at the level of genetic interaction and how the environment factors trigger the expression of these pathologies will be examined.

PMC4893437

pubmed-83

cellular adaptation requires biochemical processes including post-translational mechanisms to modify existing proteins. catalyzed by opposing kinases and phosphatases, reversible phosphorylation of serine, threonine, and tyrosine residues is now appreciated as a fundamental regulatory mechanism with the majority of phosphorylation (> 99%) occurring on serine and threonine residues [1, 2]. due to their untapped therapeutic potential, protein phosphatases have been identified as promising targets for xenobiotic manipulation through rational drug design (reviewed in [36]). in particular, the ubiquitously expressed protein phosphatase 2a (pp2a) has been proposed as a target for the treatment of a number of pathologies ranging from neurodegenerative diseases such as alzheimer disease to a variety of neoplasias [79]. compared to other members of the phosphoprotein phosphatase (ppp) superfamily of serine/threonine phosphatases, a detailed understanding of the mechanism by which pp2a recognizes substrates and mediates site-specific dephosphorylation remains to be developed. sequence and structural homology of the catalytic subunits of ppp family members has revealed a conserved catalytic mechanism in which a divalent metal cation activates a water molecule to hydrolyze phospho-serine/threonine without the formation of a phosphoenzyme intermediate (reviewed in [1012]). despite a shared catalytic mechanism, substrate specificity within the ppp family for example, the ppp calcineurin (also known as protein phosphatase 2b) has been shown to interact with two consensus sequences, pxixit and lxvp, found on nonsubstrate-interacting proteins and target substrates (reviewed in [11, 13]). for protein phosphatase-1 (pp1), substrate specificity is conferred by incorporation of pp1-interacting proteins via a conserved docking motif with a general consensus sequence of rvxf (reviewed in [11, 12]). at present, consensus sequences in pp2a substrates have not been identified. this review will focus on our emerging understanding of pp2a substrate specificity, which appears to involve additive effects of multiple discrete interactions. pp2a is a highly conserved serine/threonine phosphatase which, depending on the tissue of origin and cell type, may account for up to 1% of cellular protein and the majority of serine/threonine phosphatase activity. the physiological functions of pp2a have been implicated in all facets of cellular existence (reviewed in). further, pp2a functions as a critical tumor suppressor whose interruption leads to proliferative diseases. the heterotrimeric holoenzyme is composed of a catalytic subunit (c) a scaffold subunit (a) and one member of four families of regulatory subunits (b) (figure 1). the diversity of pp2a heterotrimers is achieved through expression of two c subunits, two a subunits and approximately fifteen b subunits in vertebrates. the b subunits are derived from four diverse gene families (b, b, b, and b) that have little sequence similarity between families but maintain high sequence similarity within families. the b family (b55, pr55, ppp2r2) of regulatory subunits consists of four genes (, ,,), the b-family (b56, pr61, ppp2r5) is comprised of five isoforms (, ,,,), the b family (pr72, ppp2r3) includes three isoforms (/pr72/130, /pr59, /pr48), and the b family (pr93/pr110) is made up of three proteins (sg2na, striatin, and mmob1). there is some controversy as to whether the b family members, most notably sg2na, are bona fide pp2a regulatory subunits that always associate with the ac dimer or whether they are merely regulated by association with the pp2a dimer. given the large number of pp2a subunits, it is thought that each cell expresses a dozen or more distinct holoenzyme complexes which act on a diverse array of substrates. pp2a holoenzyme diversity has been the subject of several excellent papers [14, 15, 18]. like pp1, regulatory subunit incorporation is thought to dictate the substrate specificity of the pp2a complex, however, only recently have molecular studies begun to develop insight into the mechanism by which the regulatory subunit acts. the results from recent studies suggests a multitiered mechanism wherein pp2a substrate specificity arises from (1) subcellular localization of pp2a defined by the b subunit, (2) selective holoenzyme assembly by posttranslational modification, (3) interaction with specific endogenous inhibitors, (4) interactions between the b subunit and phosphosubstrates at sites distant from the active site, and (5) b-subunit residues which infiltrate the catalytic cleft of the c subunit. this paper will provide a summary of these studies and how the understanding of the determinants of pp2a substrate specificity has advanced. the heterotrimeric holoenzyme is targeted to discrete subcellular locales dictated in part by which b-regulatory subunit is incorporated. the localization imparted by the b-regulatory subunit dictates the spatial sphere of influence of the holoenzyme complex for potential substrates. this mechanism of targeting pp2a activity is highlighted by extensive studies of the b family of regulatory subunits. for instance, the b family regulatory subunits target the holoenzyme to different cellular compartments in the brain. specifically, b and b are primarily cytosolic where as the b-regulatory subunit associates with a detergent-resistant protein fraction consistent with an interaction at the cytoskeleton. similar diversity has been observed in the b family of regulatory subunits. a c-terminal nuclear export signal common to b, b, and b which, when these regulatory subunits are incorporated into the pp2a holoenzyme, results in cytoplasmic localization of the heterotrimer. b and b isoforms lack a similar sequence and are found primarily in the nucleus. in cardiomyocytes, b interacts with the protein ankyrin-b through its c-terminus which leads to localization at the cardiac m-line. b on the other hand has been shown to target the holoenzyme complex to subnuclear structures in cardiomyocytes where pp2a/b may regulate gene expression. loss of proper subcellular targeting of pp2a has been implicated in the biogenesis and aggressive phenotype of neoplastic growths. specifically, a truncated form of b (b), has been isolated from a melanoma cell line wherein the pp2a/b complex is targeted to the trans-golgi network, blunts p53 responsiveness, contributes to genetic instability and increases metastatic motility [2427]. this was first observed in a member of the b family, b. two neuron-specific isoforms of b, b1, and b2, are generated by the use of an alternative 5 exon which results in the production of a divergent n-terminal extension on b2 [28, 29]. the n-terminal extension of b2 directs the holoenzyme complex to the outer mitochondrial membrane (omm) by targeting the mitochondrial translocase complex and forming an abortive complex resistant to import into the mitochondrial matrix [28, 30]. the omm-directed pp2a/b2 complex promotes fragmentation of the mitochondria reticulum and increases cell susceptibility to proapoptotic insults through an unknown mechanism. alternative splicing of b-regulatory subunits directing subcellular localization of the pp2a holoenzyme has also been observed in both the b and b families. two b isoforms differ by the inclusion or exclusion an n-terminal nuclear localization signal leading to isoform-specific nuclear or cytoplasmic localization. the murine-specific b family member, b, is also subject to alternative splicing leading to isoform-specific nuclear or cytoplasmic localization through an as yet unidentified mechanism. for example, the interaction between pp2a/b and shugoshin during meiosis is crucial for spatial and temporal regulation of sister chromatid disjunction. during meiosis, the cohesin complexes, which link the arms of bivalent chromosomes and the centromeres of sister chromatids, must be released in a stepwise fashion by the protease separase; the cohesin complex is firstly hydrolyzed along the arms of the bivalent chromosomes for completion of anaphase i and secondly at the centromeres of sister chromatids for completion of anaphase ii. during anaphase i, the centromeric cohesin complex is protected from separase-dependent proteolysis by shugoshin [34, 35]. shugoshin recruits pp2a/b to the centromere which likely results in dephosphorylation of the cohesin complex leading to protection of the cohesin complex from separase-dependent proteolysis [36, 37]. through cocrystallization, xu and colleagues, have revealed that dimeric human shugoshin 1 interacts with pp2a/b through a coiled-coil region across a broad composite surface of the c and b subunits. incorporation of specific regulatory subunits is influenced by reversible posttranslational modification of the c subunit. many groups have shown that the c-terminus of the c subunit is modified through phosphorylation and methylation on y307 and l309, respectively [3946]. phosphorylation of y307 is catalyzed by src kinase and is likely opposed by pp2a-catalyzed autodephosphorylation of this phosphotyrosine. phosphorylation of y307 selectively inhibits recruitment of the b family and some b family members to the dimeric ac complex whereas b recruitment is not effected. methylation of the c subunit at the c-terminal l309 is catalyzed by the protein phosphatase methyltransferase (ppm1) and is opposed by the phosphatase methylesterase (pme-1) [4751]. reversible methylation of pp2a is absolutely critical as knocking-out pme-1 in mice changes the phosphoproteome and results in early perinatal lethality. further, methylation of the c subunit is a dynamic process which plays a role in cellular response to acute stimuli. the recruitment of the b subunit to the ac dimer has been postulated to require methylation of the c subunit for some of the b-subunit families. however, conflicting results have been reported that may reflect differences in experimental design and will be discussed further. studies wherein pp2a/b is isolated from intact cells have revealed that methylation of the c subunit at l309 is required for incorporation of the b family of regulatory subunits into the holoenzyme complex [39, 40, 42, 44, 54, 55]. conversely, in vitro assembly of the pp2a/b holoenzyme complex does not require methylation of the c subunit for incorporation of the b family of regulatory subunits [56, 57]. similarly controversial, the requirement for c subunit l309 methylation was observed to be study-specific in in vitro pp2a/b timer formation [58, 59]. methylation was dispensable for isolation of the pp2a/b holoenzyme complex from intact cells. the role of methylation of the c subunit in recruitment of b and b families of regulatory subunits is less controversial with methylation of the c subunit being dispensable [44, 54]. for more information, posttranslational modifications which influence formation of the pp2a holoenzyme complex also occur on the b subunit. pp2a/b negatively regulates the erk map kinase signal transduction pathway. through formation of a ternary complex of the early response gene product iex-1, b1, and erk, erk mediates its own disinhibition by phosphorylation of b1 on s327 leading to b1 disassociation from the pp2a holoenzyme. since s327 is conserved among b-subunit family members, it is likely that other b subunits are regulated similarly. additionally, b is likely phosphorylated on s167 to disrupt the b subunit from the ac dimer in early mitotic stages. however, pp2a/b activity is necessary to resolve the mitotic spindles and conclude mitosis; therefore, autodephosphorylation may occur on b to allow efficient pp2a/b heterotrimer formation and cell cycle progression. thus, phosphorylation of the b-regulatory subunits also influences holoenzyme assembly and, therefore, substrate specificity. phosphorylation of the b subunit of heterotrimeric pp2a also potentiates the catalytic activity of the holoenzyme complex. in response to activation of d1 dopamine receptors on striatal neurons, camp-dependent protein kinase a (pka) phosphorylates b at s566 increasing activity of the pp2a/ b holoenzyme towards dopamine- and camp-regulated neuronal phosphoprotein (darpp-32) [64, 65]. dephosphorylation of t75 on darpp-32 by pp2a/b disinhibits pka-mediated phosphorylation of darpp-32 at t34 which converts darpp-32 into a potent pp1 inhibitor leading to changes in neuronal signaling. this circuit acts to attenuate phospho-t75 inhibition of t34 phosphorylation of darpp-32. this circuit has been shown to be differentially regulated by psychomotor stimulants and antipsychotics acting on different striatal neuron subpopulations. while assembly of the many trimeric pp2a holoenzymes directs cellular localization and substrate specificity, further regulation is afforded through binding of specific protein inhibitors of pp2a. one such inhibitor set (i2/taf-1) is upregulated during the progression of chronic myelogenous leukemia through bcr/abl activity and results in decreased pp2a activity. an additional pp2a inhibitor is the protein cip2a (cancerous inhibitor of pp2a), overexpression of which is associated with several human malignancies. cip2a associates with c-myc to protect its phosphorylated s62 from pp2a-directed activity stabilizing the c-myc protein and allowing it to promote oncogenesis. recently an interplay between the drosophila serine/threonine kinase greatwall (gwl) and pp2a/b was observed during mitotic entry in two separate studies [69, 70]. pp2a/b activity prevents mitotic entry by maintaining cdc25 in a dephosphorylated and inactive state. gwl reverses this inhibition through the phosphorylation of s67 of both -endosulfine (ensa) and cyclic adenosine monophosphate-(camp-) regulated phosphoprotein-19 (arpp-19). phosphorylation converts ensa and arpp-19 into very specific inhibitors of pp2a/b activity and produces activation of cdc25 leading to cell cycle progression. similar cell cycle regulatory activity has been observed with the mammalian ortholog of gwl, mastl; however, the mastl-pp2a interaction has yet to be characterized. once targeted to specific subcellular locales, the pp2a holoenzyme must recruit and dephosphorylate target substrates. recent structural studies have begun to suggest the mechanism by which the regulatory subunit of pp2a mediates initial binding to target substrates. other -propeller proteins have been shown to bind ligands in the central depression on the top surface of the toroid. crystallization of pp2a/b revealed a cluster of acidic residues in this depression that is available to recruit potential substrates containing a basic motif. in this same study, the acidic central depression of b was experimentally confirmed to bind the microtubule-associated protein tau, an established pp2a/b substrate. several conserved aspartate and glutamate residues in b engage in weak, electrostatic interactions across a large basic portion of tau and support dephosphorylation of tau at multiple sites through cycles of binding and unbinding. structurally divergent b family members may recruit substrates in a similar fashion as the b family. the crystal structure of pp2a/b shows that the b subunit contains 18 stacked -helices which adopt 8 huntingtin elongation a subunit tor- (heat-) like repeats [58, 59]. a portion of these heat-like repeats interact with the a subunit of the holoenzyme to mediate regulatory subunit incorporation into the holoenzyme complex. like the b subunit, an acidic patch is exposed in the b family of regulatory subunits and may mediate protein-protein interactions and substrate recruitment. structural studies of pp2a have revealed a conserved loop in the b family of regulatory subunits which infiltrates the catalytic core of the holoenzyme [58, 59] (figure 2(a)). at the tip of this loop is a conserved glutamate residue, e153 (b numbering), which contacts through its carbonyl oxygen the catalytic subunit and through its carboxyl group a cocrystallized microcystin molecule in the active site. mutational analysis revealed that e153 is an absolute requirement for efficient dephosphorylation of tyrosine hydroxylase (th), a known pp2a/b substrate, as well as other as yet unidentified cellular substrates of this pp2a holoenzyme. further, it was determined that e153 of b interacts with r37 and r38 of th to mediate dephosphorylation of both s31 and s40 on th. positively charged residues in the vicinity of target phospho-serine/threonine residues could represent a consensus sequence for b-subunit-mediated dephosphorylation. further, the infiltrating loop is likely a conformationally dynamic structure which is not sterically hindered by surrounding portions of the a, c, or b subunits. since r37/38 are important for dephosphorylation of both upstream (s40) and downstream (s31) sites, it appears that the orientation of phosphopeptides relative to the catalytic cleft is not constrained by additional interactions. collectively, the above observations support a model in which the sites of interaction between the substrate and the b regulatory subunit that are distant and near the active site together control substrate specificity. first, the interaction occurring at sites distant from the active site increases the local substrate concentration. following this initial substrate recruitment, although divergent in its sequence, an analogous structure from the -propeller fold of the b family of regulatory subunits extends to the catalytic core of the holoenzyme (figure 2(b)). this loop places conserved residues of the b-family subunits very near the holoenzyme active site. unpublished observations generated in our lab suggests that of these loop residues k87 of b2 may play a similar role as e153 of b in site-specific dephosphorylation of target substrates; however, further characterization of this substrate specificity loop is required. pp2a is a ubiquitous protein phosphatase responsible for the dephosphorylation of many different intracellular targets. the diverse repertoire of potential substrates for pp2a is imparted by the incorporation of one of fifteen unique b-regulatory subunits. recent studies have increased our understanding of the mechanisms by which the b subunit imparts specificity to the holoenzyme complex. through selective incorporation of the b-regulatory subunit, the holoenzyme complex is recruited to discrete subcellular locales which define the sphere of influence for the phosphatase. secondly, interactions between endogenous inhibitors and specific pp2a heterotrimers further restrict phosphatase activity. as shown for the b family, regulatory subunits mediate low-affinity interactions with substrates to increase the local concentration of substrates. through a flexible substrate selectivity loop which contacts the catalytic subunit, interactions between the regulatory subunit and phosphosubstrate may mediate multiple nearby dephosphorylation events. with the current structural information available for the pp2a complexes, future high-resolution studies will further define the molecular mechanism of pp2a substrate specificity. as general, inhibitors of pp2a are either clinically irrelevant or toxic, as in the case of the small molecule inhibitor microcystin, novel methods to increase the specificity of pp2a inhibition or activation must be developed. a clearer understanding of the pp2a substrate specificity mechanisms will serve as the foundation for rational drug design of selective inhibitors and activators of specific pp2a holoenzyme complexes.

protein phosphatase 2a- (pp2a-) catalyzed dephosphorylation of target substrate proteins is widespread and critical for cellular function. pp2a is predominantly found as a heterotrimeric complex of a catalytic subunit (c), a scaffolding subunit (a), and one member of 4 families of regulatory subunits (b). substrate specificity of the holoenzyme complex is determined by the subcellular locale the complex is confined to, selective incorporation of the b subunit, interactions with endogenous inhibitory proteins, and specific intermolecular interactions between pp2a and target substrates. here, we discuss recent studies that have advanced our understanding of the molecular determinants for pp2a substrate specificity.

PMC3132988

pubmed-84

dysphagia is the subjective awareness of difficulty in the passage of solids or liquids from the oropharynx to the stomach. dysphagia can be classified into an oropharyngeal or an esophageal location, and it is caused by neuromuscular motility disorders and mechanical obstruction.1) mechanical dysphagia is associated with intrinsic or extrinsic compression, resulting in progressive intolerance to solids. the term, dysphagia aortica, has been used to describe difficulty in swallowing caused by external compression from an ectatic, tortuous, or aneurysmal aorta as a result of age-related degeneration.2) dysphagia aortica is classically seen in elderly women with short stature who have hypertension and kyphosis.3) we report herein a patient with dysphagia associated with an aortic aneurysm. an 86-year-old woman presented with worsening nausea and vomiting. because of her progressive dysphagia to solids for the last 6 months, she had ingested only semisolids and liquids. three days before seeking evaluation at our hospital, she had difficulty in swallowing liquids, along with nausea and vomiting. the medical history revealed that she had been diagnosed with primary hypertension, an ascending aortic aneurysm, congestive heart failure, moderate aortic regurgitation, and moderate mitral regurgitation 6 years previously. because of old age she had undergone vertebroplasty due to multiple compression fractures of the thoracic and lumbar vertebrae 5 years ago. on admission to the hospital the blood pressure was 130/90 mmhg, the pulse rate was 64 beats/min, the respiratory rate was 28 breath/min, the body temperature was 36.0, the height was 1.43 m, and the body weight was 37 kg. the physical examination showed a diastolic murmur at the right upper sternal border and a pansystolic ejection murmur at the left lower sternal border. the laboratory findings were as follows: the white blood cell (wbc) count was 5,100/mm, the hemoglobin was 11.8 g/dl, the platelet count was 151,000/mm, the blood urea nitrogen (bun) was 39.8 mg/dl, the creatinine was 1.5 mg/dl, the total protein was 5.8 g/dl, the albumin was 3.2 g/dl, the lactate dehydrogenase (ldh) was 546 her chest radiograph revealed blunting signs at both costophrenic angles, cardiomegaly with a cardio-thoracic ratio of 0.8, and an enlarged, tortuous aorta (fig. 1). computed tomography (ct) of the chest demonstrated an enlarged, tortuous aorta (fig. the diameters of the ascending thoracic aorta, the descending thoracic aorta, and the proximal abdominal aorta were 7 cm, 6 cm, and 5.3 cm, respectively. the upper gastrointestinal barium study revealed marked extrinsic compression of the distal esophagus just above the esophagogastric junction (fig. we concluded that the symptoms and the results of the imaging studies were consistent with dysphagia aortica. although we recommended surgical correction of the aortic aneurysm or percutaneous endoscopic gastrostomy, the patient declined any invasive procedures and she was transferred to a nursing home on the 12 hospital day. the esophagus normally begins on the right side of the thoracic aorta and then descends. then, the esophagus lies on the left side of the aorta and penetrates the diaphragm through the diaphragmatic histus.4) the aging process and the accompanying degenerative changes with the loss of elasticity causes a dilated, elongated, and distorted aorta, which may result in a so-called reverse c- or reverse s-shaped aorta.3) as a result, the esophagus is pushed and compressed by the aorta against the cardiac chambers, which are anterior in location.5) there is no gold standard diagnostic procedure for dysphagia aortica. the association of suggestive symptoms, such as progressive intolerance to solids with concomitant weight loss along with the results of imaging and other diagnostic studies provide a high index of suspicion.6) the diagnostic work-up includes radiologic, endoscopic, and manometric studies. on a standard chest radiography and ct scan, the enlargement of the aortic arch and the tortuous dilated aorta can be observed. a barium swallow test may show partial esophageal obstruction and pulsatile movement of the barium synchronous with aortic pulsation.7) endoscopy reveals pulsatile extrinsic compression and stenosis of the lower esophagus with proximal dilatation. esophageal manometry may demonstrate a localized high pressure band with superimposed pounding that is synchronous with the cardiac pulsation.8) however, the typical findings of dysphagia aortica can be inconsistent. the radiographic findings are often inconclusive because a dilated, tortuous thoracic aorta is frequently encountered in elderly patients with dysphagia. although a ct scan is useful for evaluating not only the aortic lumen, but also the aortic wall,9) occasionally this is of no value in assessing compression of the esophagus by the aorta.2) false negative results are common for barium swallow studies. in addition, the classical manometric features suggestive of dysphagia aortica also occur in normal subjects.8) the differential diagnosis of dysphagia aortica includes various common structural and neuromuscular abnormalities.1) gastroesophageal reflux disease and motility disorders are common causes of dysphagia. the co-existence of these conditions and the lack of sensitivity and specificity of the usual diagnostic tests make it difficult to diagnose dysphagia aortica with certainty. wilkinson et al.2) demonstrated that a video solid bolus swallow test could be useful in determining the manometric findings that are suspicious for dysphagia aortica when the standard evaluation fails. mild cases may be treated conservatively, such as avoiding sticky solids and feeding on a liquid diet. the surgical procedures include transposition of the distal esophagus, separation of the distal esophagus from the aorta, esophagomyotomy, division of the right crus of the diaphragm, aortic resection, and repair of an aneurysm. for patients who are not candidates for surgery, insertion of a feeding tube via percutaneous endoscopic gastrostomy (peg) although we did not perform esophageal manometry, the patient's symptoms and imaging studies were consistent with the classic findings of dysphagia aortica. although the patient declined any invasive procedures, a peg for a feeding tube might have been helpful for nutritional support. dysphagia aortica is an uncommon type of dysphagia that is caused by extrinsic mechanical compression. it should be differentiated from other causes of dysphagia, such as gastroesophageal reflux disease or motility disorders, because dysphagia aortica often requires surgical intervention that can significantly reduce the morbidity and these interventions can be curative in some situations.

dysphagia aortica is difficulty in swallowing caused by extrinsic compression of the esophagus due to an ectatic, tortuous, or aneurysmatic atherosclerotic thoracic aorta. this condition is very uncommon, and it is usually associated with old age, women with short stature, hypertension, and kyphosis. we report herein a case involving a patient with dysphagia who had an aortic aneurysm.

PMC2771838

pubmed-85

from now on, molecular study regarding female sexuality has focused on female sexual hormone, like estrogen receptor (er) and er derivatives.12 but flibanserin is an agent of 5-hydroxytryptamin (5-ht) type 1a receptor and an antagonist of 5-ht type 2a and thus a new molecular entity.3 flibanserin was originally developed as antidepressant drug. in clinical stage 2a as antidepressant, flibanserin could not prove its effectiveness, but almost no sexual function disorder was reported in subjects. for this reason, the arizona sexual experiences scale (asex) was used for making a comparison of the sexual function effectiveness of flibanserin on antidepressant vs. placebo proven in stage 2b studies among four depression programs.4 stage 2b studies have failed to prove its general effectiveness on depression, but in answering this question " how intense is your sexual desire? " in the asex, flibanserin turned out to be more excellent in both placebo and active-comparator. since then, drug development has changed its directivity toward being a potential medicine for treating hypoactive sexual desire disorder (hsdd). hsdd is chara or absence of sexual fantasy and sexual desire associated with personal pain in the 4th edition of the diagnostic and statistical manual-text revision (dsm-iv-tr).5 determining lack or absence is done by clinicians by considering the factors influencing sexual activitie such as age and contexts of personal life. sexual function disorder is not explained well by other axis 1 disorders (except other sexual function disorders) and also it is not that it is caused only by direct physiological effects of materials (i.e., drug overuse, drug) or overall medical status. the dsm-5 completed in 2013 addresses new illness called as female sexual interest/arousal disorder (fsiad), which has both properties of hsdd and another illness of dsm-iv known as female sexual arousal disorder. flibanserin underwent new drug application (nda) as original drug in 2009. the two central phase 3 studies (violet6, daisy7) all used e-diary and did not show big statistical improvement compared to placebo in the pre-specified co-primary efficacy endpoint that assessed daily sexual desire. in both violet6 and daisy7 studies, flibanserin showed an increase in satisfying sexual events (sse) and female sexual function index (fsfi) and reduction in female sexual distress scale-revised (fsds-r), but did not show a meaningful increase in e-diary scores compared to placebo. in both studies, the most commonly reported adverse effects in women prescribed with flibanserin include drowsiness (11.8%), vertigo (10.5%), and fatigue (10.3%).67 these studies show a statistically significant improvement in secondary end points that measured sexual desire with other tools known as fsfi compared to placebo. applicants told that the effectiveness of flibanserin against sexual desire is better explained with fsfi, but most advisory committees did not agree to alteration in the e-diary evaluation method set as the one designed to evaluate sexual desire. the safety consciousness expressed by the advisory committees includes adverse effects such as fatigue and drowsiness as well as drug-drug interactions (ddis) and flibanserinalcohol interactions. another central stage 3 study is begonia,8 which set sse, fsfi, fsds-r as major evaluation methods rather than using e-diary in the existing viole and daisy studies and reported that flibanserin shows a statistically significant increase in sse and fsfi and reduction in fsds-r compared to placebo. however, many problems with safety were brought up in the second submission, too. vertigo, drowsiness, and vomiting are the most commonly reported adverse effects, and these occurred 2% to 3%, respectively if using placebo during stage 3 placebo-control premenopausal hsdd, whereas occurred close to 11% in subjects who used flibanserin as 100 mg every night at bedtime. events matching central nervous system depression (fatigue, drowsiness, and vertigo) occurred close to 21% in subjects who used flibanserin as 100 mg every night at bedtime and this proportion is three times higher than in placebo group. flibanserin is very difficult to endure if taking in combination with fluconazole and strong cytochrome p450 3a4 (cyp3a4) suppressant and this combination may increase the risk of swoon and symptomatic low blood pressure. and ethanol administered in combination with flibanserin greatly increases the risk of drowsiness, fatigue, orthostatic hypotension, and swoon. additionally, these studies were limited to general healthy women who did not take benzodiazepines, sleep aid, narcotics and many other drugs. the effectiveness was not so big in general, and considering these concerns, fda refused to approve it again and recommended that additional safety studies are required. according to the latest fda review, there is no data indicating new efficacy. instead, flibanserin sponsors submitted additional safety data next day including research data proving that there is no obstacle to driving, data comparing the commercialized products and the side effects profile of these products, and analysis clarifying the potential effects of alcohol on side effects.9 despite the trustfulness of highly influential studies, fda product review is not a fundamental comparison, in fact, and so comparison of safety between individual products may be misunderstood. in particular, alcohol interaction study was conducted in 25 healthy volunteers as a sample, among which only two were female. since the refusal from fda in 2013, a civic group called as even the score was formed to support what was called as " gender equality " in the manner approaching sexual function disorder treatment.10 this group insisted that there is no treatment for women although even 26 items for male sexual function disorder were already approved. this argument was rejected by fda on the ground that there are no approved products for low sexual desire in men and the 26 items for treatment contain various mixtures of testosterone. despite the fact that flibanserin is supported by consumer advocacy groups and consequently not the first product supported by pharmaceutical companies, fda's argument on gender bias while regulating is worthy of notice in that the range of making efforts against this argument is from campaigns via social media even to letters from the members of the national assembly.11 another noteworthy feature in application for flibanserin to fda is use of the findings on sexual desire reported by patients as primary efficacy variable for approval. sexual desire can be seen by those who experience it and the results reported by patients can be measured without confounding this concept from others. the results reported by patients have become more important in studies and other drugs that have such results as primary end point have been approved (although none were based on sexual desire). among all new molecular entities and biological permit applications approved by fda from 2006 to 2010, gnanasakthy et al.12 found that among which, 17% (20 in 116) used the results reported by acknowledged patients as primary outcome in approval labels. the complex element in evaluating flibanserin lies in recalling the frequency of desire and extent of intensity and then measuring with fsfi index four weeks after the daily records of the most intense desires when applying for the primary desire end point first time. fda raised some concerns about optimizing fsfi as desire assessment tool, including the validity of contents and the period of recall, but the recent advisory committees did not focus on changing the primary end points or consulting the validity of fsfi. the final concern with flibanserin is associated with its use in clinical settings and this was an important issue for the committees. despite little reliable estimate of hsdd prevalence, this product will be used obviously in women in a broader sense than had been studied so far like others, many will not satisfy the official diagnostic criteria of hsdd, and many will increase the risk of adverse effects or be administering other drugs. the concerns with its use without labeling were reemphasized again by speakers who argued that they need flibanserin, but at the same time, it was reported that those who were diagnosed with cancer or in menopause should be excluded from treatment with flibanserin by labeling when sold. on june 4, 2015, fda called a committee meeting to review the efficacy and safety of flibanserin, and after the meeting, the committee approved flibanserin under the condition that risk management tools should be enforced mandatorily. despite the presence of many problems with flibanserin, fda finally approved it under the condition that risk evaluation and mitigation strategies (rems) should be enforced for flibanserin. in the future, flibanserin is expected to gradually increase in amount with changes from off-label to on-label. if prescription with flibanserin is determined as necessary, before using it, it is important to check prescription criterion, possible adverse effects after prescription, and precautions during administration. to get a prescription with flibanserin, the first criteria is premenopausal women aged 18 year or more who met the stages of reproductive aging workshop (straw) criteria. the straw criteria are that she has a regular menstrual cycle for 21 to 35 days in the previous twelve months and a normal level of follicle stimulating hormone (fsh). in addition, it should be prescribed if primary diagnosis is hsdd according to dsm-iv-tr criteria or if there is any secondary sexual arousal disorder or female disposition disorder accompanied by hsdd. besides, its prescription is likely to be considered if the fsds-r (range, 0-52) score13 used in clinical studies is 15 points or higher. in addition, in case that sexual desire disorder comes from disease, psychiatric problems, and interpersonal problems or in case that it does so from medications or other substances taken in conjunction, these cases are not the diseases for which medicine is efficacious. besides, postmenopausal women and sexual desire decline disorder male patients are not adapted during use and i t is not intended for reinforcing sexuality.1415 the adverse effects most common in prescribing flibanserin are reported as vertigo, fatigue, vomiting, insomnia, and mouth dryness. precautions in prescription include avoiding the use of other substances associated with cyp3a4 and cytochrome p450 2c19 (cyp2c19) which are associated with drug metabolism, for example, administration with ketoconazole as cyp3a4 inhibitor and grapefruit juice and fluconazole as cyp3a4 or cyp2c19 inhibitor, which are likely to increase the risk of orthostatic hypotension side effects. furthermore, selective serotonin reuptake inhibitors and selective norepinephrine reuptake inhibitors (ssri/snri) may cause anxiety, drowsiness, fatigue, insomnia, and vertigo, if used. so carefulness is required. alcohol is contraindication because if administered with alcohol, it is highly likely to cause central nervous system depression (fatigue, drowsiness, and vertigo), low blood pressure, and swoon. hormonal contraceptives are known as weak cyp3a4 inhibitors, and likely to cause side effects such as vertigo, drowsiness, and fatigue if coadministered with flibanserin. triptans is used for treating migraine as agonist of 5-ht type 1b and 1d receptor and may cause side effects of drowsiness if administered with flibanserin. in addition, if flibanserin is administered to patients with decline in liver failure, increase in concentration of drug in blood is observed and thus it's considered that adverse effects are likely to occur. besides, taking it with herb extracts and digesters and gastroprotective drugs requiring no doctor prescription is likely to cause the risk of adverse effects and the use of flibanserin to women who are breast-feeding is contraindication. as we discussed above, flibanserin was refused to approve by fda in 2009 for its adverse effects and discordance of its efficacy from different evaluation methods. most common side effects were central nervous system depression, like fatigue, drowsiness, and vertigo. ddis with fluconazole, ethanol, and many other drugs were mainly discussed and fda refused to approve it again recommending additional safety data. with the additional profile and analysis which can prove their safety compared with other products, on june 4, 2015, fda finally approved it on the grounds that rems should be enforced, and soon we can get flibanserin on-label under the strict prescription. flibanserin can be prescribed to premenopausal women aged 18 years or higher who has a regular menstrual cycle, but if liver function fails, if breast-feeding, and if drinking alcohol, the use of flibanserin is contraindication. also, its administration with fluconazole, ketoconazole, ssri/snri, triptans, hormonal contraceptives, herb extracts, and several other drugs requiring no doctor prescription is prohibited because it is likely to increase the risk of adverse effects. we are waiting for patients ' reaction, considering the cost and effectiveness, in countries where flibanserin will be on sale, and more agents like unicenta (uncnt) and melsmon, which proved as having a efficacy of menopausal symptoms, have to be studied as treating for female sexual dysfunction.16

there have been several products developed for male sexual dysfunction. however, developing agents for female sexual dysfunction is lagging behind for various reasons. sildenafil citrate (viagra) and tadalafil (cialis), which have been prescribed for male sexual function disorders, are known to act on vessels.[1] on the other hand, flibanserin is thought to act on brain. flibanserin has been approved by u. s. food and drug administration (fda) for treatment of hypoactive sexual desire disorder (hsdd) of premenopausal women in 2015, and is expected to be released in south korea soon. authors wrote this article to acknowledge flibanserin to sexologists for females or physicians for menopausal medicine, so that this agent can be safely used for females who have hsdd.

