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pubmed-901

south east asia is home to one-third of the global burden of tuberculosis (tb), with an estimated 5 million prevalent cases and an annual incidence of 3 million tb cases. five of the 11 countries in the region are among the 22 high-burden countries, with india accounting for over 20% of the global burden of tb disease [1, 2]. using the current strategy of passive case finding, the case detection rate in the region has improved from around 40% in 2002 to 65% in 2008. however, it has stagnated since 2006 and remains below the target of more than 70%. a lower than expected case detection rate indicates that tb cases in the community are not being adequately identified and treated, which means ongoing transmission of tb infection. the risk of transmission increases with the closeness of contact, overcrowded living conditions, and the degree of infectiousness of a tb case as determined by the positivity of sputum smear microscopy of acid-fast bacilli (afb) and degree of lung field involvement in the chest x-ray (cxr) [5, 6]. close contacts to a tb case such as those living in the same household are at higher risk of infection than casual contacts. among those that are infected, young children (< 5 years) or those with immunodeficiency (e.g., hiv infected) are at increased risk of developing tb disease, usually within two years following infection. therefore, the world health organization (who), the international union against tuberculosis and lung diseases (iuatld) and the national tb control programs (ntps) recommend screening of all children who are household contacts of sputum smear-positive tb case. screening and management of child contacts has great potential to reduce tb-related morbidity and mortality in children [4, 9]. it also can identify contacts of any age with suspected tb disease at an earlier stage than they otherwise may have presented to health care services. it, therefore, has the potential to increase case finding and reduce transmission. finally, though the contribution of young children to transmission may be small, they may form a pool of infection from which future adult cases arise [11, 12]. despite the benefits, contact investigation for child contacts is rarely implemented and reported in resource-limited tb endemic settings, such as in the south east asia. this paper aims to collate published data reporting the prevalence of tb infection and tb disease among child household contacts of tb in the south east asia region. the search strategies were developed using a combination of subject headings and keywords, including tuberculosis, mycobacterium tuberculosis, contact tracing, contact investigation, contact screening, household contact, the primary studies were searched electronically using databases pubmed, embase, and web of science. manual searching of the reference lists of the primary studies was performed to identify other eligible studies. published studies were included if they included children and adolescents (015 years), reported the yield of household contact investigation in children or provided data to calculate the prevalence of tb infection or tb disease in children, and were conducted in countries in the south east asia region. the data extracted included the following information: study site, design, description of index cases, description of household contacts, definition of household contacts, investigations performed (tuberculin skin test (tst), cxr, sputum smear microscopy of afb, and culture of mycobacterium tuberculosis), outcomes among child contacts (healthy, tb infection, or tb disease), and the criteria used to determine the outcomes. there have been systematic reviews and a meta-analysis on contact investigation of tb, but none specifically assessed the yield from household contact investigation among children in the south east asia region [13, 14]. eleven eligible studies were conducted in seven countries in south east asia: india (four studies) [1518], thailand (two studies) [19, 20], and one study each from cambodia, indonesia, lao people's democratic republic, pakistan, and philippines. not all of the studies evaluated the prevalence of both tb infection and tb disease: five studies provided data on both; five studies evaluated the prevalence of tb infection only and one study of tb disease only. there was heterogeneity among studies with regards to epidemiology background, study design, the characteristics of the index case and child contact, and the criteria used for determining tb infection and tb disease (table 1). most studies were cross-sectional in design, and only one study conducted in india in 1960s performed a prospective followup for a period of 5 years. the index case in most studies was a case of sputum smear-positive pulmonary tb (ptb). the study from indonesia evaluated household contacts of an index case with sputum smear-negative ptb, and two studies in india included sputum smear-positive and smear-negative cases with abnormal cxr [15, 16]. with regard to child contacts, three studies involved children under five only [15, 21, 22], the others included older children up to 1418 years of age [1620, 2325]. there was no uniform definition of a household contact across the studies, but the most common definition was a child living in the same house as the index case. four studies specified a period of at least 3 months of living at the same house to define household contacts [19, 22, 24, 25]. a study in india defined close contact as living, cooking, and eating in the same house as the index case for the period of three months immediately preceding the start of treatment for the index case. all studies which provided data on tb infection defined it as a positive tst, evaluated 4872 after administration of tuberculin solution. four studies used a cutoff of 10 mm, whereas the philippines study used 5 mm and the thailand study used 15 mm. the study from indonesia used the local scoring system, whereas most other studies used clinical and radiological features. the number of child contacts investigated in the studies ranged from 61 to 790 children of 50 to 342 index cases. in general, the prevalence of tb infection among child contacts under 15 years of age was higher (24.469.2%) than that of active tb disease (3.35.5%). tb disease was more commonly found among children aged less than 5 years, whereas tb infection was more common in older children (table 2). the results of household contact investigation across the eligible studies can not be compared directly due to the heterogeneity, particularly in outcome definitions (tb disease or tb infection). figure 1 presents the yield of tb infection from studies which used a cutoff of 10 mm of tst result for tb infection among sputum smear-positive index cases. it is shown that tb infection is more common in children aged more than 5 years. the prevalence of infection in all children ranged from 24.4% to 38.8%, with a weighted yield of 31%. the prevalence of tb infection and disease among children living in the same house as a case of pulmonary tb in south east asia varies between settings. this variation may be due to the different epidemiology features amongst the countries or to the heterogeneity among studies with regards to the characteristics of the index case and the criteria used for determining tb infection and disease. nonetheless, tb infection among child contacts was common and would support recommendations for routine screening and management of child household contacts. the finding that tb disease is more prevalent among young children (< 5 years) is as expected given that young age is a well-established risk factor for disease following infection. various approaches and criteria were used to assess the outcome of contact investigation, indicating that there has not been a universal method accepted or implemented in south east asia. for example, the study in the philippines used a cut-off point of 5 mm induration to define positive tst, which is lower than the recommended 10 mm in a bcg-vaccinated population. the use of the cutoff may explain why this group reported the highest prevalence (67.2%) of tb infection amongst the studies. for studies which defined a positive tst as 10 mm induration, the proportion of tb infection ranged from 24% to 48% [6, 15, 22, 24, 2731]. a meta-analysis of contact investigation in low- and middle-income countries by morrison and colleagues revealed a prevalence of tb infection of 40% in children aged under 15 years. despite the evidence of high rates of infection and disease in child contacts in south east asia, screening a cross-sectional study in india reported that only 31 of 220 (14%) children younger than 14 years living in the same house as adults with pulmonary tb were screened for tb. a higher yield of 52% was reported by tornee and co-workers in a prospective study in thailand.. the potential of contact screening upon identification of an infectious case in the community is emphasised by a recent study from south africa which reported that most cases of young children with confirmed tb represented a missed prior opportunity for preventive therapy. isoniazid preventive therapy (ipt) which has proven efficacy in preventing tb disease in infected and uninfected child contacts is recommended for child contacts<5 years of age who have no evidence of tb disease [8, 34, 35]. a study in india reported that, of 84 child contacts aged younger than 6 years, only 16 (19%) were initiated on ipt. in malawi, of 365 child household contacts under five, only 33 (9%) were actually screened for tb: 23 (6%) received ipt, 6 (2%) received anti-tb treatment, and, in 4 (1%), no action was taken. when ipt is prescribed, poor adherence is another problem that is likely to reduce the effectiveness of this intervention. adherence rates of 15 to 28% have been reported from south africa [38, 39]. a reason for the poor implementation of contact investigation and ipt provision in resource-limited countries is likely to be the lack of human resources. health workers in tb endemic areas are overburdened by the identification and management of sputum smear-positive cases, which are the priority for treatment in any national tb programs. in addition, the awareness and knowledge of families and healthcare workers of the rationale and potential of ipt are lacking [33, 40]. in malawi, only 21% of tb patients who had child contacts aged 5 years or below were informed about the need for screening their children. a more recent study in malawi in 2006 reported that only 8% of sputum smear-positive cases brought their children to the clinic for screening despite provision of clear information. guidelines that are difficult to implement may also be a barrier for the implementation of contact investigation. as long as tst and cxr remain mandatory tests for screening, coverage in resource-limited settings can not be expected to improve, and its impact on tb control is likely to be limited. tst and cxr are not readily available in primary health clinics in the region, where index cases are commonly identified and treated. these tests are usually performed at main hospitals located in cities, which increases transport and time costs. the use and interpretation of tst are also problematic: unaffordable for most families in resource-limited setting; require skill to perform, and a second hospital visit is necessary. a more simple, cheap, feasible, efficient and patient-friendly method is required. the current who guideline of symptom-based screening recommends symptom evaluation alone to decide whether the contact requires further investigation for tb disease or can be prescribed ipt directly. asymptomatic child contacts aged less than 5 years can be provided ipt without further investigation. if tb is suspected at initial assessment or at subsequent followup, further investigation should be performed to establish or exclude a diagnosis of tb disease. referral to a district or tertiary hospital may be necessary when there are uncertainties about the diagnosis. a limitation of this systematic review is that it only includes published studies reported in english. more precise yields from contact investigation among child household contacts in the south east asia would have been gained if unpublished studies and the grey literature such as reports from ntps of each country were included. contact investigation studies in south east asia indicate the potential of screening and ipt to reduce the risk of tb disease in child contacts, yet it is rarely implemented. research is required to determine patient and health service barriers to screening to enable targeted effective intervention programs to be developed. one such research strategy could include a qualitative review of problems around the implementation of contact investigation and ipt provision for child contacts in the region. this will provide valuable information for the design of community-based interventions to improve the management of child contacts.

background. screening of children who are household contacts of tuberculosis (tb) cases is universally recommended but rarely implemented in tb endemic setting. this paper aims to summarise published data of the prevalence of tb infection and disease among child contacts in south east asia. methods. search strategies were developed to identify all published studies from south east asia of household contact investigation that included children (015 years). results. eleven studies were eligible for review. there was heterogeneity across the studies. tb infection was common among child contacts under 15 years of age (24.469.2%) and was higher than the prevalence of tb disease, which varied from 3.3% to 5.5%. conclusion. tb infection is common among children that are household contacts of tb cases in south east asia. novel approaches to child contact screening and management that improve implementation in south east asia need to be further evaluated.

PMC3235894

pubmed-902

tetralogy of fallot (tof) is a cyanotic congenital cardiac defect characterized by pulmonary outflow tract stenosis or obstruction, ventricular septal defect (vsd), overriding aortic root, and right ventricular hypertrophy.1 left without surgical correction, the overall survival was poor and over 90% of the patients die by 40 years of age.2,3 there are some subsets of tof patients with advanced chronic kidney disease (ckd); however, the association between tof and renal failure severe enough to require dialysis treatment has been rarely reported in the literature.4,5 in this report, we describe the case of an end-stage kidney disease patient with tof. we also discuss the complex processes of how and why peritoneal dialysis (pd) was selected as a mode of chronic renal replacement therapy in this case. a 33-year-old female with tof managed by palliative surgery was admitted to our hospital with complaints of general fatigue and appetite loss at the end of december 2013. taussig shunt at 2 years of age and palliative right ventricular outflow tract reconstruction, which is a procedure used to enlarge the ventricular outflow tract and main pulmonary artery, without inducing the closure of the concurrent vsd,6 at 4 years of age. there was no known family history of congenital cardiac disease. at 24 years of age, the patient s serum creatinine (scr) levels were increased at approximately 1.3 mg/dl, and her scr levels thereafter continued to increase gradually, but slowly. in 2012, at 32 years of age, she became hypertensive, and her scr levels were approximately 3.1 to 3.4 mg/dl with 3+for urine protein when the additional surgical correction for closure of the vsd was planned. she was then referred to our hospital. according to the clinical pictures and renal sonographic findings, which showed decreases in the renal long axis dimensions (right: 80 mm, left: 85 mm) with grade ii renal cortex echogenicity, the patient was diagnosed with ckd7 and subjected to contemporary and comprehensive renal care. despite the successful control of her blood pressure to the ranges of 130140/7080 mmhg with losartan potassium and azelnidipine, her renal function steadily worsened during the last year, and she finally became aware of the symptoms at the end of december 2013 when her scr and blood urea nitrogen (bun) levels elevated up to 8.25 and 99 mg/dl, respectively. as a result, she was admitted for further workup. a physical examination completed on the patient s blood pressure was 134/59 mmhg, her pulse was 78 beats/minute, and her temperature was 37.0 c. although the patient s oxygen saturation was 85% while she breathed ambient air, a chest x-ray film demonstrated neither an accumulation of fluid nor a sign of pulmonary infiltrates. a laboratory evaluation revealed the following results: white blood cell count, 9,000/l; red blood cell count, 407 10/l; hemoglobin (hb), 12.1 g/dl; hematocrit, 36.4%; platelet count, 19.5 10/l; bun, 111 mg/dl; scr, 8.03 mg/dl; total protein, 7.7 g/dl; albumin, 4.7 g/dl; sodium, 139 mmol/l; potassium (k), 5.3 mmol/l; chloride, 108 mmol/l; calcium (ca), 10.5 mg/dl; phosphorus (pi), 7.2 mg/dl; aspartate aminotransferase, 13 u/l; alanine aminotransferase, 12 u/l; c-reactive protein, 0.02 mg/dl; and brain natriuretic peptide, 55.4 pg/ml. her urine was 3+for protein and+for occult blood and contained 1.3 g of protein in a 24-hour specimen, while the patient s creatinine clearance was 5.02 ml/minute. transthoracic echocardiography disclosed a perimembranous vsd (maximum diameter of 21 mm), a bidirectional shunt with left-to-right shunting (2.55 m/second) predominance, and well contracting left ventricle (lv) with an lv ejection fraction of 63.8% (fig. 1a), while chest computed tomography (ct) scans demonstrated right pulmonary artery stenosis (fig. the patient was subjected to a transient session of hemodialysis (hd) treatment with repeated femoral vein puncture8 between hospital days 2 and 14, during which time she began to feel well along with the decline in her scr level. finally, a tenckhoff pd catheter was placed on hospital day 17 through a classic transverse surgical incision with a favorable postoperative course. pd using two daily exchanges (1.5 l of midperiq135 2, terumo co ..) with a total dwell of 12 hours was then initiated (fig., the patient is still doing well with a daily urine output of approximately 1,100 to 1,300 ml. considering the current therapeutic guidance for pd adequacy,9 her solute clearance status expressed in terms of kt/vurea, where k is the clearance of urea, t is the treatment duration, and v is the urea distribution volume,10 also appears to be favorable (a total kt/vurea: 1.9 [peritoneal kt/vurea: 0.8, residual renal kt/vurea: 1.1]). along with the decline in the mortality of patients with congenital heart diseases, it has become clear that various types of disturbed physiologies occur beyond the cardiovascular system.5,11 chronic renal insufficiency is one such late complication, and it may also be common even in adult subjects after obtaining the palliation of previous tof.5,11 several morphological alterations, including glomerular enlargement, mesangial hypercellularity, glomerular capillary congestion, and segmental glomerular sclerosis, have been focused on as pathologic bases of the disease.5,12,13 in the current patient, the absence of any pathological information precluded us from precisely evaluating the cause of end-stage kidney disease. however, we believe that numerous conditions, including previous surgical palliation and long-standing cyanotic conditions, could play a role in the establishment of her renal manifestations.5,11 alternatively, or in addition, the long-term use of various analgesics for relieving menstrual pain and a tension headache, which was revealed by our thorough clinical interview, might have played a role in our patient through their nephrotoxic nature.14 thus, the combination of tof and ckd may not be surprising; however, the clinical significance of the current patient should be evaluated carefully in terms of the impact of the circulation disturbance due to cardiac anomaly on the therapeutic managements for end-stage kidney disease, including the choice of dialysis modality. for many patients with end-stage kidney disease, a renal transplant is the treatment of choice as it replicates the standard renal physiology much more closely than dialysis treatments and offers an improved quality of life as well as survival benefits.15,16 despite the high risk of cardiac death before and after renal transplantation,17 patients with cardiac disease or who are at high risk for cardiac disorders are still eligible for this procedure.18 moreover, it has been stated that patients with severe irreversible heart dysfunction should not be listed for kidney transplantation alone, while there may be select patients who are candidates for combined heart kidney transplants.18 the scarce information regarding renal transplantation among patients with unrepaired congenital heart disease precludes us from precisely evaluating the validity of such a strategy in the current case; however, the lack of readily available cadaveric or living renal transplantation in a timely manner, which leaves many subjects requiring dialysis,15,16 obliged us to start dialysis treatment. considering the pathophysiologic characteristics of tof,1,19,20 we felt that avoiding the use of dialysis catheters as a means of either temporary or permanent vascular access for hd and the execution of periodic hd with peripheral vascular access should be mandatory in the current patient. indeed, these devices may predispose patients with tof to the detrimental pathologies such as pulmonary embolism, septicemia, infectious endocarditis, and paradoxical embolism,2127 although the available literature has seldom discussed pulmonary embolism in patients with tof.28 we must also recognize that in patients with an intrinsic cardiac anomaly, the creation of peripheral vascular access may result in severe cardiac failure.29 otherwise, it should be noted that bacteremia is less frequent in pd patients than in chronic hd patients with endovascular catheter, and no reports on infectious endocarditis in pd patients were available, despite the fact that septicemia is not exceptional in pd subjects30,31; therefore, pd does not appear to be an additional risk of infectious endocarditis among patients with end-stage kidney disease.31 alternatively, or in addition, the therapeutic nature of pd, including minimal variation in the intravascular volume status, reduction in cardiovascular stress, avoidance of peaks and troughs in uremic toxins, arrhythmia prevention, and the better preservation of residual renal function,10,32,33 also encouraged us to promote the procedure as a good modality of renal replacement therapy for the current patient. one may argue against our concerns about the potential risks associated with a dialysis catheter and hd. moreover, no one knows the impact of starting hd with repeated femoral vein puncturing for a transient vascular access on the risks of the concurrent development of thromboembolic and/or infectious events.8 nevertheless, it is necessary to take a proactive approach and not let lethal events become apparent. we believe that our policy regarding the application of tailored renal replacement therapy should be an appropriate therapeutic choice for the current patient. apparently, we are always facing, as do most physicians at various times, different types of clinical challenges, as described herein. there may be substantial variation in the type and intensity of managements provided to end-stage kidney disease patients with congenital heart disease, stemming from the uncertainty of the advantages and disadvantages of renal care in this population. a lack of prospective data suggests that numerous therapeutic decisions among such subjects are potentially empirical. indeed, only a few anecdotal reports describing patients with tof who began dialysis treatments during the observation periods for the disease are available.4,34,35 pd with reduced dialysate exchanges may be less advantageous than full-dose pd in terms of ultrafiltration and dialytic clearance, while such a pd protocol may permit early incremental dialysis and provide favorable clinical significance.33,36,37 at present, our patient appears to tolerate the pd program well with two daily dialysate exchanges; however, the validity of our strategy should be determined only when more experience with additional cases similar to ours has been accumulated. thus, the establishment of an optimal management program for end-stage kidney disease patients with congenital heart disease should be a matter requiring continuous and careful attention .

in this report, we describe the case of an end-stage kidney disease patient with tetralogy of fallot (tof). a 33-year-old female with tof was admitted to our hospital with complaints of general fatigue and appetite loss probably due to uremic milieu. she was ultimately treated with peritoneal dialysis (pd) with a favorable clinical course. tof patients with chronic kidney disease are not exceptional, although the currently available information regarding the association between tof and renal failure severe enough to require dialysis treatment is limited. we also discuss the complex processes of how and why pd was selected as a mode of chronic renal replacement therapy in this case.