PMC4854663

pubmed-86

hydroxyapatite biomaterials are materials that are very widely used in several health purposes, including as a source of calcium for the manufacture of toothpaste and as an important material in the formation/bone repair. the chemical properties of hydroxyapatite are bioactive and compatible with the adjacent bone and teeth. hydroxyapatite is a calcium phosphate ceramic that is totally biocompatible and nontoxic and becomes an integral part of living bone and teeth tissue [18]. the raw material for the production of hydroxyapatite biomaterial is very easily available and abundant in indonesia. the production process was easy and the cost is also relatively inexpensive if done on a large scale. among the abundant raw materials are the shells of crabs, which are one of indonesia's main export commodities. export of crabs commodity by indonesia amounted to 604.215625.000 tons/year without the shell form, while domestic consumption is expected to be so much more, as in makassar, carrying 292.5 tons exported in the form of crab without shell with the main export destination being singapore. if the mass of crab shells 2550% of the total mass, it can be estimated that in 2012 produced the shells of crabs around 151,053.75 to 302,107.5 tons in indonesia and 73.125 to 146.25 tons in makassar (a region of indonesia), there was just from crabs production were exported. this value is of course even more if the crab's consumptions in the country are also taken into account. this suggests the existence of crab shells is abundant in indonesia, including in makassar. as known in indonesia, the shells of crabs have not been used, so it will only be a waste disturbing environment [7, 8]. crabs shells containing calcium carbonate (caco3) are very abundant; amount 4070%, varies according to the species. calcium carbonate can be further processed into calcium hydroxyapatite [ca10(po4)6(oh)2] [4, 5]. according to strassler, hydroxyapatite is one of the active ingredient materials that are widely used in toothpaste products for protection against teeth demineralization [1113]. the crystals structure of hydroxyapatite will be better by using cao as a precursor of calcium. however, the use of these compounds also produces carbonate apatite [ca10(po4)6co3] in a fairly large percentage. this is because the calcination process can not completely eliminate carbon dioxide (co2) in caco3 so that there can be reaction with the precursor phosphate. however, the carbonate apatite heating at a temperature of 700900c for 25 hours, followed by washing using distilled water, the carbonate apatite can be hydroxyapatite [1113]. the instrument were used in this research were glass apparatus, ohaus analytical balance, petri dish, porcelain cup, buchner flask, buchner funnel, vacuum pump sargent-welch co. model 1400, magnetic stirrer, magnetic bar, hotplate idealife, ph meter, furnace thermolyse 6000-barnstead, dessicator, thermometers, spnisosfd oven, stopwatch, shimadzu x-ray diffractometer (xrd) model 6000, x-ray fluorescence (xrf) shimadzu, fourier transform infra-red (ftir) prestige-21 shimadzu, scanning electron microscopy combined with the ability to generate localized chemical information (sem-edxa) variant, and uv-vis shimadzu model 6105. this is because the carbonate ion compounds are able to inhibit the crystallization process ca10(po4)6(oh)2 so that the results will be dominated by an amorphous phase. if the caco3 burned at temperature calcinations, decomposition reaction of caco3 into cao will occur and co2 emissions are dominant and will be issued as a result of the combustion reaction [1315]. waste of crabs shells portunus pelagicus species was taken from an exporter crabs company in industry areal, makassar city, south sulawesi province, indonesia. wahidin hospital, and (nh4)2hpo4 was obtained from fluka chemical, ch3cooh glacial 100%, ch3coona3h2o from merck, (nh4)6mo7o244h2o from aldrich, nh4vo3 from aldrich, hno3 and naf from fluka chem., (nh4)2c2o4, distilled water, aluminum foil, and whatman filter paper numbers 40 and 42. the instruments were used in this research that glass as commonly used in laboratories, ohaus analytical balance, petri dish, porcelain cup, buchner flask, buchner funnel, vacuum pump sargent-welch co. model 1400, magnetic stirrer, magnetic bar, hotplate idealife, ph meter, furnace thermolyse 6000 barnstead, dessicator, thermometers, oven spnisosfd, stopwatch, shimadzu x-ray diffractometer (xrd) model 6000, x-ray fluorescence (xrf) shimadzu, fourier transform infrared (ftir) prestige-21 shimadzu, scanning electron microscopy combined with the ability to generate localized chemical information (sem-edxa) variant, and uv-vis shimadzu model 6105. crabs shells portunus pelagicus waste was cleaned with distilled water and dried at room temperature. furthermore, to transform crabs shells into caco3 and then into cao, calcinations were performed on the samples at 1000c temperature for 5 hours at a rate of temperature rise 5c/minute. calcium oxide (cao) obtaining dominated as result of calcinations and then made suspensions in 100 ml of distilled water with a calcium concentration of 0.3 m. the suspensions reacted by dropwise with a 100 ml 0.2 m of (nh4)2hpo4 solution through the coprecipitation method, at temperatures around 40c while the solution was stirred for 25 hours. the precipitation allowed stand overnight or 24 hours at room temperature, and the precipitate is filtered with a whatman filter paper number 40 and dried at 110c for 5 hours. the pure hydroxyapatite obtained by sintering to the dried precipitate at various temperatures of 500900c for 4 hours [16, 17]. the characterization of the compounds was performed by using x-ray diffraction (xrd), ftir, and sem-edxa. the proven in vitro demineralization of teeth was conducted through evaluating the concentration of phosphate in solution by using the uv-vis spectroscopy. the effectiveness of hydroxyapatite to protection against teeth demineralization was tested in acetate buffer ph 5.0 solutions, with 1 m of acetic acid concentration with the addition of hydroxyapatite in varying concentrations and immersion time. each of 5 beakers was filled with 300 ml of acetate buffer ph 5.0 with acetic acid concentration of 1 m. an acetate buffer was left without adding anything as a comparison. an acetate buffer was then added 10 ppm of naf left without addition of ca10(po4)6(oh)2. other three pieces beaker of acetate buffer containing 10 ppm of naf are adding of ca10(po4)6(oh)2 with variation concentration of 25 ppm, 50 ppm, and 100 ppm. the immersed times of tooth samples in solution are 3, 6, 9, 24, and 48 hours, respectively. furthermore, the phosphate concentration in each solution was measured by using uv-vis spectrophotometer with the wavelength for phosphate (maks) being 432 nm. the calcinations aim to eliminate the organic component in the shells of crabs and convert caco3 compound which is the dominant compound in the shells of crabs into cao through the elimination of co2 in gas form. characterizing the calcinations results the results can be seen from the diffraction pattern of the crab shells before and after calcinations at a temperature of 1000c for 5 hours (figure 1). in this figure can be observed changes in the diffraction pattern of crab shells, where the change in the diffraction pattern is due to the chemical change from mixture of caco3 with organic matter to pure cao. the appearance of the sharper peaks in figure 1 after calcinations (b) is a result of the crystallinity of the cao. identification by ft-ir as shown in figure 2 showed there is a reduction process of -co3 groups and some of ir-spectra were missing after calcinations. the elimination of -co3 groups and organic components can also be seen from the data of mass reduction of sample calcinations. the determinations of calcium contained in sample was conducted by using x-ray fluorescence, where obtained calcium is 66.62% after calcinations. these results are then used to calculate the stoichiometry in determining the number of results calcinations which is needed to react with (nh4)2hpo4 as the precursor phosphate. the precipitation reactions aiming to produce ca10(po4)6(oh)2 used phosphate, (nh4)2hpo4, as the precursor and then reacted with cao as calcinations results. side results ca10(po4)6co3 also occur as a product of reaction of (nh4)2hpo4 and caco3 presence in the calcinations results. dried precipitate further sintered on temperature variations of 4001000c for 2 hours; it is intended to determine the optimum temperature where the ca10(po4)6(oh)2 is formed, furthermore characterized by using xrd, ft-ir, and sem-edxa. the diffractograms of each sintering results compounds indicate that the temperature is closely related to the formation of crystals. this is due to the nature of the vibrating atoms moving faster in higher temperatures. the optimum temperature formation of hydroxyapatite was determined by calculation of the probability of the sample phase from the xrd results analysis according to jcpds standard data, which, jcpds; 24-0033 is standard data for ca10(po4)6(oh)2; 09-0169 for -ca3(po4)2; 29-0359 for -ca3(po4)2, 35-0180 -ca3(po4)2; 35-0180 for ca10(po4)6co3(oh)2 and 19-0272 standard data for ca10(po4)6co3(oh)2. figures 3 and 4 showed that the temperature is closely associated with the formation of hydroxyapatite phase. in the both graphs it can be seen that the maximum intensity of the phase formation of hydroxyapatite has been found by sintering at temperature of 800c, it means the optimum temperature of hydroxyapatite formation is 800c, and then this result will be used for another application. the formations of hydroxyapatite phase had been dominated at 800c confirmed by percentage probability sample phase (figure 5), in which the percentage of hydroxyapatite phase formed around 46.61%, while phases of -ca3(po4)2 and phase -ca3(po4)2 are 17.76% and 19.37%, respectively. however, also there is still presence of a phase ca10(po4)6co3 and ca10(po4)6co3(oh)2 with a range of 11.84% and 4.43%, respectively, which indicates the presence of carbonates. the x-ray diffractograms of ca10(po4)6(oh)2 synthesized at a 800c temperature sintering can be seen in figure 6, where the peaks of ha are symbolized by the peak of the crystal ca10(po4)6(oh)2, while peak -tkf is symbolized crystalline peaks of -ca3(po4)2, the symbol -tkf for crystalline -ca3(po4)2, aka for ca10(po4)6co3, and akb for ca10(po4)6co3(oh)2. the highest intensity peak at 31.2572 deg corresponding to crystalline -ca3(po4)2 is seen, the second highest peak intensity is 31.7783, and the third highest peak intensity is 28.0565 crystals suitable for ca10(po4)6(oh)2. ftir results showed that the sintering temperature variation affects the absorption band shapes which generally all sintering results showed absorption band of -oh, absorption band 1, 2, 3, and 4 of po4, and co3 groups. infrared spectra in figure 7 show the -oh groups at 633 cm which are characteristic of hydroxyapatite appearing on the sintering temperatures of 4001000c. additionally spectrum also showed higher sintering temperature causing the sharper peaks phosphate group (po4) because the nature of the vibrating atoms moves faster at higher temperatures. the ft-ir spectra of sample were sintered at a temperature of 800c (figure 8) showing that hydroxyapatite is the dominant compound formed. the stretching frequencies of po4 group are indicated by 1120.64 cm, 1091.71 cm, 1043.49 cm (3), 993.34 cm, 877.61 cm (1), 603.72 cm, 565.14 cm, (4), and 370.33 cm (2). and a sharp spectrum in the area of 3570.24 cm indicates the presence of free -oh and 3427.51 cm indicating -oh bounded, and this is indicating that the dominant compound is ca10(po4)6(oh)2. while area of 1654.92 cm, 1458.18 cm, and 1421.54 cm indicates the presence of carbonate groups (co3) it can be identified as ca10(po4)6co3 and ca10(po4)6co3(oh)2 which has not been transformed into ca10(po4)6(oh)2 during the sintering process. the results of characterization by sem-edxa shown in figure 9(a) show that the size of hydroxyapatite formed from the synthesis tends to be small and only a few are large. while figure 9(b) shows that the surface of hydroxyapatite is smooth and nonporous, this shows that hydroxyapatite which has synthesis from crab shells can function well as inhibiting tooth demineralization. while in figure 10 the edxa spectrum shows the composition of the synthesis yield was dominated by oxygen (o) up to 59.52%, calcium (ca) up to 23.76%, and phosphorus (p) up to 13.32%. demineralization of teeth is a process of decomposition of the crystal of ca10(po4)6(oh)2 due to the acidic conditions by releasing ca and po4 ions. demineralization of tooth causing increased levels of ca and po4 in saliva in direct contact with the tooth. in vitro, the rate of tooth demineralization can be observed through the concentrations of ca and po4 ions in solutions where the tooth was soaked each unit of time. therefore, increase of po4 ions concentrations in solution a soaked gear can be one of indicators to measure the rate of tooth demineralization. figure 11 shows the relationship between the soaking time of teeth versus the increase of the ion po4 levels in solution where the tooth was soaked as well; it appears that with the increasing addition of ca10(po4)6(oh)2 into the acetate buffer equals to the rate of demineralization decrease. it is can be altered by the amount of po4 ions in solutions; however the addition of ca10(po4)6(oh)2 ions showed lower amount of po4 ions compared to solutions without the addition of ca10(po4)6(oh)2. this proves that the ca10(po4)6(oh)2 were synthesized from the crab shell effective for protection against tooth demineralization. the decrease in the rate of tooth demineralization with the addition of ca10(po4)6(oh)2 can also be observed through analyzing the tooth mass reduction in the fifth variation of acetate buffer solution as shown in table 1. the greater concentrations of ca10(po4)6(oh)2 in the acetate buffer solution were teeth immersed exhibit the smaller mass of teeth in the solution. based on these results it is concluded as follows.waste of shells crabs (portunus pelagicus) proved to be used as raw material for the synthesis of ca10(po4)6(oh)2 due to high calcium levels which amounted to 66.62% in addition to the abundant existence as waste.the optimum temperature formation of ca10(po4)6(oh)2 is at 800c.ca10(po4)6(oh)2 were synthesized from waste of shell crabs (portunus pelagicus) in vitro effectively inhibiting the rate of demineralization of the tooth where the greater the addition of ca10(po4)6(oh)2 in the solution, the more the inhibiting demineralization of the tooth or the smaller the rate of tooth demineralization in solution. waste of shells crabs (portunus pelagicus) proved to be used as raw material for the synthesis of ca10(po4)6(oh)2 due to high calcium levels which amounted to 66.62% in addition to the abundant existence as waste. ca10(po4)6(oh)2 were synthesized from waste of shell crabs (portunus pelagicus) in vitro effectively inhibiting the rate of demineralization of the tooth where the greater the addition of ca10(po4)6(oh)2 in the solution, the more the inhibiting demineralization of the tooth or the smaller the rate of tooth demineralization in solution.

crab's shells of portunus pelagicus species were used as raw materials for synthesis of hydroxyapatite were used for protection against demineralization of teeth. calcination was conducted to crab's shells of portunus pelagicus at temperature of 1000c for 5 hours. the results of calcination was reacted with (nh4)2hpo4, then dried at 110c for 5 hours. sintering was conducted to results of precipitated dried with temperature variations 4001000c for a hour each variation of temperature then characterized by x-ray diffractometer and ftir in order to obtain the optimum formation temperature of hydroxyapatite is 800c. the hydroxyapatite is then tested its effectiveness in protection against tooth demineralization using acetate buffer ph 5.0 with 1 m acetic acid concentration with the addition of hydroxyapatite and time variation of immersion. the results showed that the rate of tooth demineralization in acetate buffer decreased significantly with the provision of hydroxyapatite into a solution where the addition of the magnitude of hydroxyapatite is greater decrease in the rate of tooth demineralization.

PMC4508471

pubmed-87

brain injury is caused by intrinsic or extrinsic factors and it can result in various disabilities such as motor, sensory, behavioral, or cognitive dysfunction depending on the area of the brain lesion1. cognitive impairment due to brain injury is an important factor affecting patients independent functions and participation in activities2, interfering with their return to daily living and work3. it can also influence motivation and the ability to participate in rehabilitation programs and interfere with a return to the community. therefore, for successful rehabilitation, accurate and comprehensive cognitive assessment and treatment are required4. for cognitive rehabilitation of patients with brain injury, traditional treatment and computer-based cognitive therapy (vr) technology is gaining recognition as a useful tool for cognitive research, evaluation, and rehabilitation5. vr systems allow users to interact in various sensory environments and to obtain real-time feedback on their performance using computer technology6. the virtual environment offered via vr technology makes it possible for patients to participate in activities in settings and environments similar to those encountered in real life7, 8. in addition, vr tools can be used to record accurate measurements of the subject s performance9 and to deliver greater therapeutic stimulation to users5. vr has been used as a tool to diagnose cognitive impairment and as a vehicle to provide new treatments5. although the use of vr in cognitive rehabilitation has been increasing, few systematic reviews have investigated the use of vr programs in cognitive rehabilitation and the overall effect of these programs on cognition. therefore, this systematic review investigated the different types of vr programs used for cognitive evaluation and interventions for patients with brain injury. studies using vr programs for cognitive intervention were reviewed according to pico (patient, intervention, comparison, and outcome) methods. (virtual reality or virtual or game based virtual reality or computer based virtual reality) and (stroke or cerebral vascular accident or hemiplegia or brain injury or traumatic brain injury) and (cognition or cognitive or memory or attention or executive function). inclusion criteria were: (1) subjects over the age of 19 years with brain injury; (2) articles written in english; and (3) studies that used vr in cognitive rehabilitation. exclusion criteria were: (1) subjects who were animals or children; (2) review articles; and (3) 2d computer-based cognitive rehabilitation. randomized controlled trials (rcts) and crossover studies were scored on the physiotherapy evidence database (pedro) scale12. two authors independently assessed the methodological quality of the included studies, and disagreements were resolved by reaching consensus. memory assessment was the most common study topic, followed by assessments of executive function and attention. a variety of vr programs were used (table 1table 1.analysis of studies of cognitive assessment using vrauthor, yearassessment areatype of vrbrooks et al. three were rcts24,25,26, one was a crossover study27, and one was a case report28. excepting the case report, four studies were assessed using the pedro scale. two studies24, 25 scored 4, and the remaining two studies scored 326 and 127, respectively. table 2table 2.analysis of studies with cognitive interventions using vrstudypatientinterventioncomparisonoutcomenmean agetype of vrintensityoutcome measure(s)findingsakinwuntan et al., 20102469eg 55 cg 54stisim drive system60 min/day, 3 times/week, total 5 weeksnon-computer based cognitive therapyufov testsignificant within group improvements but no significant difference between two groupscaglio et al., 201228124midtown madness 290 min/day, 3 times/week, total 5 weeks-fdst, bdst, ravlt-ir, dr, rbmt-ir, dr, corsi s block tapping test, corsi s supraspan test-ir, dr, tmt-a, b, phonemic fluency mmse, adassignificant improvements in ravlt-ir and corsis supraspan-ir, drgrealy et al., 1999271332.38nonimm-ersive vr exercise bicycle25 min/day, 3 times/week, total 4 weeksno treatmentfdst, bdst, digit symbol test, tmt-a&b, auditory learning test, velt, vilt, logical memory, complex figure testeg showed significant improvements in digit symbol test, velt, vilt compared to cgjacoby et al., 20132512eg 27.83 cg 30.67irex45 min/day, 34 times/week, total 3 weeksconventional otmet-sv, efpteg showed significant improvements in all outcome measures compared to the cgkim et al., 20112628eg 66.5 cg 62.0irex30 min/day, 5 times/week, total 4 weekscomputer based cognitive therapyk-mmse, tol, vcpt, acpt, word-color test, fdst, bdst, fvst, bvst, vilt, velt, tmt-asignificant difference between experimental group and control group in vcpt and bvsteg: experimental group, cg: control group, met-sv: multiple errands test-simplified version, efpt: executive function performance test, fdst: forward digit span test, bdst: backward digit span test, tmt-a: trail making test-type a, tmt-b: trail making test-type b, velt: verbal learning test, vilt: visual learning test, ufov: useful field of view test, k-mmse: korean version of the mini-mental status examination, tol: tower of london test, vcpt: visual continuous performance test, acpt: auditory continuous performance test, fvst: forward visual span test, bvst: backward visual span test, ravlt: rey auditory verbal learning test, ir: immediate recall, dr: delayed recall, rbmt-the rivermead behavioural memory test, adas: alzheimer s disease assessment scale presents the characteristics of the five studies. flowchart of the article search and study selection eg: experimental group, cg: control group, met-sv: multiple errands test-simplified version, efpt: executive function performance test, fdst: forward digit span test, bdst: backward digit span test, tmt-a: trail making test-type a, tmt-b: trail making test-type b, velt: verbal learning test, vilt: visual learning test, ufov: useful field of view test, k-mmse: korean version of the mini-mental status examination, tol: tower of london test, vcpt: visual continuous performance test, acpt: auditory continuous performance test, fvst: forward visual span test, bvst: backward visual span test, ravlt: rey auditory verbal learning test, ir: immediate recall, dr: delayed recall, rbmt-the rivermead behavioural memory test, adas: alzheimer s disease assessment scale in this review, the types of vr programs that have been used in cognitive evaluations of patients with brain injury were identified and studies of cognitive interventions were reviewed according to pico methods. in the included studies, the vr programs could distinguish the cognitive disability of patients in comparisons with healthy subjects. thus, vr could be used as a new assessment method of the cognitive function of patients with brain injury. therefore, in contrast to conventional cognitive assessments, vr programs can provide consistently accurate measurements of cognitive function. however, some methodological problems were found in the reviewed articles. in most of the studies, the vr tool used was not compared with a standardized assessment tool, and the inter-rater reliability was not measured. the five studies of cognitive therapy using vr all reported positive effects. in the assessment of cognitive function, the vr interventions resulted in improvements in the areas of memory and attention but not executive function. ben-yishay et al.29 stated that to effectively raise cognitive function, normal attention is needed. if the ability to concentrate on external information is impaired, memory, problem-solving skills, and appropriate behavior may be difficult. thus, they suggested that the impairment of attention due to brain injury may interfere with the recovery of other cognitive functions, such as memory, executive function, and planning. the results of this systematic review indicate that the cognitive improvement of attention using vr programs will have a positive impact on the recovery of general cognitive function. the advantage of cognitive rehabilitation using vr is that it provides a variety of environments similar to those encountered in real life30. the results of this review suggest that patients are more motivated in virtual environments than they are in conventional settings. therefore, vr programs can be expected to lead to an improvement in cognitive function. in vr interventions, patients can be treated in a safe environment compared to real settings. in addition, vr programs can be tailored to the type of injury and easily adjusted to the level of cognitive disability, the complexity of a task, the reaction conditions, and the characteristics and patterns of feedback30. as vr systems are constantly evolving and becoming smaller and more easily adjustable, they can be expected to provide specialized therapy in new settings, such as patients homes or clinics. these advantages of vr systems can benefit patients who find it difficult to visit health care organizations5. the results of this systematic review suggest that vr is an effective cognitive therapy for patients with brain injury compared to control therapy., there was a significant risk of bias with regard to allocation concealment and blinding. given the heterogeneity of the included studies, the ability to draw conclusions is limited. well-designed rcts and blind studies will be needed to provide evidence of the benefits of vr on cognitive function.

[ purpose] the purpose of this review was to investigate various types of vr programs and their use in cognitive evaluations and interventions for patients with brain injury. [subjects and methods] pubmed, cochrane, and otseeker electronic databases were searched with the search terms. at of 350 titles and abstracts were retrieved, and 17 articles were selected for this review. selected articles were assessed on the level of evidence using the physiotherapy evidence database (pedro) scale. [results] articles assessing the impact of cognitive impairments in memory were most commonly found, and vr interventions elicited positive effects in patients with brain injury. [conclusion] vr can be considered a new tool for cognitive rehabilitation after brain injury. vr interventions also have a number of advantages, e.g. cost-effectiveness, compared to other interventions.

PMC4616145

pubmed-88

coronary artery disease (cad) is a major public health problem worldwide and the single largest cause of mortality in the united states, responsible for one of every six deaths (aha heart disease and stroke statistics, 2010). cad is caused by atherosclerosis, which is an inflammatory disease that involves multiple cell types, including circulating cells and cells in the vessel wall. despite advances in risk factor management on an epidemiological level, various blood markers associated with increased risk for death and cardiovascular endpoints have been identified, but currently very few have been shown to have a diagnostic impact or important clinical implications that would affect patient management. therefore, there is a great need for innovative biomarkers that can assess risk for cad, assess activity of the atherosclerotic process, and guide evaluation of therapy. several recent studies have suggested that circulating micrornas could be useful as biomarkers for various human disease states, including cancer, acute myocardial infarction [47], heart failure, and chronic vascular disease [8, 9]. micrornas (mirnas) are a recently recognized class of short (1925 nt), single-stranded, noncoding rnas that regulate an array of cellular functions through the degradation and translational repression of mrnas that contain complementary sequences. more than 1000 human mirnas have been identified, and, in tissues, mirnas regulate the expression of genes involved in critical cellular processes, including differentiation, growth, proliferation, and apoptosis. importantly, mirnas are now regarded as rheostats that fine-tune expression of proteins involved in just about every process in human cells. mirnas have been found in tissues, whole blood, serum, plasma, and other body fluids in a stable form that is protected from endogenous rnase activity [3, 12]. although the biological function of mirna is yet to be fully understood, tissue levels of specific mirnas have been shown to correlate with pathological development of different diseases. mirnas function as managers in gene regulatory networks, and they are distinct from other biomarkers because they have a pathogenic role in the disease process and are not merely byproducts of the disease state. thus, mirna expression signatures in tissues and blood have a potential role in the diagnosis, prognosis, and assessment of therapy. in this study, we sought to compare mirna expression in whole blood of patients with angiographically significant cad to that of healthy aged-matched controls. we performed an initial exploratory microarray analysis in 5 cases and controls and then further examined the most highly expressed mirnas in an additional 15 cases and controls. study participants were recruited as part of the emory cardiology biobank, consisting of 3492 consecutive patients enrolled prior to undergoing elective or emergent cardiac catheterization across three emory healthcare sites, between 2003 and 2008. patients aged 2090 years were interviewed to collect demographic characteristics, medical history, and lifestyle habits. risk factor prevalence was determined by physician diagnosis and/or treatment for hypertension, hyperlipidemia, and diabetes. coronary angiograms were evaluated independently by two operators, who made visual estimation of luminal narrowing in multiple segments based on a modified form of the aha/acc classification of the coronary tree. using these data, significant cad was defined as at least one major epicardial vessel with>50% stenosis, assessed by quantitative coronary angiography. additionally, we collected whole blood samples from patients with 2 risk factors for cad, but did not have angiographically significant cad. the study was approved by the institutional review board at emory university, atlanta, ga, usa. all subjects provided written informed consent at the time of enrollment. the healthy subject cohort was a random sample of employees from emory university who were fully employed, productive people. these subjects were aged 18 and older, not taking medications, and had not been hospitalized for at least one year prior to participation. venous blood samples were drawn via antecubial venipuncture into paxgene blood rna tubes and stored at 20c within 24 hours before rna extraction. mirna was isolated using the preanalytix paxgene mirna isolation kit (qiagen) according to the manufacturer's protocol. mirna microarray analysis was performed by asuragen, inc., which uses the affymetrix manufactured genechip mirna arrays. once labeled, the mirna targets were hybridized overnight onto the microarrays following which the arrays were washed and stained using streptavidin-phycoerythrin (sape). expression analysis was performed for all small rnas for homo sapiens (e.g., mirna, small nuclear rnas like snorna and scarna) and separately for sanger mirna registry content (mirbase 11.0, http://microrna.sanger.ac.uk/) for homo sapiens. mirna reverse transcription was performed using the taqman microrna reverse transcription kit (applied biosystems) at 16c for 30 min, 42c for 30 min, and denaturation of the enzyme at 85c for 5 min. the rt reaction was performed at 37c for 1 hour followed by 5 min at 95c. taqman microrna assays (applied biosystems) were performed using the 7500 fast real-time pcr system at the 9600 emulation run mode. ct values were converted into copy numbers (copy no.=2) and normalized to rnu44. unpaired student's t-tests were used to compare data. a p value<.05 (two sided) was considered significant. for analysis of microarray expression data, a two- sample t-test was carried out for every gene, followed by multiplicity correction to control the false discovery rate (fdr) at .05. we initially performed expression profiling of all small rnas (1770 genes, figure 1) in the whole blood samples. three mirnas passed the fdr cutoff of 0.05: mir-584 (7.9-fold higher in healthy versus cad patients, p=.000103, t-test), mir-542-5p (3.9-fold higher in healthy versus cad pts, p=.000168, t-test), and mir-187*(2.77-fold higher in healthy versus cad pts, p=8.1 10, t-test). however, signal intensities for mir-542-5p and mir-187*were very low and not detected in most samples. separately, we performed an analysis of sanger registry mirnas (848 for homo sapiens) in the whole blood samples, but none of the mirnas passed the fdr cutoff of 0.05. ten mirnas passed a fdr cutoff of 0.10, but only one (mir-129-5p, 1.57 fold higher in healthy patients, p=.000044) of these mirnas had consistently detectable signal across the 10 blood samples. although we were able to detect some differences in whole blood mirna levels between healthy subjects and cad patients (mir-584, in particular), our microarray data suggest that, similar to other reports, levels of mirnas in the blood are low and microarrays may lack the sensitivity to adequately identify mirnas that might serve as vascular disease biomarkers. interestingly, among the mirnas that tended to show consistent differences between healthy and cad blood, mirna expression was generally higher in the blood of healthy subjects, a finding previously observed by others. next, we performed qrt-pcr on rna isolated from whole blood of another 10 patients with angiographically significant cad and 15 healthy subjects. we evaluated the levels of mirnas that, based on our microarray data, were highly expressed in blood and tended to have different levels of expression between the two groups. this analysis included mir-150, mir-584, mir-21, mir-24, mir-126, mir-92a, mir-34a, mir-19a, mir-145, mir-155, mir-222, mir-378, mir-29a, mir-30e-5p, mir-342, and mir-181d. among these we found that mir-19a, mir-584, mir-155, mir-222, mir-145, mir-29a, mir-378, mir-342, mir-181d, mir-150, and mir-30e-5p were significantly downregulated in the blood of patients with cad compared to healthy subjects (figure 2). furthermore, we also assessed expression of these 11 mirnas in a cohort of patients who had 2 risk factors for cad, but did not have angiographically significant cad. we found that there was no difference in whole blood mirna expression of this latter group compared to that in patients with significant cad (not shown), suggesting that these mirnas are markers for vascular inflammation rather than markers specific for significant cad. there has been one report that mirna expression in blood may be influenced by medications. indeed, one of the mirnas that we found to be downregulated in the blood of patients with cad is mir-155, which is known to target the at1 receptor. therefore, we assessed the impact of medications on expression of mir-155, mir-145, mir-181d, mir-222, mir-19a, mir-342, mir-29a, mir-30e-5p, and mir-378. we compared the mirna expression levels in the blood of patients with angiographically significant cad or 2 risk factors for cad but not on acei or arb to that in blood of similar patients who were taking at least one of medications. interestingly, levels of mir-155, mir-19a, mir-145, mir-222, mir-342, mir-30e-5p, and mir-378 (figure 3) were significantly decreased in patients taking acei or arb compared to those who were not, suggesting that these medications may directly modulate expression of these mirnas, or they may influence inflammatory factors that otherwise regulate their expression. importantly, we did not find that statin use had a significant effect on the levels of mirnas identified in this study (not shown). several recent studies have indicated that there is a potential role for circulating mirna levels as valuable biomarkers for different disease processes, including cancer, cardiomyopathy, and acute myocardial infarction. in this study, we wanted to address the hypothesis that mirna expression levels in blood could predict the presence of significant coronary artery disease in human subjects. we identified 11 mirnas whose expression was significantly downregulated in patients with angiographic evidence of significant atherosclerosis compared to healthy subjects that were matched for age and gender. in addition, our data suggest that inhibition of the renin angiotensin system by acei or arb use further influences mirna expression in blood. this study confirms previous reports showing differences in circulating mirna levels of patients with cad compared to those of healthy subjects [8, 16]. our study differs from the other studies in the content of the group of mirnas that were downregulated in patients with cad. we performed this analysis using the paxgene blood rna system because it provides a way to stabilize rna immediately after sample collection and facilitates storage of the samples for a relatively long period of time without compromising rna integrity [17, 18]. importantly, mrna profiling of whole blood or peripheral mononuclear cells has been previously applied to cardiovascular disease [19, 20], and a relationship has been identified between mrna levels in whole blood and extent of coronary artery disease. our study extends this work by providing insight into the whole blood expression of mirnas that are potentially involved in regulating these cad genes. in our qrt-pcr analysis of whole blood samples from cad patients, we found similar changes in expression of mir-155 and mir-145 as what has been reported previously. however, we failed to detect changes in other mirnas that were described in this previous report, namely mir-126 and mir-92a. first, we studied whole blood samples, whereas plasma was studied previously. thus our mirna profile likely reflects intracellular and extracellular mirnas levels in contrast to exclusively extracellular mirnas that would be detected in plasma. another difference in our study is that we normalized mirna expression to expression of rnu44, a small nucleolar rna that, based on our microarray analysis, we found to be highly and consistently expressed across blood samples from cad patients and controls. this study confirmed the observation made by others regarding the difficulty of using microarray analysis to profile mirna expression in blood samples. undoubtedly, this is in part due to the limitation of a relatively small sample size for our microarray analysis, as well as reduced sensitivity of the microarray method compared to qrt-pcr. in addition, principal component analysis of our data suggested that two of the mirna profiles for healthy subjects were actually similar to that of the cad patients, suggesting that these individuals may in fact have subclinical vascular inflammation., we believe that careful selection of patients and well-matched control subjects from a larger group of well-characterized individuals reduced some of the variability in our qrt-pcr analysis. we can only speculate as to why expression of circulated mirnas is generally decreased in patients with cad. it has been hypothesized that levels of circulating mirnas are decreased in vascular disease because they have been taken up into atherosclerotic lesions. the levels of circulating mirnas may be affected by multiple factors, including transcription, processing and stability of the mirnas within circulating cells, as well as the ability of these cells to release mirnas into the plasma. circulating mirnas may be delivered to cells in the heart or blood vessels through microvesicles, exosomes, or apoptotic bodies. because our study assessed mirna expression in whole blood, our findings are likely more reflective of changes in mirna transcription or processing rather than release from the circulating cells. it remains to be determined whether downregulation of mirnas in cad patients is directly involved in inflammation or a compensatory response to this process. based on our observed changes in mirna expression in response to acei/arb therapy, we believe that circulating mirna levels reflect a compensatory response to inflammation. further experimental studies are necessary to explore the mechanisms by which cad and therapies affect tissue versus circulating mirna levels. in summary, the present study provides insight into whole blood levels of mirnas in patients with cad compared to healthy subjects and demonstrates their potential utility as biomarkers for vascular disease. validation of the changes in mirna expression observed here in larger studies will be a necessary step to confirm their candidacy as biomarkers and therapeutic targets. we believe that further elucidation of the role that these mirnas play in the pathogenesis and progression of chronic cad will contribute to our understanding of the disease process and lead to new therapeutic and preventative strategies .

coronary artery disease (cad) is the largest killer of males and females in the united states. there is a need to develop innovative diagnostic markers for this disease. micrornas (mirnas) are a class of noncoding rnas that posttranscriptionally regulate the expression of genes involved in important cellular processes, and we hypothesized that the mirna expression profile would be altered in whole blood samples of patients with cad. we performed a microarray analysis on rna from the blood of 5 male subjects with cad and 5 healthy subjects (mean age 53 years). subsequently, we performed qrt-pcr analysis of mirna expression in whole blood of another 10 patients with cad and 15 healthy subjects. we identified 11 mirnas that were significantly downregulated in cad subjects (p<.05). furthermore, we found an association between acei/arb use and downregulation of several mirnas that was independent of the presence of significant cad. in conclusion, we have identified a distinct mirna signature in whole blood that discriminates cad patients from healthy subjects. importantly, medication use may significantly alter mirna expression. these findings may have significant implications for identifying and managing individuals that either have cad or are at risk of developing the disease .

PMC3137987

pubmed-89

among eusocial insects, paper wasps (hymenoptera: vespidae: polistinae) are notable for the diversity of their nest architecture (jeanne, 1975; wenzel, 1991). many swarm-founding polistine wasps, including most species of the neotropical tribe epiponini, construct envelopes around their brood combs. comparative and experimental studies suggest that, among other functions, nest envelopes reduce rates of predation and parasitism on wasp brood (london and jeanne, 1998; smith et al., 2001). however, little is known about the adaptive significance of variation in the materials that wasps use to construct nest envelopes (hansell 1984; wenzel, 1991; cole et al., 2001). polybia emaciata is unusual among the polistine wasps because it uses mud, rather than wood pulp or plant fibers, as the main raw material for nest construction (schremmer, 1984). the genus polybia includes 56 described species; of these, only the other three species in the subgenus pedothoeca, and one species in the subgenus furnariana, share the derived trait of mud nest construction (richards, 1978; jeanne, 1991; cooper, 1993). no other swarm-founding polistine wasps are known to build primarily mud nests, although small amounts of inorganic material are sometimes incorporated into the nest paper of other species (wenzel, 1991). the nest of p. emaciata is otherwise typical of the genus (jeanne, 1975; wenzel, 1991), being phragmocyttarus (comprising horizontal layers of comb that are conjoined and enclosed by a continuous envelope) with a single small entrance hole near the bottom of the nest. numbers of adults in mature colonies are relatively small for the genus, ranging from fewer than 100 up to about 500 adults (richards, 1978; chadab, 1979; strassmann et al., 1992). polybia emaciata nests are hard and much more durable than most paper nests of similar size and shape (rau, 1933; richards, 1978). for example, skutch (1971) noted that nests of this species in costa rica were unlike paper nests in that they persisted for several years. the high durability of p. emaciata construction material suggests that the nest itself may provide greater brood protection than paper nests. if so, we hypothesized that the wasps ' defensive responses to vertebrate attacks have changed to take advantage of nest durability. we predicted that the nests ' protective properties reduced the need for behavioral defenses by p. emaciata workers, relative to paper-nesting polybia species with similar colony and nest sizes. we tested this prediction by disturbing p. emaciata colonies, and comparing the workers ' responses to those of simultaneously observed paper-nesting p. occidentalis colonies in the same location. chadab (1979) surveyed the defensive responses of swarm-founding paper wasps to vertebrate-like disturbances, including tapping on the nest and/or on the supporting vegetation. the most frequently observed response was for dozens to hundreds of workers to rush out onto the nest envelope, sometimes adopting aposematic postures (see also o'donnell et al., response was seen in the genera angiopolybia, charterginus, leipomeles, parachartergus, polybia, pseudopolybia, and synoeca, and accords with our observations of what appears to be typical epiponine defensive behavior. weaker exit-nest responses (either requiring several taps, or involving fewer wasps) were seen in species of leipomeles, metapolybia, and protopolybia. interspecific variation in mature colony size was not a good predictor of the type of defensive response, as species with colony sizes smaller and larger than p. emaciata performed nest exiting defensive behavior. an attack response often coincides with, or quickly follows, the nest exit response and involves workers flying from the nest, striking the intruder, and attempting to sting (hermann and blum, 1981). when paper-using polybia nests are tapped, some of the workers on the nest surface may immediately perform attacks, while large numbers of workers rush out of the nest entrance and stand in alert posture on the envelope (jeanne, 1981; jeanne et al., 1992). further tapping elicits additional attacks. breathing on the nest surface or into the nest results in immediate attack by several to dozens of workers. we discuss the possible adaptive significance of deviations from the typical nest exit that have been documented in several paper wasp species, including p. emaciata. this study was conducted in and near the town of gamboa, panama province, republic of panama (0907n 7942w; 50 m elevation). observations and manipulations were conducted during the wet season from 1 august to 15 october 1990 and from 3 to 5 july 1998. surveys of eusocial wasp colonies during these periods indicated that p. emaciata was among the most abundant swarm-founding wasps in terms of nest density (s.o'd., unpublished data). most of the data presented were collected from four colonies of p. emaciata and five colonies of p. occidentalis that had been moved approximately 0.5 km to 1 km from their natural nesting sites to artificial supports to facilitate observation. all subject colonies were moved into a small area (< 0.25 ha), with similar amounts of tree cover (one p. occidentalis colony was additionally sheltered by building eaves). all colonies were in place at least 2 weeks before observations on defense behavior were initiated, and all continued to forage and to raise brood (visible through the nest entrance in the lower combs) throughout the observation period. the p. emaciata observation colonies had nests ranging in size from 9 cm long to 22 cm long. the numbers of adult wasps and colony age probably varied positively with nest size, though we did not measure these variables. we also collected data opportunistically from p. emaciata colonies in situ in 1990 and again in 1998. these nests were located in the gamboa area on thin tree branches from 1.5 m to 5 m above the ground. we disturbed p. emaciata and p. occidentalis colonies in an effort to evoke defensive behavior, using stimuli that elicited nest exit and attack responses in costa rican p. occidentalis (s.o'd. and we disturbed the polybia nests by first tapping on the side of the outer envelope near the middle of the nest for 30 sec while wearing a full bee suit, and we noted the responses of on-nest and in-nest workers. if there was no attack response to tapping, we blew into the nest entrance for 30 sec. each polybia observation colony was disturbed 10 times over the course of the study period in 1990. the in situ nests of p. emaciata were tested once each in a similar fashion. we used a non-parametric wilcoxon test for species differences in the probability of immediate attack responses using the four p. emaciata and five p. occidentalis observation colonies. trials were pooled within colonies and each colony was used as a single data point in the analysis. all observation and in situ polybia colonies contained brood (larvae and/or pupae were visible in the lower combs) when they were tested. based on forager traffic and on the numbers of wasps exiting the nests during attacks polybia emaciata only rarely (5 occasions out of 40 trials on the observation nests and 2 occasions out of>15 trials on in situ nests) exhibited a typical polybia attack response within 30 sec when the nest was tapped. polybia emaciata workers never rushed out of the nest and onto the nest envelope. in all trials where the wasps did not attack in response to the initial mechanical disturbance (within the first 30 sec), some of the workers on the nest surface flew off the nest and departed; the remainder rapidly entered the nest. after the initial disturbance, the nest surface and the lowest layer of comb (partially visible through the nest entrance, fig. breathing into the nest entrance did not elicit rapid (within 10 sec) attack responses. after continued disturbance within a trial (breath and tapping), p. emaciata workers often attacked violently. in approximately 75% of trials, several dozen individuals rapidly exited the nest, took flight, and struck and attempted to sting the observer's bee veil. the p. emaciata nests were not abandoned following these attacks, and workers later returned to the nests. in contrast, all 50 nest-tapping trials with p. occidentalis resulted in immediate (within 1 sec) exit-nest and attack responses. the species difference in the per-colony probability of nest exit and attack within 30 sec of disturbance was significant (fig. 2; wilcoxon 2-tailed test, z=2.60, p<0.01). breathing in or on p. occidentalis nests always elicited immediate attack responses, therefore, habituation to tapping and breathing stimuli was not evident over the course of the study in p. occidentalis. the four p. emaciata observation nests suffered no damage while exposed to nearly daily heavy rains for 3 weeks; four of the p. occidentalis observation nests in the same location were visibly damaged by rain during the same period. even during heavy downpours, the mud nests repelled water much longer than nearby p. occidentalis nests. during one rain shower, the mud nests did not appear waterlogged until 15 min of rainfall had elapsed, while p. occidentalis nests stopped shedding water and became waterlogged after<5 min. the nest fell 2 m to the ground, with only minor damage to its envelope, and the nest was still occupied by adult wasps when it was collected within 12 h of falling. rather than exiting and attacking within a few sec of vibrational disturbance, p. emaciata workers usually either departed or fled into the nest interior. unlike many other paper wasps (s.o'd. and r.l.j., personal observation), p. emaciata failed to respond aggressively to human breath, even when we blew directly into the nest entrance. polybia emaciata behavioral responses to nest disturbance were unusual among their congeners, and among paper-nesting epiponini with similar colony sizes (s.o'd and r.l.j. personal observation; chadab, 1979). however, a few species of paper wasps in other genera exhibit similar responses to human disturbance, which chadab (1979) labeled richards (1978) collected a p. emaciata colony in brazil, and the wasps did not exit the nest until 1.5 h after it was collected. residents of costa rica from several rural communities reported collecting and moving active p. emaciata colonies without being stung, by placing a finger over the nest entrance and plucking the nest from its attachment point. chadab (1979) noted that one colony of p. emaciata in ecuador had durable nest paper and exhibited defensive retreat responses like those that we recorded; two other p. emaciata colonies had fragile nests and performed exit-nest and attack behavior when jarred. we did not observe qualitative variation in p. emaciata nest durability in panama (> 50 colonies observed; not all were tested for defensive responses). predation on swarm-founding wasp nests by bats (jeanne, 1970a), birds (skutch, 1971; windsor, 1976), and primates (vecht, 1967) has been documented. we hypothesize that the unusual defensive response of p. emaciata is a behavioral adaptation to its use of mud in nest construction, which may make the nest more resistant than paper to entry by vertebrate predators. it appears that the wasps initially rely on the nest itself, rather than on exit and attack behavior, to thwart vertebrate predators. we suggest that this represents a special type of architectural defense (hermann and blum, 1981), where the defensive behavior of a species has been modified to exploit the properties of its nest material skutch (1959) observed a red throated caracara (daptrius americanus) removing combs from a p. emaciata nest and feeding on the brood in costa rica. our subject nests often responded with attack behavior after extended disturbances, suggesting that attack can be effective for this species. when attack did come it was sudden and massive, suggesting that it was coordinated by an alarm pheromone, as has been demonstrated for p. occidentalis (jeanne, 1981). although army ants are among swarm-founding wasps ' most frequent predators in wet tropical habitats, preliminary tests on the reaction to eciton army ants did not suggest that p. emaciata's mud nest is effective in resisting these predators (chadab, 1979; s.o'd. personal observation). the use of mud as a nesting material may have evolved in response to predation pressure, particularly by vertebrate enemies. similar advantages against hornet (vespa) predation may have favored the evolution of mud construction in hover wasps of the genus liostenogaster (hansell 1984; turillazzi 1991). however, mud construction appears to provide other benefits. our observations suggest that p. emaciata mud nests were more water repellent, and more resistant to mechanical damage, than similarly sized paper nests. even if increases in general durability were the original selective advantage driving the evolution of mud construction, the workers ' defensive behavior has apparently been secondarily modified. there is variability in average colony size and in the details of nest architecture among species within the mud-nesting polybia subgenus pedothoeca, and in the independently evolved mud-nester p. furnaria (richards, 1978; carpenter et al., 2000). of special value to our understanding of the evolutionary significance of the mud-nesting habit will be field studies on the defensive behaviors of the other mud-nesting polybia species. in particular, such studies could establish whether retreat behavior in response to vertebrate disturbance always accompanies the use of mud in nest building. useful comparisons of defensive behavior could also be made to epiponine paper-nesters with relatively strong nest paper, such as epipona and chartergus. retreat behavior in response to vertebrate disturbance is not restricted to mud-nesting species of epiponini. similar responses to human approach or nest vibration have been observed in paper- and secretion-nesting species (protopolybia emortualis, protopolybia exigua, and protopolybia (formerly pseudochartergus) fuscatus) and in cavity-nesting agelaia cajennensis (jeanne, 1970b; chadab, 1979; carpenter et al., 2001). paper-nesting polybia jurenei colonies retreated when their nest tree was rubbed, but exited and attacked when the substrate was jarred (s. o'd. these patterns suggest that retreat behavior has evolved convergently in several lineages of epiponini, perhaps in response to particular types of predators. photograph of a mud nest of polybia emaciata collected july 1998 in gamboa, panama. box plots showing the range of probabilities of response to nest disturbance with attack within 30 sec by polybia emaciata and p. occidentalis colonies.

the swarm-founding wasp polybia emaciata is unusual among eusocial vespidae because it uses mud, rather than wood pulp, as its primary nest construction material. polybia emaciata nests are more durable than similarly sized paper nests. we tested the hypothesis that the defensive behavior of this wasp may have been modified to take advantage of their strong nests in defense against vertebrate attacks. we simulated vertebrate disturbances by tapping on, and breathing in, p. emaciata nests and similarly sized p. occidentalis paper nests in the same location at the same time. polybia emaciata responses to disturbance were qualitatively different from those of p. occidentalis. the latter exit the nest and attack, while p. emaciata workers typically fled or entered the nest, attacking only after repeated and extended disturbances. we conclude that durable nest material may permit predator avoidance behavior in p. emaciata. we compare the defensive responses of p. emaciata workers with those of other swarm-founding vespidae, and discuss several selective forces that could cause the evolution of species variation in nest defense behavior.