PMC4648563

pubmed-903

a 45-year-old man with a lifelong history of atopic dermatitis had a year-long unremitting exacerbation for which he had started systemic therapy. after treatment with cyclosporine for several weeks, laboratory abnormalities and nonspecific neurologic signs prompted a switch to methotrexate. within 4 weeks, he was hospitalized (in an overseas us military hospital) for generalized umbilicated papulopustules accompanied by profound hypothermia, hypotension, and mental status changes. he had large pustules on his trunk, inner thighs, and upper extremities (figure 1, panel a). he was transferred to our intensive care unit with widespread umbilicated pustules and normal mental status. the pustules were deep seated, monomorphic, dome shaped, and firm and were distributed densely on the patient s forearms and abdomen (figure 1, panels b and c). clinical photographs of the patient. a) patient with generalized pustules, which were deep seated, monomorphic, dome shaped, and firm and were distributed densely on forearms and abdomen. c) umbilicated papulopustules in the same stage of evolution; no herpetiform clusters or red areolae are seen around the lesions. at our hospital, his oral temperature fluctuated dramatically, from 89.3f to 101.3f, with rectal confirmation<95f (< 35c), indicating hypothermia (5). we performed a tzanck preparation, which showed multinucleated giant keratinocytes with nuclear molding and margination (appendix figure). a direct fluorescent antibody (dfa) test was positive for varicella zoster virus (vzv). a biopsy specimen showed epithelial necrosis with cellular ballooning and multinucleated giant cells, plus intranuclear inclusion bodies (figure 2, panels a and b). subsequently, special immunohistochemical stains were positive for herpes simplex virus (hsv) (figure 2, panel c), and a viral culture grew hsv type 2. his illness was diagnosed with disseminated hsv concurrent with underlying atopic dermatitis (i.e., eczema herpeticum). c) positive immunohistochemical stain for herpes simplex virus. within minutes of the tzanck smear evaluation, our patient was given intravenous acyclovir. when cutaneous improvement was evident, he was switched to oral valacyclovir. within days, his skin lesions largely resolved without conspicuous crusting or scarring, but he remained intermittently hypothermic for several weeks. this patient was markedly ill on admission and had a distinctive varioliform eruption with lesions in a uniform stage of evolution. tzanck preparation promptly confirmed herpetic etiology, but we nevertheless used cdc s algorithm for evaluating agvpri, and our patient s illness stratified to high risk. cdc has 3 major diagnostic criteria to designate a case as high risk for smallpox (table) (6). the first is febrile prodrome, which typically lasts 14 days before cutaneous lesions appear and must include>1 of the following: prostration, headache, backache, chills, vomiting, or severe abdominal pain. although our patient s illness eventually met the fever criterion, his 101f temperature occurred only after he began antiviral treatment. prolonged hypothermia is associated with severe illness (7) and is equivalent to fever in determining critical illness (8), which we believe satisfies cdc s first major criterion. the second criterion requires classic cutaneous lesions that are deep seated, firm, round, well-circumscribed vesicles or pustules that may become umbilicated or confluent. the third criterion requires the same stage for most cutaneous lesions on an affected area. our patient s illness met all 3 criteria; however, laboratory tests confirmed herpesvirus infection. smallpox was declared eradicated by the world health organization in 1977; nevertheless, some health organizations consider this illness a bioterrorism threat. clinical smallpox typically starts with a prodrome of high fever, headache, myalgia, backache, nausea, vomiting, and diarrhea. an oropharyngeal enanthem is followed by cutaneous eruption of erythematous macules that quickly become papules. the papules evolve over days into vesicles and then pustules, often developing central umbilication. classic smallpox lesions occur in the same stage of evolution on a body segment, which differentiates it from varicella. smallpox pustules have been called pearls of pus to help distinguish them from the more delicate histopathologically, cutaneous smallpox lesions may resemble herpetic lesions except that smallpox has intracytoplasmic inclusions (guarnieri bodies) instead of intranuclear inclusions (lipschutz bodies) of herpetic lesions. also, multinucleated giant keratinocytes are uncharacteristic of smallpox (9). eczema herpeticum, described by kaposi in 1887, is most common in patients with atopic dermatitis but can occur in other conditions that disrupt epidermal integrity. in eczema herpeticum, lesions are typically monomorphic vesicles that evolve into pustules (10). fever, malaise, lymphadenopathy, and tender skin may accompany cutaneous eruption (11). fever is a well-recognized sign of infection; however, hypothermia can also signal serious disease, including bacterial sepsis or viral encephalitis (12), and may be more dire than fever in severely ill hospitalized patients (13). we propose that our patient s hypothermic temperature dysregulation is equivalent to fever, thus serving as a major diagnostic criterion. when a patient with agvpri is evaluated for possible smallpox, rapid laboratory tests are necessary. viral culture does not yield results quickly enough to avert infection control measures expected with a smallpox case. indeed, cdc reports 7 incidents when patients with agvpri prompted emergency department diversions or hospital closures (1). also, rapid confirmation of nonvariola etiology can help avert public panic, a potential problem in a suspected smallpox outbreak and a probable intended consequence of a terrorist attack. the tzanck smear must be performed by someone experienced in using the technique and interpreted by someone who can confidently and correctly distinguish herpesvivus nuclear inclusions from poxvirus cytoplasmic inclusions. dfa for hsv and vzv is relatively rapid, but in our case, the dfa result was positive for vzv, although viral culture and immunohistochemical staining later showed that the patient s infection was due to hsv-2. had we been unable to confirm a nonvariola etiology, we would have proceeded to poxvirus testing. with no commercially available tests for smallpox, the algorithm advises close coordination among local, state, and federal public health authorities. some state and federal reference laboratories can provide confirmatory tests, including pcr, for orthopoxviruses such as smallpox and monkeypox. although not performed in this case, we recommend such testing if a simultaneous infection with an orthopoxvirus can not be ruled out.

eczema herpeticum can clinically resemble smallpox. on the basis of the algorithm for rapid evaluation of patients with an acute generalized vesiculopustular rash illness, our patient met criteria for high risk for smallpox. the tzanck preparation was critical for rapid diagnosis of herpetic infection and exclusion of smallpox.

PMC2744234

pubmed-904

gene therapy is now a realistic prospect for the treatment of a variety of musculoskeletal disorders because of the emerging knowledge concerning proteins that govern the processes of growth and regeneration of tissues [1, 2]. research has identified numerous growth factors and other gene products with the ability to promote regeneration. the most extensively studied are members of the transforming growth factor (tgf) superfamily, collectively known as bone morphogenetic proteins (bmps). because gene therapy provides the gene rather than just a degradable protein, this technique may result in higher and more reproducible levels of protein production with the potential for long-term protein delivery. this would allow either local protein production for tissue healing and so forth or potentiate long-term systemic delivery. the most commonly used in the orthopaedic field have been viral vectors and ex vivo manipulation with considerable preclinical success [5, 6]. there are concerns regarding viral recombination/infectivity, immunogenicity, and possible carcinogenicity [79]. they are expensive to produce and as vectors have biophysical and genetic limitations. also many gene therapy strategies prove ineffective, suffering from gene silencing. non-viral therapy has not been extensively studied in orthopaedic research [11, 12]. in theory, non-viral therapy lacks some of the drawbacks associated with viral systems. in this study, the long-term viability of murine skeletal muscle plasmid gene expression in vivo was assessed using live whole body imaging of luciferase expression, along with the potential for expression control using an inducible model. finally an ex vivo model was developed in murine musculoskeletal tissue and conditions replicated to examine plasmid gene expression in human ex vivo mesenchymal tissue. the commercially available plasmid pcmv-luc was purchased from promega (wisconsin, usa). david gould (bone&joint research unit, barts and the london, queen mary's school of medicine and dentistry, university of london, london, england). b-alb c mice were obtained from harlan laboratories (oxfordshire, england). they were kept at a constant room temperature (22c) with a natural day/night light cycle in a conventional animal colony. female mice in good condition, without fungal or other infections, weighing 1622 g and of 68 weeks of age, were included in experiments. all in vivo and ex vivo animal experiments were approved by the ethics committee of university college cork. administration of 200 g xylazine and 2 mg ketamine. for liver transfection, a 1 cm subcostal incision was made over the liver and the peritoneum opened. the right lobe of the exposed liver was administered plasmid by electroporation as described below. the wound was closed in two layers peritoneal and skin using 4/0 prolene sutures (promed). all ex vivo human tissue experiments were approved by the clinical research ethics committee of the cork teaching hospitals. informed consent was obtained from patients scheduled for lower limb amputation and the surgeon's permission obtained. upon removal of sample samples were cut to a size approximating the medial thigh muscle of balb-c mice (1.5 cm). for plasmid delivery by electroporation, a custom-designed applicator with 2 needles 4 mm apart was used, with both needles placed through the skin central to the tissue. tissue was injected between electrode needles with plasmid dna in sterile injectable saline in an injection volume of 50 l. plasmid concentration was determined using the nanodrop spectrophotometer (nd-1000 spectrophotometer, labtech int, east sussex, uk). the number of plasmid copies was determined using the following calculation: (1)mass of plasmid dna (g) =no. of gene copies (bp) (plasmid size1.09610(21)). plasmid sizes for pcmv luc and pgtrtl were 6.9 kbp and 7.5 kbp, corresponding to a mass of 6.05 10 g and 6.576 10 g, respectively. after 80 seconds, square-wave pulses (1200 v/cm 100 sec 1 and 120 v/cm 20 msec, 8 pulses) were administered in sequence using a custom-designed pulse generator (cythorlab, aditus, lund, sweden). in all cases, a drop in impedance measurement was taken as evidence for successful tissue cell poration. replication incompetent recombinant adenovirus 5 particles encoding the luciferase gene under the transcriptional control of the cmv promoter were a kind gift from prof. andrew baker, university of glasgow; they were generated and titrated as described previously. viral vector particles were administered by direct intramuscular injection in a volume of 50 l. 1 10 vp of replication incompetent recombinant adenovirus 5 particles was used per administration. the entire medial thigh muscle tissue was harvested from sacrificed b-alb c mice (1.5 cm). samples were stored as per the experimental protocol to assess survival in either rpmi (roswell park memorial institute medium) or dmem (dulbecco/vogt modified eagle's minimal essential medium) at 37c for 24 hours. the effect of electroporation on cell survival was assessed by administering the plasmid as described previously and assessing viability at 24 hours. cell viability was assessed using the propidium-iodide-based nucleocounter kit as previously described. samples were washed and processed through a fine mesh filter (bd biosciences, oxford, uk), using a pestle and pbs (phosphate buffered saline) until a suspension was obtained. the suspension was made up to a standard volume, and cell viability was assessed. solutions were prepared by adding 200 g/ml of doxycycline (sigma) to distilled water containing 10% sucrose as previously described. all drinking bottles were wrapped in aluminium foil and were renewed every 2-3 days. a 20 g/ml solution was added to wells with pgtrtl ex vivo samples as the 200 g/ml resulted in excessive cell death (data not shown). in vivo luciferase activity from tissues was analysed at set time points after transfection as follows: 80 l of 30 mg/ml firefly luciferin was injected intraperitoneally. ten minutes after luciferin injection, live anaesthetised mice were imaged for 3 min at high sensitivity using an intensified ccd camera (ivis imaging system, xenogen). the exposure conditions were maintained at identical levels so that all measurements would be comparable. all data analysis was carried out on the living image 2.5 software package (ivis imaging system, xenogen). luminescence levels were calculated using standardised regions of interest (rois) for all 3 anatomical areas. actual levels were obtained by subtracting the corresponding roi of an untransfected mouse to account for background luminescence. for comparison between plasmids, flux was calculated per gene copy number. at each time point, either one-way a two-sampled t-test was used to compare mean luminescence within each experimental group. luciferase-coding plasmid dna featuring the cmv (pcmv-luc) promoter (figure 1) was delivered to liver or thigh muscle by electroporation. ivis live whole body imaging was used at time points over 370 days to determine luciferase expression (figure 2). expression driven by the cmv promoter in liver was initially high but reduced rapidly to background levels by day 14. a different temporal pattern of expression was observed in muscle tissue, with no gross reduction in expression over time observed. total luminescence for the period was 1.33 10 p/sec/cm/sr/plasmid copy. unlike liver, muscle therefore has the potential to act as a long-term conduit for gene expression. luciferase-coding plasmid dna featuring the cmv (pcmv-luc) promoter or adenovirus 5 particles encoding the luciferase gene under the transcriptional control of the cmv promoter was delivered to murine thigh muscle as described. ivis live whole body imaging was used at time points over 18 days to compare luciferase expression between the two constructs. maximum expression was significantly higher using adenoviral delivery when compared to that of plasmid delivery. expression following adenoviral delivery, however, decreased over the study period to background levels at 18 days (figure 3(b)). luciferase expression from an inducible tet-on promoter was examined in muscle tissue using the pgtrtl plasmid, both in the presence and absence of induction by doxycycline (figure 4). there was a low initial expression from reporter gene preinduction from the time of delivery. however, following induction with doxycycline at day 8, expression was increased by a factor of 11 (p=0.0001) at day 11. following withdrawal of doxycycline at day 12, expression was reinduced at day 105 and had increased by a factor of 9.2 by day 108 (p=0.001) and by a factor of 10.7 at day 116. expression remained constant at a maximal level between days 108 and 116 with no statistically significant change in magnitude observed (p=0.77). following withdrawal of doxycycline at day 117, there was no difference in maximal expression between the two induction periods (p=0.28). it can also be reinduced on a temporal basis to give reproducible levels of expression. cell viability following incubation in dmem at 37c for 1, 4, and 24 hours was 85%, 82%, and 38%, respectively (figure 5). this was significantly better than cell survival following incubation in rpmi (22%, p<0.05). there was no difference in cell survival following plasmid electroporation when compared to untreated samples (34% and 38%, resp., p=0.5). to assess translation of the murine findings to a clinical setting, samples of muscle, tendon, and bone with intact periosteal tissue were harvested from a human leg amputated for end-stage peripheral vascular disease. 4 samples were taken of each tissue type and transfected with pcmv-luc or pgtrtl as previously described. these were incubated overnight in conditions described above and luminescence assessed at 24 h (figure 6). luciferase gene expression was observed in all tissues electroporated with plasmid, indicating successful plasmid delivery and subsequent gene expression in this setting. localised gene expression offers certain advantages in the setting of tissue healing. it may provide a more accurate system of protein delivery to the desired tissue, whilst lessening systemic side effects. in addition, it has the potential for sustained protein delivery, both intracellularly and extracellularly without the need for repeated administration. however, inactivation of gene expression in specific cell types has important therapeutic implications, as does the prospect of uncontrolled protein production. here, we have demonstrated that plasmid-based gene expression in murine muscle does not decrease with time. long-term plasmid gene expression has been observed in muscle previously, although not using an in vivo luminescence system for a period greater than one year [15, 16]. in vivo luminescence it is also an ideal method of assessing an inducible agent as an on-off switch to control protein expression, by allowing repeated measurements in the same subjects over a long time period. successful bone and tendon healing in animal models have been reported following adenoviral transfer of bmp 2 and bmp 14 [5, 6, 18]. here, we have compared luciferase expression between a plasmid construct and viral particles. although maximum expression was significantly greater with adenovirus, this was rapidly silenced to background levels by day 18. the attraction of the plasmid system would therefore lie in its capacity for sustained levels of predictable expression over time. electroporation-assisted intramuscular delivery of plasmid dna has been shown to efficiently transfect cells of myogenic origin with no evidence of transgene expression in infiltrating inflammatory cells. this is broadly in keeping with our observation of long-term expression, with transfection of a nontransient cell population in muscle and the absence of a significant immune response. silencing has previously been observed with adenovirus, with the mechanism postulated as methylation of the cmv promoter. why this does not occur when the promoter is delivered using a plasmid construct, however, is unclear. for instance, in the clinical use of recombinant bone morphogenetic protein (rhbmp) for augmentation of fracture healing and joint fusion, heterotopic ossification and other complications have been reported. in this instance, the protein is eventually cleared from the body. following gene delivery, however, it would be difficult to arrest protein production in the event of unwanted side effects. it is, therefore, desirable to have a plasmid with an on-off switch. we have demonstrated that this can be achieved using pgtrtl, a tet-on inducible promoter. after plasmid delivery, gene expression initiated with the commencement of oral doxycycline administration and promptly ceased upon withdrawal. this strategy has also proven to be successful in a preclinical arthritis study. in the clinical setting, should side effects occur, gene expression could be turned off simply by omitting the oral agent. in addition, growth factors may not be clinically beneficial until days after intervention, while the delivery of rhbmp at the time of surgery has been shown to impair tissue healing by inducing antagonist factors [18, 22]. by using pgtrtl, we have demonstrated that the timing and duration of gene expression can be tightly controlled in vivo. this would decrease the chance of antagonist induction, whilst optimising the timescale of protein production. this would allow us to repeatedly treat tissue with a known quantity of protein in recalcitrant situations. as the system is orally administered, there would be no need for interference with the physical environment of tissue healing after the initial application. one issue with tetracycline compounds is that they do have an effect on the tissue healing environment and as such may not be the ideal tool for the investigation of therapeutic plasmid use. in terms of principle, however, it demonstrates the potential applicability of an inducible plasmid-based system in a clinical setting. finally, we have demonstrated, for the first time, successful plasmid-based gene delivery to human ex vivo muscle tissue. levels of luciferase expression in ex vivo tendon and muscle were similar when examined at 24 hours. although the exact reasons for the similar levels of expression are unclear, it may be related to a threshold effect or differential tissue survival in the ex vivo environment. expression may differ in the human in vivo setting, particularly with regard to increased metabolic activity in muscle tissue. nonetheless, we believe this to be an important step in the demonstration of the applicability of this technique in the in vivo setting, as expression capability can not be definitely extrapolated from the murine model to the human setting. the expression of plasmid-based delivery in human ex vivo musculoskeletal tissue is encouraging on two levels; firstly, it proves that the system can function in human tissue, and secondly, it raises the possibility of autograft or allograft transfection, which could lead to the acceleration of graft incorporation. the luminescence imaging strategy utilised here provided a rapid, robust and reliable method for ex vivo assessment of transgene expression in patient samples. the method of culturing tissue ex vivo has been demonstrated to provide high viability for up to 1 week post resection, while bioluminescence imaging provides a highly sensitive readout for real-time transgene expression in viable cells. other reporter gene imaging techniques may also be applicable in this context (fluorescence, pet etc.) these results indicate, when administered using electroporation, that plasmid constructs result in long-term in vivo gene expression and that this expression can be reliably induced with oral agents. finally, for the first time, successful plasmid gene transfection with electroporation in human ex vivo mesenchymal tissue has been demonstrated.

purpose. in vivo gene therapy directed at tissues of mesenchymal origin could potentially augment healing. we aimed to assess the duration and magnitude of transene expression in vivo in mice and ex vivo in human tissues. methods. using bioluminescence imaging, plasmid and adenoviral vector-based transgene expression in murine quadriceps in vivo was examined. temporal control was assessed using a doxycycline-inducible system. an ex vivo model was developed and optimised using murine tissue, and applied in ex vivo human tissue. results. in vivo plasmid-based transgene expression did not silence in murine muscle, unlike in liver. although maximum luciferase expression was higher in muscle with adenoviral delivery compared with plasmid, expression reduced over time. the inducible promoter cassette successfully regulated gene expression with maximum levels a factor of 11 greater than baseline. expression was re-induced to a similar level on a temporal basis. luciferase expression was readily detected ex vivo in human muscle and tendon. conclusions. plasmid constructs resulted in long-term in vivo gene expression in skeletal muscle, in a controllable fashion utilising an inducible promoter in combination with oral agents. successful plasmid gene transfection in human ex vivo mesenchymal tissue was demonstrated for the first time.

PMC3395381

pubmed-905

rickettsia are obligatory, intracellular, small gram-negative bacteria associated with eukaryotic hosts. they are traditionally divided into three groups: the spotted fever group, the typhus group and the scrub typhus group. the spotted fever results from a large group of tick-, mite- and flea-borne zoonotic infections that are caused by closely related rickettsiae. rickettsia conorii has been identified as the agent causing mediterranean spotted fever (msf). a variety of geographically distinct types of r. conorii have been described that cause variable clinical presentations different from typical msf. a 27-year-old primigravida with a 16week history of amenorrhea and respiratory failure was transfered to a tertiary care center. she had high-grade fever for 8 days prior to the transfer and had developed an erythymatous maculopapular rash on day 4 of the illness, involving the trunk initially, later extending to the palms and soles [figure 1]. erythematous maculopapular rash in a hand a persistant dry cough, not associated with hemoptysis, was noticed on day 5. she developed a progressively worsening dyspnea with bilateral ankle edema not associated with oliguria or orthopnea. her condition deteriorated rapidly, warranting intubation for respiratory failure and transfer to a tertiary care center. on admission to the tertiary care center icu, the patient also had tachycardia (128 beats/min) without any significant st segment changes on the ecg. the blood urea measured 8.7 mmol/dl with a serum sodium of 140 meq/l and a pottasium of 5.0 meq/l. full blood count showed neutrophilia of 78% in a total of 14.4 10 l. hemoglobin was 7.4 g/dl with a mild thrombocytopenia of 160,000 l. the blood picture revealed normochromic normocytic anemia with no evidence of disseminated intravascular coagulation (dic). the international normalized ratio (inr) was 0.96. the serum glutamate oxaloacetate transaminase (sgot) activity was 42 u/l and serum glutamate pyruvate transaminase (sgpt) activity was 28 the c-reactive protein (crp) level was 6 mg/dl and the antinuclear antibody (ana) was negative. chest x-ray on day 1 at icu, showing diffuse bilateral opacities the patient was started on oral azithromycin, iv cefotaxime, iv hydrocortisone 50 mg 6 hourly. the patient's condition deteriorated rapidly on day 3 of icu stay and needed adrenaline, noradrenaline, dobutamine and vasopressin to maintain a blood pressure of 70/50 mmhg. the urine output dropped and the urine microscopic examination revealed granular casts and dysmorphic red blood cells. blood urea rose to 12.9 mmol/dl and the electrolytes showed pottasium level of 5.6 meq/l. on the 4 day at the icu and 12 day of the disease, she devoloped anuria with further increase in blood urea and serum creatinine. she was hemodynamically unstable with a blood pressure of 60 mmhg even with total inotrope support. endocardium of the heart showed vegitations on a narrowed mitral valve [figure 4]. the kidneys were of normal size, but the cortico-medullary demarcations were less clear. the grossly hemorrhagic lung the heart showing narrowing and vegetations at the mitral valve the histology of the lung showed gross hemorrhage and foci of pneumonia. the vegetations of the heart were reported as fibrinous with entrapped white cells, but with no bacteria or fungi. histology of the liver revealed centrilobular necrosis with a tendency to confluence, and a mild fatty change. a final diagnosis of disseminated r. conorii infection with pulmonary hemorrhage, endocarditis and tubulointerstitial nephritis was made. r. conorii is an obligate, intracellular, slow-growing, gram-negative bacterium belonging to the spotted fever group of rickettsiae. unusual rickettsial strains related to r. conorii have been described as belonging to an r. conorii complex which includes the indian tick typhus rickettsia (atcc vr-597) with r. conorii subsp. it is transmitted to humans through the bite of dog ticks (rhipicephalus sanguineus) widely prevalent among the old world. it is also known to be transmitted by haemaphysalis ticks, especially in pakistan and kenya. the infection is transmitted via larvae and nymphs, and the tick bite is usually not felt. the incubation period ranges from 3 to 15 days depending upon the route of rickettsial entry and the rickettsial load. after introduction into the skin at the site of the tick bite or through the conjunctiva contaminated by blood or excretions from an infective tick, the primary multiplication occurs. in the skin, the localized multiplication of the rickettsiae in the endothelial cells of the capillaries leads to the formation of a raised red papule. the inflammation and thrombosis of the affected capillaries lead to necrosis of the center of the papule and the formation of the typical red lesion with a black center, the tache noire. this is followed by the spread of infection through lympho-hematogenous routes throughout the body, causing disseminated vascular lesions in multiple organs. about 6% of the cases are severe, and fatal cases occur even in young, healthy adults, with a reported death rate of about 2.5%. old age, alcoholism and glucose-6-phosphate dehydrogenase (g6pd) deficiency are known risk factors for severe disease. the patient described here is from kotagla, a tea estate village in central sri lanka. the patient reported a history of significant exposure to stray dogs, abundant in any sri lankan village, but not of any tick bites. the absence of the tachy noire is significant in this case as the clinical diagnosis of msf (caused by r. conorii subsp. however, the absence of tache noire has been noted in israeli spotted fever caused by r. conorii subsp. the fever starts after an incubation period of 7 days followed by a febrile period (up to 40c, usually continuous) associated with a maculopapular rash. the papules of the rash are first noticed on the 3 to 5 day of illness, and they come out in crops and are palpable as small nodules in the skin. in severe cases, characteristically, the rash involves the palms of the hands, the soles of the feet and, to a lesser extent, the face. in this patient, the rash involved the palms and soles, sparing the face, and was of dusky cyanotic appearence with prominant macules. the cough and the dyspnea reported in our patient were relatively rare in a case series of msf, with a representation of 10% and 21%, respectively. renal insufficiency is a known complication in around 6% of the patients and is caused by tubulointerstitial nephritis. myocarditis had been obseved in 11% of patients and might explain the severe tachycardia with poor response to inotropes in this patient. the diagnosis of r. conorii infection in this patient is justfied as there is a compatible clinical picture along with a single titer of>1/512. endocarditis is not described as a complication of r. conorii infection. identifying r. conorii as the causative organism of endocarditis in this patient is supported by the absence of previous heart disease and lack of clinical features to suggest subacute bacterial endocarditis on admission and during prenatal clinics. the vegetations isolated from this patient as well as the blood cultures did not reveal bacteria or fungi. this patient was correctly started on oral azithromicin and intravenous chlorampenicol at the tertiary hospital. a diagnostic scoring system with microbiological, epidemiologic, and clinical parameters has been proposed for msf and it had shown good sensitivity and specificity. this case highlights the need of clinical suspicion of uncommon diseases based on the geographic and socioeconomic background of a patient. relatively benign diseases may have severe manifestations resulting in fatality; therefore, physicians should consider there factors for early diagnosis and intervention.

rickettsial diseases are common in srilanka. the spotted fever group of rickettsiae presents in many ways, including very severe disease causing significant morbidity and mortality. a regional variation of the rickettsia conorii subspecies and differences in clinical presentations are reported. this case describes disseminated rickettsia conorii infection in a pregnant woman presenting with endocarditis.