PMC355903

pubmed-90

human gliomas represent 50% to 60% of all intracranial tumors. according to the world health organization (who) guidelines, gliomas are histologically classified into four grades: pilocytic astrocytoma (grade i), low-grade diffuse astrocytoma (grade ii), anaplastic astrocytoma (grade iii), and glioblastoma multiforme (gbm, grade iv). both diagnostic technologies and therapeutic strategies the 5-year survival rates of low-grade (grade i~ii) and high-grade (grade iii~iv) glioma patients in china are 75.4% and 18.2%, respectively. especially, the median survival time for patients with gbm is still only 12 months. indeed, early diagnosis and prolonging survival in glioma patients remains a great challenge for clinicians in the field of neurooncology. there have been several prognostic factors for glioma patients, such as age, preoperative duration of symptoms, karnofsky performance status (kps) score, histologic grade, tumor necrosis, surgical resection extent, use of postoperative radiation therapy, and, probably, adjuvant chemotherapy. however, these clinical parameters can not completely account for the observed variation in survival because of the heterogeneity of glioma patients. thus, there is an urgent need to further investigate the molecular mechanisms of glioma and to identify the effective prognostic indicators for survival prediction.the dna-base excision repair (ber) pathway is responsible for the repair of exogenous and endogenous alkylating and oxidative dna damage, which may lead to carcinogenesis, cell death, and aging if left unrepaired this pathway involves the recognition and removal of damaged bases by a dna glycosylase, followed by incision of the resulting abasic (ap) site by ap endonuclease, dna synthesis by polymerase, and strand ligation by dna ligase. thus, the ber pathway is an important candidate for intervention into the cellular responses to dna change. n-methylpurine-dna glycosylase (mpg) as a dna repair enzyme is a main component in the ber pathway. in previous study aimed at understanding the significance of initiating lesions removed by the ber pathway, kaina et al. identified the over-expression of the human mpg in chinese hamster ovary cells. in the n-alkylpurine repair process, mpg is responsible for the glycolytic removal of the modified base, which leads to the formation of apurinic sites. although n-alkylpurines have not been found to be directly mutagenic, apurinic sites left by this repair process can block replication and lead to mutation. mpg also participates in the repair of 8-hydroxyguanine and hypoxanthine. because of the potential role of dna base lesions in mutagenesis and carcinogenesis, a number of studies have been performed to investigate the association of mpg with various human cancers. (1998) detected the increased mpg gene and protein expression in the breast cancer cells versus normal breast epithelial cells by northern analysis, southern blots, immunofluorescence, immunohistochemistry, and western blot analysis. in 2001, sohn et al. reported that the expression of mpg was increased in high-risk hpv-infected cervical neoplasias and the intracellular distribution of mpg protein was altered, suggesting a role of mpg in carcinogenesis. in an effort to improve the efficacy of cancer chemotherapy by intervening into the cellular responses to chemotherapeutic change, many researchers have been interested in the effects of mpg in tumor sensitivity to the clinical chemotherapeutic agents. as their results, mpg-overexpressing ovarian cancer, osteosarcoma, and breast cancer cells are significantly more sensitive to the clinical chemotherapeutic agents, suggesting that the overexpression of mpg may be a possible gene therapy approach to sensitize tumor cells to the cell-killing effects of chemotherapeutic alkylating agents. the biological mechanism behind the increase of sensitivity to the chemotherapeutic agents in mpg overexpressing cell lines may be that the balance between glycosylase activity, leading to apurinic sites and formation of strand breaks, and subsequent excision repair processes may play an important role in determining cellular cytotoxicity and resistance to alkylating agents. of our interests, we focus on the involvement of mpg in human gliomas. recent study has demonstrated that mpg mrna expression was higher in astrocytic tumor tissues than in brain tissues adjacent to tumor, and mpg protein localization in immunohistochemical study was detected only in the nucleus of all tumor tissues, suggesting an mpg's role in human astrocytic tumors and raise the possibility that the altered mpg expression and intracellular localization could be associated with astrocytic tumorigenesis. tang et al. further demonstrated that mpg overexpression, together with the inhibition of ber, could sensitize glioma cells to the alkylating agent. however, little is known about the expression level of mpg in human gliomas with different clinical grades and its prognostic significance. to address this problem, we used quantitative real-time pcr, immunohistochemistry assay, and western blot analysis to investigate the expression pattern of mpg gene and protein in glioma specimens and normal control brain tissues. next, we analyzed the relationship between mpg expression and the glioma stage as well as the survival of patients. this study was approved by the research ethics committee of the institute for functional neurosurgery p.l.a, tangdu hospital, fourth military medical university, xi'an, p.r. china. written informed consent a total of 128 formalin-fixed, paraffin-embedded specimens of gliomas resected between 2000 and 2010 were retrieved from the archives of the pathology department of tangdu hospital, fourth military medical university, p.r. all the slides were reevaluated according to who classifications by two pathologists, with differences resolved by careful discussion. a total of 76 males and 52 females (1.46: 1) were enrolled in this study, and the median age was 42 years (range, 1271). thirty-two of the 128 gliomas were classified as low-grade [18 pilocytic astrocytomas (who i) and 14 diffuse astrocytomas (who ii)], and 96 were classified as high-grade gliomas [38 anaplasia astrocytomas (who iii), and 58 primary glioblastomas (who iv)]. the clinicopathological features, and the treatment strategies of all the patients were indicated in table 1. paraffin and snap-frozen sections of nonneoplastic brain tissues from 10 patients with intractable epilepsy were also included as controls. five-year followup was performed, and all patients had complete followup until death. overall survival time was calculated from the date of the initial surgical operation to death. patients, who died of diseases not directly related to their gliomas or due to unexpected events, were excluded from this study. in addition, 20 glioma specimens [5 pilocytic astrocytomas (who i), 3 diffuse astrocytomas (who ii), 3 anaplasia astrocytomas (who iii), and 9 primary glioblastomas (who iv)] were snap-frozen in liquid nitrogen and stored at 80c following surgery for mrna and protein isolation. total rna purified from all 20 glioma tissues and 10 control brain tissues real-time monitoring of polymerase chain reactions (pcrs) was performed using the abi 7900ht (idaho technology, idaho falls, i d, usa). cdna was synthesized using random primers and oligo 18dt and superscript ii reverse transcriptase (invitrogen). gene expression was determined using the sybr green i dye (biogene), which binds preferentially to double-stranded dna, and 10 ng of template. fluorescence signals, which are proportional to the concentration of the pcr product, are measured at the end of each cycle and immediately displayed on a computer screen, permitting real-time monitoring of the pcr. the reaction is characterized by the point during cycling when amplification of pcr products is first detected, rather than the amount of pcr product accumulated after a fixed number of cycles. the higher the starting quantity of the template, the earlier a significant increase in fluorescence is observed. the threshold cycle is defined as the fractional cycle number at which fluorescence passes a fixed threshold above the baseline. the primers 5-gtc cta gtc cgg cga ctt cc-3 and 5-ctt gtc tgg gca ggc cct ttg c-3 were used to amplify 603-bp transcripts of mpg, and the primers 5-ggt ggc ttt tag gat ggc aag3 and 5-act gga acg gtg aag gtg aca g3 were used to amplify 161-bp transcripts of -actin. the pcr profile consisted of an initial melting step of 1 min at 94c, followed by 38 cycles of 15 s at 94c, 15 s at 56c, and 45 s at 72c, and a final elongation step of 10 min at 72c. fluorescence data were converted into cycle threshold measurements using the sds system software and exported to microsoft excel. thermal dissociation plots were examined for biphasic melting curves, indicative of whether primer dimers or other nonspecific products could be contributing to the amplification signal. twenty glioma tissues and 10 control brain tissues were homogenized in lysis buffer [pbs, 1% nonidet p-40 (np-40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (sds), 100 ug/ml aprotinin, 100 g/ml phenylmethylsulfonyl fluoride (pmsf), sodium orthovanadate] at 4c throughout all procedures, and sonicated for 70 s, then add 300 g pmsf per gram of tissue and incubate on ice for 30 min, followed by centrifugation at 15,000 rpm for 20 min at 4c. the protein content was determined according to bradford's method, with bovine serum albumin used as a standard. protein samples (30 g) were boiled with 2 sample buffer containing 5% -mercaptoethanol for 5 min, separated by size on 15% polyacrylamide gel under sds denaturing conditions, and transferred to a nitrocellulose membrane at 90 v for 2 h. the nitrocellulose membranes were stained with ponceau s to assess the efficiency of transfer. nonspecific binding was blocked by incubation in block buffer (5% nonfat dry milk, 0.05% tween-20, 1 tris-cl-buffered saline) overnight at 4c, the membranes were hybridized with a mouse monoclonal antibody to mpg (ab55461; abcam), then incubated with a horseradish peroxidase-labeled goat anti-mouse igg (1: 500). the bound secondary antibody was detected by enhanced chemiluminescence (amersham life science, little chalfont, uk). positive immunoreactive bands were quantified densitometrically (leica q500iw image analysis system) and expressed as ratio of mpg to -actin in optical density units. immunohistochemical assay was performed using the conventional immunoperoxidase technique according to the protocol of the department of neurosurgery, institute for functional neurosurgery p.l.a, tangdu hospital, fourth military medical university, xi'an, p.r. briefly, following peroxidase blocking with 0.3% h2o2/methanol for 30 min, specimens were blocked with phosphate-buffered saline (pbs) containing 5% normal horse serum (vector laboratories inc., all incubations with a mouse monoclonal antibody to mpg (ab55461; abcam) at 1: 500 dilution were carried out overnight at 4c. then the specimens were briefly washed in pbs and incubated at room temperature with the anti-mouse antibody and avidin-biotin peroxidase (vector laboratories inc., the specimens were then washed in pbs and color developed by diaminobenzidine solution (dako corporation, carpinteria, ca, usa). after washing with water, specimens were counterstained with meyer's hematoxylin (sigma chemical co., st louis, mo, usa). nonneoplastic brain tissues were used as control tissues, and nonimmune igg was also used as negative control antibody for immunohistochemical staining. immunostaining was scored by two independent experienced pathologists, who were blinded to the clinicopathologic parameters and clinical outcomes of the patients. 5% scored 0, 625% scored 1, 2650% scored 2, 5175% scored 3,>75% scored 4; (2) intensity of stain: colorless scored 0, pallide-flavens scored 1, yellow scored 2, brown scored 3. the staining score was stratified as (0 score, absent),+(14 score, weak),++ (58 score, moderate), and+++(912 score, strong) according to the proportion and intensity of positively stained cancer cells. all computations were carried out using the software of spss version13.0 for windows (spss inc, il, usa). randomized block design anova was used to analyze the statistical difference among different tissue types. in the analysis of glioma morbidity for all patients, we used the kaplan-meier estimator and univariate cox regression analysis to assess the marginal effect of each factor. a spearman's analysis was carried out to analyze the correlation between mpg mrna and protein expression levels. the gene expression of mpg normalized to -actin in 20 glioma and 10 control brain tissues was detected by real-time pcr. as shown in figure 2, the expression levels of mpg gene in human glioma tissues were conspicuously higher than those in control brain tissues (p<0.001). interestingly, mpg gene expression increased with the advancement of who grades from i to iv (p<0.001). the protein expression of mpg normalized to -actin in 20 glioma and 10 control brain tissues was also detected by western blot analysis. as the results, the expression levels of mpg protein tended to increase from the glioma to the normal tissue (figures 3(a) and 3(c)). mpg expression was highest in grade iv and lowest in grade i (figures 3(b) and 3(c)), indicating a close correlation of mpg protein expression with who grade. there was a significant positive correlation between the expression of mpg gene and protein expression levels from the same glioma tissues (rs=0.82, p<0.001). the expression and the subcellular localization of mpg protein in 10 nonneoplastic brain and 128 glioma tissues were detected by immunohistochemistry assay. as shown in figure 4, the positive staining for mpg was mainly observed in the nuclei of tumor cells in glioma tissues, which was consistent with the previous study of wang et al. the representative photographs were shown in figures 4(a) and 4(b). among the glioma specimens, 102 (79.7%) glioma specimens were positively stained for mpg, whereas its immunoreactivities in nonneoplastic brain sections ranged from undetectable to low (figure 4(c)). additionally, the immunostaining of mpg in nonneoplastic brain tissues is below detection levels of both real-time pcr and western blot. according to the statistical analysis, the expression level of mpg protein in glioma tissues was significantly higher (p<0.001) than that in nonneoplastic brain tissues, which was consistent with the results of western blot analysis. in addition, mpg expression was not significantly affected by the gender and age (both p>0.05) of the patients. in contrast, the mpg expression was the closely correlated with who grade, as well as kps. the expression levels of mpg protein in glioma tissues with higher who grade (table 1; p=0.002) and lower kps (table 1; p=0.01) were significantly higher than those with lower who grade and higher kps. moreover, we reviewed clinical information of these mpg-positive or -negative glioma patients. during the follow-up period, 100 of the 128 glioma patients (78.1%) had died (24 from the mpg-negative group and 76 from the mpg-positive group). as determined by the log-rank test, the survival rate of patients with positive mpg staining was lower than those without mpg staining (p<0.001; figure 5(a)). the median survival time of patients with negative expression of mpg could not be estimated by statistical analysis because all patients survived better than the overall median level, and those patients with strong positive (+++), moderate positive (+ +), and weak positive (+) of mpg were 9.1 1.5 months, 12.3 1.1 months, and 22.6 2.2 months (log-rank test: p<0.001). furthermore, figure 5(b) shows the postoperative survival curve of patients with glioma and mpg expression after adjusting for age, gender, who grade, and kps. by multivariate analysis (table 2), the loss of mpg expression was a significant (p<0.001) and independent prognostic indicator for patients with glioma besides age, who grade and kps. the cox-proportional hazards model showed that mpg over-expression was associated with poor overall survival. because of the dismal survival in the patients with glioma, especially in gbm patients, there is an urgent need to find novel specific and effective prognostic markers in order to apply individualized treatments to specifically target the pathophysiologic and molecular properties of glioma patients. in the current study, we investigated the expression of mpg in 128 cases of human glioma and compared the expression with tumor grade and survival rates of patients. our data demonstrated that mpg gene and protein were both increased in glioma compared to nonneoplastic brain tissue. we found an increased trend of both mpg protein level and gene level from who grade i to who grade iv glioma. these results suggest that the transcriptional and translational activation of human mpg might participate in the tumorigenesis and progression of glioma. dna in all somatic and germ cells in the body are continuously exposed to exogenous and endogenous alkylating and oxidative agents that result in damage to dna. if dna damage was not repaired, it may lead to genetic mutation, chromosome aberration, aging, and carcinogenesis. the lack of precise dna repair activities may lead to defective embryogenesis, tissue-specific dysfunction, hypersensitivity to dna-damaging agents, premature senescence, genetic instability, and elevated cancer rates. the repair of dna bases damaged by alkylation is initiated in mammalian cells by mpg, as an alkyladenine dna glycosylase. the majority of repair that is initiated by mpg occurs via short-patch ber, a mechanism whereby only one nucleotide is replaced. the majority of base damage induced by methylation is removed by mpg, including the principal adducts 3-mea and 7-meg. it has been reported that the expression of mpg could be induced by viral damage and a variety of dna-damaging agents. for example, the increased expression of mpg gene was studied in breast cancer, cervical neoplasia, and thymic carcinoma in sv 40 t-antigen-expressing transgenic mice. as previous investigation, mammalian cells actively regulated dna repair enzymes and genes during cell proliferation. dna repair enzymes are expressed in the cell cycle in a defined temporal pattern relative to the induction of dna replication. in brain cells that are not undergoing dna replication, kim et al. indicated that mpg expression in the brain was relative high in 1 week after birth, and the level remained low in day 400 mature adults, suggesting that brain tissue is terminally differentiated and nonproliferating tissues. it has been demonstrated that dna repair enzymes play an important role in the carcinogenesis of several cancers. of our interests, mpg was overexpressed in breast cancer up to 24-fold as compared to normal primary breast epithelium, suggesting the role of mpg in breast carcinogenesis. in astrocytomas, kim et al. in 2003 reported that mpg mrna level was significantly higher than that in tumor-adjacent brain tissues. with the similar results, we in this study also found the upregulation of mpg protein and gene in glioma tissues. additionally, we provide evidence that mpg protein is found in nuclear localization of the glioma tissues, which was consistent with the results of kim et al.. the nuclear localization of mpg in tumor cells of glioma tissues may be suggestive of a role of mpg in the regulation of cell division and cell cycle. furthermore, the most important finding of this study was the correlation of mpg expression and survival rates of patients. kaplan-meier and multivariate analysis both showed a significantly worse overall survival for patients whose tumors had high mpg levels, indicating that high mpg protein level is a marker of poor prognosis for patients with gliomas. this is the first investigation to demonstrate the prognostic value of mpg in human cancers. our data showed the over-expression of mpg gene and protein in human gliomas and also suggested for the first time that mpg be an unfavorable independent prognostic indicator for glioma patients.

aim. to examine the expression of n-methylpurine-dna glycosylase (mpg) gene and protein in glioma samples with different who grades and its association with patients ' survival. methods. immunohistochemistry assay, quantitative real-time pcr and western blot analysis were carried out to investigate the expression of mpg gene and protein in 128 glioma and 10 non-neoplastic brain tissues. results. mpg gene expression level in glioma tissues was significantly higher than that in non-neoplastic brain tissues (p<0.001). immunohistochemistry also showed that mpg protein was over-expressed in glioma tissues, which was consistent with the resutls of western blot analysis. additionally, the expression levels of mpg gene and protein both increase from grade i to grade iv glioma according to the results of real-time pcr, immunohistochemistry and western blot analysis. moreover, the survival rate of mpg-positive patients was significantly lower than that of mpg-negative patients (p<0.001). we further confirmed that the over-expression of mpg was a significant and independent prognostic indicator in glioma by multivariate analysis (p<0.001). conclusions. our data showed the over-expression of mpg gene and protein in human gliomas, and also suggested for the first time that mpg be an unfavorable independent prognostic indicator for glioma patients.

PMC3303893

pubmed-91

the prevalence of overweight and obesity among children and adolescents has widely increased worldwide [1, 2], making it one of the most common chronic disorders in this age group and in adulthood. the use of body mass index (bmi) for age to define being overweight and obese in children and adolescents is well established for both clinical and public health applications, because of their feasibility under clinical settings and in epidemiological studies [3, 4]. in children and adolescents, the natural increases in bmi that occur with age necessitate the use of age-sex-specific thresholds. the most widely used growth charts are the centers for disease control and prevention (cdc-2000), the international task force (iotf), and the 2007 growth references for 5 to 19 year olds produced by the world health organization (who-2007). the cdc-2000 growth charts were developed to evaluate the nutritional status of us children and were originated from five cross-sectional representative surveys carried out in the us between 1963 and 1994. these growth charts are routinely applied to identify children and adolescents with a bmi greater than the 85th or 95th percentiles following the advice of the us expert committee on childhood obesity. however, the appropriateness of an american dataset for defining overweight in young people from other countries is questionable. the iotf reference also uses age-sex-specific bmi percentiles, and overweight and obesity definition corresponds to an adult bmi of 25 and 30 kg/m, respectively, and reflects values in children tracking to overweight and obesity in adults. this reference is based on six large international cross-sectional representative datasets, identifying the bmi values that extrapolate to childhood. the who-2007 growth references were created to replace the national center for health statistics (nchs) references [10, 11]. this reference was constructed using data from the 1977 nchs/who growth reference (1 to 24 years old) merged with data from the 2006 who child growth standards for preschool children (under 5 years of age) using state-of-the-art statistical methods. although valuable information has been appearing in the literature or online, such as works from the health behaviour of school-aged children study which is mainly related to social determinants of health and well-being among young people, no systematic review has been conducted to understand the worldwide magnitude of the overweight and obesity problem among the adolescent population. thus, the objective of this study was to systematically review the literature regarding the prevalence of overweight and obesity in adolescents (1019 years old) of both sexes published in the past 12 academic years (19992011). a systematic literature search was performed which ended on june 7, 2012 (see figure 1). the literature search was conducted in medline and scopus using the following mesh terms: overweight; obesity; prevalence; adolescent. in total, 2537 articles were selected. we also reviewed the data on the prevalence of childhood overweight and obesity on the international obesity task force website at http://www.iaso.org/iotf/. to find the articles included in this review, the following inclusion criteria were used: (1) cross-sectional studies conducted in the last 12 years (19992011)when the original study did not report the survey year, it was not included; (2) national and regional representative samples, but articles published on the prevalence of overweight in towns, urban, or rural areas in a country were excluded; (3) weight and height objectively measured; (4) results presented by sex; (5) and for both overweight and obesity prevalence; (6) the definition of overweight and obesity using the (i) cdc-2000, (ii) iotf, and (iii) who-2007 growth references; and (7) studies written in english, spanish, italian, or portuguese. moreover, if there were more than one national or regional study in the same country, the most recent one was included in the prevalence tables (except for usa and canada, countries in which the most recent data was not included in the tables due to differences in the representativeness of the samples and the impossibility to calculate a single prevalence of overweight and obesity for adolescents ' boys and girls; however, no differences in prevalence were observed between studies as it has been indicated in the discussion). the final number of articles included in this review was 39 articles related to overweight and obesity and also a study on the latest statistics on the prevalence of overweight and obesity in south africa. potentially relevant papers were selected by (1) screening the titles; (2) screening the abstracts, and (3) if abstracts were not available or did not provide sufficient data, the entire article was retrieved and screened to determine whether it met the inclusion criteria. full-text articles were assessed by 2 authors (m. m. bibiloni and j. a. tur). any matter of doubt was discussed by at least two of the reviewers (m. m. bibiloni, a. pons, and j. a. tur). a total of thirty-nine articles and a national health report were eligible according to the inclusion criteria established for this review. table 1 presents a description of the forty studies selected for this review including the continent and the country where it was performed (and region for not national studies), year of publishing, total number of participants in the study, number of adolescents, age range, proportion of girls, and number and definition for overweight and obesity classification used. nationally representative data were obtained in twenty-five countries (including northern ireland) [1539], and ten countries were represented only by regional data [40, 42, 44, 45, 47, 5054]. table 2 shows overweight and obesity prevalence from the twenty-five national studies (one of them including data from northern ireland) that were included in this review according to the continent and the country where it was performed, year of survey, study population, age range, criteria used for classifying overweight and obesity used, and along with total data by sex. there were thirty-two different prevalence levels described in the included articles, because five countries presented data using at least two different criteria for overweight and obesity classification [18, 23, 27, 36, 39]. the iotf cut-off was used to classify overweight and obesity in twenty-three of the twenty-five national studies considered in the present review. in general, the prevalence of overweight plus obesity was higher in america [1820], oceania [38, 39] and europe [3037] and lower in africa [1517] and certain parts of asia [2129] (in china and iran the total prevalence was less than 10% by the iotf cut-offs). overall, about 30% of american adolescents and 22%25% of european adolescents (excepting the czech republic and italian adolescents ' which showed a prevalence of 13.7% and 17.9%, resp.) were overweight or obese. among oceanian adolescents the prevalence ranged from 23.2% in australia in 2004 to 34.2% in new zealand in 2007. in africa, the overall prevalence of overweight and obesity was lower than 20%. among asian adolescents there was a broad range of overweight plus obesity. using iotf cut-off, the prevalence of being overweight or obese for asian boys and girls ranged from 5.2% in china in 2002 to 36.4% in bahrain in 2000. specific prevalence from all the geographic regions was included in this review from three countries: south africa (nine provinces), usa (fifty two states), and italy (five regions). in europe, data from islands of greece (crete) and italy (sicily and sardinia) [48, 49] and spain (balearic islands ' archipelago; and the grand canary island) were also included. on the other hand, regional but not national data was found for eleven countries (italy, brazil, india, jordan, denmark, france, hungary, poland, spain [51, 52], switzerland, and turkey). the iotf cut-off was used to classify overweight and obesity in fourteen of the eighteen selected studies that included regional data. in one study, data was presented using only the who-2007 growth charts and in two studies using only the cdc-2000 growth reference [20, 43]. in south africa and usa, substantial geographic variations in adolescent overweight and obesity prevalence varied in south africa from 13.5% in limpopo to 25.5% in kwazulu-natal. in 2007, overweight and obesity varied in usa from 23.1% in utah and minnesota to 44.5% in mississippi. in 2002, the prevalence of overweight and obesity in southern italy and italian islands was higher among boys. in southern italy, the overweight prevalence among girls also was higher than in the other geographic regions. comparison between the islands from greece (crete), italy (sicily and sardinia), and spain (balearic islands and grand canary island) which were included in this review showed that crete had the highest prevalence of overweight and obesity despite data were presented using different definition. in spain, using the iotf cut-off (data not shown for balearic islands but given by authors), the prevalence of overweight plus obesity was higher in the grand canary island (29.1%) than in the balearic islands (24.7%). according to national data, the prevalence of overweight among boys was 10% higher than girls in nine countries (canada, qatar, taiwan, cyprus, czech republic, germany, greece, italy, australia, denmark, and hungary) and among girls 10% higher than boys in seven of the twenty-five countries (south africa, seychelles, tunisia, mexico, bahrain, saudi arabia, and sweden). the obesity prevalence was 10% higher among boys in seventeen countries (canada, usa, china, iran, israel, qatar, saudi arabia, taiwan, cyprus, czech republic, germany, greece, italy, portugal, sweden, australia, new zealand, denmark, and hungary) and 10% higher among girls in four of the twenty-five countries (south africa, seychelles, tunisia, and bahrain). the aim of this study was to review systematically the literature on overweight and obesity prevalence among adolescents worldwide. thirty-nine articles and one national health report that met the inclusion criteria were considered. the overweight and obesity prevalence in the included studies ranged widely. in sixteen of the twenty-three countries with national representative data using the iotf cut-off, overweight and obesity prevalence higher than 20% were found, five countries showed prevalence above 30%, and just in two countries prevalence was lower than 10%. regarding national data, when prevalence was analysed according to sex, it was observed that boys showed a higher prevalence of overweight in almost half of the countries and a higher prevalence of obesity in almost all countries. these results are consistent with previous studies that pointed out a high prevalence of abdominal obesity among boys. differences of prevalence of overweight and obesity between genders have been related to geopolitical and cultural conditions. eight articles compared data between 1980s and/or 1990s with 2000s [16, 19, 20, 22, 28, 32, 37, 50] and pointed out an increased prevalence of overweight and obesity in both sexes over this period. however, among australian adolescents the overweight and obesity prevalence increased significantly among boys but not among girls over the period 19972004. in the australian national children's nutrition and physical activity survey 2007 (ncnpas07), 25% of boys and 30% of girls aged 9- to 13-year-olds and 25% of boys and 23% of girls aged 14- to 16-year-olds were overweight or obese using the iotf criteria. a comparison of the 1985, 1995, and 2007 australian national surveys of 7- to 15-year-olds indicated that australian children are changing body shape to a more central fat distribution. in usa, overweight and obesity prevalence increased by 3% and 18% among usa girls over this period. however, a cross-sectional analyses of a representative sample (n=4111) of the usa child and adolescent population (birth through 19 years of age) with data from the national health and nutrition examination survey 2009-10 (nhanes) indicated a prevalence of overweight and obesity among adolescents aged 12 through 19 years of 15.2% and 18.4%, respectively. analyses of trends in obesity prevalence for the last two nhanes surveys (2007-08 and 2009-10) indicated that the prevalence of obesity in children and adolescents has not changed in 2009-10 compared with 2007-08. on the other hand, since 2004 the overweight and obesity trends were stabilized or decreased among german adolescents. in usa, substantial geographic disparities in adolescent overweight and obesity were found, with an apparent shift toward higher prevalence in 2007 for several states. reported that children in northern europe countries generally tended to have lower overweight and obesity prevalence (1020%) than in southern europe (2035%). also within the same country, the prevalence and trends of overweight and obesity may not be homogeneous according to different geographic regions. in italy, a north-south gradient in overweight and obesity prevalence among boys but also in overweight prevalence among girls was also reported. a higher prevalence of overweight and obesity has been reported in southern spain in both children and adults. it is important to note that the choice of a reference and a cut-off point will determine the absolute prevalence of overweight and obesity and its trends, and hence different information will be obtained from the papers. argued that the reference they published, supported by the iotf, is less arbitrary and more international than others and recommended its use in international comparisons. lately, monasta et al. suggested that the iotf reference and cut-offs could be preferable to identify overweight and obesity both at individual and population levels because they are at least based on a crude association with ill and health later in life, namely, the definition of overweight and obesity at age 18 years. however, the iotf cut-offs have been not recommended for clinical use when assessing an individual child's growth [9, 6264]. furthermore, recent findings suggested that a universal bmi classification system for childhood and adolescent overweight and obesity may not correspond to a comparable level of body fatness in all populations. the prevalence estimates may not accurately characterize the population groups most at risk of health disadvantages because the correlation of bmi with adiposity is highly variable and dependent on ethnic group [9, 60, 65, 66]. the comparisons of overweight and obesity prevalence need interpretation with caution due to the difference in survey sampling methods, sample sizes, age range of subjects, quality of data in terms of height and weight measurement, and whether national programmes or strategies to tackle overweight and obesity are in place. even within the same country, the prevalence and trends of overweight and obesity may not be homogenous in view of different ethnicities, geographic regions, and socioeconomic status. only articles in english, spanish, italian, and portuguese were included in this review. the results of this review allow the following conclusions: (1) overweight and obesity prevalence is high; (2) obesity is higher among boys, although it is not clear which sex has a higher proportion of adolescents with overweight; (3) despite that there is no consensus about criteria to be used to classify adolescents as overweighed or obese, the most frequently used was the iotf reference. however, the international reference charts for monitoring the secular trends in childhood obesity need to be continually refined and evaluated. the results of this study would contribute to guiding health planners and administrators to develop proper tools for adolescent obesity management.

objective. to review the extant literature on the prevalence of overweight and obesity in adolescents (1019 years old) of both sexes. design. the search was carried out using medline and scopus considering articles published from the establishment of the databanks until june 7, 2012. data on the prevalence of children being overweight and obese from the international obesity task force (iotf) website was also reviewed. only original articles and one national health report were considered. forty studies met the inclusion criteria. results. twenty-five of these studies were nationally representative, and ten countries were represented only by regional data. conclusions. the prevalence of overweight and obesity among adolescents worldwide is high, and obesity is higher among boys. the iotf criterion is the most frequently used method to classify adolescents as overweighed or obese in public health research.

PMC3901970

pubmed-92

sacubitril/valsartan is a combination of a neprilysin inhibitor and an angiotensin ii receptor blocker, indicated to decrease the risk of cardiovascular death and hospitalization for heart failure in patients with chronic heart failure.a patient previously stable on atorvastatin developed severe rhabdomyolysis and an elevation of transaminases within 26 days of initiation of sacubitril/valsartan. rhabdomyolysis is a syndrome characterized by skeletal muscle cell damage, leading to elevated creatine kinase (ck), lactate dehydrogenase (ldh), and aspartate aminotranferase (ast), which can result in severe sequelae, including renal failure, cardiac arrhythmia, hyperthermia and death. associated elevations in transaminases may be seen, even in the absence of significant liver injury. clinically, rhabdomyolysis classically presents with muscle pain, weakness and dark, red or tea-colored urine; however, less than 10% of patients will demonstrate all three components of this triad. rhabdomyolysis may be triggered by hereditary and/or acquired mechanisms, with approximately 75% of initial episodes being a result of acquired causes. in particular, a large number of prescription drugs and drugs of abuse can cause rhabdomyolysis, with statins being one of the top three prescription drugs most commonly responsible for rhabdomyolysis for this syndrome. myopathy and myositis have been reported in conjunction with all of the currently available statins and are considered to be a class effect of these medications. in addition, it has been recognized that statin-associated muscle complaints are dose-related and drug drug interactions that cause an elevation of statin levels have been identified. subgroups of patients at greater risk of statin-associated muscle pathology have been identified and include age>70 years, impaired renal function, and impaired hepatic function. entresto is a combination of sacubitril, a neprilysin inhibitor, and valsartan, an angiotensin ii receptor blocker (arb), indicated to decrease the risk of cardiovascular death and hospitalization for heart failure in patients with chronic heart failure (new york heart association [nyha] class ii sacubitril/valsartan is usually administered in conjunction with other heart failure drug regimens instead of an angiotensin-converting enzyme inhibitor (acei) or another arb. the commonly occurring (5%) adverse reactions reported are hypotension, hyperkalemia, cough, dizziness, and renal failure [6, 7]. sacubitril/valsartan has not been associated with rhabdomyolysis and is commonly used in patients who are also receiving statin therapy. a 63-year-old guyanese woman with hypertension, hyperlipidemia, congestive heart failure (chf), atrial fibrillation, and peripheral vascular disease presented to her primary care provider s office with a complaint of an episode of weakness at home associated with general malaise. she was receiving atorvastatin 40 mg daily, carvedilol 25 mg twice daily, digoxin 0.125 mg daily, furosemide 40 mg daily, spironolactone 25 mg daily, rivaroxaban 15 mg daily, and sacubitril/valsartan 24/26 mg twice daily. she was initiated on sacubitril/valsartan 22 days before her clinical presentation and had been on all of her other concurrent medications for more than 1 year. on the review of systems, the patient reported generalized weakness, pain in her knees, and an ill-defined stinging pain in the skin of her arms and legs. her physical examination was non-focal, with no muscle weakness or tenderness of either the skin or muscles noted. laboratory tests performed on presentation were significant for elevated ast/alanine aminotransferase (alt) of 351 u/l/131 be having a reaction to her statin therapy prescribed for hyperlipidemia and therefore the atorvastatin was withheld. she was scheduled to return in 4 days for repeat laboratory testing and re-evaluation. when the patient returned in 4 days, she complained of dark urine, generalized weakness, and increased muscle and skin pain throughout her body. she was then found to have rhabdomyolysis, with ck at 58,349 u/l (> 50 the upper limit of normal). sacubitril/valsartan was stopped at this time as it was the only new medication in her regimen. the ck level decreased steadily and her symptoms resolved over the course of a 5-day hospitalization (fig. 1). her renal function was preserved throughout and her bilirubin and transaminases briefly increased and also subsequently steadily decreased (figs. 2, 3). barr virus, hepatitis a, herpes simplex virus, hiv, and cytomegalovirus (cmv). barr virus, and herpes simplex virus, and had negative hepatitis a and b, hiv, hsv. and cmv tests. fig. ast aspartate aminotransferase, alt alanine aminotransferase creatine kinase values during and post hospitalization. ast aspartate aminotransferase, alt alanine aminotransferase by 23 days after her initial presentation, the patient no longer had any muscle or skin pain and her ck and liver function test (lft) levels were completely normalized. the patient has not had any reoccurrence of rhabdomyolysis and elevated transaminases for 46 days, and her bilirubin and transaminase levels also returned to normal (figs. 2, 3). one week later, the patient was started on an alternate statin at a low dose (rosuvastatin 5 mg) and the ck levels and lfts have remained stable. this patient had been on a variety of statins for more than 7 years, including atorvastatin, simvastatin, and rosuvastatin. her baseline ck and lfts were normal prior to the initiation of sacubitril/valsartan. she had previously experienced two transient elevations in ck, to a maximum of 1608 u/l, while hospitalized for an implantable defibrillator infection and explanation complicated by an acute chf exacerbation and a recurrent chf exacerbation in the 3 years prior to the current admission. her ck levels returned to normal after her acute illnesses, without change in her lfts. in this case, the immediate cause of her rhabdomyolysis was unknown, but the new medication in her profile seemed to be the likely trigger. due to the severity of her rhabdomyolysis the naranjo adverse drug reaction probability scale score was 5, making it probable that the adverse drug reaction was precipitated by sacubitril/valsartan. the drug interaction probability scale score was 3, consistent with a possible interaction as a cause for the reaction, with sacubitril/valsartan as the precipitant drug and atorvastatin as the object drug. of note, one of the concurrent medications, carvedilol, is a p-glycoprotein inhibitor that may increase the serum concentration of atorvastatin, a p-glycoprotein substrate; however, the patient was stable on this combination for 7 years before sacubitril/valsartan was initiated. none of the other medications taken by this patient are known to cause rhabdomyolysis or an elevation in transaminases. a literature search regarding a drug interaction between sacubitril and statins yielded no reports of rhabdomyolysis with statins. as per a recent study, coadministration of sacubitril/valsartan with atorvastatin led to a twofold increase in the maximum concentration (c max) of atorvastatin and its metabolites. in addition, the us and canadian package inserts of sacubitril/valsartan cite in vitro data showing sacubitril inhibiting organic anion transporting polypeptides (oatp) 1b1 and 1b3, resulting in increased c max and area under the curve (auc) of atorvastatin with coadministration of sacubitril/valsartan [6, 7]. the canadian version of the product insert further states that sacubitril/valsartan may increase the systemic exposure of oatp1b1 and oatp1b3 substrates, such as statins. sacubitril/valsartan increases the c max of atorvastatin and its metabolites by up to twofold, and auc by up to 1.3-fold. the canadian insert recommends that sacubitril/valsartan must be coadministered cautiously with statins, especially simvastatin, a sensitive oatp1b1/1b3 substrate, and a decrease in dose of simvastatin and atorvastatin may be considered when coadministered with sacubitril/valsartan. the interaction between atorvastatin and sacubitril is mentioned in the us product package insert as a theoretical possibility. there is currently no recommendation to increase monitoring or empirically decrease the dose of any statin on initiation of sacubitril/valsartan. a number of medications are known to inhibit oatps and have been shown to increase statin plasma concentrations. many single nucleotide polymorphisms (snps) have been found within the slco1b1 and/or slco1b3 genes encoding oatp1b1 and oatp1b3 proteins, respectively, and were shown to effect the pharmacokinetics and/or pharmacodynamics of statins [12, 13]. the clinical consequences of the drug interactions may vary based on the genetic variation in oatp-encoding genes and inhibition of oatp function. the slco1b1*1b haplotype appears to be associated with enhanced hepatic uptake and reduced plasma concentrations of oatp1b1 substrates. discontinuation of the statin alone did not result in a decrease in the patient s lfts. it was not until sacubitril/valsartan was also discontinued, and treatment for rhabdomyolysis was initiated, that the patient s symptoms began to improve and her ck and lfts returned to baseline. this could be due to either a direct effect of sacubitril/valsartan or the increased serum concentration of atorvastatin, as described in the literature. because of the above-stated pharmacodynamics, we believe that the more likely mechanism is the increased serum concentration of atorvastatin. currently, there is no recommendation regarding the initiation or continuation of statins in patients with nyha class ii iv heart failure as there is insufficient information on which to base recommendations for or against statin treatment. in these patients, the choice of statin dose, and using a statin for cardiovascular risk reduction benefit, must be weighed against adverse effects, drug drug interactions, precautions and contraindications to statin therapy. we would have liked to have arranged slcob testing for the patient but were unable to. further research will be useful in identifying patients at risk for this adverse reaction, and will likely include genetic testing. this case of severe rhabdomyolysis should stimulate further investigation of the potential negative effects of the initiation of sacubitril/valsartan in patients receiving statin therapy. since sacubitril/valsartan is targeted for patients with heart failure, we predict that this would comprise a large number of the candidates for treatment. we recommend consideration of obtaining baseline ck levels and lfts and close observation of patients. in this case, rhabdomyolysis developed within 3 weeks of initiation of sacubitril/valsartan and evaluation within a shorter time frame may be appropriate. future reports of this reaction may help to define high-risk patients, but it should be noted that our patient had no known high-risk characteristics for the development of rhabdomyolysis, except for prior mild elevations of ck during hospitalizations for chf exacerbations. we encourage the reporting of any similar reactions to the us fda, the manufacturer, and the medical community. eve s. faber, madhavi gavini, ronald ramirez and richard sadovsky declare that they have no conflicts of interest that are directly relevant to the content of this report. a copy of the written consent may be requested for review from the corresponding author.

a 63-year-old woman previously stable on a regimen of atorvastatin 40 mg daily, carvedilol 25 mg twice daily, digoxin 0.125 mg daily, furosemide 40 mg daily, spironolactone 25 mg daily, rivaroxaban 15 mg daily, and enalapril 20 mg twice daily for heart failure developed rhabdomyolysis 26 days after enalapril was stopped and sacubitril/valsartan (entresto) started. the patient received sacubitril/valsartan at 24/26 mg twice daily for heart failure; however, after 26 days she developed muscle and skin pain. investigations revealed elevated creatine kinase and liver function tests, and rhabdomyolysis with raised transaminases was diagnosed. sacubitril/valsartan and atorvastatin were discontinued and the patient was hydrated. she returned to baseline in 23 days and has not had any reoccurrence of rhabdomyolysis and elevated transaminases for 46 days. a naranjo assessment score of 5 was obtained, indicating a probable relationship between the patient s rhabdomyolysis and her use of sacubitril/valsartan. the drug interaction probability scale score was 3, consistent with a possible interaction as a cause for the reaction, with sacubitril/valsartan as the precipitant drug and atorvastatin as the object drug.

PMC5089965

pubmed-93

isolated involvement of the appendix in crohn's disease is reported to be 0.2% to 1.8%, and is usually associated with ileocaecal crohn's disease in 25% of ileal and 50% of caecal disease. while appendicitis in a patient who was previously diagnosed to have ileocaecal crohn's may be managed with appendicectomy and ileocaecal resection, appendicectomy alone when performed for appendicitis in a patient with unsuspected ileocaecal crohn's disease could lead to postoperative complications including enterocutaneous fistula. a young female patient who underwent appendicectomy elsewhere for acute appendicitis presented to us with a persistent enterocutaneous fistula of 6 weeks duration. she had complained of general ill health and occasional altered bowel habits for 6 months prior to the acute appendicitis presentation. our investigations, including a ct scan, suggested the possibility of ileocaecal crohn's disease. she underwent excision of the enterocutaneous fistula and ileocaecal resection, and histopathology of the resected specimen confirmed crohn's disease. in the postoperative period she received mesasalazine. when last seen 2 years later during her regular follow-up, she was found to be in good health. the possibility of ileocaecal crohn's disease should be considered in patients presenting with unexplained postoperative enterocutaneous fistula following appendicectomy. a high index of clinical suspicion is required to make a prompt diagnosis and institute appropriate further treatment in form of ileocaecal resection. crohn's disease is a chronic inflammatory condition that may involve the entire gi tract. histological features include granuloma, lymphoid aggregates, fissures and ulcers extending into muscularis propria, and transmural inflammation. crohn's disease has been reported to be the cause of acute appendicitis in 0.2% to 1.8% of all the appendicectomies in some series. operations described in the literature for crohn's disease of the appendix include appendicectomy with caecectomy and ileocaecal resection. however, patients who undergo appendectomy alone are at the risk of complications including enterocutaneous fistula in 34% to 58% of the cases a 24-year-old unmarried female patient presented to us with a persistent fistula in the right iliac fossa following an appendicectomy done in another hospital 6 weeks previously. on further questioning, she informed us that she had presented to this hospital with a 2-week history of abdominal pain, confined to the right lower abdomen. she complained of general ill health and occasional altered bowel habits for 6 months prior to this acute episode. an enquiry with the doctors who had initially seen her revealed that at the time of presentation for acute appendicitis she was quite unwell with fever, tachycardia, and dehydration. her abdomen was tender all over with guarding, more pronounced over the right lower half. an x-ray of her abdomen showed a few fluid levels and an ultrasound scan revealed free fluid in the abdomen with an associated right iliac fossa mass. a diagnosis of pelvic peritonitis secondary to acute appendicitis was made by the physicians, and she was taken up for surgery after adequate resuscitation. there was pus in the abdomen with an appendicular mass, and the appendix appeared oedematous, thickened, and congested. the abdomen was closed after placing a drain, which was removed on the 3 post-operative day. in spite of antibiotic administration she was managed conservatively with antibiotics and anti-inflammatory medication and eventually improved, except for a persistent purulent discharge from the wound. she then decided to seek our opinion and was admitted under our care for further investigation and management. the appendicular histopathology was retrieved from the previous hospital and revealed transmural inflammation with granulomas suggestive of crohn's disease. a computerized tomography (ct) scan of the abdomen carried out in our hospital showed pericaecal collection with an inflammatory mass in front of the caecum (fig. the abdomen was surgically explored through the previous incision after excising the fistula leading into the caecum. an inflammatory mass associated with the caecum was noted. the appendicular stump had not healed, and was draining into a cavity which was communicating with the wound, indicating a complex enterocutaneous fistula. a limited right hemicolectomy was carried out and continuity established with a primary anastomosis of macroscopically-appearing healthy bowel of the ascending colon and terminal ileum. a specimen of the resected caecum revealed the cobblestone appearance of the mucosa, strongly suggesting the possibility of underlying caecal crohn's disease (fig. the histology was reported as inflammatory bowel disease (ibd) consistent with crohn's disease (fig. she was referred to a gastroenterologist and was being treated with mesasalazine and a regular annual colonoscopy. when last seen at two years post-surgery she continued to remain in good health. limited right hemicolectomy specimen showing the cobble stone appearance of the mucosa typical of crohn's disease. crohn's disease may involve the appendix by extension from the terminal ileum or the caecum and present as an acute or sub-acute appendicitis[15]. crohn's disease limited to the appendix usually affects young adults in their 20s and 30s, although it is not limited to this age group and can occur at any age. the clinical presentation is variable, with acute right iliac fossa pain suggestive of acute appendicitis in about 85% of patients, and chronic pain and a palpable mass in the right iliac fossa in about 25% of patients. when the preoperative diagnosis is acute appendicitis, crohn's disease should be suspected when an atypical or protracted clinical course is present in a patient prior to the appendicitis, particularly in areas where crohn's disease is prevalent. crohn's disease is diagnosed by a combination of clinical manifestations and radiological findings including barium studies, showing string strictures, fissures, and fistula in the ileum and caecum. recently, gray scale sonography and color doppler-flow features have been used in diagnosing crohn's disease. when the disease is located atypically in the appendix, macroscopically, the appendix will be seen to be enlarged, swollen and adherent to the surrounding structures, these findings being secondary to chronic inflammation. histologically, the disease is characterized by transmural inflammation with thickening of the appendiceal wall, epitheloid granulomas, lymphoid aggregates, and mucosal ulceration. other histological features are multinucleated giant langherhan's cells, crypt abscess, neural hyperplasia and lymphangiectasia. differential diagnosis includes the presence of foreign bodies and diverticulitis of the appendix, which also give rise to chronic granulomatous inflammation with induration and fibrosis of the appendiceal wall, and granulomatous disease of unknown aetiology such as appendiceal sarcoidosis, which is rare and often accompanies systemic disease. differential diagnosis should also include infectious diseases such as tuberculosis, actinomycosis and yersenia infection. a rare fungal infection such as histoplasmosis or blastomycosis, or a parasitic infestation such as schistosomiasis or enterobius vermicularis infestation can elicit an appendiceal granulomatous reaction. the diagnostic difficulty arises when a patient undergoes an appendicectomy for suspected acute appendicitis and the surgeon unexpectedly encounters ileocaecal pathology whose nature is difficult to ascertain in an emergency situation. in view of this, a definitive treatment in the form of ileocaecal resection for conditions like crohn's disease may be difficult to carry out. while a frozen section may be helpful to differentiate some of these conditions it may not always be feasible in an emergency situation. if crohn's disease of the appendix is suspected and is limited to the appendix then appendicectomy alone is a routine surgical procedure with very low intraoperative or postoperative mortality and a low rate of fistula formation.the postoperative enterocutaneous fistula incidence rate in crohn's disease restricted to appendix alone has been reported to be 3.5%, whereas in patients with crohn's disease of the ileocaecal segment, as in our patient, the postoperative enterocutaneous rate rises to 34% to 58%. weston et al reported that the majority of the patients whose first presentation of crohn's disease simulates appendicitis and who undergo appendicectomy alone leaving the ileocaecal segment in place to be treated medically, returned postoperatively within 3 years with symptoms. a significant percentage (38%) of these patients returned within one year and most of them (77%) were frequently ill with their disease in the interim period. patients who had an ileocolic resection at the time of the initial operation did not require early reoperation, did not develop short bowel syndrome, and did not have significant postoperative complications. in the postoperative period these patients receive medical therapy to keep the disease quiescent such as salazopyrine, 5-asa, prednisilone, and azothioprine.crohn's disease-related fistula have also been treated with infliximab with some success. despite improvements in medical therapy, between 70% to 90% of the patients with crohn's disease will eventually require surgical intervention, and approximately half of these will require additional operations. because of the high risk of reoperations and a relatively young age at the time of first operation, a minimally invasive approach has recently been used to carry out the ileocolic resections. a recent meta-analysis of laparoscopic ileocolic resection has revealed a shorter hospital stay compared to open resection, while the morbidity rates were equal and conversion rates were acceptable. the average interval between surgery and recurrence after appendicectomy for crohn's disease of the appendix is 4 years, after which some claim the recurrence rate to be almost nil. however, others have reported a recurrence rate of 10% over an 8-year follow-up and is much higher in patients with crohn's disease of the appendix associated with ileocaecal crohn's disease. most authors recommend periodic surveillance visits with radiological and endoscopic examination of the small intestinal and colon for a period of at least 3 years to promptly detect recurrence. however, others claim that patients with crohn's disease should be followed for 10 years, and if disease free by then are considered cured. crohn's disease of the appendix is usually associated with crohn's disease of the ileum and caecum. appendicectomy will suffice in those who present subacutely, are diagnosed preoperatively by clinical signs, and radiographic evidence shows that the disease is restricted to the appendix alone. however, in the presence of ileocaecal involvement, resection of the ileocaecal segment may be required to prevent postoperative complications of enterocutaneous fistula. while patients who develop enterocutaneous fistula post-appendicectomy in an unsuspected crohn's disease case have been treated with infliximab with some success, the majority of the patients required ileocaecal resection. moreover ,

context: isolated involvement of the appendix in crohn's disease is reported to be 0.2% to 1.8%, and is usually associated with ileocaecal crohn's disease in 25% of ileal and 50% of caecal disease. while appendicitis in a patient who was previously diagnosed to have ileocaecal crohn's may be managed with appendicectomy and ileocaecal resection, appendicectomy alone when performed for appendicitis in a patient with unsuspected ileocaecal crohn's disease could lead to postoperative complications including enterocutaneous fistula. case report: a young female patient who underwent appendicectomy elsewhere for acute appendicitis presented to us with a persistent enterocutaneous fistula of 6 weeks duration. she had complained of general ill health and occasional altered bowel habits for 6 months prior to the acute appendicitis presentation. our investigations, including a ct scan, suggested the possibility of ileocaecal crohn's disease. she underwent excision of the enterocutaneous fistula and ileocaecal resection, and histopathology of the resected specimen confirmed crohn's disease. in the postoperative period she received mesasalazine. when last seen 2 years later during her regular follow-up, she was found to be in good health. conclusion:the possibility of ileocaecal crohn's disease should be considered in patients presenting with unexplained postoperative enterocutaneous fistula following appendicectomy. a high index of clinical suspicion is required to make a prompt diagnosis and institute appropriate further treatment in form of ileocaecal resection.

PMC3354430

pubmed-94

the random sample comprises 267 caucasian preadolescents (121 girls, 146 boys) and 330 caucasian adolescents (164 girls, 166 boys) of a population belonging to the swedish part of the european youth heart study (eyhs). the eyhs is a cross-sectional school-based study of risk factors for future cardiovascular disease among preadolescents (910 years old) and adolescents (1516 years old). the mean ages in the swedish sample for preadolescents and adolescents were 9.6 years and 15.6 years, respectively. height, weight, and birth weight were measured by internationally accepted standardised procedures (22). body mass index (bmi) was calculated as weight/height2 (kg/m). identification of sexual maturity was assessed according to tanner and whitehouse 1976. a researcher of the same gender as the child recorded the pubertal stage after brief observation. on account of ethical reasons the direction of one school preferred not to take part in the assessment of sexual maturity. fifty subjects did therefore not participate in this assessment (3 preadolescent girls, 7 preadolescent boys, 25 adolescent girls, and 15 adolescent boys). the consumption of milk was assessed by an interviewer-mediated 24-hour recall. in preadolescents, a qualitative food record completed the day before the interview with the help of parents served as a checklist for the data obtained during the recall. dietary data were processed by stormats (version 4.02, rudans lttdata, sweden) and analysed using the swedish national food database (version 99.1). as part of a broad-ranging questionnaire, parents of the participants were asked if their child had a chronic illness or adhered to a special diet. since under-nourishment is practically non-existent in a nutritionally replete population such as sweden and milk intake accounts in part for daily energy intake, the bmi was taken as proxy for energy intake. furthermore, bmi is traditionally used to validate energy intake data (23). for the genetic analysis of lp and lnp, genomic dna was isolated from edta whole blood samples from the individuals with the qiaamp dna blood mini kit spin procedure. the dna fragment spanning the -13910-c/t polymorphic site was amplified using a biotinylated forward-primer (5 gggctggcaatacagataagata-3) and an unbiotinylated reverse-primer (5 agcagggctcaaagaacaatcta-3). the applied sequencing primer was: 5-ctttgaggccaggg-3. sequencing was performed using a psq96 snp reagent kit and a psq 96ma system (pyrosequencing ab) psq 96ma 2.0.1 software. the procedure has been previously described in detail (24, 25). for the determination of socio-economic status (ses) we used the dichotomous variable, below versus above the mean income level in the catchment areas of the sample (below or above the mean in their municipality) on account of the relatively equal distribution of income in sweden (26). for four subjects, data for ses student's t-test was used to determine differences in milk intake (g/d) between lp and lnp subjects. milk intake (g/d) was somewhat skewed to the right and therefore split in quintiles of milk intake. stepwise backward multiple linear regression analysis was performed in order to study the relationship between milk intake in quintiles and body height (cm) after adjustments for sex, birth weight (g), father's and mother's height (cm), bmi, tanner stage (15), and ses. the study was approved by the research ethics committees of rebro county council and huddinge university hospital. parents and 15-year-olds gave specific written informed consent to participate in the study. the variables included in the backward multivariate regression model were: milk intake, lct c>t-13910 polymorphism (lp=ct, tt vs. lnp=cc), sex, birth weight, father's and mother's height, bmi, tanner stage, and ses. characteristics of preadolescents and adolescents by quintile of milk intake are given in table 2. basic characteristics of covariates to body height in the study population of swedish preadolescents and adolescents variables associated to body height by quintiles of milk intake fifty-six (9%) of the subjects of the whole sample were lnp. where lnp may restrict the intake of milk in these subjects and can thereby have an effect on the dependent variable, body height as well as the exposure variable, i.e. milk intake. milk intake tended to be lower in lnp compared to lp but the difference did not reach statistical significance (p=0.067). the studied exposure variables in this model were milk intake in quintiles and the lct c>t-13910 polymorphism, and the outcome variable was body height. the model explained 90% of the observed variance of body height in preadolescents and adolescents (adjusted r =0.89). non-significant variables were removed in a stepwise manner by elimination when p(f) 0.10. model 1 included the variables: milk intake in quintiles, lct-13910 c>t polymorphism, sex, birth weight, parents height, tanner stage, bmi, and ses. in a first step the variable bmi was eliminated and in a second step ses (table 3). milk intake (g/d) in quintiles remained significantly different (=0.46; 95% ci: 0.040, 0.87 and p=0.032) in the final model. in addition, the lct c>t-13910 polymorphism (lp vs. lnp), remained significant in the final model (=2.05; 95% ci: 0.18, 3.92 and p=0.032), showing a positive association of lp with height. main effect by quintiles of milk intake and lct-13910 c>t (exposure variables) on body height in swedish preadolescents and adolescents (n=542); stepwise backward multiple linear regression models were used included variables in model 1: birth weight, sex, mother's and father's height. bmi, tanner stage, ses excluded variable: bmi (p[f] 0.10). excluded variables: ses (p[f] 0.10). we found that milk intake and lct c>t-13910 polymorphism, known to modulate milk consumption, were significant contributors to the observed variance of body height in the studied population. we primarily did not expect to find our hypothesis confirmed in a nutritionally replete country with already one of the highest intakes of milk and dairy products per capita in the world. the lct c>t-13910 polymorphism might contribute with a small effect to phenotypic variation in height. as far as we know no genome wide association studies or meta-analysis have been performed previously addressing the question whether lp or lnp affects body height. increasing evidence suggests that cow's milk consumption exerts a positive effect on longitudinal growth in preadolescents and adolescents, for instance, by affecting the igf-i-axis (8, 11, 2730). but not all studies show a positive effect of milk on height, especially in nordic countries, where average milk intake per capita traditionally is high (31). the lct c>t-13910 polymorphism, known to modulate milk intake (32, 33), remained significant in the final model. by including the lct c>t-13910 polymorphism in our model, we subtract for hidden heritability in an already predominantly lp population (18). in a recent prospective study, evidence of an association between increased infant size at birth and cow milk consumption during pregnancy it is known that lp subjects consume on average more milk than lnp subjects. where lp might thus lead to an increased susceptibility towards already prenatally programmed increased body sizes, mediated by the higher capacity of lp mothers to consume milk and dairy products. our study accounts for parental height as regards milk intake and its potential effects on body height and growth in preadolescents and adolescents. despite the circumstance that body height is a highly heritable complex trait; corrections for parental height continue to be an exception, also shown in recent studies (31). even twin growth studies have been performed without consideration of parental height (35). it has been demonstrated that at the current stage a simple prediction based on phenotype of relatives obviously outperforms sophisticated genomic predictions as regards body height (36). limitations of this study are the sample size, and the limits inherent to cross-sectional studies as regards causal inference. cow's milk is an evolutionary food constructed to promote growth and development in calves and appears to affect growth in human beings also (13, 14, 28). milk has become a normative food for preadolescents and adolescents beyond weaning age, even in asian populations and developing countries, which traditionally did not have home fare based on domesticated dairy animals (8, 37). the long-term effects of this changed nutritional normative are yet poorly understood, even in western countries. we conclude that actual milk intake and the genetic lp trait is positively associated with body height in preadolescents and adolescents. nevertheless, higher milk consumption in childhood might exert both negative and positive effects, since greater height has been associated with higher risk of some cancers (29, 3841). this study was supported by grants from rebro lns landstings forskningskommitt and by nyckelfonden, rebro, sweden.

backgroundbody height is a classic polygenic trait. about 80%90% of height is inherited and 10%20% owed to environmental factors, of which the most important ones are nutrition and diseases in preadolescents and adolescents. objectivethe aim of this study was to explore potential relations between the lct (lactase) c>t-13910 polymorphism, milk consumption, and body height in a sample of swedish preadolescents and adolescents.designin a cross-sectional study, using a random sample of preadolescents and adolescents (n=597), dietary intakes were determined. anthropometric measurements including sexual maturity (tanner stage) and birth weight were assessed. parental body height and socio-economic status (ses) were obtained by questionnaires. genotyping for the lct c>t-13910 polymorphism that renders individuals lactase persistent (lp) or lactase non-persistent (lnp) was performed by dna sequencing. stepwise backward multivariate linear regression was used. resultsmilk consumption was significantly and positively associated with body height (= 0.45; 95% ci: 0.040, 0.87, p=0.032). adjustments were performed for sex, parental height, birth weight, body mass index (bmi), ses, and tanner stage. this model explains 90% of the observed variance of body height (adjusted r2=0.89). the presence of the -13910 t allele was positively associated with body height (= 2.05; 95% ci: 0.18, 3.92, p=0.032). conclusionsmilk consumption is positively associated with body height in preadolescents and adolescents. we show for the first time that a nutrigenetic variant might be able to explain in part phenotypic variation of body height in preadolescents and adolescents. due to the small sample size further studies are needed.