PMC3162761

pubmed-906

the waste arising from>60 years of civil and military nuclear operations around the world contains long-lived radionuclides that must be contained and isolated from future populations. deep geological disposal facilities (gdf) proposed by us and european waste management organisations employ an engineered multibarrier approach (figure 1) to retard the release of radioactive species from the waste in quantities that could be detrimental to life and the environment. the multibarrier design concept typically combines reducing conditions with high ph with the purpose of limiting the solubility and mobility of radionuclide species within gdf if (or when) primary containment fails. general design features of the multibarrier geological disposal facility (gdf) concept proposed for the long-term (10 year) storage of high-level nuclear wastes in the deep subsurface (bgl=below ground level). a significant obstacle to implementation of gdf is public and political concern around risks and consequences of failure against design criteria over the 10 to 10 year required lifespan of the facility, highlighted by several failures to site gdf repositories, e.g., in the uk and at yucca mountain in the usa. should gdf performance be compromised, it is possible that long-lived, mobile radionuclides will be transported through the engineered backfill into groundwater and pose a long-term hazard to the biosphere and water resources. thus, it is critical to the safety case for the gdf not only to be able to demonstrate that the design performance is well understood but to show that conditions arising from design failures are also accounted for and mitigated as far as possible. one potentially problematic radionuclide is technetium-99, a high-yield fission product of u, which has a long half-life (2.1 10 years) and high solubility in oxic conditions as the pertechnetate anion [tc(vii)o4]. while the conditions within the gdf are expected to be reducing, such that insoluble tc(iv) should be the dominant oxidation state, the uk nuclear authority estimates suggest that a significant proportion of the uk tc inventory is expected to be present as the tc(vii) pertechnetate species. performance assessment analysis of tc mobility has shown that the potential risk to future populations from tc critically depends upon its oxidation state, such that tc(vii) presents a significantly greater risk than tc(iv) over the one million year lifetime of the gdf, even when reducing conditions are applied. therefore, a robust design for the engineered barrier concept should be able to account for the risk arising from the presence and behavior of the mobile tc(vii), separately from the specific probability of oxic conditions occurring or persisting within any given gdf scenario. an improved understanding of the behavior of pertechnetate in proposed barrier materials is also necessary to evaluate the potential of different design specifications to mitigate or remove the potential hazard. understanding the spatial and temporal dynamics of geochemistry within and surrounding a gdf is essential in this task. the importance of (bio)geochemical gradients on radionuclide mobility is the focus of substantial current research, e.g., refs (911). such studies ideally require noninvasive, nondestructive measurement of the distribution, migration, and chemical transformation of radionuclides within a physical model of the barrier material. this should be considered over time as internal conditions respond to controlled changes in boundary conditions. quantitative imaging techniques offer a means of achieving this information and have been developed to study reactive transport in porous media for a range of materials. imaging techniques include visible light transmission and fluorescence imaging, nuclear magnetic resonance (nmr), and x-ray computed tomography (see ref (14) for a recent review). gamma attenuation techniques with external americium-241 or cesium-137 sources have been used to determine fluid transported within a column. a key methodological step remains: the extraction of quantitative geochemical information from image data, particularly in three dimensions and opaque materials. techniques have been developed to quantify, from image data, ph and oxygen gradients in two dimensions (2d) within porous media and to extract transport, deposition, and remobilization rate parameters from time-lapse image sequences of colloidal particles in translucent quartz sand. recent work demonstrated that gamma-emitting radioisotopes can be used as an effective imaging tracer within opaque sediment and mineral systems, both in static batch experiments and in flow-through columns. these studies utilized ultratrace concentrations of a gamma-emitting technetium isotope, technetium-99 m (commonly used in medical and industrial imaging applications), to demonstrate qualitatively the immobilization of technetium on fe(ii)-bearing sediments and minerals, via an fe(ii)-mediated reduction of tc(vii) to tc(iv). in this study, we report the use of 2d gamma-imaging to quantify tc transport parameters in a simple granular porous media model. uniform, saturated one-dimensional flow through ottawa quartz sand, as a model test material, is used to demonstrate the ability of gamma imaging to obtain reproducible data sets at the mesoscale (millimeters to decimeters), which can be used to yield transport parameters by fitting standard convection dispersion models. furthermore, we apply this methodology, for the first time, to investigate the feasibility of direct noninvasive quantification of radionuclide migration within opaque cementitious gdf candidate material (crushed nirex reference vault backfill (nrvb)) under circum-neutral and alkaline ph. the technique represents a base for development of model systems for noninvasive study of radionuclide migration in complex physicochemical environments, critical to establishing the design specifications and safety case for future gdfs, and also for application to other contaminant transport in the subsurface. replicated flow cells enabled aqueous solutions with and without a tc tracer to be pumped through saturated quartz sand at a steady flow rate with continuous monitoring using a gamma camera. tc is a controlled radioactive substance; therefore, experiments were performed with appropriate risk assessment in specialist facilities at a hospital which routinely produces and handles the material for use in clinical nuclear medicine. all other chemicals used were obtained from fisher scientific (uk) unless otherwise indicated. bench-scale flow cells were constructed of two perspex plates separated by viton seals and bolted tightly together (figure 2). the rear plate was solid, while the front plate had an indent creating a void space to hold the porous material. an upper port in the rear plate allowed for input of the aqueous phase to the cell, while a lower port in the same plate allowed for removal of the aqueous phase for control of flow rate and sampling. flow was from top to bottom along a distance of 70 mm between the ports. tc tracer was injected through a needle immediately below the inlet port (figure 2). schematic of the experimental setup showing construction of the flow cells, the pumping system, and image acquisition geometry. the flow cells were filled with 70 g of ottawa sand (99.5% sio2, particle diameter 500700 m) or 45 g of crushed nirex reference vault backfill (nrvb), sieved to 24 mm particle size, ensuring maintenance of appropriate flow rates. crushing and experimental setup were conducted in air several hours prior to experimentation, in which time some carbonation of the exposed surfaces may have taken place. the possible chemical changes that might be expected for degraded backfill are outside the scope of the current study and are thus not addressed in this model gdf system. the sand was washed and sonicated in ultrahigh quality water (18 m) five times to remove any existing impurities and oven-dried for 24 h prior to use. the nrvb material was prepared by mixing 130.1 g of ordinary portland cement, 49.12 g of ca(oh)2, 143.1 g of caco3, and 177.76 ml of water in a hobart mixer, giving a w/s ratio of 0.552. it was cured at room temperature for 28 days and kept sealed prior to use. duplicate flow cells containing sand were saturated with 16 ml of ph 5.7, deionized water (18 m) so that the material plus aqueous phase filled the cell above the inlet port. a 3 mm depth of solution was maintained above the top of the material to ensure a uniform pressure head across the flow field. identical flow cells were prepared using a ph 10.7 buffer solution (0.05 m nahco3, 0.1 m naoh). a flow cell containing nrvb was saturated with 25 ml of deionized water at ph 5.7 (18 m). the porosity calculated from the ratio of solution volume to total saturated pack volume was 0.37 for sand and approximately 0.77 for the crushed nrvb, taking into account the internal porosity of the material itself (estimated as 0.55). bulk densities of the porous materials as packed were 1.68 and 1.57 kg dm for sand and nrvb, respectively. fully constructed, prefilled flow cells were transported to the nuclear medicine department of the royal hallamshire hospital (sheffield, uk) for imaging. flow was maintained in the cells at 0.33 ml min using a multichannel peristaltic pump (watson marlow, uk), yielding a calculated pore velocity equal to 4.29 10 m s for the sand and 2.05 10 m s for the nrvb (due to the greater porosity). the darcy flux in both cases was 1.5 10 m s. flow cells were flushed with tc-free solution for 20 min to establish uniform flow conditions prior to injection with tc and subsequent imaging. imaging was performed on a dual-headed ge medical systems infinia gamma camera (ge medical, milwaukee, wi, usa) fitted with a high resolution collimator. a dynamic acquisition with 30 s frame intervals was initiated a few seconds prior to injection of the tc into the flow cells. images were acquired with a matrix size of 256 256 resulting in a pixel size of 2.2 mm. the spatial resolution of the imaging system was measured at the collimator face using standard nema testing techniques and was found to be 4.6 mm fwhm. this equates to a spatial resolution of 6 mm at the location of the flow cells. due to the low spatial resolution tc as pertechnetate [tc(vii)] was produced on-site via saline-based elution of a ge medical systems drytec tc generator. this volume gave an activity of 1520 mbq at the time of the experiment. this corresponds to tc concentrations of<1 mm. the activity in each syringe was accurately measured in a capintec crc-15r radionuclide calibrator. following injection of the tc into the flow cells as instantaneous pulses (< 1 s injection), the residual activity in each syringe was measured and this reading was subtracted from the full reading to determine the exact activity injected into each cell. in all cases, tc activity readings were decay-corrected to the time the gamma camera acquisition was started (eq 1, 2):12where a0 is the corrected activity (mbq), at is the uncorrected activity (mbq), k is the decay constant (s), t is the time elapsed (s), and t1/2 is the half-life of tc (21 636 s). for each flow cell, a sensitivity value (counts per mbq) was determined so that image counts could be related directly to tc activity. this sensitivity value was calculated by using region of interest (roi) analysis to determine the image counts per frame within the region of the cell, averaged over the first 4 frames following injection of the radioisotope. during this early period, this averaged count value was then divided by the known activity injected into the cell to yield a sensitivity factor (eq 3):3where sf is the sensitivity factor, xci is the mean of the counts from the first four frames of image acquisition (counts pixel), and a0 is the initial activity (mbq). this sensitivity factor was applied to all decay-corrected gamma counts throughout the experiments to yield the concentration c (mbq pixel, normalized by the volume of pores in each pixel to give mbq ml) of tc at any location in each time step. raw image data were calibrated using the sensitivity factor (eq 3) to give 2-d planar spatial arrays of tracer concentration data at 30 s intervals for up to 3 h during and after transit of the main mass of tc through the flow cell. spatial moments in the direction of travel were calculated at the center of mass of the plume using imagej software. calibrated concentration maps showing contours of tc mass within the flow cells were produced by interpolation of the 2-d data arrays using surfer 9.0 software (golden software, ca). the transport of the tc through the uniform saturated flow field was modeled using a one-dimensional (1-d) convection dispersion equation for reactive solute transport (eq 4):4where, subject to specified initial and boundary conditions, c is the aqueous concentration of a tracer at a given distance along the center of mass from inlet x (m) and elapsed time t (s), (s) is a first-order decay coefficient describing irreversible removal from the mobile aqueous phase, r is a retardation factor describing equilibrium interaction with the solid phase, and d is a dispersion coefficient equal to the product of the longitudinal dispersivity (m) and mean pore flow velocity, vp (m s). a numerical solution to eq 4 was implemented in inverse (parameter-fitting) mode in excel-cxtfit software to yield transferable parameters describing the transport of the radionuclide in the ottawa quartz sand and nrvb. figure 3a d shows the calibrated concentration distribution data for tc transport through the ottawa sand at ph 5.7, at 8 (0.29), 16 (0.58), 24 (0.87), and 32 (1.16) minutes after injection. values in parentheses and all time data thereafter are expressed in pore volumes (pv), where time is normalized by the transit time of a volume of solution equal to the volume of void spaces in the sand. under the conditions of these experiments, 1 pv was equivalent to 1650 s (27.5 min) of travel time. the tc tracer passed through the saturated sand as well-defined plumes with peak concentrations in the center approximately 10 2 mbq ml. figure 4 shows the total tc activity measured in the sand as a function of time for experiments at ph 5.7 and 10.7. this behavior was highly reproducible for experimental runs at both ph 5.7 and 10.7. residual activities measured after 2 pv (not shown) were less than 1% of the total activity injected and were not significantly different from zero, taking into account the assumed measurement error quantified by the standard deviation (1%) for total activity measurements made between 0.25 and 0.5 pv. calibrated concentration distribution from gamma camera images of tc activity in ottawa quartz sand in a ph 5.7 solution at (a) 0.29 pv (8 min), (b) 0.58 pv (16 min), (c) 0.87 pv (24 min), and (d) 1.16 pv (32 min) and in nirex reference vault backfill at (e) 0.3 pv (20 min), (f) 0.6pv (40 min), (g) 0.9 pv (60 min), and (h) 1.2 pv (80 min). total measured activity of tc (normalized to input activity) as a function of time (expressed as pv, normalized to flow rate) during transport through ottawa quartz sand s1 and s2 at ph 5.7 and s3 at ph 10.7 and nrvb. figure 3e h shows calibrated concentration distributions for tc transport in nrvb at 0.3, 0.6, 0.9, and 1.2 pv. due to the lower pore velocities in nrvb than in sand, 1 pv was equivalent to 4300 s (71.5 min) of travel time. the peak concentration at 0.3 pv was 12 mbq ml, measured at 3 cm from the injection point. after 0.6, 0.9, and 1.2 pv, the peak concentrations were measured as 10, 9, and 8 mbq ml, respectively, at 4, 5.5, and 6 cm from the tracer injection point (figure 3). the total tc activity measured in the nrvb as a function of time is shown in figure 4. as in sand, total activity decreased broadly symmetrically around 1 pv, indicating conservative transport with longitudinal dispersion. residual activity at the end of the measurement period was less than 2% of the initial activity and not significantly different from zero, taking into account variability in the image data quantified as noted above. tc activity was summed across horizontal pixel rows (normal to the vertical direction of transport through the cells) to yield concentration profiles which could be expressed as a function of distance from inlet or time since tracer injection. these data were fitted with the 1-d convection dispersion model (eq 4). in individual experiment runs s1s3 and n1n2 (table 1), the model was regressed to concentration profiles measured at five distances from the inlet simultaneously, using the linear least squared error method. the best fit model parameters for each experiment condition are shown in table 1; irreversible sorption parameter was never larger than zero and is therefore not tabulated. figure 5a compares data from an individual experiment run in sand with the output from the model run in forward mode for the same distance intervals, using averages of the parameter values shown in table 1. the correlation between the model and data is very strong (r=0.98). figure 5b shows equivalent data and model output (r=0.99) for nrvb. dispersion model (lines) to measured tc concentrations (data points) in (a) ottawa quartz sand and (b) nrvb material at several distances from the inlet during uniform saturated flow. the parameters obtained from numerical modeling of tc transport in sand (table 1) strongly indicate conservative transport (r=1) of the tc through the ottawa quartz sand. this conclusion is supported by independent spatial moments analysis of the calibrated image data (figure 6), which yields a mean velocity for the center of mass of the tc plumes in sand of 4.32 10 m s. the transport velocity for the tc was therefore not significantly different from the pore velocity in the quartz sand (4.29 10 m s) calculated a priori. in contrast, both spatial moments and numerical modeling yielded the same mean transport velocity for tc transport through nrvb, 1.64 10 m s, which was slower than the calculated pore velocity based on the internal and boundary conditions of the experiment (2.05 10 m s). this may be due to errors in estimation of the internal pore structure of the nrvb which may be discontinuous, creating regions of low flow or immobile pore water. while r remained close to 1 indicating conservative transport, the longitudinal dispersivity,, for nrvb was 0.0033 m, more than three times that modeled in sand. tracer plume center of mass (com, first spatial moment in the direction of flow) plotted as a function of calculated water movement for tc transport through ottawa quartz sand and nrvb. data are averages of experimental replicates. the sorption of solutes to a solid phase is often described by an equilibrium linear sorption coefficient kd (m kg) estimated from batch experiments, which contain a known volume of solution, concentration of solute, and mass of solid phase, yielding (eq 5):5where c0 and cs are, respectively, the initial solute concentration in solution and final equivalent concentration on the solid phase, m (kg) is the mass of solid phase, and v (m) is the volume of the fluid. we approximated these parameters by normalizing the known input activity and the observed retained activity by the volume and mass of porous media in the flow chamber, to obtain an estimate for kd (we denote this method m1). for reactive transport through porous media and assuming that surface reactions occur sufficiently rapidly relative to transport that equilibrium can be achieved, kd can also be related both to the retardation factor r in the convection dispersion equation (denoted method m2) and to the ratio of mean transport velocities obtained from spatial moments analysis (method m3) by eq 6:6where is the porosity, b is the bulk density (kg m) of the porous media, and vtc is the mean velocity of mass flux (m s). estimated sorption coefficients for both sand and nrvb were small, of the order 10 m kg, which is consistent with the transport parameters obtained from the convection dispersion modeling and the observed low retention of tc in the sand after 2 pv. although the model-derived errors associated with nrvb were relatively large, kd as estimated by all three methods was consistently greater in nrvb (approximately an order of magnitude) than in sand (table 1). we reiterate that kd as calculated assumes equilibrium in the underlying sorption reactions; however, we can not confirm this with the data reported here and, as such, our values may be biased toward underestimation. we do note, however, the empirical observation that after a relatively short period of flushing of the mobile tc plume from the flow chamber, less than 12% remained suggesting that the sorption that does occur within the transit time of the plume may be readily and rapidly reversible when solute concentrations return to zero, for both materials under these experimental conditions. we are also aware of the possibility that some irreversible sorption (12%) may occur as it is hard to rule out this condition without sorption capacity measurements. in the interpretations that follow, we recognize that several assumptions and simplifications have been made in the model gdf systems investigated. these have been made in order to demonstrate the applicability of the gamma imaging technique to radionuclide transport in a gdf and, as such, provide the basis for future detailed experimentation. the limited retardation (r 1) interpreted using the model, very low estimated kd obtained with the different methods, and the minimal retention of tc in the sand at the end of the experiments imply closely conservative transport of tc through ottawa sand. although this is the first time that this has been confirmed directly, it is not an unexpected result. the point of zero charge (pzc) of ottawa sand is between ph 2 and 5, so at the ph of these experiments (> 5.7), the sand surface is negatively charged. since the pertechnetate anion (tco4) is also negatively charged, chemical sorption is therefore impeded by repulsive electrostatic interactions between the tc and sand. this indicates that tc(vii) may be transported freely in environments where the substrate has only negatively charged surfaces. such pure-phase interactions are a simplification of natural environments, especially where significant quantities of fe(ii) or other minerals capable of reducing tc(vii) to the less mobile and less soluble tc(iv) are present or in environments where microbially mediated reactions may take place to alter the oxidation state of technetium. nevertheless, these results highlight the utility of quantitative measurements of transport parameters for tc(vii) in opaque porous media, that may be applied to substrate related to gdf concepts (e.g., clay, host rock). we have shown that it is possible to obtain quantitative transport data using the gamma imaging technique in opaque engineered backfill material. pertechnetate transport in nrvb in our gdf-proxy experiments was closely conservative, i.e., our data pertaining to tc transport in this material showed no significant retardation and very low sorption coefficients. previous studies have suggested low sorption coefficients for tc(vii) in batch experiments using aged, crushed nrvb, and our study indicates, for the first time using quantitative imaging, that such observations may translate into a significant potential for transport of tc(vii) through a backfill candidate material in a model flowing groundwater system. despite the low spatial resolution of the gamma images, tc transported through nrvb exhibited a greater dispersivity and slower transport velocity than in sand. discontinuities in the internal structure may create significant immobile (very low flow) zones within the pore space. further work on nrvb will explore the use of a mobile-immobile (mim) transport model (e.g., tang et al.), to better elucidate the dynamics of solute transport through this material. the quantitative gamma imaging technique described in this paper represents a rapid and convenient method for obtaining transport data for tc. the main advantage of this technique is that quantitative images can be obtained in opaque media; it is possible to see the retained mass as a function of time and space, allowing for a direct visual quantification of transport parameters. furthermore, in experiments where the sorption can be controlled for, it may be possible to visualize and quantify sorption. this multitude of information is such that transport models, such as cxtfit used here to test and validate the methodology, may not be required to derive transport parameters. the spatial resolution presented in this methodology was relatively low (6 mm), largely as an artifact of the spatial constraints placed upon using a working hospital camera. however, higher spatial resolution, and thus accurate dispersivity measurements, should be possible, depending upon the quality of the instrument and proximity to the collimator. quantitative gamma imaging has several potential applications to contaminant transport in opaque media. in the context of geological disposal of nuclear waste, the transport of i from the waste and through the engineered barrier is a key concern due to its long half-life (15.7 10 years), high solubility, and poor sorption. gamma imaging coupled with the -emitting i radiotracer could be used to develop an understanding of iodine transport behavior and thus support engineered barrier material design. because the gamma camera can detect ultratrace concentrations of radionuclides, several gamma-emitting isotopes could also be used to nondestructively quantify the transport of environmental contaminants in soil, such as chromium (cr) or mercury (hg). this highlights the potential versatility of the technique, applicable to a wide range of scenarios as a novel tool to understand the spatial and temporal dynamics of the geochemistry of a variety of radiotracers in opaque media.

we present a novel methodology for determining the transport of technetium-99 m, a -emitting metastable isomer of 99tc, through quartz sand and porous media relevant to the disposal of nuclear waste in a geological disposal facility (gdf). quartz sand is utilized as a model medium, and the applicability of the methodology to determine radionuclide transport in engineered backfill cement is explored using the uk gdf candidate backfill cement, nirex reference vault backfill (nrvb), in a model system. two-dimensional distributions in 99mtc activity were collected at millimeter-resolution using decay-corrected gamma camera images. pulse-inputs of 20 mbq 99mtc were introduced into short (< 10 cm) water-saturated columns at a constant flow of 0.33 ml min1. changes in calibrated mass distribution of 99mtc at 30 s intervals, over a period of several hours, were quantified by spatial moments analysis. transport parameters were fitted to the experimental data using a one-dimensional convection dispersion equation, yielding transport properties for this radionuclide in a model gdf environment. these data demonstrate that 99tc in the pertechnetate form (tc(vii)o4) does not sorb to cement backfill during transport under model conditions, resulting in closely conservative transport behavior. this methodology represents a quantitative development of radiotracer imaging and offers the opportunity to conveniently and rapidly characterize transport of gamma-emitting isotopes in opaque media, relevant to the geological disposal of nuclear waste and potentially to a wide variety of other subsurface environments.

PMC3871888

pubmed-907

obstructive sleep apnea (osa) is characterized by frequent episodes of termination of respiratory airflow caused by upper airway collapse during sleep, followed by oxyhemoglobin desaturation, persistent inspiratory attempts against the obstructed airway and arousals from sleep. the inspiratory airflow can either decrease (hypopnea) or be completely absent (apnea), and an apnea that lasts 10 seconds or longer, associated with ongoing ventilatory effort, characterizes a patient with osa. the airflow limitation episodes are usually accompanied by decrease in hemoglobin level, which are usually terminated by quick micro-arousals as a result of excessive respiratory drive caused by the continuing hypoxemia [23]. to establish a definite diagnosis, the diagnosis of osa is confirmed when a person has an apnea/hypopnea index (ahi: number of apneas and hypopneas per hour of sleep) of more than five events per hour, associated with symptoms of excessive daytime sleepiness. although known to be a benign disease, numerous studies have shown that osa is usually correlated with increased morbidity and mortality because of cvd such as systemic and/or pulmonary hypertension as its most common consequence, heart failure, myocardial infarction and stroke [69]. oxyhemoglobin desaturation-resaturation, which is common in osa patients, results in free radical production, release of proinflammatory cytokines and prothrombotic mediators and endothelial dysfunction and leads to sympathetic nervous system activity and increased blood pressure, as well as impairment of cerebrovascular auto-regulation. continuous positive airway pressure (cpap) has been shown to have cardio protective effects, decreasing mortality and morbidity among these patients [1214]. numerous community-based studies have examined the relationship between osa and cvds [10, 1821]. based on these studies, age, gender, smoking status, bmi, ahi, total sleep time and day sleepiness were some of the clinically important predictors of cvd outcomes in osa patients. we hypothesized that the ahi, currently used to determine the severity of osa, is not by itself enough to accurately predict cardiovascular outcomes in individuals with osa. we also hypothesized that several factors such as patients demographic and clinical characteristics would have greater accuracy for predicting cvds. based on our electronic search, there is no published data about the prevalence of cvd among the iranian population with osa. our study aimed to resolve conflicting evidence on the impact of severity of osa, gender, age, bmi, smoking status and socioeconomic status on the association between osa and development of cvds. medical files of 385 patients (from 2009 to 2011) with definite osa, confirmed by overnight polysomnography were selected in noor sleep clinic, tehran, iran. participants were eligible if they only had osa (not other types of sleep apnea such as central or mixed sleep apnea) with ahi of equal or more than five events/hour. demographic data were collected from patient files and included gender, age (as reported by patients), smoking status and educational status (last academic degree reported by patients). three modes for smoking status were considered: never-smoker, ever-smoker and ex-smoker. bmi (weight in kilograms divided by the square of height in meters) and ahi (events per hour) were derived from the polysomnography reports. several questions were designed in a questionnaire to detect cvds such as presence of cvd, type of cvd and medications used. if there was any controversy in the answers, the patients were questioned about their disease status to clarify their health/disease state. the data were analyzed using single regression analysis followed by multiple regression analysis (by backward method) to identify variables that were independently associated with cvd. the majority of patients were males (71.9%). among the studied individuals, 26.5% showed at least one sign/symptom of cvd and hypertension was the most commonly reported sign (74.5%). demographic characteristics and polysomnographic data of patients in cvd positive and cvd negative groups (values are presented as meansd) of patients, 17.9% had hypertension, 2.6% had coronary artery disease, 0.5% had arrhythmia and 0.3% had stroke; 2.3% of the patients had both hypertension and coronary artery disease, 0.3% had both hypertension and history of myocardial infarction and 0.3% had both hypertension and history of stroke; 0.8% of patients had myocardial infarction and one of them had hypertension as well. all variables had a significant association with cvd, except for smoking status (p=0.77). we only selected variables with p-values less than 0.20 in simple regression analysis and entered them into the multiple logistic regression model. multiple logistic regression analysis showed that the odds ratios for one grade increase in bmi and one year increase in age were 1.13 and 1.12, respectively (p<0.001). in presence of these variables, severity of apnea, gender and level of education (as a socioeconomic index) had no significant correlation with cvd (p=0.36, p=0.83 and p=0.79, respectively). this study provided information on the association between osa and cvds. in our osa population, 26.5% showed at least one sign/symptom of cvd, with hypertension being the most common sign (74.5%). while ahi was found to predict cvd in simple regression analysis, no significant association was found in multiple model adjusted for potential confoundders. multiple regression analysis showed that aging and obesity were significant predictors of the occurrence of cvd in osa patients. lavie and lavie in a case-control study on seventy of osa showed that severity of osa affected biochemical markers associated with cvds only in severe stage and not mild or moderate stage of osa. shahar et al, in a cross sectional study also showed modest to moderate effects of sleep disordered breathing on various manifestations of cvds within a range of ahi values that were considered normal or only mildly upraised. kendzerska et al, in a decade-long cohort study showed that osa-related factors other than ahi were important predictors of composite cardiovascular outcome. our findings were in agreement with the afore-mentioned studies indicating that osa-related factors other than ahi (bmi and aging) are important predictors of cvds. the mean of bmi in cvd+ group was calculated to be approximately 32 kg/m2, which put them in obese group of bmi classification. in parallel with our study, dacal quintas et al, found that the prevalence of osa in normal weight patients was lower than that in overweight and obese patients. seetho et al, reported that patients with osa and severe obesity had increased arterial stiffness, which would possibly affect cardiovascular risk independently of metabolic abnormalities. carlson et al, showed that age, sleep apnea and obesity represented both independent and additive risk factors for development of systemic hypertension. similarly, lenfant described the correlation among obesity, hypertension and osa as a triangular relationship; osa and obesity have an interactive relation and both of them have increasing effect on incidence of hypertension. our study showed that aging was another significant factor to explain the presence of cvd among osa patients. pywaczewski et al, showed that the frequency of cardiovascular complications occurring in osa population increased with age. evidence has been inconsistent on the effect of gender on the association between osa and cardiovascular events. in our study, women with osa were more susceptible to develop cvd than men. drummond et al, and quintana-gallego et al, found that women with osa had hypertension more frequently than men. faulx et al, found that ahi was inversely associated with flow-mediated dilation and peak blood flow in women. in contrast, no relationship between ahi and flow-mediated dilation was found in men. these results raise the possibility that women with sleep-disordered breathing are more vulnerable to related cvds than men. on the other hand, mohsenin et al, showed that markedly obese men with osa may have a nearly two-fold greater risk for hypertension than women. gaines et al, showed that although women have naturally higher levels of inflammatory and metabolic markers than men, men with sleep apnea appear to have a more severe inflammatory profile compared to women. it is important to consider the role of bmi in this study and its conclusion: male sex is more susceptible for developing cvd only at the highest quartile of bmi. in a case-control study by lavie and lavie, a comparison was made between smoker and non-smoker patients with osa and they showed a significant interaction effect between smoking and severity of apnea on ceruloplasmin and high-density lipoprotein (hdl) levels. smokers with severe sleep apnea had the highest level of ceruloplasmin and the lowest level of hdl. they concluded that smoking and sleep apnea had a synergistic effect on some of the biochemical cardiovascular risk markers. patients with severe sleep apnea who smoked were at a greater risk for developing cvds than smokers with mild-moderate sleep apnea and patients who did not smoke. in our study, no significant correlation was observed between smoking and cvds (p=0.77). one probable reason may be that most ex-smokers stopped smoking before doing the sleep test according to the doctor s advice. some studies categorized ex-smokers in the non-smoker group [3536]. based on the electronic search, the current study is the first to provide comprehensive information about the role of severity of osa as a cause of cvd and its relationship with other important risk factors in an iranian population. we included patients with a wide range of osa severity and a relatively large number of females. our findings are based on patients referred to a single center, which may reduce the generalizability of our findings. our study showed that ahi was significantly associated with cvd in simple regression analysis; however, this association was not significant after controlling for other predictors. other osa-related predictors, such as bmi and age were significantly and independently correlated with increased risk of cvds, respectively. in presence of these variables, other phenotypic and socioeconomic factors like severity of apnea, thus, health care providers should implement bmi control strategies and improve the quality of care for the elderly patients.

objectives: obstructive sleep apnea (osa) can lead to various cardiovascular disorders (cvd) such as hypertension. there is no documented data about this relationship among the iranian population. the aim of this study was to obtain comprehensive information about the severity of osa as a cause of cvd and its relationship with other important risk factors. materials and methods: in this cross sectional study, we studied patients with osa and apnea/hypopnea index of 5 or more. the data were collected from the patients polysomnography report and medical files. data analysis was done with spss version 18. results:the majority of patients were males (71.9%). among the studied individuals, 26.5% showed at least one sign of cvd, and hypertension was the most common condition (74.5%). multiple regression analysis showed that the odds ratios for one unit increase in bmi and one year increase in age were 1.13 and 1.12, respectively (p<0.001). conclusions: in our study, bmi and age had the strongest relationship with cvd. thus, public health care providers should implement weight control strategies and improve diagnostic and treatment procedures for the elderly patients.

PMC5376541

pubmed-908

in functional neuroimaging, and particularly in pet and fmri, study design and analysis have been dominated by the concept of functional segregation, which emphasizes the specialization of a brain structure for a specific part of a cognitive function. this has resulted in a large number of studies in which differences in cognitive states are linked to the differential activation of separate brain areas. the concept of functional integration, on the other hand, has long been recognized as an equally important principle of brain organization. functional integration refers to the interaction between brain areas and has been studied with two categories of analyses: functional connectivity analyses and effective connectivity analyses. in functional connectivity analyses [24], the covariance structure of a measure of brain activity is studied, from which differences in cognitive states are linked to differences in correlations between regions. these analysis methods are thus limited in their capacity to make inferences about the directionality of these correlations, which makes it difficult to address, for instance, the functional hierarchy of the brain structures under investigation. in effective connectivity analyses, on the other hand, models are defined a priori, comprising the brain structures of interest and assumptions about the afferent/efferent connections between them [5, 6]. these models are then fitted to the activity of these brain areas to obtain the strength of these connections, which enables inferences on changes in connection strengths in relationship to cognitive states. effective connectivity has been defined as the influence one neural system or region exerts over another. there are two ways by which this influence can be mediated: via a direct path between two regions, or via an indirect path in which a third region is involved. the analysis methods that have been applied to functional neuroimaging data, structural equation modeling (sem) and dynamic causal modeling (dcm), differentiate between these two possibilities, provided that the underlying model (regions and their paths) is completely specified. if two regions are connected via a third region and this third region has furthermore no other influence on the rest of the network, one can choose to incorporate only an indirect path between the two regions and to refrain from explicitly modeling the third region and its direct paths. we are working here, however, on the premise that most of the specified paths should reflect veridical direct paths, because we believe that too many indirect paths, involving these third party regions, which are not explicitly taken into account in the model, will seriously decrease the biological validity of the model. sem has also extensively been applied in the social sciences, where these paths represent abstract causal connections between variables. when this method is applied to functional neuroimaging however, these paths should ultimately correspond to the white matter connectivity of the brain regions under investigation. furthermore, mcintosh and gonzalez-lima have studied the effect of erroneous model specification on the estimation of the path coefficients and have found that it could seriously impinge on the estimation of these coefficients. therefore, it would be highly valuable to incorporate all available knowledge regarding anatomical connections into effective connectivity models. it should be noted that we do not imply that each existing anatomical connection should result in an effective connection in each and every cognitive task. however, it is the role of the model estimation to indicate which anatomical connections have become effective for which task manipulation. therefore, leaving an anatomical connection, which is known to exist, out of a model specification can only be justified when one has strong beliefs about the functioning of this path in the present context, possibly combined with a requirement for a decrease in model complexity. unfortunately, our knowledge about human anatomical connectivity is relatively sparse, because a number of techniques that are used in other species (active tracers) are highly invasive. therefore, only two classical anatomical methods can be used: dissection studies, which only provide information on a relatively coarse scale, and passive tracer studies, which are not very well suited to investigate long-range connectivity. most effective connectivity studies thus can only validate their connections with information from nonhuman primates, which in turn raises problems with the homology of brain structures between different species. in the last decade, a technique known as diffusion tensor imaging (dti) [9, 10] has emerged as a good candidate to resolve this situation. in dti, the sensitivity of the (diffusion-weighted) mr signal to the self-diffusion of water on a microscopic scale is employed to characterize the anisotropic structure of white matter in vivo. it is assumed that the direction in which diffusion is largest is collinear to the direction of the axonal bundle in the voxel, because diffusion is assumed to be hindered in directions perpendicular to this direction. with this information, fiber tracking [11, 12] can be performed in which the main diffusion directions of voxels are followed throughout white matter. this method already has provided useful insights in, for instance, the anatomy of the thalamus and the striatum [13, 14]. first, dti provides no information about the afferent or efferent character of the axons, because the diffusion of water does not differentiate between these two situations. second, the tensor model can only provide one main direction per voxel, which considerably increases the likelihood of erroneous tracking results through a region of crossing fibers. finally, in cerebral gray matter there is generally no dominant fiber direction, making it difficult to track fibers to their cortical origin. the aim of the current study is to investigate to what extent dti-based tractography can provide support for the anatomical basis of the networks, proposed in effective connectivity studies. furthermore, we have investigated whether dti-based tractography is able to reveal any connections that go beyond the ones proposed in the original analysis. to address these questions, we have chosen eight effective connectivity studies all using structural equation modeling. sem was introduced at an early stage of the development of pet and fmri, with the consequence that a body of sem studies is to be found in the literature. dcm, on the other hand, has only relatively recently been introduced, but is gaining a rapid popularity. it should be noted that any conclusions we are able to draw in this study within the context of sem models can be readily generalized to dcm studies as there is no difference in the role of the underlying anatomical model in both frameworks. we have chosen networks spanning a number of different cognitive domains, including learning [15, 16], cognitive control [17, 18], working memory, visual and auditory perception, major depression, and the thalamocortical network involved in general-anaesthetic-induced unconsciousness. we performed a standard dti experiment on 6 subjects and used the coordinates of the network nodes as seed regions for a fiber tracking analysis. in this analysis, we have established whether dti-based fiber tractography provides evidence about the direct nature of every possible connection, whether or not it was proposed in the original studies. we studied 6 healthy subjects (2 females, age range 2532 years) after informed consent was given according to institutional guidelines of the local ethics committee (cmo protocol region arnhem-nijmegen, the netherlands). dti was performed using a twice refocused pulsed gradient spin echo epi sequence at 1.5 t(sonata system, siemens, erlangen, germany)with a standard head coil. axial slices were obtained using the following imaging parameters: repetition time=9900 milliseconds, echo time=88 milliseconds, flip angle=90, 128 128 matrix, 320 mm 320 mm field of view, and slice thickness=2.5 mm with no gap (2.5 2.5 2.5 mm isotropic voxels). diffusion weighting was obtained along sixty noncollinear directions using a b-value of 700 s/mm. each subject the five reference images with no diffusion weighting were averaged and normalized to the mni t2 template in spm2 (statistical parametric mapping, http://www.fil.ion.ucl.ac.uk/spm). the matrix of normalization parameters was inverted to obtain the transformation matrix from standard space to world space. we have done this to avoid the extensive resampling and reorientation of the data that is involved in the normalization of dti data [25, 26], because we hypothesize that this would lead to a degradation of the finer details in the fiber tracts.only linear terms were used in the normalization to ensure that the transformation matrices could be inverted. diffusion tensors and fractional anisotropy (fa) maps were calculated using the diffusion toolbox in spm2. the fa maps were used for displaying the anatomical location of the roi coordinates. fiber tracking was performed in the dti-studio package using the fact algorithm. tracking was terminated when the angle of two consecutive eigenvectors was larger than 85, or when a voxel was reached with an fa value smaller than 0.20. in most of the original studies [1519, 22], the rois that were used for effective connectivity analysis were all spheres of 8 mm radius. however, in two studies [20, 21] only the peak voxels of a partial least-squares analysis were used. because these voxels represented larger clusters of voxels, we have used rois with roughly the same size (spheres of 8 mm radius) as a starting point for all networks. we have chosen not to transform the whole original roi into subject space, as this might lead to extensive seeding of the white matter adjacent to the seed coordinate, and thus to many false positives. instead, we have drawn rois on the individual subject s fa maps, taking care that the borders of the roi were at the border of gray and white matters (as visible on the fa map) and that the roi would approximately be of the same size as the original roi. all possible combinations of regions were tracked, including the connections that were not proposed in the original effective connectivity studies. the results for all networks under investigation are visualized in figures 18, in which the thickness of the connecting lines indicates the number of subjects in which a particular connection was found. in the appendix, we have also listed these results in table 1. the most striking class of paths which are not supported by our findings contains frontal interhemispheric paths. this can however convincingly be explained by methodological shortcomings in regions with crossing fibers in the frontal parts of the brain. we will discuss these issues further in section 4. in half of the studies, we have also found connections indicating paths that were not included in the original studies. in the following this not only leads to the situation that this nomenclature is inconsistent between studies, but may also give the incorrect impression that similar connections are under investigation in different networks. it is therefore important to note that the regions of interest normally are spheres of approximately 8 mm in diameter, and thus two regions with the label the network of this study can be separated into a dorsal stream (v1, de, pp, and lp) and a ventral stream (v1, itp, and ita) of visual areas. the paths within these streams are supported in the majority of subjects by our dti results. however, the crucial path under investigation is the path between the two streams (pp-itp), which is hypothesized to mediate the learning effect under investigation. interestingly, we have found no evidence for this path, but we have found support for a path (de-ita), which was not included in the proposed network and which could be a potential candidate to mediate this effect. whereas we do not wish to suggest that the original path (pp-itp) should be dismissed, it would be interesting to investigate whether a part of the learning related effects, reported in the original study, is mediated by the new path we have reported. in this network, a series of regions (occ, par, and pfc) is proposed in both hemispheres with symmetrical paths within the hemispheres and extensive interhemispheric connections. the paths between occipital and parietal cortices are supported by our findings as are the interhemispheric connections between these regions. furthermore, we have found evidence for paths that were not included in the original model, namely, interhemispheric connections between occipital and parietal cortices. this is quite remarkable, because interhemispheric connections between nonhomologous regions are rarely found in this kind of analyses. also of interest are the cases in which the proposed paths are not supported by our results. the interhemispheric connections we did not find (lpfc-rpfc, lpfc-rpar, and rpfc-lpar) fall into the aforementioned problematic class of frontal interhemispheric connections. the most interesting negative result is the lack of connections between right parietal and right prefrontal cortices, in contrast to the presence of these connections in the left hemisphere. in this case, methodological shortcomings are not likely to affect the results, as this would indicate that these shortcomings would exist for the right hemisphere but not for the left hemisphere. the same argument makes the consideration of connections via an extra region, which has not been included in the network, also unattractive. therefore, we tentatively interpret these findings as a support for an asymmetry in connectivity between these areas in parietal and prefrontal cortices. this network contains a set of motor and prefrontal regions in both hemispheres which are symmetrically connected within the hemispheres. this last set of paths falls again in the class of frontal interhemispheric connections and thus it is not surprising that we have found no support for these paths. have proposed an alternative network in their study, which contained also paths directly from premotor cortex to rostral prefrontal cortex. this extra network did not result in significant changes in the original paths, which is in agreement with the lack of support for these paths in our results. kondo et al. have proposed four network nodes (pfc, acc, spl, and ifc). however, two of these nodes (spl and ifc) can be further split up in four anatomically distinct regions (spl1, spl2, ifc1, and ifc2). we have thus decided to treat each of these regions as a separate node and have studied the connections between these nodes. our results show that the regions which constitute each original node (e.g., ifc1 and ifc2) are connected with each other, but they show differential connectivity patterns with the rest of the network, which suggest that they also have a different role within this network. the lack of support for paths from and to the acc was surprising, especially given the fact that the acc is known to connect extensively with the prefrontal cortex in the macaque. in this network, a set of regions is proposed ranging from occipital, temporal, premotor, and prefrontal regions. given our results in the other studies, it was not surprising that there was hardly any support for the (nonhomologous) interhemispheric connections originating in frontal and prefrontal cortices. support was found for paths between occipital cortex and frontal and superior temporal cortices. however, there are also two intrahemispheric paths for which no support was given (a6-a18r, a10-a6). furthermore, there was no evidence found for paths which were not included in the original analysis. therefore, within the context of this network, dti did not deliver any extra information. rowe et al. have proposed a network with bilateral parietal, prefrontal, and prestriate areas and a motor area in the left hemisphere only. we have found only scarce support for the paths to and from prestriate area, even after lowering the fa threshold. in the left hemisphere, we have found consistent evidence for all other proposed paths. however, as in the network of fletcher et al., we have found support for parietal-frontal connections in the left but not in the right hemisphere. based on the same arguments as in the network of rowe et al., we again interpret this as an evidence for an asymmetry in these connections. this is even more remarkable, because the locations of parietal and especially prefrontal areas in both studies are quite widely separated. the network of seminowicz et al. consists predominantly of prefrontal subcortical areas in the right hemisphere, with the lateral prefrontal cortex as the only region in the left hemisphere. this might directly explain why we have found only scarce evidence for paths from this region to the rest of the network, because of the aforementioned methodological problems with frontal interhemispheric connections. in contrast to the study of kondo et al., we have found connections supporting the proposed paths from the anterior cingulate cortex. furthermore, we have found many connections from the thalamus to the rest of the network which do not conform to proposed paths. this is however not surprising, as it is well known that the different nuclei in the thalamus extensively connect to different parts of the cortex. connections are thus most probably the result of the fact that the seed region in this case was too large to specifically select the nucleus of the thalamus involved in this network. further evidence for paths that were not proposed in the original network was found for connections from medial prefrontal cortex to the hippocampus and the subgenual cingulated cortex. the hippocampus is a region of low fa and thus in an initial analysis showed only very limited connectivity with the rest of the network. we have therefore repeated the analysis with a lowered fa threshold (0.15) which yielded support for the proposed paths from the hippocampus. white et al. have proposed a model with the left motor cortex and supplementary motor area, the thalamus, and two areas from the right cerebellum. we have found support for the intrahemispheric paths but not for the interhemispheric ones. this last finding contradicts findings from the macaque literature in which there are connections found between the primary motor cortex and the contralateral cerebellum deep nucleus. moreover, there is evidence that the pons, which is a region through which these fibers have to pass, contains crossing fibers. we, therefore, conclude that it is valid to include these connections in an effective connectivity analysis. in this study, we have for the first time used dti-based fiber tractography to investigate the anatomical basis of effective connectivity models. first, we wanted to establish that dti-based tractography is able to resolve the connectivity between rois of the size typically used in effective connectivity studies. we hypothesized that the majority of the proposed paths were indeed valid, and compared the results of our dti-based analysis with these paths. however, we believe that the greatest potential advantage of using dti-based tractography in the context of effective connectivity models lies in establishing paths that are not suggested by the available knowledge (e.g., macaque tracer literature). our second aim was therefore to investigate the evidence for connections which were not proposed in the original studies. we have found evidence for such connections in half of the models. in the following, we will discuss these findings in the context of current methodological limitations and we will evaluate their implications for the proposed models. when there was no evidence found for a proposed path, there are two explanations possible: there is no direct anatomical connection; in this case, the proposed path can only be supported by an indirect connection with a third region;there is a direct connection, but it has not been found due to methodological limitations (false negative). the second explanation is especially relevant to a class of frontal interhemispheric connections. as mentioned in the introduction, dti-based tractography has profound difficulties when tracking through voxels with multiple fiber populations. the frontal interhemispheric connections pass through the corona radiata, which is well known to contain such voxels. therefore, any negative findings about this class of connections have to be interpreted as being inconclusive. there is no direct anatomical connection; in this case, the proposed path can only be supported by an indirect connection with a third region; there is a direct connection, but it has not been found due to methodological limitations (false negative). there is another set of negative findings which merits further discussion: in both networks of fletcher et al. and of rowe et al., we have found evidence for a connection between parietal and prefrontal cortices in the left hemisphere but not in the right hemisphere here, an explanation in terms of methodological limitations seems unattractive, because this would suggest that these limitations occur in the right hemisphere but not in the left hemisphere. therefore, we tentatively conclude that these connections are indeed absent in the right hemisphere and that this is an evidence for an asymmetry in parietal-frontal connectivity. we should point out, however, that this asymmetry seems counterintuitive at first, since the right hemisphere is hypothesized to be dominant for visuospatial processing and one would expect that these connections would be especially strong for this hemisphere. however, as we are dealing with relatively small rois within the frontal and parietal lobes, this does not rule out the possibility of there being any connections between these lobes in the right hemisphere. there are a number of paths for which no evidence was found and which can not be interpreted directly in terms of methodological limitations. as in many neuroimaging studies, these negative findings are difficult to classify and it would certainly be imprudent to conclude that these paths do not exist. moreover, it is also possible that a path is mediated by connections from and to a third region, which was not included in the network. this situation can be relatively harmless if the function of the missing region is known to be restricted to message passing. however, we hypothesize that, if there is a substantial number of such indirect connections present in a network, the inclusion of these other regions, which mediate these connections, becomes necessary to ensure the biological validity of the network. given the methodological issues discussed above, it is clear that the potential contribution of dti to connectivity studies lies not in disproving the existence of postulated connections, but in the unique potential for detecting hitherto unconsidered direct anatomical connections. this is because dti may be prone to type ii errors, but it is far less likely to consistently produce type i errors when connections are averaged across subjects. it is hence highly significant that we have also found evidence for a number of paths which have not been taken into account in the original studies. incorporating these paths in a new analysis of these models can potentially have a significant impact on the interpretation of these models, since they point to improvements in the anatomical validity of the models, which in turn leads to more veridical path coefficients. at the current state of technology in effective connectivity, one has considerable freedom to choose the connections in a model and to evaluate different models with different connectivity profiles against each other. this model selection procedure can be augmented significantly with dti-based tractography, because models in which the connections to a large extent overlap with the connections found in a dti analysis should in turn be more likely. one could potentially formalize this in a bayesian framework by designing priors on the connections and by subsequently making the priors on the known connections high and sharp and the priors on the unknown connections relatively noninformative. if this would be a veridical variability, it would be a surprising and new finding, since the intersubject variability of anatomical connections is generally considered to be low and is furthermore difficult to assess with either dti or tracer methods. in dti, the normalization of findings still makes it difficult to compare findings across subjects, whereas tracer studies are normally performed in very few animals because of ethical considerations, which makes any discussion about differences between animals extremely difficult. although we have not normalized the fiber tracks to a template, we do believe that normalization problems still play an important role in our studies because they might cause the erroneous placement of seed regions in some subjects, which in turn would lead to misleading tractography results and the above-mentioned intersubject variability. the smoothing strategy, employed in functional studies to reduce the effect of anatomical differences between subjects, can not be applied in our framework because the directional information, used in the tracking procedure, would be smeared out over other voxels and potentially other tracks, with unpredictable implications for the veridicality of the tracks found. currently, there is no convincing way of solving this problem, as the basic anatomical landmarks vary substantially over subjects. recently, advances have been made towards the estimation of multiple fiber directions within one voxel and also in probabilistic tractography. we will now discuss each of these developments and their potential use for our framework. the estimation of multiple fiber compartments per voxel, in general, brings this technique closer to producing veridical anatomical connections, and a number of techniques have been proposed to achieve this [33, 3639]. there is, however, one problem which can not be solved by this technique alone, and that is the kissing/crossing fiber problem: when a fiber has to track through a voxel with multiple compartments, it is uncertain which compartment has to be used to determine the direction in which the track is to be continued. in a number of studies, the direction that is most collinear with the incoming fiber was chosen, but this does not necessarily have to be the true direction. whereas the single tensor model is probably too conservative in the connections it yields, multiple compartment models might thus yield a number of false positives. in probabilistic fiber tracking, a measure of uncertainty of the local fiber direction is estimated per voxel [4044]. fiber tracking is now done in a monte carlo type experiment: the tracking is performed multiple times, each time with a different orientation drawn from the local fiber direction distributions. subsequently, the number of times a target voxel was hit by this procedure is calculated, which then is converted to an informal measure of probability of connection. while this procedure by itself seems valid, in practice this leads to widespread patterns of connectivity and it is uncertain at which level of probability the map should be thresholded. moreover, the probability of connection tends to decrease with increasing distance. in a sem network, both relatively local and long connections can be included, which makes the comparison of these connections difficult. thus while both techniques (multiple direction estimation and probabilistic fiber tracking) can potentially alleviate some of the problems, we have encountered (e.g., interhemispheric connections), unresolved issues remain. in conclusion, we have shown that dti-based tractography can be used to explore the anatomical connections between regions, used in effective connectivity studies, notwithstanding the current limitations of this method. we have observed evidence for the proposed paths in a large number of cases and, more importantly, we have shown in several cases direct connections that were not included in the original models. we therefore conclude that dti-based tractography is a valuable tool for exploring the anatomical basis of functional networks.