PMC3169089

pubmed-95

primary hyperparathyroidism (php) is a hypercalcemic disease stemming from an abnormal increase in parathyroid hormone (pth) secretion by one or more parathyroid glands. the hallmark of this condition is the presence of high levels of calcium and high or inappropriate levels of pth. primary hyperparathyroidism is more common in women than in men and increases with aging in both genders. the goal of parathyroidectomy is the excision of the abnormal parathyroid gland(s), preserving the normal ones in order to achieve and maintain a postoperative normocalcemic state. success rates for surgical treatment depend on the skill and experience of the surgeon in finding and recognizing the pathologic changes and excising the correct amount of hyperfunctioning parathyroid tissue. the surgical treatment of php has undergone substantive changes since the first successful parathyroidectomy was performed by felix mandl in 1925. it is now expected that the vast majority of patients will be cured during initial surgical exploration at a low probability of morbidity. the conventional time-honored operation employing general endotracheal anesthesia and bilateral cervical exploration is safe and effective when performed by experienced surgeons. however, recent technical innovations, including improved preoperative localization and availability of rapid intraoperative pth assays (iopth), have yielded focused approaches with excellent outcomes. sporadic primary hyperparathyroidism is caused by a single enlarged parathyroid gland (parathyroid adenoma) in approximately 85% of the cases, whereas multigland hyperplasia occurs in 15% and parathyroid carcinoma is found in less than 1% of patients. unlike the previous dogma that required surgical identification of both enlarged and normal parathyroid glands, the current paradigm in many centers is to identify and excise the incident enlarged gland and to confirm operative cure, employing a rapid intraoperative pth assay. due to the relatively short half-life of pth (4-5 min), a dramatic drop in circulating hormone can be detected once the abnormally secreting gland or glands have been removed. a curative drop in pth allows the surgeon to terminate the operation and obviate additional exploration, whereas failure of the pth levels to demonstrate an adequate decrement asks for additional exploration because of the presence of presumed additional hypersecreting gland(s). the aim of the present study was to evaluate our 10-year experience in employing a rapid intraoperative pth assay for php. this is a prospective study on a cohort of operated patients treated at a university referral center. this investigation was approved by the unifesp/epm ethics committee. from june 2000 to april 2011, 96 surgeries for php were performed at hospital so paulo unifesp/epm, so paulo, brazil. these 96 procedures were performed in 91 previous unexplored patients who had at least 6 months of postoperative follow-up with enough reported data to be eligible for the study. the php diagnoses were established in the presence of high levels of calcium and high or inappropriate levels of pth. all patients had their age, gender, symptoms (bone and kidney), preoperative localization tests, serum ionized calcium (ica), serum total calcium and intact pth recorded before parathyroidectomy, as well as iopth dosages. the laboratorial tests were repeated 1 month, 6 months, and in 1-year increments after surgery. a series of 91 consecutive patients with primary hyperparathyroidism underwent parathyroidectomy guided by intraoperative pth at federal university of so paulo, brazil, from june 2000 to april 2011. focused parathyroidectomy guided by intraoperative pth was the initial procedure when preoperative localization tests were positive and when there were no suspicion of malignant disease. a bilateral cervical exploration guided by intraoperative pth was performed when preoperative localization tests were negative. a baseline peripheral venous blood sample was obtained just after anesthesia induction as well as 10 minutes after the abnormal parathyroid tissue removal. the intraoperative criterion used to predict successful parathyroidectomy was a decrease in the intact pth levels exceeding 50% from the preincision hormone level. if this criterion was met, surgical exploration of the neck would be completed and the incision closed. otherwise, further surgical exploration of the neck would have to be carried on. intraoperative pth was measured using elecsys pth immunoassay (elecsys 1010 system, roche, mannheim, germany). the test is an immunometric assay based on monoclonal antibodies, magnetic particles as solid phase, and ruthenium complex as chemiluminescent label. total time to perform the assay is 9 minutes; reference values are 1065 pg/ml. to validate the rapid pth assay, 170 samples from the study the iopth accuracy calculation was based on the following definitions: a true-positive (tp) result of iopth was defined as the correct prediction of postoperative normal calcium levels for at least 6 months; true negative (tn) was the correct prediction of incomplete excision by either resection of an additional gland(s) or operative failure; false positive (fp) was the incorrect prediction of normocalcemia with subsequent postoperative persistent hypercalcemia and high pth levels; false negative (fn) was the incorrect prediction of incomplete excision followed by postoperative normocalcemia. persistent disease was considered when serum calcium and pth levels remained above normal range just after surgery. recurrence was defined when, after reaching normocalcemic levels, serum calcium and intact pth measurements start to rise to abnormal values at least 6 months after surgery. serum ionized calcium (1.151.32 mmol/l), serum total calcium (8.510.5 mg/dl) and intact pth (1167 pg/ml), were measured using standard automatic assays. the demographic, clinical, and biochemical aspects of 91 patients with php are shown in table 1. among all cohort of 91 patients, 69 (75.8%) had solitary adenoma, 10 (11.0%) had multiple endocrine neoplasia type 1 (men1), 6 (6.6%) had double adenomas, 4 (4.4%) had carcinomas, and 2 (2.2%) patients are still waiting for remedial surgery for multiglandular disease (mgd), 1 of whom had recurrence disease after 10 years of follow-up and the other persistent disease after a false positive iopth (table 2). we had 85 (93.4%) successful parathyroidectomies 6 (6.6%) failed parathyroidectomies in 91 previous unexplored patients, and 5 (100%) successful remedial surgeries. among the 85 successful patients, 69 (81.2%) had solitary adenoma, 3 (3.5%) had carcinoma, 4 (4.7%) had double adenomas, 8 (9.4%) had men1, and 1 (1.2%) still has mgd (table 2). the mean decay of iopth in the successful group of patients was 80.5% (34.3% to 96.0%). in these 85 patients, we had 80 tp results (67 adenomas, 3 carcinomas, 8 men1 and 2 recurrences we also had 3 cases of double adenoma in which the iopth resulted in a tn value, requiring additional exploration to prevent persistent disease (table 2). there were only 2 fn results among the successful group patients (table 4). the mean decay of iopth in the 80 patients with tp results was 81.7%, with a minimum drop of 55.0% (55.0% to 96.0%). operative failure of the initial surgery occurred in 6 patients: 2 double adenoma, 2 men1, 1 carcinoma, and 1 mgd who is still waiting for remedial surgery (table 5). both cases of double adenoma had a tn decay of iopth, but the second adenoma wasnot found during the initial operation. with the aid of new localization exams, successful remedial operations were performed with an iopth tp result. the patient with parathyroid carcinoma developed hypercalcemia and high levels of pth two months after surgery. in search of distant metastasis, this patient underwent a pulmonary computerized tomography and multiple pulmonary nodules were found. for this reason, she was classified as iopth false-positive. in both men1 patients, one patient already had men1 diagnosis at the time of initial surgery and we failed to find the fourth gland. the other patient was first operated as a solitary adenoma and men1 diagnosis surfaced just in the follow-up. there was only one case in the operative failure patients; the iopth had an fp result (table 4). the surgical treatment for primary php has undergone some changes in recent years, evolving from the standard bilateral neck exploration technique to a less time-consuming procedure of unilateral neck exploration. taking into account the increasing number of asymptomatic and/or oligosymptomatic primary hyperparathyroidism diagnosis in the recent years, the need for a safe and less time-consuming procedure with low perioperative morbidity is clear. preoperative parathyroid localization imaging study and iopth are essential components of the focused parathyroidectomy that allow the excision of all abnormal parathyroid glands without the examination of the normally secreting glands. the clinical utility of rapid iopth measurements in parathyroidectomy was first reported in 1988 using a modified intact pth irma assay. since then, rapid assays have been developed by means of radioactive [4, 11, 12] as well as nonradioactive formats [12, 13]. the predominance of a solitary adenoma disease in 85% to 96% of cases of php and the short half-life of intact pth (184) of only 1.4 to 4 minutes [1420] combined with the remaining suppressed normal parathyroid glands after removal of all hyperfunctioning tissue allows the measurement of iopth to evaluate its decline rates. several studies have demonstrated the utility of iopth monitoring in the treatment of single-gland primary hyperparathyroidism [3, 8, 2127]. most experts agree that iopth assay is the most useful intraoperative adjunct to assist the surgeon in php surgical treatment [28, 29]. it is worth highlighting the useful employment of preoperative localization imaging study in conjunction with iopth: the former points out where the surgeon should start exploration from, and the latter assures that hypersecretory parathyroid tissue removal was accomplished. the current usual criteria for iopth measurement describe a decrease of 50% or over from either the baseline (preincision) or the highest preincision or preexcision value within 10 minutes following hyperfunctioning parathyroid resection, pointing out surgical cure and predicting normocalcemia. by resorting to those criteria, high accuracy in intraoperative prediction of cure is achieved. mostly for practice and cost reasons, we have used just two samples as our criteria since we started making use of the iopth: the preincision and 10 minutes postexcision of hyperfunctioning parathyroid. our series of 96 php consecutive surgeries over 10 years reflects a complex tertiary referral center and has its limitations. most of these patients were referred specifically due to their severe signs/symptoms and comorbid medical conditions, which are related to their high bone and kidney disease and the 4.4% incidence of carcinoma. furthermore, all patients were first evaluated by an endocrinologist group specialized in osteometabolic disease who elected the patients for surgical treatment, reflecting the 11% incidence of men1 and the absence of familial hypocalciuric hypercalcemia case in our surgical series. the php diagnose has not been made in a routine practice in most brazilian centers, and many patients have their diagnoses made just after severe signs/symptoms. we have noticed an increase in the percentage of asymptomatic patients over the years in the number of php cases, but probably such increase has not been enough to raise the number of our small solitary adenomas (78.3%). we get an inferior number of successful parathyroidectomy (93.4%) in previous unexplored patients compared to some large series [9, 32], but it can be considered a satisfactory result, taking into account the high number of patients with carcinoma and men1 in our series. on the other hand, we have obtained good results (100%) in 5 successful second surgical explorations so far: first, we removed a second adenoma in three patients (two persistent and one recurrent disease); second, we were able to remove a not found fourth gland in a men1 patient, and finally, we removed three glands in a men1 patient previous operated as an sporadic php. in our series of 91 patients, iopth had true results in 87 patients (95.6%). however, considering our 96 surgical procedures (5 remedial surgeries), iopth was able to obviate or to ask for additional exploration because of the presence of presumed additional hypersecreting gland(s) in 92 (95.8%) procedures. the iopth had a mean decay of 81.7% in the tp patients, and the minimum drop in our series that results in patient cure (operative success) was 55.0%. one of these cases occurred in a ruptured parathyroid cystic adenoma that may have resulted in a substantial elevation of the hormone levels after the preincision sample, and it should be related to an inadequate iopth decay. the second patient had the others three parathyroid glands identified in normal conditions. based on such scenario, the iopth decay was 50.9%, dropping from 216 pg/dl to 106 pg/dl. as observed by other authors, the fp cases are usually represented by a marginal pth level decrease with a final pth level above the normal range. for these reasons, stricter criteria for the iopth dynamics have been suggested, such as the return of the 10-minute pth to within normal range. however, these stricter criteria were estimated to increase the operative success only by 0.3%, with significant increase in the false negative results, bringing on more unnecessary bilateral neck dissection [9, 33]. using these stricter criteria in our patients would have brought about 21 (24.7%) unnecessary bilateral dissections, and just one additional diagnose. we classified a patient with parathyroid carcinoma that revealed metastatic pulmonary disease two months after surgery as an fp result because she certainly had metastatic disease at the surgery and iopth (84.1% decay) failed to predict the presence of presumed additional hypersecreting tissue. however, it can be controversial to suppose that iopth has failed in this patient, once iopth was not defined to predict distant disease. all 4 patients with parathyroid carcinoma had very suggestive signals of malignant disease at presentation (very high pth levels, severe hypercalcemia and palpable neck mass) and were not elected to the focused approach. they were submitted to an en bloc tumor resection, removing the parathyroid tumor, the ipsilateral thyroid lobe, and the lymph nodes related to a central neck dissection. during the follow-up period, 2 (2.1%) one patient removed a second adenoma and had a successful remedial surgery with iopth decay of 78.4%. the second patient recurrence occurred almost 10 years after initial surgery and is still waiting for remedial surgery. published a large series of 1,650 php patients and found 5 (0.3%) recurrent cases. one of our series limitations is the absence of vitamin d dosages, one important factor that should be related to the genesis of php recurrence. the iopth revealed to be an important technological adjunct in the current parathyroid surgery for php.

introduction. primary hyperparathyroidism (php) is characteristically determined by high levels of calcium and high or inappropriate levels of parathyroid hormone (pth). technological advances have dramatically changed the surgical technique over the years once intraoperative parathyroid hormone (iopth) assay had allowed for focused approaches. objective. to evaluate our 10-year experience in employing a rapid intraoperative pth assay for php. methods. a prospective cohort of 91 php-operated patients in a tertiary institution in so paulo, brazil, from june 2000 to april 2011. results. we had 85 (93.4%) successful parathyroidectomies, 6 (6.6%) failed parathyroidectomies in 91 previous unexplored patients, and 5 (100%) successful remedial surgeries. the iopth was true-positive in 88.5%, true-negative in 7.3%, false-positive in 2.1%, and false-negative in 2.1% of the procedures. iopth was able to obviate additional exploration or to ask for additional exploration in 92 (95.8%) procedures. conclusion. the iopth revealed to be an important technological adjunct in the current parathyroid surgery for php.

PMC3317101

pubmed-96

although enhanced cardiovascular reactivity is generally associated with future development of hypertension and other cardiovascular events [15], there are studies that have failed to show any relationship between reactivity to stress and future elevation of blood pressure [1, 612]. emerging evidence suggest that some stress tests may be better predictors of future cardiovascular events than other stressors [4, 5, 10]. for example, blood pressure response to arithmetic and star tracing stress tests predicted high blood pressure while reactivity to cold pressor stress test did not [13, 14]. furthermore, metanalysis of studies that assessed mental stress tests and hypertension development revealed variable success of mental tests in predicting hypertension. among the different types of mental stressors, cognitive mental stressors were more consistent in predicting hypertension compared to emotion evoking, interview, and public speaking stressors. the inconsistencies in prediction do not appear to be explained by differences in the type (mental, physical, or psychophysical) of stress tests for there is inconsistent predictability even among the types of stressors. another possible explanation for the inconsistencies in reactivity prediction of adverse cardiovascular outcomes is the interaction of psychosocial factors with cardiovascular responses to acute laboratory stressors. metanalysis of over 700 studies revealed that chronic (trait) anxiety is associated with decreased cardiovascular reactivity. in contrast, a study of young european population revealed that acute (state) anxiety was associated with significantly increased reactivity to cold pressor test but not mental stress test. these observations, when taken together, suggest that individuals may be inaccurately identified as hyperresponsive if anxiety is not considered as a confounder in the reactivity response to acute laboratory stress tests. consequently, inaccurate assessment of increased reactivity due to the interaction of anxiety with acute stressors may explain the inconsistent reports of increased risk of hypertension with increased reactivity. this study sought to investigate whether (1) anxiety determined the blood pressure response to stress tests and (2) anxiety differentially influenced blood pressure response to anger recall and cold pressor stress tests in african americans. we chose to study african americans for several reasons: (1) this group is characterized as hyperresponsive to stress [7, 1720], (2) several reports have failed to find increased reactivity in this population [4, 8, 2123], and (3) psychosocial factors, including anxiety, are significantly associated with blood pressure in this population [2429]. we report that state (in the moment) anxiety was significantly associated with blood pressure response to both stressors (anger recall and cold pressor stress tests) in this population. these results support the idea that identification of hyperresponders to acute stress tests among african americans must take into account anxiety levels before determining whether an individual has increased reactivity to acute stress and/or that anxiety may play an important role along with reactivity response in hypertension development. however, our results do not support the idea that anxiety differentially impacts reactivity response to psychological, psychophysical, and physical stressors. a sample of 179 (116 males, 63 females) participants of african descent were recruited to the study. all study procedures and materials were approved by and in compliance with the north carolina central university institutional review board. eligibility criteria for entry were (1) be 18 to 65 years old (2) being a student or employee at north carolina central university or living in the surrounding regions of durham, orange and wake counties, (3) having no diagnosed cardiovascular disease (self-reported), and (4) not taking any hypertensive medication. these regions of durham, orange, and wake counties make up the north carolina triangle region that is in the stroke belt (e.g., a geographic region with a higher occurrence of stroke). of these 179, only 50 are reported in the current report; these were selected based on the type of mental stress used. the 50 participants reported here met the following criteria: (1) completed both the trait anxiety scale and the state anxiety scale, and (2) were between the ages of 18 and 40 years old. study participants were scheduled at either 9 am or 1 pm for the three-hour study protocol. after receiving informed consent, trained staff measured blood pressure by sphygmomanometer method with a ge dinamap pro 100 automatic model and a cuff size appropriate for the body size. each participant was allowed five minutes to sit quietly before taking the first resting parameters. the dinamap was set to assess systolic blood pressure (sbp) and diastolic blood pressure (dbp) at one-minute intervals for the resting measurements as well as during the two acute stressor tasks. following resting blood pressure and heart rate measurements, participants were administered the cold pressor test, consisting of submersion of the hand in ice cold water for three minutes followed by a five-minute recovery period. a psychological stressor, anger recall, was given only after blood pressures and heart rate returned to baseline resting values. anger recall stress consisted of 5 minutes of contemplating an event that evoked anger, 5-minute discussion about the event, and 5-minute recovery period. cardiovascular reactivity was calculated as the difference between the average baseline prestressor blood pressure and the average change in blood pressure over the 5-minute stress period. the study protocol consisted of state anxiety assessment, resting bp measurement, second resting bp measurement, cold pressor stressor, third resting bp measurement, anger recall stressor, trait anxiety assessment, recording medical history, body mass index measurement, and completing a demographics questionnaire. state anxiety is defined as an acute response to a threatening or challenging situation, while trait anxiety is defined as a stable and enduring tendency to be anxious. each item is rated on a four-point scale (1=almost never, 4=almost always). items from each subscale are summed to create a total state anxiety score and a total trait anxiety score. higher scores on the state anxiety subscale indicate greater anxiety at the present time; higher scores on the trait anxiety subscale indicate greater anxiety, in general. the state anxiety subscale has an alpha coefficient of .87, and the trait anxiety has an alpha coefficient of .88, indicating good (since .80 or greater) internal consistency in this sample. scoring of the psychosocial scales spielberger state trait anxiety inventory utilized scoring protocols documented in prior research as indicated above and were confirmed with factor analysis. mean, standard deviation, standard error, median, and quartile calculations provide data reductions for sbp, dbp, and other clinical measures with multiple measurements. regression models, goodness of fit, multivariate parameter estimates, and confidence intervals were evaluated for each stressors impact on sbp, and dbp. two participants did not complete the cold pressor stressor; thus, the sample size is 48 for the cold pressor cardiovascular reactivity and 50 for the anger recall cardiovascular reactivity. the african american study samples are relatively young (median age of twenty-one years) with normal bmi (median bmi was 26.2 kg/m; normal bmi is 2530 kg/m) and waist circumference normal values of less than 102 for males and 88 cm for females [3335]. this group also had normal cholesterol (less than 200 mg/dl), triglycerides (less than 150 mg/dl), glucose (less than 126 mg/dl), and insulin (< 10 miu) levels. this group was normotensive with median systolic (sbp) and diastolic (dbp) blood pressures of 114 and 70 mmhg, respectively. a summary of the cardiovascular reactivity responses to ar and cp is shown in table 2. cardiovascular reactivity was defined as the change in cardiovascular parameters (sbp, dbp, mean arterial pressure (map), and heart rate (hr)) following the induction of a stress stimulus compared to baseline cardiovascular parameters. both the cp stressor and the ar stressor produced significant rises in sbp, dbp, and hr. all of the values returned to baseline during the recovery period except for sbp during the cp recovery period. repeated measures anova and the multiple comparison test, student-newman-keuls test, verified that the two stressor tasks (cp and ar) produced statistically significant increases (p<0.0001) in the cardiovascular parameters in comparison to the resting measurements. we examined pearson's correlations of state anxiety, trait anxiety, age, bmi, and resting cardiovascular measures with the cardiovascular reactivity parameters. trait anxiety had a statistically significant, positive correlation with state anxiety (n=50; pearson's r=0.47; p<0.001, two-tailed pearson's correlation). state anxiety had statistically significant, positive correlations with cp reactivity response for sbp (average change; n=48; pearson's r=0.37; p=0.01), dbp (average change; n=48; pearson's r=0.40; p=0.005). similarly, state anxiety was highly correlated with the ar reactivity response for sbp (n=50; pearson's r=0.34; p=0.015) and dbp (n=50; pearson's r=0.35; p=0.013). specifically, state anxiety was significantly associated with hr change to cp (average change; n=48; pearson's r=0.37; p=0.009) but was not related to hr changes to ar (n=50; pearson's r=0.14; p=0.30). trait anxiety had a nonsignificant correlation with sbp (n=48; pearson's r=0.23; p=0.12) but a positive, significant correlation for dbp (n=48; pearson's r=0.34; p=0.02) reactivity response to cp. as for ar, trait anxiety was positively and significantly correlated with sbp (n=50; pearson's r=0.35; p=0.012) but was not significantly correlated with dbp (n=50; pearson's r=0.23; p=0.11). age, bmi, and resting cardiovascular measures had nonsignificant correlations with both cp reactivity and ar reactivity as measured by sbp, dbp, and hr changes. resting sbp was only associated with map changes with cp and ar stress tests (pearson's r=0.33; p<0.02 and r=0.38; p<0.01, resp.). the following variables were evaluated in the stepwise procedure: state anxiety, trait anxiety, resting cardiovascular measures, body mass index, and age. the variable selection results of the stepwise algorithm suggested the use of state anxiety for a parsimonious model of both cold pressor cardiovascular reactivity as well as anger recall cardiovascular reactivity. our next step was to evaluate regression models to predict cardiovascular reactivity with state anxiety as the independent variable. the model for predicting the cp increase in sbp (see table 3) had an r of 0.14 and state anxiety as a significant parameter (p=0.009). state anxiety was also a significant independent variable (p=0.005) in the model of the cp change in dbp (r=0.16). similar results were found with the model for predicting the change in cardiovascular reactivity for the ar stressor as shown in table 4. state anxiety was a significant parameter for the change in sbp (p=0.015, r=0.12) and dbp (p=0.013, r=0.12). each model was scrutinized to verify adherence to the assumptions of regression modeling (linear relationship between independent variables and dependent variables, homoscedasticity of the errors, and errors are independent and normally distributed). shapiro-wilke statistics did not reject the null hypothesis of normal distribution of the residuals. this study investigated whether anxiety differentially affects cardiovascular reactivity to cold pressor and anger recall stress tests in a sample of young, healthy, community dwelling african american adults, a population prone to develop cardiovascular disease. importantly, we show that the state (at the moment) anxiety was significantly associated sbp and dbp responses to both cold pressor and anger recall laboratory stress tests in this population. in contrast to blood pressure, state anxiety differentially predicted hr response to cp but not ar. on the other hand, chronic (trait) anxiety was not a significant predictor of reactivity in our statistical models. we interpret these results to mean that the state of anxiety at the time of the stressor must be considered when assessing cardiovascular reactivity to laboratory stress tests. failure to consider state anxiety as a confounder of reactivity responses may lead to misidentifying some individuals as hyperresponders when compared to others. misidentification of individuals may contribute in part to the inconsistent findings of increased reactivity in african americans and to the inconsistent prediction of hypertension in those with increased vascular reactivity. alternatively, these results can be interpreted to mean that the interaction of state anxiety and cardiovascular reactivity may be important determinants of hypertension development in african americans. anxiety, chronic anxiety in particular, has been linked to the development of disease. contrary to what would be expected, chronic anxiety has been shown to be negatively associated with cardiovascular reactivity. although chronic anxiety and cardiovascular reactivity associations have been studied, few studies have investigated the role of state anxiety in determining blood pressure response to stress. white coat response and is effective in predicting ambulatory evening systolic blood pressure in young black males. studies that have investigated the effect of state anxiety on reactivity did not include african americans [16, 37]. our study provides evidence that young healthy african americans who are anxious prior to the stress tests are likely to have higher blood pressure responses to the stress. thus, variability in hyperresponsiveness response to laboratory stress tests in african americans may be due in part to a failure to consider state anxiety as a confounder. although many studies have shown that enhanced cardiovascular reactivity predicts hypertension development and other cardiovascular events [15], there are several studies that failed to show any relationship between reactivity to stress and hypertension development [1, 612]. the inconsistency may be a consequence of the type of stressors used [4, 5, 38, 39] and the interaction of psychosocial factors with blood pressure response to the stressor [4042]. psychological stress tests may be better than cold pressor stress tests at predicting future cardiovascular events [4, 5, 22, 43, 44]. this differential effect of stressor type and hypertension development may be a consequence of differential psychosocial factor interaction with stressors. in a study of a european population, psychosocial factors appear to have a greater impact on reactivity to cold pressor than reactivity to mental stress. our study compares the impact of anxiety on reactivity to cold pressor and anger recall in african americans. another explanation for the inconsistent findings of increased risk of hypertension development with increased reactivity is the interaction of psychosocial factors with cardiovascular reactivity to promote hypertension development. for example, studies show that hostility, depression, and anger contribute to increased reactivity [40, 45, 46]. psychosocial factors also are associated with increased incidence of cardiovascular events [4750] as well as the development of cardiovascular disease [51, 52]. we show that the psychosocial factor, anxiety, can influence the reactivity response to acute stress. however, because the study design was cross-sectional, it could not be determined whether state anxiety interaction with cardiovascular response to acute stress predicts hypertension development. this study shows that the current state of anxiety was significantly associated with blood pressure response to laboratory stress tests. consequently, anxiety levels should be assessed when using acute laboratory stress tests for identification of those with increased cardiovascular reactivity. further studies are needed to determine whether reactivity normalized to anxiety increases the accuracy in identifying hyperresponders and, subsequently, predicting future hypertension development. these findings need to be validated in a larger cross sectional population of african americans. additionally, our study design did not allow us to determine whether the impact of anxiety on blood pressure response to acute stressor is unique to anxiety or whether other psychosocial factors similarly influence blood pressure response to acute stress in our cohort. another limitation of the study is that adrenergic system activation was not measured; consequently, it could not be determined if the two stressors differentially activated the beta or alpha-adrenergic receptor pathways. this information would be helpful in future longitudinal studies that will address how activation of the alpha and beta adrenergic receptors pathways ultimately leads to hypertension development and the attending cardiovascular disease. a longitudinal study design will also help to address the question of whether inclusion of anxiety enhances the ability of increased reactivity to predict future elevations of blood pressure .

although several studies have shown that enhanced cardiovascular reactivity can predict hypertension development in african americans, these findings have not been consistent among all studies examining reactivity and hypertension susceptibility. this inconsistency may be explained by the influence of anxiety (state and trait) on the blood pressure response to stress. therefore, this study sought to determine whether anxiety is associated with blood pressure response to cold pressor (cp) and anger recall (ar) stress tests in young healthy african americans. modeling using state and trait anxiety revealed that state anxiety predicts systolic (sbp) and diastolic blood pressure dbp response to cp and ar (p 0.02). interestingly, state anxiety predicted heart rate changes only to cp (p<0.01; p=0.3 for ar). although trait anxiety was associated with sbp response to ar and not cp, it was not a significant predictor of reactivity in our models. we conclude that anxiety levels may contribute to the variable blood pressure response to acute stressors and, therefore, should be assessed when performing cardiovascular reactivity measures.