diffusion tensor imaging (dti) is considered to be a promising tool for revealing the anatomical basis of functional networks. in this study, we investigate the potential of dti to provide the anatomical basis of paths that are used in studies of effective connectivity, using structural equation modeling. we have taken regions of interest from eight previously published studies, and examined the connectivity as defined by dti-based fiber tractography between these regions. the resulting fiber tracts were then compared with the paths proposed in the original studies. for a substantial number of connections, we found fiber tracts that corresponded to the proposed paths. more importantly, we have also identified a number of cases in which tractography suggested direct connections which were not included in the original analyses. we therefore conclude that dti-based fiber tractography can be a valuable tool to study the anatomical basis of functional networks.

PMC2377327

pubmed-909

a 48-year-old female patient 158 cm in weight weighing 53 kg was admitted to receive functional endoscopic sinus surgery and septoplasty for a diagnosis of chronic sinusitis and nasal septal deviation. the patient was a non-smoker and her medical history stated that she had received an appendectomy one year prior to her visit and had a benign tumor removed from her bladder six months before her visit. there were no abnormalities other than light anemia with an hb of 9.7 g/dl and hct at 30.7% from the blood test. glycopyrrolate 0.2 mg was i m injected as premedication, and preoxygenation was sufficiently achieved with 100% oxygen before induction of anesthesia. propofol 100 mg and rocuronium 50 mg was slowly iv injected and after fully confirming loss of consciousness and muscle relaxation, oral endotracheal intubation was attempted with a curved laryngoscope (macintosh size3 blade). the patient was class 1 based on the cormack and lehane laryngeal endoscopic classification, and endotracheal intubation was performed smoothly in the first attempt with a preformed ring, adair, elwyn tube (rae, mallinckrodt) 7.0 mm in diameter. proper placement of the tube was confirmed by chest auscultation and it was fixed at a tube length of 20 cm. a tidal volume of 450 ml and respiration rate of 12 times/min were maintained with mechanical ventilation (aestiva/5, datex-ohmeda, madison, usa); airway pressure was 14-16 cmh2o and positive end expiratory pressure was 1 cmh2o. anesthesia was maintained with o2 2 l/min, n2o 2 l/min, and sevoflurane 2.0-2.5 vol%. during surgery, the blood pressure was 105-150/60-95 mmhg, heart rate 75-100 beats/min, and oxygen saturation 99-100%, and end-tidal carbon dioxide tension was maintained at 33-37 mmhg. the duration of the surgery was 2 hours and 30 minutes, and there were no abnormalities during surgery. after surgery, pyridostigmine 15.0 mg and glycopyrrolate 0.4 mg were iv injected to reverse the muscle relaxation. when spontaneous respiration was sufficiently recovered and extubation of the endotracheal tube was about to be performed, suddenly, the patient severely shook her head and started to struggle so the bandage fixing the tube was immediately removed; air was removed from the balloon, and extubation was performed. during extubation, the patient was severely bucking while raising her neck and upper body so surrounding staff were called for help in safely holding down the patient. after extubation, the patient was soon stabilized after a few coughs and was moved to the recovery room. a blood pressure of 120-140/70-80 mmhg, heart rate of 65-76 beats/min, and oxygen saturation of 99-100% were maintained in the recovery room, and other than weak pain in the operated area, there were no specific complaints of symptoms so she was moved to general ward without abnormalities. three hours after surgery, the patient started to feel nauseous and five hours after surgery, she vomited severely several times thus metoclopramide 10 mg was iv injected. seven hours after surgery, the patient's neck and face swelled up with complaints of pain, and physical examination showed crepitation. vital signs at the time was blood pressure 100/60 mmhg, heart rate 80 beats/min, respiration rate 20 times/min, temperature 36.3, and oxygen saturation 98%. arterial blood gas analysis was immediately performed as well as chest and neck x-ray, and neck ct. the patient was put in semi-fowler's position to provide oxygen 2 l/min through a nasal cannula, and was put on a fast. results of arterial blood gas analysis showed ph 7.42, paco2 31.7 mmhg, pao2 80.2 mmhg, hco3 20.7 meq/l, and sao2 96.8%. in the chest x-ray pneumothorax, subcutaneous emphysema in the chest wall, pneumomediastinum, and pneumoperitoneum were observed (fig. the neck ct showed extensive subcutaneous emphysema in the neck and face area, and emphysema in the retropharyngeal space (fig. the patient was immediately transferred to icu and oxygen 5 l/min was provided through a mask in fowler's position. no abnormalities were discovered in the fiberoptic laryngoscope examination performed by the ent department when investigating the cause. 4 ml of air were extracted from the retropharyngeal space with an 18 g needle using the intraoral approach, and the thoracic surgery performed insertion of chest tube on the right chest and incision of the subcutaneous emphysema on the chest wall. the patient received esophago gastro duodenoscopy (egd) and bronchoscopy for differential diagnosis, and there were no abnormalities found in the egd. in the bronchoscopy, there were no abnormalities in the laryngopharynx, vocal cords, and carina, but mucosal damage and bloody secretion were observed in the right second carina (fig. the patient was positive for mycobacterium tuberculosis polymerase chain reaction, and was diagnosed with tuberculosis. the patient received conservative treatment and anti-tubercular treatment, and was discharged from the hospital 2 weeks later with improved symptoms (fig. clinical symptoms of pneumomediastinum are usually chest pain with accompanying radiating pain from the neck or back, breathing difficulties, tachypnea, and throat discomfort. physical findings include subcutaneous emphysema, the loss of cardiac dullness to percussion, and mediastinal crepitation (hamman's sign). diagnosis can be achieved by the above mentioned clinical symptoms and radiological observations. in a simple chest x-ray, usually a line due to an air shadow can appear along the left heart disinsertion and between the heart and diaphragm (continuous diaphragm sign), while in the lateral view of chest x-ray, there can be subcutaneous emphysema of the chest and neck, air shadows that appear along the back of the sternum or aortic arches, and air shadow around the outer pericardium of the right pulmonary artery (ring around the artery sign). fifty percent of pneumomediastinum can not be found just with chest pa x-rays; therefore, a lateral chest x-ray must be taken together. chest ct can clarify the diagnosis or help confirm lesions that did not appear in simple chest x-rays. explain the occurrence of pneumomediastinum by the elements inside the thorax and by the elements outside the thorax, depending on the place where the air originated. elements inside the thorax are rupturing the pulmonary alveoli from asthma, vomiting, giving birth, lifting heavy objects, and applying force with the glottis closed, as well as secondary rupture of pulmonary alveoli or damage to the trachea and bronchus from external physical impact. elements outside the thorax are cases where anatomical boundaries surrounding the mediastinum is pierced and air is injected, such as in surgery or injury to the head and neck area, tooth extraction, infection or penetration in the abdominal or peritoneal cavity. from these various causes, those that are associated with surgery and anesthesia can be complications from the surgery itself, pressure damage from positive ventilation during anesthesia, airway damage from endotracheal intubation, and airway damage from movement of an endotracheal tube. one case of pneumomediastinum that occurred after functional endoscopic sinus surgery was reported by sohail et al., where a few hours after receiving functional endoscopic sinus surgery under general anesthesia, pneumomediastinum occurred together with subcutaneous emphysema in the face, neck, and chest wall, and the patient recovered after conservative treatment. the authors inferred that the surgery itself could not be seen as the direct cause of pneumomediastinum, and that pulmonary alveoli rupture or airway damage from endotracheal intubation were possible causes. also in our case, the fiberoptic laryngoscope examination performed by the ent department showed no abnormalities from the operated area to the glottis, so there is low possibility that the surgery itself was the direct cause for pneumomediastinum. the possibility of pressure damage from positive ventilation during anesthesia is also low since various indicators such as ecg, oxygen saturation, airway pressure, positive end expiratory pressure, and end-tidal carbon dioxide tension were all in the normal range. the possibility of mucosal damage due to endotracheal intubation while inducing anesthesia is also low since there were no difficulties in the intubation process and the 7.0 mm diameter rae tube forms a curve at a 20 cm length so it is difficult to have deeper intubation., when mean height of korean females is 157.8 3.7 cm; the mean length from the front teeth to carina was 25.3 0.9 cm, and the height of the patient in our case was 158 cm so the possibility of deep intubation is low. additionally, if the tube had been inserted into right second carina, there should have been a clear reduction in breath sound in the non-ventilated lung, increased airway pressure, and a decrease in oxygen saturation, but these situations did not occur up to the end of surgery. therefore, the authors believe that there is low possibility of mucosal damage due to endotracheal intubation. in addition, since a stilet was not used, damage from a stilet can also be ruled out. next, there is the possibility of mucosal damage due to the movement of the endotracheal tube. in a study regarding the movement of the endotracheal tube, conrardy et al. reported that the tube moves 1.5 cm towards the carina when bending the neck, and moves 2.4 cm towards the vocal cords when the neck is extended. in our case, there were no excessive position changes to the neck area during surgery, but in the process of regaining consciousness and spontaneous respiration, there were several bouts of severe struggling and coughing before extubation. the tube could have moved during the neck bending and the position could have changed during this incident. especially immediately before extubation, the air was removed from the balloon of the tube so the resistance between the tube and airway mucous membrane was largely reduced, and as the bandage was removed and the rae tube was about to be extracted, the patient suddenly lifted her upper body and coughed while bending her neck. hence, it is conjectured that the tube was inserted much deeper than in the experiment where the neck was simply bent, and the end jabbed the mucous membrane of the airway. macklin has shown the course of air which causes pneumomediastinum in an animal experiment, and also in our case, the air inflow through the damaged mucous membrane of the airway slowly expanded along the bronchovascular sheath to develop pneumomediastinum, and then expanded along the aorta and membrane surrounding the esophagus to develop pneumoperitoneum. in addition, it is considered that it detached the carotid perivascular sheath to develop subcutaneous emphysema in the head and neck, and ruptured into the pleural space causing the pneumothorax. according to the report by hood and sloan, in patients who suffered injury to the trachea or bronchial tubes, the time taken from damage to diagnosis was diverse, and a delay of 24 hours or more appeared in 66%. in our case, it took about 7 hours from the symptoms to appear from bronchial damage to be diagnosed, and such delay in time is because when there is partial damage to the tracheal wall, the air enters by detaching the adventitia and expanding to form an air sack. this expansion of air can take many hours, and there could be no symptoms until it ruptures into the mediastinum or pleura space. mccoy et al. reported that causes for endobronchial intubation were head and neck surgery and laparoscopy which can affect the location of the tube during surgery, incapacity of the anesthesiologist, the movements of the surgeons, and usage of the rae tube. the rae tube has a relatively large elasticity and structurally forms a sharp angle; thus, it is easy to damage the bronchial tubes when deep intubation occurs, hence there is need for more caution. it is conjectured that also in our case, these structural characteristics of the rae tube caused more damage to the bronchial tubes than the generally used single lumen endotracheal tube. in our case, the patient severely vomited several times 5 hours after surgery and the symptoms occurred 2 hours later, and for pneumomediastinum caused by vomiting, there are 2 reasons for esophagus rupture and pulmonary alveoli rupture. differential diagnosis is performed with fiberoptic esophagoscopy or esophagography, and in our case, an egd was performed which confirmed that there were no abnormalities in the esophagus. hence, it is considered that the pulmonary alveoli ruptured from the valsalva maneuver during vomiting, and the inflow of air expanded through the bronchovascular sheath to cause the pneumomediastinum. treatment for pneumomediastinum caused by tracheal damage is conservative treatment consisting of bed rest, a supply of oxygen, and use of antibiotics when the symptoms are not severe and the vital signs are satisfactory. when there is severe damage, the vital signs are unstable, and there is a possibility of dyspnea or septicemia, it is better to perform surgical suturing in the early stages. in addition, to reduce pressure, extraction of air through subcutaneous dilacerations and insertion of a subcutaneous catheter are performed; chest tube insertion is performed for a pneumothorax larger than 15-20% of the thoracic volume, and endotracheal intubation or tracheostomy can be performed when there is severe circulatory disturbance or respiration difficulty. the patient in our case received conservative treatment consisting of bed rest, oxygen therapy, use of antibiotics, and administration of analgesics together with prompt investigation of the cause after presenting symptoms. the vital signs were not bad so the patient could be observed and monitored, but under the decision that the accompanying pneumothorax and subcutaneous emphysema were severe, chest tube insertion, dilacerations of the subcutaneous emphysema, and aspiration in the retropharyngeal space were performed. although invasive, it is considered that these prompt decompression procedures prevent cardiac tamponade and an increase in pulmonary vascular pressure caused by air in the vascular sheath and hence, contribute to the faster recovery of the patient. as a method to prevent excessive excitement of the patient while waking up, continuous iv injection of a low concentration of remifentanil has the effect of hemodynamic stability, cough reflex suppression, and pain relief, which can lead to smooth awakening and reduce serious complications as in our case. the patient subsequently received conservative treatment and antitubercular treatment, and recovered enough to be discharged 2 weeks later. before discharge, we wanted to check what happened to the lesions in the bronchial mucous membrane, but the patient refused so it is a pity that we were unable to follow-up. from the various cases and literature reviewed above, it is believed that in our case, the struggling and coughing of the patient during regaining consciousness pushed the endotracheal tube into the bronchial tubes, and the end of the tube damaged the bronchial mucous membrane to gradually develop emphysema. the valsalva maneuver during severe vomiting additionally increased the airway pressure, leaking a large amount of air through the damaged area in a short period of time, which accelerated the emphysema. pneumomediastinum can be completely treated with conservative treatment, but according to the circumstances, it can proceed to mediastinitis or septicemia and lead to the death for the patient, so diagnosis, treatment, and methods of prevention must be well informed. in addition, the possibility of pneumomediastinum from anesthesia should also be considered and caution should be taken for positive ventilation and endotracheal intubation, and there should be close observation of the patient's state before and after surgery. especially, there is need for meticulous care after regaining consciousness since the patient's struggling or coughing during extubation can push the endotracheal tube inside to damage the trachea or bronchial tubes, or cause unintended extubation and damage the glottis causing difficulties in maintaining respiration.

the occurrences of pneumothorax and pneumomediastinum are rare, but considered to be potentially life-threatening conditions in patients undergoing functional endoscopic sinus surgery under general anesthesia. tracheobronchial rupture may results in serious complications, such as pneumothorax and pneumomediastinum. it may occur accidentally by endotracheal tube when the patient's neck is flexed or extended. we report the case of a 48-year-old female patient who developed massive subcutaneous emphysema, pneumothorax, pneumomediastinum and pneumoperitoneum seven hours after functional endoscopic sinus surgery under general anesthesia.

PMC3640172

pubmed-910

recent evidence suggests that failure to recognize insular cortex seizures could be responsible for some cases of surgical failure in patients with temporal lobe epilepsy (tle) [15]. insular seizures may mimic tle or may coexist with temporal seizures, an entity referred to as temporal plus epilepsy [68]. clinical observation of patients with insular seizures proven by depth recordings and after cortical stimulation using insular contacts has revealed a high prevalence of somatosensory and pharyngolaryngeal auras (ssa and pla, resp.), including a characteristic sensation of laryngeal constriction (lc) [13, 915]. in this study, we sought to determine the prevalence and prognostic significance of ssa and pla in tle patients undergoing epilepsy surgery. we performed a retrospective chart review of all patients who underwent surgery for refractory tle at our institution between january 1980 and july 2007. all patients underwent a comprehensive epilepsy surgical workup, including complete anamnesis and neurological examination, neuropsychological evaluation, and video-electroencephalographic (veeg) monitoring with scalp electrodes. magnetic resonance imaging (mri) single photon emission computed tomography (spect) and f fluorodeoxyglucose positron emission tomography (pet) was performed in more recent cases. invasive veeg recordings were obtained in the majority of patients before the availability of mri. since then, electrode implantation has been performed only in well-selected cases following evaluation by our epilepsy multidisciplinary team. collected data included patient demographics, cause of epilepsy and risk factors, seizure type and clinical features, presence and characteristics of ssa and/or pla, number of antiepileptic drugs tried, type of resective surgery, histopathological findings, and final outcome in seizure control. ssas were defined as a perceptual experience of tingling, numbness, electric-shock sensation or pain, occurring in isolation as a simple partial seizure or as an early manifestation (i.e., aura) of a complex partial seizure [1618]. plas were defined as ictal pharyngolaryngeal symptoms of paresthesia (tingling or burning), discomfort, or sensation of throat constriction of varying intensity [13, 1922]. other early ictal symptoms suggestive of operculoinsular involvement were also documented, including motor, epigastric viscerosensory, cephalic, auditory, and dysphasic symptoms. we compared subgroups of patients with ssa/pla to those without ssa/pla. categorical (binomial) variables were compared using boschloo exact unconditional test and continuous variables using the wilcoxon rank sum test. statistical analysis was performed using r [24, 25] and pasw statistics 18 (spss inc., chicago, il, usa). during the study period, a total of 158 patients underwent surgery for pharmacoresistant tle in our institution. this patient population consisted of 74 women (47%) and 84 men (53%) with an average age of 33.9 years (range 1362) and an average duration of epilepsy of 21 years (range 146). risk factors for epilepsy included cns infection in 20 (12.7%), head trauma in 12 (5%), positive family history for epilepsy in 33 (21%), febrile seizures in 34 (22%), perinatal complications in 10 (6%), and developmental delay in 10 (6%). mri was obtained in 114 (72%) of these patients, revealing abnormalities in most cases (87%), hippocampal atrophy (ha) without sclerosis in 22 (19%), hippocampal sclerosis (hs) in 54 (47%) and other temporal lobe abnormalities in 51 patients (32%). sixty-five (67%) ictal spect scans localized the seizure focus to one temporal lobe. fdg pet was only obtained in 15 patients, and it localized the seizure focus to the temporal lobe in 13 of these patients (87%). twenty-seven underwent selective amygdalo-hippocampectomy (sah) (17%), 75% underwent anterior medial temporal lobectomy (amtl) (n=118), 4% amtl and lesionectomy (n=6), and 4% lesionectomy (n=7). amtl involved resection of 3.57 cm t2, t3, most (two-thirds) of the amygdala, and a radical hippocampectomy. pathology confirmed hs in 69 (44%), hs and another pathology in 9 (5.7%), ganglioglioma in 6 (3.8%), glioma in 5 (3.2%), cavernoma in 4 (3%), focal cortical dysplasia in 4 (3%), gliosis in 3 (2%), dysembryoplastic neuroepithelial tumor in 2 (1%), was normal in 24 (15%), and was inconclusive in 16 (10%). only 11 (7%) experienced symptoms of ssa and/or pla as part of their seizures: ssa (n=8, 5%), pla (n=2, 1.2%), or both (n=1, 0.6%) (table 1). this group of patients was composed of 3 men and 8 women with mean epilepsy duration of 27 years (range 246) and mean age at surgery of 40 years (range 2155). risk factors for epilepsy included central nervous system (cns) infections in 4 patients, perinatal anoxia in 1, febrile seizures in 1, and family history of epilepsy in 1 (table 2). mri was obtained in 10 patients, revealing hs in 6, ha in 2, a posterior temporal cavernous malformation in 1, and a fusiform gyrus pilocytic astrocytoma in 1. scalp eeg recordings localized the seizure focus to the temporal lobe in 7 patients (64%), lateralized the focus to the hemisphere ipsilateral to the resection side in 3 (27%), and were non-localizing in 1 (9%) patient. seven patients underwent preoperative ictal spect, which localized the seizure focus to the temporal lobe in 2 and was nonlocalizing in 5 cases. intracranial electrodes were implanted in 6 patients, enabling localization of seizure focus to the mesial temporal lobe in all of them. surgical procedures included amtl in 7 patients, sah in 2, lesionectomy and temporal corticectomy in 1, and simple lesionectomy in 1. three patients underwent a second operation during the study period (before outcome assessment). one patient with previous cavernoma lesionectomy and corticectomy underwent second operation with electrocorticography (ecog) guided further perilesional corticectomy. two patients with initial sah underwent a second resective surgery consisting of radical temporal lobectomy, including the superior temporal gyrus. pathology demonstrated hs either alone (n=7) or with another pathology (n=1) in 8 patients, a cavernous malformation in 1, a pilocytic astrocytoma in 1, and was inconclusive in 1. compared to patients without ssa or pla, those with ssa and/or pla were older at surgery (p=0.049), had a higher prevalence of early ictal motor symptoms (p=0.022), had a higher rate of cns infections (p=0.022), and were less likely to have a localizing spect study (p=0.025) (table 3). eight patients had only ssa, 2 had only pla, and 1 had both ssa and pla. of the 9 ssa patients, 7 (77.8%) had strictly unilateral symptoms involving the contralateral hemiface in 2, the contralateral hand in 2, the contralateral hemibody in 1, the ipsilateral leg in 1, and the ipsilateral hemibody in 1. in 1 patient with contralateral hemiface ssa, symptoms progressed in a somatotopic jacksonian march to the ipsilateral hemitongue, arm, and leg. in 2 patients (22.2%), ssas were strictly bilateral, involving the legs in 1 and the forearms in the other. in the 3 patients with pla, symptoms were described as a sensation of pharyngo-laryngeal constriction (n=3), with occasional pharyngeal pain in 1 patient. outcomes were assessed in all but 1 patient who was lost to followup in the no ssa/pla group (n=157). a favorable outcome (engel i or ii) was achieved in 81.8% (9/11) of patients with ssa and/or pla and 90.4% (132/146) of those without ssa or pla (p>0.05) (table 4). a complete seizure-free outcome (engel ia) was observed in 36.4% (4/11) of patients with ssa and/or pla and 32.2% (47/146) of those without ssa or pva (p>0.05). since the study period, two patients of the ssa/pla cohort have undergone invasive reinvestigation for persistent disabling seizures (table 2). both have obtained seizure-freedom following a third resection of the temporal lobe and anterior insular cortex, respectively. insular epilepsy should be suspected whenever tle-like seizures are accompanied by early-occurring lc and/or somatosensory symptoms (sss), especially those described as unpleasant paresthesias or warmth affecting the perioral region or extending to a large somatic territory, either bilateral or ipsilateral to the seizure focus [13, 5]. although some studies have suggested that patients with pharmacoresistant tle who exhibit ssa and pla have a favorable prognosis following temporal lobe surgery [8, 21, 26, 27], others have reported a poor outcome in this patient population [2830]. the purpose of this study was to determine the prevalence and prognostic significance of ssa/pla as a potential marker of insular epilepsy and thus a predictor of poor response to surgery in tle patients. the 5% rate of ssa among surgical tle patients in the present series is comparable to the 1.7%14% rates reported in the literature [26, 29, 3135]. unlike prospective studies that have reported an 11% incidence of ssa in tle surgery patients, retrospective series may actually underestimate the real prevalence of ssa, mainly as a result of recall bias. although viscerosensory auras are frequent (45%) in patients with pharmacoresistant tle, the prevalence of pla is less well known. pharyngeal auras have been reported to occur in up to 16.9% of pharmacoresistant tle and 13% of temporal plus epilepsy cases. the higher prevalence in the series of barba et al. compared to ours may either reflect a difference in the definition of pla or a selection bias since all patients in their study had undergone intracranial electrode implantation. in this study, ssa and/or pla were not found to be negative prognostic factors in tle patients undergoing surgery. our findings are in line with previous reports suggesting that ssa [8, 26, 27] and pla [21, 22] do not necessarily indicate an extratemporal seizure onset or an independent extratemporal seizure focus in patients with pharmacoresistant temporal lobe-like epilepsy. our findings would suggest that for the majority of our patients, ssa or pla was the result of rapid spread of epileptic activity to perisylvian somatosensory structures such as the insular cortex and second somatosensory cortex (sii) [1, 2, 5, 6, 22, 26, 3537] rather than from an extratemporal seizure focus. because almost all of the patients in the ssa/pla cohort had lesional tle, it is not possible to draw any conclusion about the prognosis of ssa/pla in nonlesional tle surgery patients. for the latter, it would remain prudent to perform an intracranial study to rule out an extratemporal focus. for the former, however, temporal lobe surgery without preoperative invasive investigation would appear to result in a good outcome for most. although independent insular seizures have been known to coexist with temporal lobe seizures and ha/hs (i.e., the hippocampal mri abnormality may represent only the tip of the iceberg of a larger pathological substrate), it may be that this is a rare occurrence which does not necessarily warrant systematic invasive investigation in the presence of ssa/pla. in our series sampled the insula in 50 consecutive patients with tle on the basis of ictal symptoms or scalp veeg data suggesting an early spread of seizures either to the suprasylvian opercular cortex (e.g., lip and face paresthesiae, tonic-clonic movements of the face, dysarthria, motor aphasia, gustatory illusions, hypersalivation, and postictal facial paresis) or the infrasylvian opercular cortex (e.g., auditory hallucinations, early sensory aphasia). only five patients (10%) had seizures originating from the insular cortex while in 43 patients (86%), they propagated to the insula after a temporal onset. hopefully, further clinical observations and imaging techniques (e.g., magnetoencephalography) will allow us to better identify the small subset of patients who will most benefit from an implantation prior to surgery [38, 39]. the retrospective nature of this study may be associated with a recall bias for the incidence and characteristics of ssa/pla. these auras are very subjective and are very much dependent on the history taking skills and detailed attention of the examiner. furthermore, because they were not assessed in a standardized way, the timing of these auras during a seizure may not always be clear with regards to other subjective symptoms patients may have felt at seizure onset. whether ssa/pla occurs as the initial or only aura may be important [28, 29]. many of the patients in this report had multiple auras, some more typical of mesial temporal lobe epilepsy (dj vu, epigastric). finally, as mentioned previously, our data does not allow drawing conclusions about the prognosis of ssa/pla in non-lesional temporal lobe-like epilepsy as numbers are too small. it is possible that these patients have been selected out already by careful presurgical evaluation and found to have extratemporal or temporal plus epilepsy. most patients with pharmacoresistant lesional tle appear to have a favorable outcome following temporal lobectomy, even in the presence of ssa and pla.

purpose. somatosensory (ssa) and pharyngolaryngeal auras (pla) may suggest an extratemporal onset (e.g., insula, second somatosensory area). we sought to determine the prognostic significance of ssa and pla in temporal lobe epilepsy (tle) patients undergoing epilepsy surgery. methods. retrospective review of all patients operated for refractory tle at our institution between january 1980 and july 2007 comparing outcome between patients with ssa/pla to those without. results. 158 patients underwent surgery for pharmacoresistant tle in our institution. eleven (7%) experienced ssa/pla as part of their seizures. all but one had lesional (including hippocampal atrophy/sclerosis) tle. compared to patients without ssa or pla, these patients were older (p=0.049), had a higher prevalence of early ictal motor symptoms (p=0.022) and prior cns infection (p=0.022), and were less likely to have a localizing spect study (p=0.025). a favorable outcome was achieved in 81.8% of patients with ssa and/or pla and 90.4% of those without ssa or pla (p>0.05). conclusion. most patients with pharmacoresistant lesional tle appear to have a favorable outcome following temporal lobectomy, even in the presence of ssa and pla.

PMC3686131

pubmed-911

the long-term storage of information in the form of memory is one of the principal functions of the developed nervous system. the ability to utilize this information provides evolutionary advantages in adapting and responding to situations in a given environment. the method for the formation of memories and the process of functional specialization in the brain during development has been found to be mediated by both structural and functional plasticity, including long-term potentiation between neurons. while much attention has been given to these processes on a neuronal level, less attention has been given to what role glial cells, particularly astrocytes, may have in the underlying mechanisms. while astrocytes were formerly thought to serve mostly as housekeeping cells, they have recently gained attention as an integral part of the chemical synapse. in addition to their structural and metabolic roles, astrocytes are now thought to be heavily involved in synaptogenesis and in regulating the communication between already formed connections. several studies have demonstrated that astrocytes utilize both ionotropic and metabotropic systems in order to regulate neuron to neuron communication [35], and that they may have specific mechanisms for regulating the formation of memories. here, we review recent evidence for the importance of astrocytes in both structural and functional synaptic plasticity, specifically long-term potentiation, the key chemical transmitters that are involved (table 1), as well as the underlying mechanisms by which astrocytes may regulate these processes. glial cells are nonneuronal cells that are now believed to constitute 50% of the cells in the whole brain in humans and other primates [69], although other reports have suggested that glia may outnumber neurons 10: 1 [1012]. astrocytes, as their name suggests, appear to be star-shaped when golgi stained or immunostained for glial fibrillary acidic protein. however, the morphology and physiology of astrocytes differ depending on the type. typically, astrocytes have a complex structure that is highly branched with many small protrusions that contact the synaptic cleft [14, 15]. with their unique morphology, astrocytes form the blood brain barrier, have a role in ion homeostasis, and form the tripartite synapse. the blood brain barrier is made up of capillary endothelial cells, vascular pericytes, and the perivascular endfeet of astrocytes. together, they create a highly selective barrier that allows oxygen and hormones to permeate into the brain while preventing the passage of other molecules due to possible harmful effects. astrocytes also maintain homeostasis of various ions such as sodium, potassium, chloride, and hydrogen. when the extracellular concentration of k is high, astrocytes uptake the ion using transporters or channels and transfer it to adjacent astrocytes via gap junctions by a process called spatial buffering [1821]. due to this process, astrocytes prevent extracellular concentrations of k from reaching toxic levels. in the tripartite conceptualization of the synapse, perisynaptic astrocytes are present along with the standard presynaptic and postsynaptic neurons [15, 2224]. contact made by perisynaptic astrocytes with the synaptic cleft depends on the type and location of synapses [13, 15, 25]. in the hippocampus for instance, the intricate arborization and ramifications of astrocytes allow them to tightly enwrap the synaptic terminal in order to modulate synaptic processes [14, 15, 25]. previous studies suggest that astrocytes respond to neurotransmitter release by increasing their intracellular calcium levels and controlling neuronal excitability through the release of gliotransmitters. based on findings that explain the functioning of the tripartite synapse, more attention has been given to the potential role of how astrocytes aid memory. in areas known for synaptic plasticity, such as the hippocampus, astroglial membranes appear to surround the majority of larger axo-dendritic synapses, and around 60% of all synapses in the hippocampus [27, 28]. in astrocytes that are part of a tripartite synapse, calcium peaks, which correspond to calcium oscillations tuned to neuronal activity, excess glutamate is taken up by astrocytes and further regulated through a shunting cycle by which it is broken down into glutamine, repackaged, sent to the presynaptic-neuron, and finally converted back into glutamate. astroglial glutamatergic regulation is so widespread that it is estimated that only 20% of synaptic glutamate is taken up by transporters on the postsynaptic neuron, while the other 80% is processed by transporters such as the glutamate aspartate transporter (glast) on the membrane of the associated astrocyte. additionally, astrocytes have the ability to swell and shrink in size through the use of aquaporin channels, and this may allow them to reduce the leakage of neurotransmitters, increasing the active concentration in the synapse, and preventing spillover in the case of damage [30, 31]. however, transmitters can also be released through these channels when exposed to a hypotonic bath solution, ischemia, or a traumatic brain injury [3234]. besides their role in signaling, astrocytes have also been implicated in controlling the development of the nervous system through factors such as axon guidance and synaptogenesis, as discussed below. it is now well known that the hippocampus, located in the inferior temporal lobe, is responsible for the formation and storage of memory [35, 36]. the hippocampal structure has distinct functional areas implicated in memory formation, that is, the ca1, ca3, and the dentate gyrus. various parts of the brain display some form of synaptic plasticity, but the hippocampus is one of the structures that has received much attention due to its overall functional importance. synaptic plasticity refers to experience mediated structural and functional changes to the connections between neurons that results in changes to neural circuits [3739]. these neural circuits are often developed (synaptogenesis) and strengthened through the reinforcement of some connections and the removal of others (synaptic stripping), which can occur in response to environmental experience. during early development, plasticity can occur through large scale dendritic and axonal conformational changes, and while these processes are observed in the adult mammalian brain, the scale on which they take place and the efficacy of regulatory processes involved are inhibited. while less overall change is observed in the adult brain, early developmental plasticity in children, as well as memory formation and learning in adults, are both likely dependent on structural changes in the functioning of the synapse itself [4143]. recent evidence shows that even small structural changes to the dendritic spines can drastically alter the overall output/input of synaptic protein receptors, which in adults is likely more important in determining neuronal activity than dendritic spine density [4143]. due to a high concentration of synapses in the brain, tuning of activity could be accomplished through regulating synaptic function with relatively little conformational change, which is important in being able to learn and store large quantities of information without negatively impacting other signaling pathways. the tuning of synaptic activity associated with functional plasticity, or changes in synaptic strength, has been demonstrated through the modulation of membrane receptors by enzymatic activity such as phosphorylation [39, 4547]. however, changes to the chemical environment within the synapse are likely more influential in the associated changes to neuronal firing and receptor concentrations. the changes in dendritic spines and synaptic activity in relation to plasticity have been found to be most closely linked to synaptic glutamate receptors and changes in both internal and external calcium in neurons. it is consistently demonstrated that neuron-glia interactions are essential to this type of environmental regulation, with astrocytes being paramount in regulating signaling molecules such as glutamate which are particularly important in the plasticity and learning processes [49, 50]. as astrocytes are in part often responsible for regulating synapse formation and synaptic activity, there is a strong possibility that their activity plays an integral role in plasticity and learning. among the various forms of functional synaptic plasticity, long-term potentiation (ltp) has received much attention in the hippocampus due to its role in memory. this was first observed in electrophysiological studies, using high-frequency stimulation (100 hz) of neurons in the perforant pathway and recording the activity at the dentate gyrus. electrode recordings followed by tetanic stimulation exhibited a longer lasting excitatory postsynaptic potential (epsp) of the postsynaptic neuron in the dentate gyrus. n-methyl d-aspartate (nmda) receptor dependent ltp occurs at the schaffer collateral region while nmda receptor independent ltp is observed at the mossy fibers of the ca3 region [5357]. despite various forms of ltp that occur in the hippocampus, nmda receptor dependent ltp several studies blocking nmda receptor activity showed impairment in different types of memory in mice, implicating nmda receptors in memory formation [5861]. however, these studies do not indicate that ltp causes memory, as ltp may be an underlying process that helps form memory but does not directly cause it. for nmda receptor dependent ltp to occur, activation of nmda receptors allows calcium to stimulate cyclic adenosine monophosphate (camp) release, causing a cascade of signaling mechanisms involving protein kinase a, camp response element binding protein (creb), camp response element (cre), mitogen activated protein kinase, and calcium calmodulin dependent protein kinase ii [6264]. therefore, synthesis of new proteins underlies the mechanism for long-term memory. while much attention has been given to the regulation of hippocampal neurons by these factors, there is a growing body of evidence that astrocytic support is more critical in the regulation and function of many ltp related compounds and mechanisms than previously thought. astrocytes release and regulate several neuroactive molecules that can affect neuronal activity and modulate plasticity and ltp. these compounds (summarized in table 1) include glutamate, atp, cytokines, and several other key signaling molecules like d-serine, adenosine, and lactate. glutamate plays a key role in the regulation of synaptic activity and causes a response in astrocytes [66, 67]. importantly, astrocytes actively sequester up to 90% of glutamate that is released into the extracellular space between neurons [68, 69]. glutamate causes a wide range of effects in astrocytes via metabotropic glutamate receptors (mglur), nmda receptors, and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (ampa) receptors. although cortical astrocytes generally express functional nmda receptors, this does not appear to be the case for hippocampal astrocytes [7072]. do not exhibit activation upon standard nmda receptor agonists [70, 72]. functional ampa receptors, on the other hand, are expressed by hippocampal astrocytes [73, 74]. additionally, hippocampal astrocytes change the properties of their ampa receptors during postnatal development. at the beginning stages of postnatal development, low levels of ampa receptor currents immature astrocytes also had a prolonged activation of the ampa receptor, which induced an influx of na and ca. astrocytes are likely able to synchronize with neuronal activity and subsequently regulate glutamate transmission between neurons [7679]. for example, astrocytic glutamate release activates presynaptic nmda receptors and promotes increased excitatory communication between neurons. these nmda receptors are also subjected to further regulation by endogenous concentrations of d-serine, which serves as a coagonist, specifically in the hippocampal region, suggesting its potential importance in new memory formation [8184]. in addition to the ionotropic glutamate receptors, astrocytes also use mglurs. in the hippocampus, mglur1 [85, 86], mglur5 [87, 88], and mglur3 [89, 90] however, astrocytes of 1-week-old mice, but not older mice, express high levels of mglur5. furthermore, astrocytes of adult mice did not exhibit an increase in ca when stimulated with an mglur5 agonist. contradictory to the observations by, there has been research demonstrating mglur5 's role in ca elevation in adults. astrocytes express a neurotransmitter receptor called g-protein coupled metabotropic receptor (gpcr). specifically, the g-protein gq, coupled to phospholipase c (plc), is involved in elevating intracellular ca levels in astrocytes [91, 92]. when gq is stimulated, plc is activated to break down phosphatidylinositol 4,5-bisphosphate (pip2) into inositol 1,4,5-triphosphate (ip3) and diacylglycerol (dag) [91, 92]. by breaking down pip2, the endoplasmic reticulum can release stored ca to stimulate gliotransmitter release. in gliotransmission, astrocytes release vesicles that are packed with gliotransmitters via the process of exocytosis. astrocytes express proteins that are known to be involved in vesicle fusion such as soluble nsf attachment protein receptor (snare), synaptotagmin, complexin2, and munc18a, which are critical for gliotransmitter release [5, 92]. for example, altering the snare complex resulted in a failure of glutamate release from astrocytes. glutamate release also requires vacuolar type of proton atpase to exchange the proton gradient from the vesicular lumen with glutamate [94, 95]. in addition, in hippocampal astrocytes, synaptic-like microvesicles (slmv) were found with the r-type snap receptor (r-snare), which is known to govern exocytosis. atp released from astrocytes also interacts directly with pre- and postsynaptic neurons, serving to regulate their own glutamatergic transmission and to also enhance the concentration of ampa receptors, which facilitates the release of neuropeptides including oxytocin and vasopressin. additionally, some of the atp released by astrocytes is converted directly to adenosine, which can act as both an agonist and antagonist for specific k and ca channels. cytokines and chemokine receptors are also implicated in the regulation of ca stores, glutamatergic transmission, and synaptic plasticity as a whole. in astrocytes, the cxcr4-cxcl12 signaling axis has been implicated in both modulating glutamate exocytosis, and in causing the release of the cytokine tnf-. tnf- is also linked to regulating both glutamate release and the insertion of ampa receptors into neighboring neurons [99, 100]. finally, cytokine signaling in astrocytes, as well as microglia, plays a role in the response to physically sensing pain and responding to damage, with chemokine (c-c motif) ligand 2 (ccl2) released from astrocytes having a strong regulatory effect on the activity of nmda receptors. despite the evidence indicating the significance of ca in the release of gliotransmitters, some studies have observed that blocking ca in hippocampal astrocytes located at the ca1 region in situ does not change ca levels in neurons, change spontaneous excitatory postsynaptic current, result in astrocytic glutamate release, or nmda receptor mediated slow inward currents in pyramidal neurons [102104]. these findings may suggest that a mechanism not dependent on ca release may lead to gliotransmitter release in astrocytes. although the gliotransmitters discussed above are important in regulating ltp, another crucial gliotransmitter to postsynaptic neurons is lactate. memory formation is the result of a cascade of cellular and molecular processes and thus, to ensure the proper functionality of a neuron, astrocytes provide neurons with lactate, a usable form of energy [105107]. through glycogenolysis, astrocytes convert stored glycogen into lactate and release it into the synapse through the mct1 or mct4 transporter. the neuron is then able to take up lactate via an mct2 transporter, which has been confirmed through blocking mct2 with either 4-cin or mct2-oligodeoxynucleotides [106, 107]. rats showed memory impairment in inhibitory avoidance and spatial memory tasks when glycogenolysis, mct1, mct4, or mct2 were inhibited [106, 107]. thus, it is clear that the metabolism of astrocytes is critical in hippocampal dependent memory. ephrin signaling, consisting of ephrin-as and ephrin-bs, is known for its involvement in neural development by inhibiting axonal and dendritic growth via actin rearrangement [108114]. the interaction between ephrin-a3 and epha4, which are expressed by astrocytes and dendritic spines of neurons, respectively, is involved in decreasing levels of glast and glutamate transporter 1 (glt-1) for proper synapsing to occur [115118]. astrocytes express both ephb receptors and ephrin-b ligands, ephrinb3 being the most active during ltp. ephrinb3 enhances d-serine release by regulating serine racemase (sr), an enzyme responsible for the conversion of l-serine to d-serine, and an sr-interacting protein, protein kinase c (pkc). specifically, ephrinb3 downregulates pkc in order to increase the interaction between sr and protein interacting with c-kinase (pick1), causing d-serine release. moreover, ephrinb3 is able to bind to both ephb3 and epha4 receptors. by measuring d-serine levels in ephb3 and epha4 knockouts in cultured astrocytes, thus, while ephrin-a signaling regulates levels of glt-1, ephrin-b signaling regulates levels of d-serine release for activation of nmda receptors. nicotine influences memory by inducing synaptic transmission at acetylcholinergic synapses [121123]. in alzheimer's disease, astrocytes express nicotinic acetylcholine receptors (nachr), implicating nicotine's role in cholinergic dependent memory. this effect on memory is dependent on glutamatergic nmda receptors, which requires binding of d-serine released by astrocytes [8183, 125]. as described previously, d-serine binds to nmda receptors, therefore, nicotine binding to the nachr on astrocytes stimulates the release of d-serine by increasing internal calcium concentrations, allowing nmda receptors on the postsynaptic neuron to induce ltp [123125]. similar to nachr, activation of muscarinic achr (machr) also increases internal calcium concentrations [126128]. the adenosine a1 receptor is expressed on presynaptic neurons, and activation of the receptor activates the inhibitory metabotropic g-protein (gi) pathway. memory deficits in mice that underwent 6 hours of sleep deprivation were prevented by pharmacologically blocking the a1 receptor. furthermore, astrocytes modulate levels of adenosine during 12 hours of sleep deprivation in mice. interestingly, a1 receptor activation in astrocytes can also modulate sleep in a rodent model of inflammation. the cytokine interleukin-1 (il-1) also plays a key role in hippocampal dependent memory. blocking activity of il-1 receptors resulted in the poor performance of learning with the morris water maze and fear conditioning, as well as reduced ltp [132135]. although il-1 receptors can be expressed by many cells, it is prominently expressed on astrocytes [136140]. il-1 receptor knockout mice that did not express il-1 receptors on astrocytes exhibit memory deficits that can be rescued with transplantation of neural precursor cells from wild-type mice that express il-1 receptors. the underlying mechanism of il-1 has yet to be determined in the context of memory. research on synaptic plasticity and memory has traditionally been neuron-centric, yet it is crucial to not ignore the astrocytic role in these processes since they are now known to modulate neuronal activity. not only do astrocytes regulate the extracellular concentration of neurotransmitters, they also regulate the activity and expression of receptors on the postsynaptic neuron through gliotransmitter activity, and play a role in dampening activity and promoting the removal of nonadvantageous connections. the evidence reviewed here shows that astrocytes have an ongoing role in the regulation of neuronal activity through the release of gliotransmitters and the expression of transporters/receptors on their extracellular surface. based on these findings, we propose a mechanism of astrocyte-to-postsynaptic neuron interaction that supports the induction of ltp (see figure 1). here, the influx of intracellular ca caused by the activation of cholinergic receptors and mglurs allows multiple gliotransmitters (e.g, glutamate, d-serine, tnf-, and atp) to be released. these gliotransmitters then bind to their respective receptor to regulate the influx of ions on the postsynaptic neuron, which causes a cascade of molecular mechanisms that initiate transcription. moreover, for the cellular and molecular changes of the postsynaptic neuron, lactate must be provided by astrocytes for energy to protect neurons from cytotoxic death, and glt-1 regulates the extracellular glutamate concentration during the late phase of ltp. the purpose of the proposed mechanism is to represent how astrocytes may regulate the postsynaptic neuron during ltp. behavioral studies used to determine that the role of astrocytes are known to be hippocampal dependent tasks. however, this by no means allows us to determine which part of the hippocampus the mechanism takes place in, nor the type of ltp. more importantly, there are various kinds of memory such as episodic memory, procedural memory, associative memory, and fear conditioned memory. moreover, it is important to note that this model only examines astrocyte to postsynaptic terminal communication: it is well known that astrocytes are also able to modulate presynaptic terminal [142, 143]. although we have explained detailed evidence of how astrocytes regulate the postsynaptic neuron, we must also consider how astrocytes affect activity of the presynaptic neuron as well. hippocampal astrocytes are able to detect synaptic activity at distinct locations via mglur5 and increase intracellular ca levels for a prolonged time span, which results in alteration of basal synaptic transmission [144, 145]. the mechanism also involves astrocytic release of purines to activate a2a receptors expressed by the presynaptic neuron. calcium activity was also observed to not only be involved in gliotransmission, but neurotransmission as well. synaptic transmission in neighboring synapses was reduced when blocking ca in astrocytes, suggesting the ability of astrocytes to modulate the activity of presynaptic neurons. the engraftment of human astrocytes in mice enhances ltp and significantly increases the release of the cytokine tnf-. since a xenograft of human astrocytes can functionally modulate the activity of mice neurons, it may be possible that a xenograft from another species would facilitate ltp if placed into a human patient. there is still much to research in glial neurobiology in order to fully understand the underlying mechanisms of neural networks that are involved in plasticity and memory. for instance, since astrocytes are physically connected with other astrocytes through gap junctions to form a glial syncytium, it is crucial to further examine how astrocytic signaling may regulate neuronal activity and therefore, underlie ltp. it is now clear that astrocytes play an important part in learning and memory, and continuing to elucidate astrocytic processes that are involved in learning and memory will help advance our understanding of the dynamic role of these glial cells in modulating ltp.