PMC3259484

pubmed-97

in order to prevent and control microbial proliferation in industrial settings, cleaning and disinfection plans are applied on a regular basis [1, 2]. in food processing plants, the control of microbial contamination generally involves clean-in-place (cip) procedures which consist of running alternated cycles of detergent and disinfectant solutions with water rinses in high turbulence regimes through the plant and pipeline circuits without dismantling or opening the equipment [25]. biocides are currently used in industrial processes as the most significant countermeasure to control microbial growth and proliferation. industry moved progressively towards the use of surfactants that are less toxic and more biodegradable. surfactants are classified according to the ionic physiognomies of their hydrophilic group as anionic, cationic, nonionic, and zwitterionic [6, 8]. quaternary ammonium compounds (qacs) are cationic surfactants that are commonly used because of their hard-surface cleaning, odor removal and antimicrobial properties. besides killing bacteria, the chemical nature of qacs can cause modifications on the properties of abiotic surfaces, decreasing their tension and therefore preventing attachment of microorganisms. the antimicrobial mode of action of cationic surfactants is proposed by some authors as a sequence of events: attraction by the negatively charged cell surface; adsorption to the cell wall through the hydrophobic headgroup; reaction with the lipids and proteins that compose the cytoplasmic membrane; and cell penetration and interaction with intracellular constituents [10, 11]. thus, qacs damage the outer layers of bacteria, thereby promoting the release of intracellular constituents. antimicrobial efficacy tests require planning of an adequate strategy and should include all the parameters found in real settings. aspects such as the proper contact time under known water hardness and conditions of high or low soil content should be considered. for an effective cleaning and disinfection plan, the choice of the disinfectant must follow specific criteria such as compatibility with the surfaces to be disinfected, economic constraints, safety in the workplace, toxicological safety, and biological degradability. it should, most of all, target the type of bacteria and the type of soiling. in fact, disinfectants can be seriously affected by the presence of organic matter. interfering substances have been studied in the last years and included in cleaning and disinfection plans regulated by the authorities such as the european standard en-1276. however, most of these studies only address the effects of bovine serum albumin (bsa) and water hardness [9, 14, 15, 1921]. evaluated the bactericidal activity of disinfectants referred in the german veterinary society guidelines as references for testing disinfectants used in dairy and food industries. in order to simulate the conditions found in practice, they used low fat milk as an organic load and reported the significance in choosing an appropriate disinfectant since the inclusion of a challenging substance (organic material) is important to access the proper bactericidal activity. bessems demonstrated that a qac tested on three microorganisms (pseudomonas aeruginosa, staphylococcus aureus, and candida albicans) had a similar killing rate in the absence of interfering substances and after the inclusion of 17 dh water hardness, a strong reduction of the killing activity was found for the gram-negative bacteria assessed the effect of dried yeast and human serum on the activity of benzalkonium chloride and concluded that the bactericidal activity of the qac was inhibited by solutions of both interfering substances. this work provides information on the influence of potential interfering substances (bovine serum albumin ha) on the antimicrobial activity of two qacs (benzalkonium chloride and cetyltrimethyl ammonium bromide) against bacillus cereus and pseudomonas fluorescens, as they are two major contaminants in the food industry, particularly the dairy industry, and are a known cause of produce spoilage and foodborne illnesses [2, 2226]. some of the interfering substances used throughout the experiments are proposed in the european standard en-1276 as potential interfering agents in disinfection while the others are extracellular polymeric substances (eps) from the biofilm matrix that have an important role in antimicrobial resistance. the bacteria used in this work were pseudomonas fluorescens atcc 13525 and a bacillus cereus strain, isolated from a disinfectant solution and identified by 16s rrna gene sequencing. bacterial strains were grown at a temperature of 30 2c and ph 7, with glucose as the main carbon source. culture medium consisted of 5 gl glucose, 2.5 gl peptone, and 1.25 gl yeast extract in phosphate buffer (pb) (ph 7, 0.025 m). a bacterial suspension was prepared by inoculation of a single colony grown on solid medium into a 1 l flask containing 250 ml of sterile nutrient medium. this bacterial suspension was incubated overnight at the given temperature with agitation (120 rpm). the qacs used throughout the experiments were benzalkonium chloride (bac) and cetyltrimethyl ammonium bromide (ctab) (sigma, portugal). preliminary studies with a concentration range between 0 and 5000 mgl were initially made. in order to ascertain the behaviour of bacteria to the qac, the selected concentrations for further studies were 3, 5, 10, 20, and 35 mgl. the qacs were used individually and in combination (both chemicals were combined in equal volumes and concentrations). ha (acros organics, fisher chemical, portugal), and yeast extract ye (merck, portugal). after the growth period, the suspensions were centrifuged (3999 g, 5 minutes), washed two times, and resuspended in pb to a final cell density of approximately 1 10 cellsml. in the case of the consortium, both bacterial suspensions were washed two times resuspended in pb to a final cell density of approximately 1 10 cellsml, and combined in equal volumes to obtain the same cell concentrations of the single species tests. afterwards, all bacterial suspensions were exposed to several concentrations of qac for a period of 30 minutes. the effects of the chemicals were evaluated by the assessment of the oxygen uptake rate due to glucose oxidation, according to simes et al.. to investigate the influence of interfering substances on the antimicrobial efficacy, the same procedure was followed with the addition of 300 mgl of bsa, alg, ye, or ha to the bacterial suspension, simulating low concentrations of interfering substances according to the european standard en-1276. three independent experiments, each with duplicate samples, were performed for each condition tested. the methodology was performed according to johnston et al. for a period of 10 minutes. bac and ctab were chemically neutralized by a sterile solution of (w/v) 0.1% peptone, 0.5% tween 80, 0.1% sodium thiosulphate, and 0.07% lecithin dissolved in pb. control experiments were performed to ascertain the effects of the 10-minute exposure to the neutralization solution, and no effects were detected on the respiratory activity of b. cereus and p. fluorescens (data not shown). after the neutralization step, the bacterial suspensions were centrifuged (3999 g, 5 min) and resuspended in the same volume of pb. the respiratory activity was ascertained by measuring oxygen uptake rates in a biological oxygen monitor (yellow springs instruments 5300a). demonstrated that this procedure is more adequate and rapid than the assessment of colony forming units to characterize the antimicrobial activity of biocides against heterotrophic aerobic bacteria. samples were placed in the temperature-controlled vessel of the biological oxygen monitor (t=25 1c) each containing a dissolved oxygen probe connected to a dissolved oxygen meter. before measuring, the samples were aerated for 10 minutes to ensure oxygen saturation ([ o2]=8.6 mgl). the vessel was closed, and the decrease of oxygen concentration was monitored over time. the initial linear decrease corresponds to the endogenous respiration rate. to determine the oxygen uptake due to substrate oxidation, 12.5 l of a 5 gl glucose solution was added to each vessel. the slope of the initial linear decrease in dissolved oxygen, after glucose injection, corresponds to the total respiration rate. the difference between these two rates is the oxygen uptake rate due to glucose oxidation. the inactivation was calculated using metabolic activities according to the following equation: (1)% inactivation=(mcmt)mc100, where mc is the metabolic activity of the control experiments (without antimicrobial exposure) and mt is the metabolic activity of the bacterial solutions exposed to the antimicrobial. if% inactivation>0 there was inactivation of the microorganisms whereas if% inactivation<0 there was metabolic potentiation. the mbc for each situation was determined as the lowest concentration of qac or qac combination where no respiratory activity was detected. for each parameter the statistical significance of the results was evaluated using the wilcoxon test (confidence level 95%) to investigate whether the differences between the resulting experimental values could be considered significant. the antibacterial activity of bac, ctab, and their combination was investigated in the absence and in the presence of four selected interfering substances. in the absence of interfering substances bac caused the inactivation of b. cereus at 10 mgl, p. fluorescens at 35 mgl, and the consortium at 20 mgl. ctab at 20 mgl completely inactivated b. cereus and at 35 mgl inactivated the total population of p. fluorescens and the consortium. the combination of both qacs was synergistic in the inactivation of b. cereus (total inactivation with 3 mgl) and indifferent for p. fluorescens (35 mgl) and the bacterial consortium (35 mgl). the inclusion of the selected interfering substances influenced the antimicrobial activity of the qacs to some extent (figures 13). the inactivation of b. cereus (figure 1) was not affected by the presence of any interfering substances (p>0.05), except with ha. the antimicrobial action of the qacs against p. fluorescens (figure 2) was not significantly influenced by the presence of most potential interfering substances (p>0.05), except for ha where interference was observed (p<0.05). the antimicrobial activity of the qacs against the bacterial consortium (figure 3) was affected by the presence of interfering substances. ha reduced significantly the activity of ctab at higher concentrations (p<0.05). bsa and ye resulted in a significant reduction of the activity of the combination of qacs (p<0.05). linear correlations were determined to assess the relationship between qac concentrations and the inactivation data. the effect of increasing qac concentration on bacterial inactivation shows that there are strong linear correlations (r>0.850) for the control assays, with the exception of b. cereus (this bacterium was inactivated with low qac concentrations). the most extreme cases are the treatments with ctab to p. fluorescens with alg as an interfering substance (r=0.771) and the bacterial consortium in the presence of ye (r=0.738). likewise, this decrease of linear correlation factors was found for p. fluorescens and for the consortium exposed to ha where the lowest correlation factor was 0.153, which was obtained for p. fluorescens treated with ctab. this phenomenon only happened when the qacs were used on p. fluorescens and the bacterial consortium in the presence of ye and ha. the most significant cases of oxygen uptake rate increase were verified for p. fluorescens exposed to bac (5 to 35 mgl) and ctab (3 to 35 mgl) in the presence of ha and combination of qacs (3 to 10 mgl) in the presence of ye. a similar metabolic behaviour was found for the bacterial consortium exposed to bac (3 to 35 mgl) and ctab (5 and 10 mgl) for ha and qac combination (3 to 20 mgl) with ye. the mbc values for the different conditions tested (single and combined qacs, in the absence and presence of potential disinfection interfering substances) are shown in table 1. the presence of bsa increased the mbc of the combination of qacs for b. cereus (3 to 5 mgl) and the consortium. alg increased the mbc of bac for the consortium (20 to over 35 mgl) and qacs combination (3 to 5 mgl) for b. cereus. ye increased the mbc of bac for b. cereus (10 to 20 mgl) and qac combination (3 to 5 mgl). the mbc values for the consortium of cells increased in the presence of ye (bac20 to 35 mgl, ctab35 to over 35 mgl, and qac combination35 to over 35 mgl). ha increased the mbc for all the scenarios, except of ctab when applied to b. cereus (in this situation the mbc was reduced). the mbc was reduced in other situations such as, for b. cereus, in the presence of alg when using bac and ctab (10 to 5 mgl and 20 to 5 mgl, resp.) and in the presence of ye when using ctab (20 to 3 mgl). fluorescens inactivation by ctab was reduced by bsa (35 to 20 mgl). alg also reduced the antimicrobial activity of the combination of qacs against the bacterial consortium (35 to 20 mgl). it is assumed that the organic material can potentially interfere with the antimicrobial agents by chemical and/or ionic interactions [15, 33]. therefore, it is necessary to know the role of each potential interfering substance in the antimicrobial activity in order to develop effective disinfection strategies. the interfering substances tested are commonly found as residuals in the food industry (from food products and from microbial contaminants, biofilms) [18, 27]. in this study, higher inactivation rates were verified for b. cereus in comparison to p. fluorescens at the same qac concentration. in fact, when b. cereus and p. fluorescens are combined in a 1: 1 bacterial suspension, it is expected that the first is more affected than the second. b. cereus is more susceptible due to the fact that it is a gram-positive bacterium that lacks an outer membrane, which typically provides increased protection to gram-negative bacteria. this fact is corroborated by previous reports which stated that gram-positive bacteria are more susceptible to cationic surfactants than gram-negative bacteria [34, 35]. bsa was already studied as an interfering substance in disinfection practices [9, 14, 1921, 36]. the negative effect of bsa on the action of biocides against p. fluorescens was demonstrated by simes et al. p. fluorescens treatment with ctab with the addition of 3 gl of bsa resulted in a 10-fold increase on the mbc of this qac [9, 21]. in the present study, the efficacy of the combination of qacs against b. cereus and the cell consortium was also reduced. this effect of bsa as an antimicrobial quencher is apparently due to the strong ability of qacs to react with proteins. proteins can precipitate in the form of their anions, in this way, the negative-charged protein ions will cling to the positively charged molecules of the cationic compounds. ctab is a biocide that targets the membrane and has a strong affinity for proteins. bac is composed of a positively charged hydrophobic headgroup which clings to opposite charged surfaces [8, 37]. studied the effect of the alkyl chain of bac binding to bsa and dried yeast. their conclusions were that bac is often inactivated by organic matter, either by adsorption to the bacterial surface or by adsorption to the organic matter in general. these authors also suggested that the reduction in the activity of bac was probably related to more than one physical property of the compounds like the chain length (longer chains result in more adsorption to the bacterial surface). alg is a common constituent of the extracellular polymeric substances of the biofilm matrix [3840]. a function frequently attributed to eps is their general protective effect on biofilm microorganisms against adverse conditions. the eps matrix delays or prevents antimicrobials from reaching target microorganisms within the biofilm by diffusion limitation and/or chemical interaction with the extracellular proteins and polysaccharides [32, 41]. in this study, alg either potentiated or hindered the antimicrobial activity of the selected qacs. the presence of this interfering substance was not obvious on the inactivation of p. fluorescens. on the other hand, the inactivation of b. cereus by bac and ctab and the consortium by the combination of qacs was easier in the presence of this interfering substance. the bacterial consortium treatments with bac and b. cereus with the combination of qacs were hampered by the presence of alg. davies et al. found that the production of alg was triggered by membrane perturbation induced by ethanol stress, nitrogen limitation, attachment to surfaces, or even high oxygen tension [42, 43]. this substance is suggested as one of the main biofilm resistance vectors either by reacting with the antimicrobials or by hindering antimicrobials diffusion to the cells. the antimicrobial interference caused by alg is apparently due to electrostatic interactions between the anionic alg and the cationic-selected qacs. the presence of ye as interfering substance resulted in three different outcomes on the antimicrobial activity of the qacs: (1) no effect/indifference, (2) the respiratory activity reduced, and (3) the respiratory activity potentiated. this interfering substance worked mainly as a hinderer of the antimicrobial activity by increasing the mbc of b. cereus in all cases except for ctab, of p. fluorescens with the combination of qacs, and of the consortium of cells with ctab and the combination of qacs. ye is listed in the european standard en-1276 as an interfering substance native to the brewery industry. the constituents of ye are very similar to the components of the bacterial cells, thus, it is expected that the antimicrobial agents that target the bacterial cells are also drawn to ye. in a similar study by jon et al. it was shown that the presence of dried yeast decreased the biocidal effectiveness of bac. humic substances are found ubiquitously in the environment and can be found in the biofilm matrix [2, 46]. ha reduced the antimicrobial activity of the qacs in most of the cases, although in some cases it promoted the respiratory activity (potentiation). the presence of these compounds had the strongest effect compared to the remaining interfering substances. studied the sorption mechanisms of anionic and cationic surfactants to natural soils concluding that the dominant sorption mechanism of surfactants to clay is cation exchange. koopal et al. also verified the formation of complexes ha-cationic surfactant. respiratory activity potentiation was verified with the addition of ha to p. fluorescens and ye to the bacterial consortium. it is known that ha participates in cellular metabolism processes such as growth, respiration, photosynthesis, and nitrogen fixation. on the other hand, ha were proposed to replace synthetic surfactants such as sds, tween 80, and triton x-100 in industrial applications such as textile dying or washing. it is therefore possible that the inclusion of humic substances in a solution of qacs may interfere with the chemical characteristics of the solution. the resultant mixture, with an apparent reduced antimicrobial efficacy, seems to potentiate the respiratory activity of the bacteria, particularly of p. fluorescens. as qacs are membrane active agents, their use at sublethal concentrations could improve membrane permeability and consequently the nutrient influx, without compromising the bacterial viability. also, there is the hypothesis that the potentially interfering agents could be used as nutrients. in fact, it was found that the growth rates of anaerobic and aerobic microorganisms increased when humic substances were added, which stimulated enzyme activity [52, 53]. in a similar way, ha are likely to be used for growth in the same way as ye; these might be broken down to smaller molecules that can be used by cells as a carbon or nitrogen sources. the antimicrobial activity of the tested qacs was enhanced in some cases, where the interfering substances were present. this is an unexpected result due to the recognized potential of alg, bsa, ha, and ye to interfere with disinfection. this effect is probably due to the low concentration of interfering substances tested that caused both respiratory activity reduction and potentiation. ethylenediamine tetraacetate (edta) was reported as early as 1965 to increase the biocidal effects of bac and chlorhexidine diacetate on pseudomonas aeruginosa. reported that chitosan (a polysaccharide) potentiated the antimicrobial action of sodium benzoate on spoilage yeasts. in dairy plants, disinfection is potentiated by pre-washes with alkali or enzyme-based cleaning agents. most of these cases were observed for b. cereus (four occurrences), one was observed for p. fluorescens, and another one was observed for the consortium of cells. the mbc was improved by more than 50% in the cases of b. cereus and less than 30% for p. fluorescens and the consortium of cells. to our knowledge there are no reported cases of antimicrobial agents potentiation by bsa, ye, or alg. although the exact chemical structure of ha has not yet been determined, ha could be chemically similar to the tested qacs, presenting a positive hydrophilic head and a hydrophobic tail. with this structure ha could act as detergents in conditions such as those observed in the treatment of b. cereus with ctab. the present work shows that increasing qacs concentrations lead to an increase in antimicrobial effectiveness. this is valid mainly when the qacs were applied in the absence of interfering substances. this means that disinfection was concentration dependent, as found for most of the antimicrobial chemicals. however, the linear dependency of inactivation versus concentration is not verified for most of the tests where interfering substances were added. this result evidences that the mathematical modelling of disinfection strategies requires a case-to-case analysis when interfering substances are present. the overall results demonstrate that a disinfection process in the presence of the selected interfering substances can reduce the effectiveness of bac, ctab, and their combination. the bacteria were inactivated equally by all qacs, although in the absence of interfering substances ctab was the most efficient solution. p. fluorescens was the bacterium with the highest resistance to inactivation, followed by the bacterial consortium. the tested interfering substances, referred in the european standard 1276 (bsa and ye), and known eps constituents related with biofilm resistance (alg) resulted in mild interferences on the activity of the qacs. ha were the interfering substance that resulted in the most severe effect by reducing the activity of qacs, causing, in some circumstances, significant respiratory activity potentiation.

standard cleaning processes may not remove all the soiling typically found in food industry, such as carbohydrates, fats, or proteins. contaminants have a high impact in disinfection as their presence may reduce the activity of disinfectants. the influence of alginic acid, bovine serum albumin, yeast extract, and humic acids was assessed on the antimicrobial activities of benzalkonium chloride and cetyltrimethyl ammonium bromide against bacillus cereus vegetative cells and pseudomonas fluorescens. the bacteria (single and consortium) were exposed to surfactants (single and combined) in the absence and presence of potential disinfection interfering substances. the antimicrobial effects of the surfactants were assessed based on the bacterial respiratory activity measured by oxygen uptake rate due to glucose oxidation. the tested surfactants were efficient against both bacteria (single and consortium) with minimum bactericidal concentrations ranging from 3 to 35 mgl1. the strongest effect was caused by humic acids that severely quenched antimicrobial action, increasing the minimum bactericidal concentration of the surfactants on p. fluorescens and the consortium. the inclusion of the other interfering substances resulted in mild interferences in the antibacterial activity. this study clearly demonstrates that humic acids should be considered as an antimicrobial interfering substance in the development of disinfection strategies.

PMC4745498

pubmed-98

social scientists who explore factors mediating and moderating the relationships between social stressors and mental health, including drinking outcomes, have highlighted modes of coping [1, 2]. these studies have explored behaviors which protect people from being psychologically harmed and cognitive appraisals which influence behaviors such as problem-focused coping or using alcohol to self-medicate distress. however, studies have not considered the characteristics of the stressful situation itself that may make certain coping strategies more or less effective. in particular, psychiatric epidemiologic studies have tended to emphasize microlevel stressors (e.g., stressors in individuals ' role domains) and, until recently, have ignored the linkages between macrolevel social forces and the daily stressors in people's lives [79]. however, macrolevel social conditions can affect the magnitude of stressors experienced in people's lives and the extent to which they experience cumulative adversity. this paper focuses on coping with the fallout from one type of macrolevel social stressor: the recent great recession. this economic downturn constituted the most severe economic crisis in the united states since the great depression and had persisting economic effects (e.g., job loss, less desirable working conditions, loss of home, loss of retirement savings, lack of health care access, and social isolation) which have been linked with deleterious drinking outcomes. a key issue involving the effectiveness of alternative modes of coping with stressors derived from macrolevel social forces and protecting against deleterious drinking outcomes is the question of whether individual modes of coping outside of the political realm emphasized in the overall coping literature are most efficacious. or, alternatively, do stressors engendered by macrolevel social forces require unique forms of coping, that encompass the political realm? with respect to coping strategies employed in the wake of the great recession, politically oriented coping strategies might be of particular relevance due to the impact of governmental decision-making on the state of the economy. politically oriented coping strategies might include political activism oriented to altering economic policies or support for campaigns by politicians offering solutions to economically based hardships. scholarly work on the occupy wall street movement, for example, documents involvement by individuals sharing their own economic struggles and collectively gathering to protest their precarious economic situation and uncertain economic future. earlier work also suggests the salience of activities oriented toward changing politically based social realities such as through the act of voting or by collectively challenging community-level decisions such as school closings. by contrast, the traditional coping literature has emphasized individual, nonpolitical modes of coping, such as emotional acceptance of the stressful situation, blaming one's self for the situation, or taking individual actions such as looking for a job if unemployed. while sociologists have recently accorded greater attention to the prevalence and alcohol-related consequences of macrolevel stressors, they have yet to address the extent to which politically oriented modes of coping may be the most efficacious ways to address problems stemming from macrolevel social forces or events. prior to the more recent focus on macrolevel stressors, kaplan and liu embraced the idea of collective coping as one means for individuals who maintained stigmatized personal identities to challenge and transform conventional socionormative systems through participation in social movements. subsequently, thoits more explicitly addressed collective coping within the context of the stress paradigm involving acute and chronic stressors not limited to the specific area of stigmatized statuses. she argued that individuals who find themselves in problematic situations can deliberately work to transform the meaning of their experiences and they can additionally use these experiences as a basis for helping or effecting changes in the lives of others she proposed the concept of transformatory coping to include engagement in collective activist activities with others who share similar problems. for example, parents of autistic children have lobbied governments for social services perceived to aid in their children's development. in sum, (1) collective coping tactics (such as politically oriented coping) represent an unmeasured dimension of coping behaviors beyond that represented in the coping literature to date and (2) collective coping tactics may demonstrate a stronger association between stressors, particularly those stemming from macrolevel social forces, and deleterious drinking outcomes compared to the use of modes of coping previously emphasized in the literature on coping. the present study extends previous work by empirically addressing the extent to which politically oriented coping activities engaged in as a response to a macrolevel social stressor, the great recession, are protective against alcohol-related outcomes compared with coping strategies focused outside of the political realm. we hypothesize that politically oriented coping will be more protective against economic stressors linked with the great recession than nonpolitical modes of coping and will uniquely account for some portion of the associations between economy-related stressors and drinking outcomes. further, we also examine whether politically oriented coping and coping outside of the political realm are more protective for men versus women in the face of macrolevel engendered stressors such as those involving the economic fallout from the great recession. there is consistent evidence that women are more likely to use support-based coping strategies (e.g., seeking support from others such as partner family and friends) in response to stress in contrast to men and some indication that avoidant coping techniques are associated with greater alcohol consumption among men but not women [1, 2022]. (in contrast, parity by gender in the use of individual active coping strategies and their significance for mental health outcomes is generally reported [1, 21].) however, whether there is a corresponding propensity for men and women to differ in the use of politically oriented coping to offset the alcohol-related effects of economy-related stressors is less certain. earlier research tended to argue that women were less politically interested, informed, and efficacious compared to men. more recent work has shown that women and men differ in particular modes of participation; women are more likely to vote and engage in individual political actions such as signing petitions or donating money, whereas men are more likely to be engaged in collective forms of action such as group protest activities. thus, we hypothesize that there will be no overall differences in the extent to which women and men manifest politically oriented coping or in the effect that politically oriented coping has on drinking outcomes. following a transactional model of stress [4, 25], we model coping as a mediator of the relationship between the stressor (i.e., stressful consequences of the great recession) and the stress response (i.e., drinking outcomes). data were derived from a study conducted in the united states between june, 2010, and january, 2011, that was undertaken in order to understand life change consequences of the major downturn in the economy known as the great recession. respondents were selected by a random digit dial (rdd) phone survey of the continental united states, and those who consented to participate in the study were mailed questionnaires. eligible respondents were selected from the households using the troldahl-carter-bryant method of respondent selection which involves the means to randomly select a respondent from all eligible household members. respondents were told during the phone screen that a $50 american express gift card would be sent to the eligible respondent if he or she completed the questionnaire. respondents were mailed an initial survey, a postcard reminder to nonresponders, and a second questionnaire if they still had not responded. figure 1 encompasses a flow chart characterizing each stage in the data collection process through the completion of the questionnaires. 65.9% (n=663) of the respondents completing the screening calls returned the questionnaire. the telephone screening cooperation rate and the mail survey response rate were each calculated using the conservative aapor response rate formula 3. the overall survey response rate is the product of the phone screening cooperation rate (35.5%) and the mail questionnaire return response rate (65.9%) or 16.8%. we acknowledge that this response rate is less than ideal and further address this issue in the discussion of study limitations and note other indicators of the representativeness of the final sample. selection weights were calculated for each of the cases to weight for the different probability of selection for each case. poststratification weights were calculated for the dataset to ensure that the distribution of sample cases on important demographic variables (age, race/ethnicity, and gender) conformed to the distribution of these variables in census bureau's 2008 united states population estimates. it should be noted that estimates of alcohol consumption for the present sample did appear to conform to national estimates preweighting. for example, the average number of drinks consumed in the past month on days when one drank for the present sample is 2.16, versus the estimated average of 2.10 reported by the centers for disease control and prevention's behavioral risk factor surveillance system for 2010 (by gender, the averages are 2.35 for men and 1.89 for women in the present sample versus 2.43 for men and 1.81 for women in the cdc estimates; by race/ethnicity, the averages are 2.16 for non-hispanic whites, 2.08 for asians, 2.12 for african americans, and 2.72 for hispanics in the present sample versus 2.26 for non-hispanic whites, 2.41 for asians, 2.19 for african americans, and 2.68 for hispanics in the cdc data). it should additionally be noted that the respondents included in this sample reported an overall higher level of education than the general population based on 2008 census estimates. analysis of variance revealed no significant variation by education in either the outcomes or support coping, avoidant coping, or politically oriented coping. respondents with less than a high school degree are found to be marginally less likely (p<0.10) to use active coping strategies compared to those with a college degree or postcollege training. given this discrepancy as well as the fact that education is generally protective against problem drinking predictor variables are economy-related stressors, coping strategies enacted outside of the political realm (i.e., active coping, support coping, and avoidant coping), politically oriented coping, and gender. the sociodemographic characteristics of age, education, and race/ethnicity are controlled in all analyses. to assess past-month drinking patterns, we use the quantity-frequency-variability index (qfv) developed by cahalan et al.. frequency of drinking is measured as a count of the days on which alcohol was consumed in the past 30 days, and quantity of drinking is measured as the average number of drinks consumed on those days. variability is calculated by the greatest number of drinks consumed on any one day in the past 30 days. scores are calculated by multiplying responses to the quantity, frequency, and variability questions (= 0.87). as with the current data, this index tends to approximate a continuous scale, and ample evidence supports its use as such (see fitzgerald and mulford for a review). our measure of problematic drinking is the 10-item bmast (= 0.74), which is a count measure of difficulties related to alcohol use over the past year. respondents were asked to indicate yes or no in response to 10 items such as having an accident, losing a close friend, spouse, or loved one, being hospitalized, having trouble at work, and soliciting professional help because of one's drinking. the bmast is one of the most widely used tools for assessing alcohol dependence and problems. it correlates strongly with the full-length mast and evidence of its reliability and validity is widely available [31, 32]. moreover, we are not using the bmast as a diagnostic tool for depicting problem versus nonproblem drinking, but as it is intended, as suggestive of different degrees of problematic drinking. the measure of economy-related stressors is the life change consequences of the great recession (lccgr) instrument. this construct was developed on the basis of qualitative analyses of transcripts derived from focus groups involving both genders and diverse racial/ethnic groups. the items fall into seven categories: home ownership problems, such as difficulties in mortgage payments, difficulties in paying property taxes, or a drop in credit rating; undesirable living situation, including having to live in a less desired location to save money or having gas and electricity or heat shut off due to an inability to pay bills; problematic employment situation, including a pay-cut, furlough days, and increased feelings of competition with fellow employees; unemployment or underemployment; inadequate health insurance, including lack of medical or dental coverage, decreased quality of coverage, and inability to obtain coverage; social role constraints, such as dissolution of spouse/partner relationship, decreased social life, and increased social isolation due to finances; and inadequate sick time, including inadequate sick days and having to work despite poor health. consistent with common practice, each score for this measure is a straight count of the number of stressors reported. three dimensions of nonpolitical modes of coping are assessed: active coping, support coping, and avoidant coping. these measures of coping are derived from subscales of the brief cope instrument and have previously been validated as stand-alone indices in community samples [3436]. participants in the present study were asked whether they have used these coping strategies in response to the economic recession. confirmatory factor analysis of the present data supports the inclusion of these items as three separate coping indices, consistent with prior research. active coping (= 0.84) includes eight items measuring acceptance, positive reframing, and planning and taking action in response to the economic recession. support coping (= 0.81) includes four items measuring use of emotional and instrumental support (i.e., receiving emotional support and getting advice and help from other people). avoidant coping (= 0.75) includes 10 items measuring self-distraction, behavioral disengagement, self-denial, blame, and a tendency to vent about or make fun of the situation. all items were rated on a four-point likert-type scale ranging from 0 (i did not do this at all) to 3 (i did this a lot). the measure of politically oriented coping is assessed by a four-item instrument (= 0.79) drawn from the summed responses (i.e., not at all, a little, some, quite a bit, and a lot) to four statements asking how often respondents, in response to the economic recession, have been (1) engaging in political activities such as signing petitions, leading or participating in rallies or marches, or writing to political representatives; (2) organizing with others to challenge politicians currently in office; (3) voting in elections to support politicians who share your political beliefs; and (4) participating in groups trying to influence the policies of the government at the local, state, or national level. the questions used to construct this index were derived from analyses of focus group transcripts (see for details about these focus groups). confirmatory factor analysis reveals that these items load on a single factor, supporting their inclusion as one index. education is a categorical variable based on the educational attainment categories of (1) less than high school (n=45); (2) high school graduate (n=350); (3) college graduate (n=110); and (4) postcollege training (n=150). race/ethnicity is a dummy variable including non-hispanic whites (n=436), african americans (n=80), hispanics (n=91), asians (n=29), and individuals who identify as an other race/ethnicity (n=17). in all analyses, non-hispanic whites serve as the reference category. after examining bivariate correlations in order to assess the basic patterns of association among key study variables, we performed structural equation modeling (sem) using mplus software to examine the predictive significance of economy-related stressors for coping outside of the political realm and politically oriented coping tactics and the two drinking outcomes considered (i.e., past-month drinking and problematic drinking), net of the sociodemographic control variables. we considered the potential for nonpolitical coping and politically oriented coping tactics to mediate the associations between economy-related stressors and each of the outcomes assessed in two models. the first model tested for associations between economic stressors and the drinking-related outcomes. the second model adds nonpolitical and politically oriented coping tactics to test the full mediation model. this latter model assesses all of the direct and indirect paths between economic stressors and the drinking outcomes considered through the nonpolitical and politically oriented coping tactics investigated. we formally tested for mediation using the procedures described by muthen and muthen for mplus software, which apply the tests described by mackinnon et al.. finally, because the associations between social stress and drinking are found to vary by gender [33, 39], we examined whether any observed mediating effects of the coping strategies investigated vary by gender. for these tests, separate equations include the interaction term for gender by each coping strategy in the path models linking coping with drinking outcomes. it is noteworthy that stressors related to the economy are associated with each of the alcohol-related outcomes and all of the coping resources considered: economy-related stressors are associated with more alcohol consumption and problematic drinking, as well as higher levels of active coping, support coping, avoidant coping, and politically oriented coping. it is also noteworthy that only two of the coping strategies assessed are associated with the drinking outcomes. avoidant coping and politically oriented coping are associated with both alcohol consumption and problematic drinking, but in opposite directions. that is, greater avoidant coping is associated with greater alcohol consumption and problematic drinking, whereas greater politically oriented coping is associated with less alcohol consumption and problematic drinking. the lack of correlation between active coping and support coping and each of the drinking outcomes, respectively, provides some indication that not all of the coping strategies may be useful in understanding the associations between economic stressors and drinking-related outcomes. additionally, the possibility that coping resources may vary by gender is not strongly supported by the pattern of correlations reported, with the exception that women reported significantly more emotional support than men. however, and consistent with previous research, women are found to drink less and less problematically. the hypothesized associations between economy-related stressors, coping strategies, and the alcohol-related outcomes are further elaborated upon in the structural equation model. estimation of the first model (figure 2), including only economic stressors, the drinking-related outcomes, and sociodemographic controls, produces a just identified model and, as such, meaningful fit statistics are not provided. the standardized path coefficients demonstrate that economic stressors and each of the drinking-related outcomes considered are significantly and positively related. net of the sociodemographic controls, greater economic strain is associated with greater alcohol consumption over the past month (= 0.094, s.e.=0.004, and p<0.01) and a higher incidence of problematic drinking over the past year (= 0.181, s.e. sem analysis testing the second model, of the hypothesized associations between economy-related stressors, coping strategies, and the alcohol-related outcomes considered, is presented as figure 3. in figure 3, solid lines indicate effects that are statistically significant, and dashed lines indicate effects that are not statistically significant. the model fit criteria provided by hu and bentler (cfi>0.95; rmsea<0.06; srmr<0.08) are used to assess the measurement model. based on these criteria, there is consistent evidence of good fit for this model (cfi=0.982; rmsea=0.046; this model demonstrates that economic stress is associated with higher levels of each of the coping resources considered. that is, greater economic strain appears to predict a greater propensity to use both maladaptive coping tactics (i.e., avoidant coping) and adaptive coping tactics (i.e., active coping, support coping, and politically oriented coping). avoidant coping is associated with higher levels of past-month drinking and problematic drinking, whereas politically oriented coping is associated with less alcohol consumption and problematic drinking. formal mediation tests next reveal that the effects of economy-related stressors on the alcohol-related outcomes assessed are partly explained by variation in avoidant coping and politically oriented coping. significant indirect effects are found for the pathways from economy-related stress to both past-month drinking and problematic drinking. of the total effect of economy-related stress on past-month drinking (0.071), 0.047 is accounted for by the indirect effect of economy-related stress through avoidant coping and 0.024 is explained by the indirect influence of politically oriented coping. a similar pattern emerges with respect to the relationship between economy-related stressors and problematic drinking. the indirect effects of avoidant coping account for 0.046 of the total effect of economic stressors on problematic drinking (0.098) and politically oriented coping explains 0.032. taken together, these findings indicate that both adaptive and maladaptive coping strategies come to bear on drinking patterns associated with economic strain. on the one hand, economic strain appears to be associated with drinking more and more problematically, in the extent to which it is associated with a tendency to engage in avoidant coping strategies. but, then, again, economic strain is also associated with greater politically oriented coping, which is protective against alcohol use and misuse. moderation tests next determine whether the mediating effects of the coping strategies investigated vary by gender. however, the mediating effect of avoidant coping for the economy-related stressor problematic drinking association differs for men and women in this sample. this effect is displayed in figure 4, which presents the predicted pattern of gender contrasts in the effects of avoidant coping on problematic drinking based on the mean, plus and minus two standard deviation values of avoidant coping (as displayed in table 1). as shown in figure 4, the mediating effects of avoidant coping differ for men and women because the relationship between avoidant coping and problematic drinking is significantly less strong for women compared to men (= 0.319, s.e. thus, the observation that economic strain is associated with greater problematic drinking in the extent to which it is associated with avoidant coping strategies appears to be more pronounced among men than among women. the findings from this study support a broadened conceptualization of modes of coping in stress paradigm-oriented research to encompass politically oriented coping, in addition to modes of coping outside of the political realm which have predominated in studies of the coping-related moderators or mediators of the associations between social stressors and drinking outcomes. moreover, given our focus on economic stressors deriving, at least in part, from the macrolevel social forces producing the great recession, we suggest that politically oriented coping is particularly salient as a mode of behavioral adaptation in relation to societally engendered stressors as opposed to stressors that are less affected by macrolevel social forces. while this study specifically addressed deleterious drinking consequences of the recent great recession, social scientists have also delineated broader social-structural and political forces occurring over the last three decades which have led to pervasive job insecurity across all sectors of the us workforce and the erosion in the standard of living for most of the population [4143]. these phenomena include globalization and the outsourcing of work, the downsizing of corporate entities, the shift from secure semiskilled industrial jobs which paid a living wage to low wage service sector jobs, an increase in contingent workers with lower pay, and lack of job security and fringe benefits. thus, politically oriented coping encompassing the goal of changing governmental policies (e.g., rallying for changes in the tax structure influencing the distribution of wealth throughout society, fighting for government stimulus policies oriented toward job creation, or rallying support for raising the government-mandated minimum wage) may prove to be a more efficacious mode of coping with economic stressors and more protective against deleterious drinking outcomes compared to nonpolitically oriented active coping activities such as looking for a job when unemployed, especially if adequate numbers of jobs relative to demand do not exist and a large proportion of jobs that do exist pay less than a living wage. consistent with our gender-linked hypothesis, our data showed that males and females did not differ either in the use of politically oriented coping or in the extent to which politically oriented coping was protective in relation to drinking outcomes. however, our data showed that male but not female avoidant coping significantly predicted problem drinking. thus, with regard to nonpolitically oriented coping with economic stressors, males clearly utilized a coping mode that was maladaptive. this finding might be seen as congruent with early male socialization patterns which have been viewed as fostering men's sense of self-importance rather than connectedness in social relationships insofar as men's avoidance in dealing with economic problems may affect both themselves and others close to them. alternatively, avoidant coping may predict problem drinking in men but not women to the extent to which males are socialized to drink more heavily than women. our findings should be viewed within the context of the methodological limitations of this study. the politically oriented coping measure which we developed for this study represents an initial attempt to operationalize this concept. however, it is limited to 4 items in contrast to the much longer nonpolitically oriented coping instrument and does not differentiate politically oriented coping tactics into discrete modes, similar to the measurement of nonpolitically oriented coping. although factor analysis supported the inclusion of the indicators of politically oriented coping as a single measure, additional work on the concept of politically oriented coping would be useful, with a differentiation between alternative types of politically oriented coping. first, the differentiation between individual modes of political action (e.g., voting, signing petitions, and donating money) and collective political activities (participation and leadership roles in different types of political action groups) would be useful. secondly, some types of collective coping could be viewed as relatively more adaptive versus maladaptive. for example, the tea party social movement was motivated by both economic and cultural concerns [45, 46]. however, one might differentiate between the tea party articulation of problem-focused social policies such as its belief in the need to diminish the size of government and racially oriented collective venting, such as screams of kill him by audience members in response to sarah palin's critique of barak obama at rallies during the 2008 presidential election. second, this study utilized cross-sectional data and, thus, likely provides only a snapshot of the complex processes linking economy-related stressors, coping strategies, and drinking outcomes. while our theoretical framework postulated that exposure to economic stressors coupled with particular modes of coping leads to problematic drinking, it is possible that problem drinking coupled with maladaptive coping might make one prone to experiencing economic stressors such as losing one's job. moreover, we lack data regarding alcohol consumption prior to exposure to the stressors focused on in this study and the extent to which alcohol may have been used as a coping mechanism. thus, further longitudinal studies are necessary to more clearly delineate the causal directions of the relationships between economic stressors, modes of coping, and drinking outcomes. third, the study methodology encompassed random-digit-dialing for recruiting the sample, thus only reaching individuals with landline telephone numbers. consequently, individuals relying on cell phones only, along with households without access to any telephone, were not included in this study. this potential noncoverage error is a source of concern because comparisons of our data with the us population revealed that the sample underrepresented african americans, latinos, and younger (< age 40) and less-educated (high school or less) persons. however, our data were weighted to reflect the demographics of the overall population, and we compared weighted and unweighted estimates of each of our dependent variables to determine if nonresponse and/or noncoverage may have introduced serious bias into one or more of them. in each instance, we found that the weighted values of each measure fell well within 1 sd of the unweighted values, suggesting that the distributions of our key measures were not appreciably influenced by the underrepresentation of particular demographic groups. despite the noted limitations, this study extends prior research on the moderators and mediators of the social stressor-drinking outcome relationships to broaden notions of coping to include politically oriented coping. future studies incorporating this mode of coping may more clearly elucidate the political dynamics involved in both the macrolevel production of social stressors and the deleterious alcohol-related consequences of these stressors. this line of research would also have implications for the treatment of alcohol-related problems. in particular, considerations of more adaptive modes of coping might go beyond recommending individual behaviors such as job seeking by unemployed individuals to also suggest politically oriented coping to collectively try to influence the social conditions such as unemployment levels that may give rise to the propensity to self-medicate distress through the use of alcohol.

research derived from the stress paradigm suggests that certain types of coping (e.g., problem-focused coping instead of behavioral disengagement) are protective against problem-related drinking to deal with social stressors. going beyond the typical focus in the coping literature, we hypothesize that stressors engendered by macrolevel social forces may require coping actions within the political realm in contrast to modes of coping focused outside of the political realm. a united states sample of 663 respondents completed a mail survey in 2010, including measures of stressful consequences of the great recession, drinking patterns and problems, modes of coping encompassed in the brief cope instrument, and politically oriented coping. structural equation modeling examined whether modes of coping mediated the links between stressors and drinking outcomes. a substantial portion of the associations between stressors and drinking was explained by modes of coping. politically oriented coping was protective against problem drinking for both genders. future studies should further explore politically oriented coping in addition to modes of coping outside of the political realm when studying the relationships between macrolevel social stressors and deleterious drinking outcomes.

PMC4180206

pubmed-99

the development of titanium fixtures has brought several benefits for the rehabilitation of edentulous patients. when biological and mechanical principles are respected, this treatment modality may successfully restore the functional and esthetic impairments caused by tooth loss3. in spite of the significant evolution of a number of implant systems, implant design and features, such as those related to the mechanical behavior of implant-supported prostheses, dental prostheses do fail during function mainly due to abutment and prosthesis screw loosening and/or fracture. in addition, it has been reported that abutment screw loosening is only surpassed by loss of osseointegration as the main cause of failure on implant-supported restorations, as shown in longitudinal follow-up studies5. when two metal surfaces are in contact, adhesion and friction forces do limit the movement between them. an applied method aimed to reduce this friction and improve adhesion consists of interposing a lubricating film between these surfaces. a metal with low shear strength, such as pure gold, may act as a dry lubricant. when compared to screws without gold coating, it has been found that gold-coated abutment screws subjected to torques of 12, 20, and 32 ncm aiming a 0.0064 mm opening of the implant-abutment interface presented 26, 24, and 24% of preload increase, respectively7. in another study, it was found that when a torque force is applied to an abutment screw, a significant part of this force is lost due to friction between the contact points of metal surfaces, inhibiting the rotation of the screw. thus, decreasing of friction between metallic surfaces may increase the screw rotation and, consequently, the preload. the rotation of gold-coated abutment screws placed with torque forces of 12, 20, and 32 ncm increased 73, 76, and 62%, respectively, when compared to titanium abutment screws2. the purpose of this study was to evaluate by strain gauges the preload and torque removal values on three abutment screws (gold, titanium, and titanium with surface treatment screws) applying an equivalent torque force (30.070.28 ncm). three machined self-tapping external hex titanium screws with 4 mm in diameter and 15 mm in length were used in this investigation (master screw, conexo sistemas de prtese ltda. three transmucosal cera one type abutments (2.0 mm height, cera one, conexo sistemas de prtese ltda., reference 045022, batch #5080915121), designed for cemented single standing implant-supported restorations were also used. the transmucosal abutments were attached to the fixtures using 10 gold screws (conexo, sistemas de prtese ltda., reference 121022, batch #5073147) (figure 1 a a), 10 titanium screws (conexo sistemas de prtese ltda., reference 121024, batch #5063223) (figure 1a b), and 10 surface treated titanium screws (ti-tite, conexo sistemas de prtese ltda., reference 121026, batch #5063035) (figure 1a c). a load cell adapted from a model described in the literature this load cell presented a central void for fixture fixation in its upper portion and a horizontal plate for fixation of four strain gauges on its lower portion (figure 1 b). the abutments were placed on the upper plate of the cell (figure 1 c) in such way that the contact between abutments and fixtures was free of any interference. therefore, when the abutment was fixed to the fixture by the abutment screw, tensions generated by this fixation pulled the unit against the abutment, producing a deformation in the lower plate connected to the strain gauges. next, the force (preload) generated on the abutment screws was captured by the strain gauges in volts (v) and later converted to newton (n). the aforementioned sample was arranged into 3 different groups according to the characteristics of each abutment screw: group a was formed by gold screws, group b was formed by titanium screws, and group c was formed by surface-treated titanium screws. to determine fixation and removal torques, a digital torquemeter was used (torqueleader, model tsd150, type i, class e, part #117317), previously calibrated according with iso 6789:2003 (e) standard. a square hand wrench (1.27 mm diameter, conexo,, reference 062300, batch #5091632) was attached to the torquemeter to allow proper connection between torquemeter and screws (figure 1 d). following the proper assembly of fixture and abutment on group a, the gold screw was then attached to the fixture with an applied torque force of 30.070.28 ncm, being repeated afterwards for groups b and c. then, the initial preload value for the abutment screws was determined (in volts) and all screws were kept in their positions for 5 minutes for preload stabilization, according to the literature9. during this the preload stabilization period, preload values were obtained after 1, 2, 3, 4, and 5 minutes after fixation, and thereafter a mean value was calculated. after this evaluation, the abutment screw was removed using a digital torquemeter and the maximum value of reverse torque force required for screw removal was recorded in ncm. next, the same screw was fixed again another 4 times, obtaining the preload and torque removal mean values. nine other screws from group a as well as 10 other screws from b and c groups were subjected to the same procedure described above. the values of fixation torque forces of the 3 groups were analyzed statistically by to one-way analysis of variance (anova). comparison of preload values within groups was possible using the mean of 6 measurements (0 to 5 minutes) obtained at the first abutment fixation, using two-way anova. one-way anova was used to compare the mean preload values among the groups. tukey's test was used for multiple comparisons among the groups. for all statistical tests, all fixations for each screw (n=5) were considered for evaluation of the torque removal to obtain an average value, and the statistical analysis was performed similarly to the preload data. two-way anova was used to evaluate the mean torque removal among the groups, and one-way anova to evaluate the differences between screws. tukey's multiple comparison test was used for individual comparisons among the groups. for all statistical tests, the mean fixation torque force when all screws were considered was 30.070.28 ncm (c.i. there were no statistically significant differences (p=0.1244) among the groups regarding the fixation torque. the gold screws presented the highest preload values and the titanium screws the lowest values (figure 2). the mean preload values of the gold screws was 131.728.98 n, and presented statistically significant differences (p<0.01) compared to groups b and c. screw #3 and screw #1 presented the highest (140.48 n) and the lowest (117.73 n) preload values, respectively. the titanium screws presented statistically significant differences (p<0.01) (mean 37.035.69 n). the highest preload value was obtained for screw #7 (49.68 n), while screw #3 presented the lowest value (25.30 n). in the group of surface-treated titanium screws, the mean preload value was 97.784.68 n. statistically significant differences (p<0.01) were also observed in this group, (maximum: 104.09 n for screw #4; minimum: 90.28 n for screw #2). the removal torque presented statistically significant differences among the analyzed materials (p<0.001) (figure 3). the gold screws presented a mean removal torque of 17.641.12 ncm, i.e. they did not shown statistically significant differences (p=0.3713). titanium screws presented a mean removal torque of 18.751.89 ncm, and showed statistically significant differences (p<0.001). the group of surface-treated titanium screws present statistically significant difference (p=0.004) (mean: 16.431.33 ncm). the results of this study are similar to those of previous investigations4,8, which found statistically higher preload values for gold fixation screws in comparison to other tested materials, in spite of using different methodologies and materials. a maximum preload value of 666.4 n for gold screws and 458.2 n for titanium screws was reported8. in addition, the finds of the present study suggest that the evaluated surface treatment of titanium screws was effective, since these screws presented higher preload values than conventional titanium screws. most previously published reports in the dental literature do not specify the removal torque of abutment screws. the effect of different cyclic loads has been evaluated using a 32 ncm torque for fixation of the screws, which was repeated after 10 minutes to avoid contact relaxation6. several manufacturers have suggested this clinical approach to decrease screw loosening. in the present study, to allow a direct comparison between results of preload and torque removal for the studied groups, all screws received a similar fixation torque of30.070.28 ncm, despite the manufacturer's recommendations of using 35 ncm torque for the ti-tite screws. this was performed to eliminate a possible bias caused by elastic deformation that might occur in titanium screws during fixation, which might not only quantitatively but qualitatively influence the study outcomes and impair comparison among groups. this approach allowed that, in this investigation, only the screw was left as a study variable. another relevant issue is that up to 10% of the initial preload may be lost to smooth contact surfaces (embedment relaxation), rather than elongation stresses. however, it has been previously observed that when the same screw is fixed several times, its preload values increased4. comparison to different results obtained in other studies might be a difficult task due to variations of tested products and variables that may influence the produced preload value, such as the elastic modulus of the screws, opposing joint surfaces, abutment design, friction coefficient, lubrication, rate of the applied torque and the adaptation between the fixture hexagon and the abutment2. the findings of the present study suggest a trend for greater preload values verified for gold screws, followed by surface-treated titanium screws and titanium screws, confirming the outcomes of previous investigations4,8. finite element analysis has been used to evaluate preloading through elongation of the abutment screw. a study evaluating the preload of two different systems of the same implant manufacturer in function, found that the optimal preload produced should be 75% of the screw yield stress, and also that the preload value increases dramatically as the friction coefficient between implant and screw decreases7. as loosening of implant/abutment joint causes clinical problems, another question that may be raised is whether the torque values recommended by the manufacturers should be increased in order to obtain greater longevity of screw tightening. the preload produced by gold or titanium fixation screws can be evaluated through the measurement of elongation stresses. it has been found that the stresses were 60% lower than the strengths against the torques applied according to the manufacturer's instructions4. on the other hand, it has been reported that gold or titanium screws could support higher torques than those indicated by the manufacturers without presenting plastic deformations, although it has been recommended elsewhere that the stresses should not exceed 65% of the screw's fracture strength1. although the results of this study showed that gold screws have a clear superiority of the produced preload, application of cyclic loads would be required for a closer simulation of the masticatory function on implanted-supported restorations. it may be concluded: 1. gold should be the material of choice for abutment fixation screws, since it produced the highest preload values, followed by surface-treated titanium screws and conventional titanium screws; 2. titanium screws presented the highest torque removal values, followed by gold screws, and surface-treated titanium screws.

several authors still consider the mechanical problems of fracture and component loosening as the main causes of failure of implant-supported restorations. the purpose of this in vitro study was to compare the preload of three types of screw for transmucosal abutment attachment used in single implant-supported prosthesis through strain gauge and removal torque measurements. three external hex fixtures were used, and each received a transmucosal abutment (cera one), which was fixed to the implant with its respective screw: group a- gold screw, group b- titanium screw and group c- surface-treated titanium screw (ti-tite). ten screws of each type were attached applying a 30.070.28 ncm torque force and maintained in position for 5 minutes. after this, the preload values were measured using strain gauges and a measurement cell. gold screws presented higher preload values (131.728.98 n), followed by surface-treated titanium screws (97.784.68 n) and titanium screws (37.035.69 n). anova (p<0.05) and tukey's test (p<0.05) were applied. statistically significant differences were found among the groups for both preload and removal torque values. in conclusion, gold screws may be indicated to achieve superior longevity of the abutment-implant connection and, consequently, prosthetic restoration due to greater preload values yielded.

PMC4327281

pubmed-100

colorectal adenocarcinoma is the fourth most frequently diagnosed malignancy in korea, accounting for 12% of newly diagnosed cancer cases. according to the ministry of health and welfare, the incidence of colorectal cancer has increased in both males and females during the past 2 decades. although surgery is potentially curative, approximately one third of all newly diagnosed patients present with inoperable metastatic disease. palliative chemotherapy is more effective than the best supportive care for improving overall survival (os) as well as quality of life in advanced colorectal cancer. while significant advances have been made in recent years, cure is rarely possible in advanced colorectal cancer (acc), making further improvements in therapy imperative. for more than three decades, the treatment options for acc have been almost exclusively based on 5-fluorouracil (5-fu) and folinic acid (fa). until the early 1990s, 5-fu, often modulated by fa, was the single effective chemotherapy available, but only led to meaningful responses in a small minority of treated patients. the recent integration of oxaliplatin and irinotecan for the management of acc patients has extended median os in a meaningful way. both drugs have been shown to have synergistic effects with 5-fu and fa in colorectal cancer cell lines. since irinotecan and oxaliplatin have been used for acc treatment, the efficacy of palliative chemotherapy for acc has considerably improved. first-line treatment with irinotecan and either bolus or infusional 5-fu/fa, namely irinotecan/bolus fluorouracil/lv or 5-fluorouracil, folinic acid plus irinotecan (folfiri), significantly improved outcomes as compared with 5-fu/fa. similarly, a combination of oxaliplatin and infusional 5-fu/fa, known as 5-fluorouracil, folinic acid plus oxaliplatin (folfox), significantly increased the response rate and the time to progression compared with 5-fu/fa. these results established 5-fu-based chemotherapy, in combination with either irinotecan or oxaliplatin, as standard first-line chemotherapy regimens for patients with acc. furthermore, capecitabine has been shown to exhibit antitumor activity against acc both as a single agent and in combination with oxaliplatin [12-14], as well as with irinotecan. in korea, capecitabine was approved in january 2006 as a partner with oxaliplatin or irinotecan for the treatment of acc. a decision regarding chemotherapy regimens for factors considered include the extent of disease, potential toxicities, especially for those with impaired oral intake or with decreased performance status, activity of chemotherapy, and the patient preference. we therefore decided to evaluate various combination chemotherapy regimens as first-line chemotherapy for acc. this report describes a single-center, retrospective study. between january 2006 and december 2007, a total of 537 acc patients were treated with first-line chemotherapy for advanced disease at samsung medical center (seoul, korea). fifty-nine patients were excluded because they were treated with single-agent chemotherapy. the criteria for case inclusion were as follows; 1) histologically confirmed diagnosis of adenocarcinoma arising from colon or rectum, 2) no prior chemotherapy or radiotherapy except for adjuvant use, 3) advanced (metastatic and/or recurrent) disease, 4) availability of clinical data at the beginning of therapy and follow-up. we attempted to exclude patients who were enrolled in clinical trials to ensure the choice of chemotherapy regimen was solely at the discretion of the treating physician. all the data were prospectively recorded and only the survival data was updated at the time of analysis. written informed consent was given by all patients prior to receiving chemotherapy, according to institutional guidelines. the patients were required to have a life expectancy of 12 weeks of more; age of at least 18 years or older; eastern cooperative oncology group (ecog) performance status of 2 or lower; adequate hematologic (neutrophil count>1,500/mm and platelet count>100,000/mm), hepatic (serum total bilirubin level<1.5 mg/dl and aspartate aminotransferase/alanine aminotransferase<3upper limit normal) and renal (serum creatinine level<1.5 mg/dl) functions. our department guidelines define combination chemotherapy regimens as follows: in folfox or folfiri, fa is given at the dose of 200 mg/m followed by bolus 5-fu 400 mg/m on day 1, and a 46-hour continuous infusion of 5-fu 2,400 mg/m on days 1 and 2. oxaliplatin 85 mg/m or irinotecan 180 mg/m was administered on day 1 as a 2-hour infusion, concurrent with fa. in capecitabine plus oxaliplatin (xelox) and capecitabine plus irinotecan (xeliri), oxaliplatin 130 mg/m or irinotecan 240 mg/m was given on day 1 in combination with oral capecitabine 1,000 mg/m twice daily on days 1 to 14. folfox and folfiri were repeated every 2 weeks, whereas xelox and xeliri were repeated every 3 weeks until disease progression or unacceptable toxicity occurred, or until a patient chose to discontinue treatment. the dosages for subsequent cycles were adjusted according to the toxic effects that developed during the preceding cycle. the prophylactic use of hematopoietic growth factors was not allowed during treatment, except for patients with febrile neutropenia or grade 4 myelosuppression at the treating physician's discretion. after this combination chemotherapy had failed, second-line chemotherapy was recommended to all the patients if their performance status was preserved. according to department policies, all tumor measurements were assessed every 2 or 3 cycles of chemotherapy by using an abdominopelvic computed tomography (ct) scan and other tests that were initially used to stage the tumor. tumor response and progression were evaluated according to the response evaluation criteria for solid tumors (recist). the primary endpoint of this retrospective study was progression-free survival (pfs). the date of disease progression or death from causes other than acc was used in calculating pfs. pfs and os were estimated according to the kaplan-meier method and the statistical significance of differences in survival curves between groups was tested with a log-rank test. multivariate models were used for exploratory purposes to examine the impact of each regimen on the outcomes of chemotherapy. covariates included were age (below vs.median), gender, previous adjuvant therapy, an ecog performance status (0-1 vs.2), number of involved sites (1 vs.2), metastases (liver, peritoneum, and lung), baseline chemistry profiles (serum albumin, alkaline phosphatase, and bilirubin), combination with bevacizumab, and chemotherapy regimens. laboratory parameters were initially recorded as continuous variables and later dichotomized according to the median value of each variable (below vs.median). between january 2006 and december 2007, a total of 478 acc patients were treated with combination chemotherapy in a first-line setting: folfox (n=172), folfiri (n=95), xelox (n=155), and xeliri (n=56). in 29 patients, chemotherapy involved bevacizumab.. sixty percent of patients were male, and 33 patients (7%) had an ecog performance status of 2 or more. more than half of the patients (n=263) had received surgery for curative intent, and 206 patients received adjuvant chemotherapy or chemoradiotherapy. approximately one-third of the patients had two or more metastatic disease sites, mostly involving liver and abdominal lymph nodes. we noted that more folfiri or xeliri patients had received adjuvant therapy involving oxaliplatin than folfox or xelox patients. after first-line failure, second-line chemotherapy was administered for more than half of the patients (n=290). median follow-up duration was 40.6 months (95% confidence interval [ci], 39.3 to 41.8 months). at the time of data collection, 449 (89%) median chemotherapy durations for folfox, folfiri, xelox, and xeliri were 4.9, 4.5, 5.7 and 5.4 months, respectively. we found no statistically significant difference among chemotherapy durations for these regimens (log-rank p=0.19). the most common reason for discontinuation of treatment was disease progression. in 172 patients receiving folfox, two-thirds of patients discontinued therapy due to disease progression (n=118), physician's recommendation or patient withdrawal (n=33), or toxicity (n=21). folfiri patients discontinued therapy due to progression (n=60), physician's recommendation or patient withdrawal (n=13), toxicity (n=20), or unknown causes (n=2). similarly, disease progression was the most common cause of therapy discontinuation in 101 xelox patients and 34 xeliri patients. we found no relevant difference in the occurrence of overall grade 3 or 4 toxicities among regimens (table 2). in brief, the main difference in grade 3 or 4 toxicities with folfiri or xeliri was diarrhea (21% for folfiri and 27% for xeliri vs. 11% for folfox and 9% for xelox; p=0.02), and with folfox or xelox the difference involved peripheral neuropathy (9% for folfox and 7% for xelox vs. 2% for folfiri and xeliri; p<0.01). although patients who were treated with irinotecan-containing chemotherapy more frequently experienced grade 3 or 4 leukopenia (13% vs. 10%; p=0.09) than those treated with oxaliplatin-containing regimens, we found no significant difference in the incidence of febrile neutropenia (5% in each arm). in addition, grade 2 or more hand-foot syndrome was more frequently observed in xelox and xeliri (14% and 12%, respectively) than in folfox and folfiri (6% and 8%, respectively). in the current retrospective analysis, one death occurred in the midst of treatment with folfox, with no clinical evidence of progression having been demonstrated. another patient died of respiratory failure shortly after completion of the second folfox cycle, in which the possibility of disease progression could not be completely excluded. the patient, initially presenting with multiple pulmonary nodules and bilateral pleural effusion, complained about increasing dyspnea and suffered a sudden respiratory arrest. five other deaths occurred in patients while receiving folfiri or xelox, and were attributed to neutropenic sepsis. of a total of 478 patients, 61 could not be evaluated for responses because of the absence of any measurable lesions or early discontinuation of therapy. objective responses to chemotherapy were noted in 200 patients (response rate, 48%; 95% ci, 43 to 53%); 42% for folfox, 49% for folfiri, 55% for xelox, and 51% for xeliri. the difference in the response rates for each regimen was not statistically significant (p=0.09). patients who had a poor performance status (2 in ecog scale) were significantly less likely to respond to first-line chemotherapy (24% vs. 50%; p<0.01) compared to those with an ecog performance status of 0 or 1. response rate was not significantly influenced by age, gender, prior treatments, baseline laboratory parameters, metastatic sites, or chemotherapy regimen. the estimated median pfs for all patients was 6.8 months (95% ci, 6.3 to 7.3 months), and the median os was not yet reached. pfs was shorter, although statistically insignificant (p=0.12), in patients receiving folfox or folfiri (6.2 months and 6.0 months, respectively) than those receiving xelox (7.9 months) or xeliri (7.1 months). one-year pfs rates for folfox, folfiri, xelox, and xeliri were 24%, 21%, 32%, and 32%, respectively (fig. 1). a multivariate regression model revealed that pfs was significantly affected by performance status (hazard ratio, 0.39; 95% ci, 0.28 to 0.57; p<0.01). we also tested whether the pfs was modified by interaction between the effect of baseline clinical parameters and the chemotherapy regimens; the first-level interaction term between these variables was entered into a separate multivariate model. however, we found no interaction between any first-line chemotherapy regimens for each clinical parameter. despite recent advances in the treatment of acc, patients treated with first-line chemotherapy have a median os rarely exceeding 24 months. in the current analysis, our observation that pfs was slightly shorter with folfox or folfiri than with xelox or xeliri is possibly related to negative prognostic factors influencing the choice of intravenous chemotherapeutic agents instead of oral agents. similarly, the choice of a chemotherapeutic agent depends on the ones previously used in the adjuvant setting. although this study is retrospective in nature, the results provide a piece of evidence that patients with acc may derive an indisputable benefit from fluoropyrimidine-based combination chemotherapy. the decision to use specific chemotherapeutic agent(s) in patients with acc should be determined by their relative merits on a case-by-case basis. the evolution of treatment using irinotecan and oxaliplatin in combination with 5-fu regimens has resulted in a significant prolongation of life in patients with acc. since both drugs have demonstrated similar activity in first- and second-line settings, the optimal chemotherapy regimen and dosing schedule in patients with acc remains undecided. randomized trials comparing first-line folfox with folfiri indicated equivalent activity and moderate toxicity differences between these two regimens, suggesting that use of either oxaliplatin or irinotecan is reasonable. in another phase iii study in which patients crossed over to the alternative regimen upon progression while on their first-line regimen, the overall pfs on first-line therapy was 8.1 months for folfox and 8.5 months for folfiri. in a combined analysis of seven phase iii trials in acc, grothey et al. reported that the strategy of making these three active drugs (fluoropyrimidine, oxaliplatin, and irinotecan) available to patients with acc could maximize os. in addition to efficacy data, patient convenience and satisfaction are assuming increased importance in cancer management. oral administration has the advantage of permitting convenient, patient-orientated therapy, providing the patient with a degree of independence and improved quality of life, while avoiding complications associated with intravenous drug administration. chemotherapy regimens involving oral capecitabine showed similar safety and efficacy profiles compared with 5-fu-based regimens. oral capecitabine was proved to have a similar quality of life profile to that of 5-fu, but seemed to be more convenient in terms of administration. in korea, a capecitabine-containing regimen was approved in january 2006 as the first-line treatment of acc. data from the current retrospective study suggest that patients are highly compliant with capecitabine, when given in combination with either oxaliplatin or irinotecan. patients treated with capecitabine experienced a higher incidence of clinically relevant hand-foot syndrome, but this toxic effect was predictable and manageable. an improved understanding of the molecular pathogenesis of cancer has lead to the development of novel agents designed to target critical cellular pathways. one of the most successful novel approaches in the treatment of acc involves therapeutic agents that inhibit the neovascularization process of growing tumors. bevacizumab is a recombinant humanized anti-vascular endothelial growth factor monoclonal antibody that is being clinically evaluated in many tumor types, including acc. because bevacizumab was only recently made available in korea as first-line chemotherapy, only 29 patients received bevacizumab. this study confirms that both oxaliplatin- and irinotecan-containing combination chemotherapy are effective for the first-line treatment of acc. capecitabine clearly represents an effective and well-tolerated oral alternative to 5-fu. while the current study was primarily focused on cytotoxic therapies, the integration of targeted agents, including bevacizumab, is a novel standard option to further improve outcomes for patients with acc.

purposefluoropyrimidine-based combination chemotherapy, in combination with either oxaliplatin or irinotecan, has demonstrated efficacy and tolerability in treatment of advanced colorectal cancer (acc). materials and methodsbetween january 2006 and december 2007, a total of 478 acc patients were treated with combination chemotherapy in first-line settings. combination therapies included: 5-fluorouracil, folinic acid plus oxaliplatin (folfox, n=172), 5-fluorouracil, folinic acid plus irinotecan (folfiri, n=95), capecitabine plus oxaliplatin (xelox, n=155), and capecitabine plus irinotecan (xeliri, n=56). folfox and folfiri were repeated every 2 weeks, whereas xelox and xeliri were repeated every 3 weeks until occurrence of disease progression or unacceptable toxicity, or until a patient chose to discontinue treatment. resultsthe median age was 58 years (range, 19 to 84 years) and the median chemotherapy durations for folfox, folfiri, xelox, and xeliri were 4.9, 4.5, 5.7, and 5.4 months, respectively. combination chemotherapy regimens were generally well tolerated. the estimated median progression-free-survival (pfs) for all patients was 6.8 months (95% confidence interval, 6.3 to 7.3 months). no statistically significant difference in pfs was found among regimens used as first-line chemotherapy. sixty percent (n=290) of patients received second or further lines of therapy after failure. conclusionfluoropyrimidine-based combination chemotherapy regimens appear to be equally active and tolerable as first-line therapy for acc.

PMC3138923