astrocytes regulate synaptic transmission and play a role in the formation of new memories, long-term potentiation (ltp), and functional synaptic plasticity. specifically, astroglial release of glutamate, atp, and cytokines likely alters the survivability and functioning of newly formed connections. among these pathways, regulation of glutamate appears to be most directly related to the promotion of ltp, which is highly dependent on the synchronization of synaptic receptors through the regulation of excitatory postsynaptic potentials. moreover, regulation of postsynaptic glutamate receptors, particularly ampa receptors, is dependent on signaling by atp synthesized in astrocytes. finally, cytokine signaling is also implicated in regulating ltp, but is likely most important in plasticity following tissue damage. despite the role of these signaling factors in regulating ltp and functional plasticity, an integrative model of these factors has not yet been elucidated. in this review, we seek to summarize the current body of evidence on astrocytic mechanisms for regulation of ltp and functional plasticity, and provide an integrative model of the processes.

PMC3867861

pubmed-912

systemic sclerosis (ssc) is characterized by early and persistent microvascular impairment leading to functional raynaud's phenomenon (rp) and clinical manifestations (i.e., digital ulcers, pulmonary arterial hypertension, etc.) (figure 1) [1, 2]. digital ulcers in ssc are considered to be related to tissue ischemia following several processes, including at the beginning persistent vasospasm (rp), but in the progression of the disease also to intimal fibroproliferation, tissue fibrosis, and thrombosis of digital arteries. progressive deficiency in vasodilatory capacity of the vessels and tissue fibrosis is proposed as a mechanism of the persistent vascular spasm; however, the mechanism of endothelial injury is still unclear. the assessment of vascular involvement is still a matter of study, and several noninvasive techniques have been proposed. peripheral microvascular impairment in ssc may be easily and safely detected by nailfold videocapillaroscopy (nvc). the morphological capillary abnormalities in ssc have been classified in 3 validated patterns (early, active, and late) of microangiopathy by nvc and scored (figure 2) [57]. nvc may partially observe the column of red blood cells moving inside the capillary, but the technique does not allow measurement of the blood flow. laser doppler flowmetry (ldf) is the best non invasive and safe technique to assess and to measure the blood perfusion at peripheral sites [8, 9]. blood flow has been found to be reduced in patients with ssc, compared with healthy subjects and patients with primary rp. patients with ssc showing the late nvc pattern of microangiopathy have a significantly lower finger blood perfusion (fbp) than patients with the active and early nvc patterns (p<.05). the question today is if capillaroscopy (and eventually ldf) may represent an outcome measure for clinical trials on the peripheral vasculopathy in ssc. we will analyze clinical conditions related to ssc in which nvc may represent an outcome measure by considering their already assessed relationship with the nvc patterns and/or eventually scores. the 3 important biological/clinical conditions are: the ssc-specific serum autoantibodies, the ssc skin digital ulcers (dus), and the pulmonary arterial hypertension (pah) associated to ssc. ssc is characterized by serum autoantibodies, including anticentromere (anti-cenp-b), anti-th/to, antitopoisomerase i (anti-topo i), and anti-rna polymerase i/iii (anti rnap iii). together, these markers account for almost 85% of autoantibodies specific for ssc and show a predictive value for clinical evaluation and prognosis [11, 12]. anti-cenp-b and anti-topo i are known predictors of progression from isolated rp to ssc. however, until recently, many of the studies on the significance of expression of these antibodies in ssc have been limited by small sample sizes, incorrect classification of patients with manifestations of connective tissue disorders as having primary rp, use of varying definitions of subsets of patients, lack of standardised methods for determining antinuclear antibodies, omission of tests for anti-th/to and anti-rnap iii antibodies, and absence of multivariable analyses. antiendothelial cell antibodies (aecas) are a heterogeneous class of antibodies whose role in the pathogenesis of autoimmune diseases with vascular involvement has been extensively studied and are present in the serum samples of many patients with ssc (2286%) but are not ssc specific. even if, among the demonstrated clinical associations, lung and peripheral vascular involvement is the most common, further research on this topic, including longitudinal studies in patients with ssc, is mandatory for a better understanding of the clinical value of aeca. however, for long time it has not been determined prospectively whether ssc autoantibodies are related to the course and type of microvascular damage detectable by nailfold capillaroscopy. leroy and medsger proposed that patients with rp who had abnormal findings on nvc and ssc-specific autoantibody should be classified as having early ssc. this set of criteria had not been validated and considered for long time, until recently. finally, koenig et al. prospectively studied a large cohort of raynaud's patients who were referred to a single centre for evaluation of rp over a period of 20 years. the objectives were to identify the strongest independent predictors of progression to definite ssc, to determine the type and time course of microvascular damage by nailfold capillaroscopy and its relationship to major ssc autoantibodies, and to validate the criteria for early ssc. of the 586 patients who were followed up for 3,197 person-years, 74 (12.6%) developed definite ssc. in fact, this study validated the criteria of leroy by demonstrating that almost all patients who were to futurely develop ssc had early ssc (raynaud's phenomenon plus a scleroderma pattern on capillaroscopy and/or ssc-specific antibodies) at the baseline visit. concerning the scleroderma pattern they reported a characteristic sequence of microvascular damage, starting with enlarged capillaries (giant capillaries) that identify the early ssc pattern, followed by capillary loss that indicates the active ssc pattern and then by capillary telangiectasias (neoangiogenesis) that might better characterize the late ssc pattern (figure 2), from the stadium of early ssc until development of definite ssc. enlarged capillaries (giant capillaries), capillary loss, and ssc-specific autoantibodies independently predicted definite ssc. interestingly, anti-cenp-b and anti-th/to antibodies predicted for the development of giant capillaries; these autoantibodies and anti rnap iii also predicted for capillary loss. each autoantibody was associated with a distinct time course of microvascular damage. at followup, 79.5% of patients with one of these autoantibodies and abnormal findings at the baseline nailfold capillaroscopy examination had developed definite ssc. patients with both baseline predictors were 60 times more likely to develop definite ssc. these data validated the proposed criteria for early ssc. in conclusion, in the presence of a secondary rp evolving to definite ssc, microvascular damage (as assessed by nailfold capillaroscopy) is dynamic and progressive, and ssc-specific autoantibodies are associated with the course and type of capillary abnormalities. it was confirmed that the microvascular damage in secondary rp evolving to definite ssc is characteristically sequential, starting with enlarged capillaries (giant capillaries, early ssc pattern) followed by capillary loss (active ssc pattern), and then by capillary telangiectasias (neoangiogenesis, late ssc pattern). since new therapeutic agents are being evaluated in patients with ssc, awareness of this sequence of microvascular damage has potential implications for future trials. of note, recently, it was established the evolution of microangiopathy in established ssc disease, by pointing out the progression of capillary loss and augmentation of ramifications in a microangiopathy score. the final message is that abnormal findings on nailfold capillaroscopy at baseline together with a systemic sclerosis-specific autoantibody indicate a very high probability of developing definite systemic sclerosis whereas their absence excludes this outcome. in ssc patients awareness of this sequence of microvascular damage and ss-associated autoantibodies has potential implications for future therapeutical trials. for example, in trials of novel angiogenic, vasculogenic, or fibrosis-modulating agents, it would appear realistic to select ssc patients at a uniform stage of microvascular damage and with similar ssc-specific autoantibodies and eventually to evaluate such damage longitudinally by nvc to assess response to treatment. skin dus represent one of the most frequent clinical manifestations of microangiopathy in patients with ssc (figure 3). on the other hand, a decreased number of capillary loops should be considered highly specific for advanced rp, and it has been estimated that the number of normal capillaries may be reduced to just 20% in patients with active ssc. recently, using a semiquantitative score that highlights the importance of the number of capillaries, an association was reported between advanced stages of capillary loss (mean score class 2 and 3) and digital trophic lesions in 49% of patients with ssc. also, loss of capillaries may be relevant in determining tissue hypoxia, and, in patients with recent onset of rp, the appearance of rapidly progressive capillary loss may represent the first capillaroscopic evidence of severe ssc with destruction of microvessels. the extensive disappearance of capillaries may generate large avascular areas giving a desert-like appearance to the nailfold bed, and progressive loss of capillaries has been associated with more extensive skin involvement (as well as diffuse ssc) and a poor prognosis. as a consequence, the early detection of ssc patients who are at high risk of developing du could allow preventive treatment of these complications with reduction of morbidity and social costs. very recently, it was found, in 130 ssc patients examined at entry and after 20 months of follow-up, that the diffuse cutaneous form of ssc with avascular areas on capillaroscopy represented, among other factors (e.g., increased interleukin- 6) the major risk factor for du development. a previous study showed that, patients with late ssc pattern at nvc showed an increased risk to have an active disease (odds ratio (or) 3.50; 95% confidence interval (ci) 1.319.39) and to present skin du (or 5.74; 95% ci 2.0815.89). another recent investigation showed that a quantitative capillaroscopic score was suggested highly predictive of the development of new skin du within 3 months after nvc. the predictive value of this index still needs to be confirmed in a validation study. a clinical history of multiple skin du is the most helpful predictor and indicator for preventive therapy, and the loss of capillary as assessed at nvc has been found the best possible nvc predictive marker to be considered. however, the routine use of nvc now seems a possible predictive tool to enable the early detection of patients at a high risk of developing skin du. in this regard, at present, the cost/effectiveness ratio for the therapy of ssc skin du is very unfavourable, and strategies for their treatment or prevention are under debate. ssc is the main connective tissue disease associated with pah and pah is estimated to affect 12% of ssc patients, being the leading cause of death in this disease. structural changes in the systemic microcirculation, consisting of a reduction of capillary density and widening of the capillaries, are considered an hallmark of ssc. it is now clear that the severity of this microvascular damage as assessed by nvc differs between patients with ssc and pah (ssc-pah) and those with ssc without pah (ssc-non-pah) and correlates with pulmonary haemodynamic parameters. interestingly, when compared to healthy controls, the same is true for patients with idiopathic pah, a condition not known to be characterized by systemic microvascular changes. a recent study suggests that capillary density reduction is a marker of the presence and severity of pah. a few studies have investigated nailfold capillary patterns in patients with ssc-pah, with only one study including patients with idiopathic pah. two studies used echocardiography and/or right heart catheterisation to confirm the diagnosis of pah., found a significant reduction of capillary density in eight patients with ssc-pah in comparison with 12 patients with ssc-non-pah. the other study, by greidinger et al. using capillary density and qualitative scoring of nailfold patterns, found no differences in capillary patterns between eight patients with ssc-non-pah and seven with ssc-pah, but capillary density in these groups was not reported. a third study, by ohtsuka et al. using only right heart catheterisation to diagnose and exclude the diagnosis of pah, showed a significant difference in semiquantitative scoring of nailfold patterns between ssc-non-pah and ssc pah patients, but, again, capillary density was not assessed in this study. practically, only one of these studies included patients with idiopathic pah and reported no differences in capillary density and capillary patterns between 13 healthy controls and 37 patients with idiopathic pah. there is, however, more recent evidence of a reduction of capillary density in both ssc-pah and, albeit to a milder extent, in idiopathic pah. the explanation for a reduction in capillary density may not be the same for the two disorders. in ssc it is generally presumed that structural changes in the systemic (micro)circulation precede changes in the pulmonary circulation, as systemic microvascular changes may precede the development of ssc by many years. therefore, nvc abnormalities might also reflect what is going on in the pulmonary circulation. this may not be true for all capillary abnormalities, because most patients with ssc demonstrate nailfold capillary abnormalities whereas only a minority develop pah. recent data confirm that only capillary density is associated with the presence of pah and is a marker of disease severity in ssc. a further suggested explanation for the more pronounced capillary reduction in ssc-pah could be that pah itself amplifies the reduction of capillary density already present in ssc. however, houben et al. recently observed an increase rather than a decrease of nailfold capillary density in patients with heart failure. the observation that circulating plasma levels of endothelin1 (et-1) are raised in patients with pah and that et-1 production is increased in the pulmonary tissue of affected individuals makes this vasoconstrictor a particularly interesting target for therapeutic intervention in pah. clinical trials with et receptor antagonists have clearly shown that such antagonists provide symptomatic benefit in patients with pah, thereby proving the clinical relevance of the endothelin system as a therapeutic target with optimised use of selective eta or nonselective eta/etb blockade. as matter of fact the highest serum et-1 levels are found in ssc with late nvc pattern and visceral involvement. recent therapeutical examples of clinical trials with nvc as measure of outcome and conclusions are presented. very recent studies represent examples of clinical trials in which nvc has been successfully included as a measure of treatment outcome. the objective of one study was to evaluate nvc pattern changes in ssc patients treated regularly on cyclic basis with iloprost and to find associations with clinical, serologic, and pharmacological variables. forty-nine patients affected by ssc underwent two ncv analyses at 3 years apart from each other. six patients showed an amelioration of nvc abnormalities who changed from active to early pattern; five of these cases (83.3%) had been given cyclophosphamide therapy and the remaining case methotrexate plus azathioprine. cyclophosphamide administration was significantly associated with regression of the nvc pattern (p<.001). interestingly, none of the ssc patients who received cyclophosphamide demonstrated worsening of the microvascular lesions; the progression of nvc pattern was inversely correlated to cyclophosphamide treatment (p=.02). therefore, cyclophosphamide treatment demonstrated to be effective in modulating the ssc microvascular damage as directly observed and monitorized by nvc. in another study cyclophosphamide treatment showed to be effective for ssc microvascular damage as directly observed by rapid improvement of the nvc pattern. confirmation of the trend was obtained by a further study on autologous stem cell transplantation that confirmed a significant regression of the nvc pattern together with the improvement of the clinical conditions. in conclusion, to the question if capillaroscopy (nvc) may represent in ssc an outcome measure for clinical trials on the peripheral vasculopathy, based on the growing evidences and our detailed studies, the answer is positive. early recent therapeutic trials in ssc are confirming this role, and the experience is growing rapidly. to definitely establish its role as an outcome measure two requirements still need to be fulfilled. first, large multicentric and longitudinal and randomized controlled trials (certainly in establishing its role as an outcome measure in therapeutical trials) are needed. recently, a first step in assessing reliability has been taken through demonstration of reliability both in qualitative and semiquantitative assessment of nailfold images of ssc patients in a bicentre setting .

peripheral microvascular impairment in systemic sclerosis (ssc) may be easily detected and scored in a safe noninvasive way by nailfold videocapillaroscopy (nvc). the paper highlights clinical conditions related to ssc in which nvc may represent an outcome measure of therapeutical interventions, by elaborating on their already assessed relationship with the nvc patterns and eventually scores. the 3 important biological/clinical conditions are: the positivity for ssc-specific serum autoantibodies, the presence of ssc skin digital ulcers (dus) and of pulmonary arterial hypertension (pah) ssc associated. in conclusion, to the question if capillaroscopy (nvc) may represent in ssc an outcome measure for clinical trials on the peripheral vasculopathy, based on the growing evidence and our detailed studies, the answer is positive. recent therapeutic trials in ssc are confirming this role, and the experience is growing rapidly.

PMC2935167

pubmed-913

autophagy is a catabolic process that degrades cytoplasmic components and organelles and is conserved in almost all eukaryotes. autophagy is initiated in response to cellular stresses such as nutrient starvation, oxidative stress, infection, or inflammatory stimuli. upon its induction, a cup-shaped double-membrane structure, called an isolation membrane (or phagophore), emerges in the cytoplasm, then the isolation membrane elongates with curvature and finally becomes enclosed, forming an autophagosome containing cytoplasmic components. subsequently, autophagosomes fuse with lysosomes/vacuoles, and lysosomal hydrolases degrade the sequestered material [15]. this process facilitates physiological processes such as survival during starvation, clearance of dysfunctional or aggregated proteins and organelles, development, differentiation, and aging [68]. in addition to the nonselective degradation of cytoplasmic components, autophagy can selectively degrade specific organelles or proteins. these include peroxisomes, endoplasmic reticulum, ribosomes, the nucleus, intracellular pathogens, protein aggregates, lipid droplets, and secretory granules. these catabolic processes are termed pexophagy, reticulophagy (erphagy), ribophagy, nucleophagy, xenophagy, aggrephagy, lipophagy, and zymophagy, respectively. similarly, the yeast cvt complex (a protein complex comprising aminopeptidase i (ape1) and alpha-mannosidase (ams1)) is delivered to vacuoles via an autophagy-like process; ape1 and ams1 are processed and activated in the vacuoles, and this autophagic process is called the cvt pathway. it has been known for some time that mitochondria are also degraded by autophagy in mammalian cells (first described by clark in 1957) and in yeast (first described by takeshige and colleagues in 1992), but this selective autophagic process has recently been described in more detail. daughter mitochondria with reduced membrane potential after a fission event are preferentially removed by autophagy in mammalian cells. photoirradiation-damaged mitochondria are selectively degraded by autophagy in hepatocytes [12, 13]. during the maturation of erythroid cells, mitochondria are preferentially degraded by autophagy in a manner dependent on the mitochondrial outer membrane protein nix [14, 15]. recently, it has been reported that there are two types of autophagy in mammalian cells: autophagy related protein 5 (atg5) and atg7-dependent (conventional) autophagy and atg5/atg7-independent (alternative) autophagy. both conventional and alternative autophagic processes are implicated in the autophagic degradation of mitochondria during erythroid cell maturation [16, 17]. similarly, during white adipose tissue differentiation, mitochondria are preferentially degraded by autophagy. when yeast cells were cultured in lactate-containing medium as the sole carbon source and the identification of the yeast mitophagy-specific protein atg32, which plays a key role in the recognition of mitochondria by the autophagic machineries, confirmed the existence of selective degradation of mitochondria by autophagy [20, 21]. atg11 is a cytosolic adaptor protein that is required for selective cargo recognition by autophagy. for example, during the cvt pathway, the cargo proteins ape1 and ams1 generate a complex with the receptor protein atg19 that is recognized and bound by atg11. similarly, during pexophagy, pichia pastoris atg30 (ppatg30) binds peroxisomal proteins pppex3 and pppex14 and is recognized and bound by atg11. finally, in both cases, atg11 transports the cargo to the pre-autophagosomal structure/phagophore assembly site (pas), where the isolation membrane emerges, and the cargo is surrounded by the autophagosome [22, 23]. atg11 is also essential for mitophagy, suggesting the presence of a receptor protein for mitophagy that corresponds to atg19 or ppatg30 in the cvt pathway and pexophagy, respectively ,. a genetic screen for yeast mutants defective in mitophagy identified such a receptor protein, which is now known as atg32 [20, 21, 25]. atg32 is a mitophagy-specific protein that is not required for nonselective autophagy or other types of selective autophagy [20, 21]. atg32 consists of 529 amino acids and localizes in the mitochondrial outer membrane with its n-terminal domain towards the cytoplasm. similarly to the cvt pathway and pexophagy, when mitophagy is induced, atg32 is bound by atg11 and the atg11atg32 complex recruits mitochondria to the pas [20, 21]. during this recruitment step, this atg32atg8 interaction is thought to increase the efficiency of mitochondrial sequestration by the isolation membrane. although the molecular processes by which the autophagic machinery selects and degrades mitochondria have been revealed, little is known about the upstream signaling pathways. recently, it was reported that the related signaling pathways of two mitogen-activated protein kinases (mapks), slt2 and hog1, are involved in the induction of mitophagy [26, 27]. in the slt2 signaling pathway, all of protein kinase c (pkc1), mapkkk (bck1), mapkk (mkk1/mkk2), slt2, and the upstream cell surface stress sensor wsc1 are required for mitophagy. in the hog1 signaling pathway, pbs2-hog1 and the upstream stress sensor sln1 the role of slt2 is, however, controversial: in the above-mentioned study, nitrogen starvation-induced mitophagy was deficient in slt2-deleted cells, whereas another study reported normal mitophagy in slt2-deleted cells cultured to the post-log phase. recently, we found that, when mitophagy is induced, ser114 and ser119 on atg32 are phosphorylated and that the phosphorylation of atg32, especially on ser114, mediates the atg32atg11 interaction and mitophagy. similarly it has been noted that phosphorylation of ser112 on ppatg30 is required for ppatg30ppatg11 interaction and pexophagy in pichia pastoris. these findings suggest that both mitophagy and pexophagy are regulated by kinase activity and/or the localization of the kinases that phosphorylate atg32 and/or ppatg30. although the mapk hog1 is required for atg32 phosphorylation, the direct phosphorylation of atg32 by hog1 was not observed in an in vitro phosphorylation assay. presumably, the unidentified kinase that phosphorylates atg32 is downstream of hog1 and slt2. atg33 is a mitophagy-related protein that was identified by a genetic screen for yeast mutants defective in mitophagy. atg33 is located in the mitochondrial outer membrane and functions in mitophagy but not in nonselective autophagy, the cvt pathway, or pexophagy. interestingly, in an atg33-knockout strain, although mitophagy was partially inhibited when induced by starvation, it was blocked almost completely when induced during the stationary phase. although the function of atg33 in mitophagy is unknown, it might be a factor for the selection or detection of damaged or aged mitochondria when cells have reached the stationary phase [25, 29]. in addition to atg33, whi2, uth1, and aup1 have also been reported as related to mitophagy [3032]. whi2 is a stress response protein that predominantly influences mitophagy and, to a lesser extent, autophagy. mller and reichert speculated that whi2 and the ras/pka (protein kinase a) signaling pathway are linked to the regulation of mitophagy. uth1 is a mitochondrial outer membrane protein and is reported to be required for mitophagy induced by rapamycin or nitrogen starvation. aup1 was identified by a screen for protein phosphatase homologs that interact with the serine/threonine kinase atg1 that is required for autophagy and is suggested to be needed for efficient mitophagy to survive in prolonged stationary phase culture in a medium containing lactate as the carbon source. interestingly, it was shown that deletion of rtg3, a transcription factor that mediates the retrograde signaling pathway, causes a defect in stationary phase mitophagy and that deletion of aup1 leads to alterations in the patterns of rtg3 phosphorylation under these conditions, implying that the function of aup1 in mitophagy may be regulation of rtg3-dependent transcription. inconsistently, both uth1 and aup1 have also been reported to be not required for mitophagy and were not identified in genome-wide mitophagy screens [20, 29]. further studies are required to clarify these discrepancies, which could be due to differences in the condition used to assess autophagy. reported that n-acetylcysteine, which increases cellular levels of reduced glutathione, prevents mitophagy. reported that the expression of atg32 is suppressed by n-acetylcysteine treatment, and, as a result, mitophagy is inhibited. because mitophagy is thought to preferentially eliminate damaged mitochondria, it is reasonable that cellular oxidative status, which is compromised by reactive oxygen species (ros) generated by damaged mitochondria, is related to the induction of mitophagy. we have summarized the above-described molecular processes and regulatory mechanisms in figure 1. it has been suggested that mitophagy eliminates damaged or aged mitochondria, thereby maintaining mitochondrial quality. there are several lines of evidence demonstrating that damaged mitochondria are eliminated by mitophagy in yeast. suggested that conditional knockout of fmc1, a gene encoding the fmc1 protein that is concerned with the folding of the f1fo-atpase, induces mitophagy under anaerobic conditions. suggested that interference with the mitochondrial k/h exchanger mdm38 causes the swelling of mitochondria and the degradation of those mitochondria by mitophagy. zhang et al. blocked mitochondrial dna (mtdna) replication using ethidium bromide or a mtdna polymerase temperature-sensitive mutant and observed rapid degradation of mitochondria via autophagy. these results indicate that mitochondrial damage is related to the induction of mitophagy, but are not direct evidence that autophagy selectively eliminates damaged mitochondria. accordingly, it is still unknown whether mitophagy contributes to mitochondrial quality control in yeast. in fact, it has been difficult to identify the physiological role of mitophagy in yeast, because mitophagy-deficient atg32-deleted cells do not show any phenotype, including phenotypes of mitochondrial dysfunction. when mitophagy-deficient atg32-deleted cells were precultured in nonfermentable medium (for instance, lactate-containing medium as the sole carbon source) and were then shifted to nitrogen starvation for long-term culture (~5 days), the atg32-deleted cells grown on nutrient-rich plates generated small colonies, while wild-type cells did not. further analysis revealed that, when wild-type cells encounter nitrogen starvation, they induce mitophagy and quickly eliminate mitochondria that have proliferated during respiratory growth. as a result, cellular ros production, which occurs mainly in mitochondria, is suppressed. on the other hand, in mitophagy-deficient atg32-deleted cells, ros damage mitochondria, and damaged mitochondria produce further ros, finally leading to mtdna deletion. ultimately, cells with mtdna deletion generate small colonies even in fermentable medium; this phenotype is called petite. this suggests that mitophagy is required to regulate the number of mitochondria to minimize ros production and, as a result, maintains the quality of mitochondria. bulk autophagy-deficient yeast strains exhibited reduced mitochondrial membrane potential, reduced activities of the electron transport chain, and higher levels of ros and oxidative stress, resulting in the loss of mtdna [38, 40]. in bulk autophagy-deficient cells, cellular ros accumulate during nitrogen starvation because the cellular amino acid pool is reduced and the expression of the ros scavenger proteins is suppressed. this finding suggests that autophagy, including mitophagy, contributes to the quality control of mitochondria. in a contrasting situation, graef and nunnari demonstrated that healthy mitochondria are required for efficient induction of autophagy under amino acid starvation. autophagic flux is regulated by atg1, target of rapamycin (tor) kinase complex i, and camp-dependent protein kinase a (pka), whereas atg8 induction is solely dependent on pka. defects in mitochondrial respiration induce pka activity, resulting in the suppression of both atg8 induction and autophagic flux. the data presented by graef and nunnari indicate that defects in mitochondrial respiration inhibit autophagy including mitophagy during amino acid starvation. they suggest that the effect of mitochondrial dysfunction on the regulation of autophagy varies according to the severity of the defect. furthermore, these authors also suggest that inordinate accumulation of mitochondria that are defective in respiration beyond a certain level decreases the capacity for autophagy and mitophagy in these cells and evokes a negative feedback that results in cellular aging or death. as described above, the molecular processes and regulatory mechanisms of mitophagy in yeast have been slowly but surely identified. since the 2008 report that a defect in mitophagy might be involved in the pathogenesis of parkinson's disease, there has been much interest in mitophagy in higher eukaryotes and, in particular, mammalian cells. most mitophagy studies in mammalian cells have focused on pten-induced putative kinase protein 1 (pink1)/parkin-dependent mitochondrial degradation by autophagy. parkin and pink1 are encoded by the park2 and park6 genes, respectively; both are responsible for familial parkinson's disease and have been reported to be associated with mitophagy [4244]. pink1 is expressed in the cytoplasm and constitutively translocates into the mitochondrial inner membrane where it is promptly degraded by the mitochondrial inner membrane rhomboid protease presenilin-associated rhomboid-like protein (parl) [43, 4547]. when mitochondria lose their membrane potential, pink1 can target to the mitochondria, but can not translocate across the mitochondrial outer membrane; therefore, it accumulates there. parkin translocates to mitochondria in a pink1-dependent manner [4244, 48, 49]. parkin triggers the ubiquitination of many mitochondrial proteins such as mitochondrial assembly regulatory factor (marf) in flies or mitofusin 1, mitofusin 2, and voltage-dependent anion channel 1 (vdac1) in mammalian cells [4954]. the ubiquitinated proteins on mitochondria are bound by the autophagy substrate p62/sqstm1, which contains a ubiquitin-associated domain, and the p62-associated mitochondria aggregate near the nucleus [49, 54, 55]. because p62 is a substrate of autophagy, it is thought that p62-associated mitochondria are eventually degraded by autophagy [49, 54, 55]. although it is accepted that p62 associates with mitochondrial proteins ubiquitinated by parkin and mediates the aggregation of mitochondria, there have been conflicting reports showing that p62 is not indispensable for mitophagy [56, 57]. the histone deacetylase hdac6, which can bind ubiquitinated proteins and facilitates the clearance of protein aggregates, is also reported to accumulate on mitochondria after parkin translocation from the cytosol and mediate mitophagy. further studies are required to clarify the precise roles of p62 and hdac6 in mitophagy. however, other studies have focused on parkin/pink1-independent mechanisms of mitophagy in higher eukaryotes. in mammalian cells, ulk1, a homolog of yeast atg1, is known to be associated with the control of autophagy by the tor signaling network. ulk1 activity is suppressed under nutrient-rich conditions by tor complex 1 (torc1). recently, it has been suggested that phosphorylation of ulk1 by adenosine monophosphate-activated protein kinase (ampk) is concerned with autophagy. loss of ampk or ulk1 resulted in deficient mitophagy and aggrephagy during starvation in mouse embryonic fibroblasts and hepatocytes, resulting in increases in the overall mitochondrial number and aberrant morphology. this finding suggests that ampk-mediated phosphorylation of ulk1 is required for mitochondrial homeostasis in nutrient-poor conditions. tectonin domain-containing protein 1 (tecpr1) has been identified as an atg5-binding protein. this protein forms a complex with atg12-atg5-atg16l1 and binds to wipi-2, which is capable of association with phosphatidylinositol 3-phosphate at an isolation membrane. interestingly, tecpr1 is required for xenophagy, which selectively recognizes and eliminates bacterial pathogens such as shigella, salmonella, and group a streptococcus. tecpr1 is also required for the autophagic degradation of misfolded protein aggregates and depolarized mitochondria but not for nonselective autophagy. these findings suggest that tecpr1 is an essential factor for specific cargo recognition in selective autophagy. it has been reported that mitophagy is induced under several conditions such as mitochondrial permeability transition, during cellular development or during hypoxia. first, nutrient starvation and photodamage, which both lead to mitophagy, cause mitochondrial permeability transition (mpt), in which the opening of the mpt pores causes mitochondria to become permeable to all solutes up to a molecular mass of approximately 1500 da, leading to mitochondrial depolarization and outer membrane rupture [62, 63]. cyclosporin a, an inhibitor of mpt through interaction with cyclophilin d, blocks mitophagy during mpt [12, 64]. these findings suggest that mpt is a trigger for mitophagy that arises from mitochondria themselves. second, recent studies have revealed that mitophagy plays an important role in cellular differentiation. during reticulocyte maturation (as with erythroid cell maturation mentioned in section 1), mitochondria are eliminated via autophagy in a nix-dependent manner [14, 17, 65]. nix, in both in vivo and in vitro assays, interacts with lc3/gabarap, which anchors to the isolation membrane and is involved in isolation membrane extension, and this nix-lc3/gabarap interaction is thought to mediate efficient targeting of mitochondria to autophagosomes [66, 67]. similarly, when autophagy was inactivated by targeted deletion of the autophagy-essential gene atg7, post-differentiated white adipocytes exhibited large numbers of mitochondria compared with the relatively few mitochondria observed in wild-type white adipocytes. this suggests that mitochondria are preferentially eliminated by autophagy during adipogenesis [18, 6871]. third, mitophagy is induced by hypoxia in a bcl-2/adenovirus e1b 19 kda interacting protein 3-(bnip3-) dependent manner; the expression of bnip3 is regulated by hypoxia-inducible factor [7274]. this indicates that mitophagy might be a survival mechanism to regulate the production of ros from mitochondria during hypoxia. as shown here, mitophagy plays a role in several aspects of cellular physiology, not just eliminating depolarized mitochondria in a parkin/pink1-dependent manner. although there are at present more than 50 publications regarding parkin/pink1-dependent mitophagy, the precise mechanisms are still unknown. recently, it was reported that parkin induces rupture of the outer membrane of depolarized mitochondria, depending on proteasomal activity, and then the ruptured mitochondria are eliminated by mitophagy. this finding implies that parkin and pink1 are not the primary factors required for mitophagy but rather that they present depolarized mitochondria to the autophagic machineries by disrupting the mitochondrial outer membrane. most of the autophagy-related genes identified in yeast are also present in mammals, suggesting that the molecular processes of autophagy are conserved throughout evolution. it is surprising then that the molecular processes of mitophagy and the essential factors identified to date are completely different between yeast and mammals. for example, in mammals, the mitochondrial receptor protein corresponding to atg32 in yeast has not been identified. in recent years, there has been significant progress in studies of mitophagy in both yeast and mammals. in particular, the molecular processes and regulatory mechanisms of mitophagy in yeast have been well described, such as the specific atg32atg11 interaction and the requirement for signaling by the two mapks slt2 and hog1. meanwhile, the physiological role of mitophagy in mammalian cells has been well understood. because mitophagy is evolutionarily conserved, it is reasonable to speculate that there will be similar molecular processes, regulatory mechanisms, and physiological roles in both yeast and mammals. the interplay of yeast and mammalian mitophagy studies will consolidate our understanding of this cellular process.

mitochondria play an essential role in oxidative phosphorylation, fatty acid oxidation, and the regulation of apoptosis. however, this organelle also produces reactive oxygen species (ros) that continually inflict oxidative damage on mitochondrial dna, proteins, and lipids, which causes further production of ros. to oppose this oxidative stress, mitochondria possess quality control systems that include antioxidant enzymes and the repair or degradation of damaged mitochondrial dna and proteins. if the oxidative stress exceeds the capacity of the mitochondrial quality control system, it seems that autophagy degrades the damaged mitochondria to maintain cellular homeostasis. indeed, recent evidence from yeast to mammals indicates that the autophagy-dependent degradation of mitochondria (mitophagy) contributes to eliminate dysfunctional, aged, or excess mitochondria. in this paper, we describe the molecular processes and regulatory mechanisms of mitophagy in yeast and mammalian cells.

PMC3312226

pubmed-914

knowledge of the anatomy of root canal systems is an essential prerequisite for endodontic treatment. many of the problems encountered during and after root canal treatment occur because of inadequate understanding of the pulp space anatomy. studies on the internal and external anatomy of teeth have shown that anatomic variations can occur in all groups of teeth and can be extremely complex. this applies to mandibular incisor teeth as well, as many dentists fail to recognize the presence of a second canal. current knowledge of pulp space anatomy is based on research findings and individual case reports. there is a lack of consistency in the reported prevalence of second canals in mandibular incisors.[15] the differences may be related to study design (in vivo versus ex vivo), technique of canal identification (radiographic examination, sectioning and clearing) or to racial divergence. it is important to be familiar with variations in tooth anatomy and characteristic features in various racial groups, since such knowledge can aid location and negotiation of canals, as well as their subsequent management. additionally, a number of studies have shown different trends in shape and number of roots and canals amongst the different races.[1368] these variations appear to be genetically determined and are important in tracing the racial origins of populations. descriptions of the frequently occurring root canal systems of permanent teeth are based largely on studies conducted in europe and north america, and relate to teeth of mainly caucasian origin. these descriptions may not be fully applicable to teeth of non- caucasian origin. there are no published reports on the root canal anatomy of mandibular incisors in north east indian population. however, some studies have examined an indian population. the north-eastern population in india is mostly comprised of indo-aryans (caucasoid) mongoloids (tibeto-burman and paleo-mangoloid sub race) and dravidian sub populations. the population of north-east india is nearly 38 million, out of which tribal mongoloid population accounts for almost 42-45% of the total population. a total of five hundred and thirty six extracted mandibular incisors were randomly collected from general dental clinics within north-eastern india.out of the collected teeth, fifty six teeth were excluded because of immature or resorbed apices and four hundred and eighty teeth were selected for the present study. the gender and age of patients was not known, and no attempt was made to differentiate between central and lateral incisors. the samples were stored in 5.25% sodium hypochlorite (organo bio tech laboratories pvt ltd new delhi, india) for 30 minutes for the removal of organic debris, and then in 10% formaldehyde until use. the teeth were cleaned under running water, access cavities were prepared and the coronal pulp tissue extirpated in the canal orifices. the samples were stored in 5% nitric acid solution (aries laboratories, india) for 5 days. demineralization was assessed by the insertion of a paper pin in the crown and with the help of radiographs. the samples were then rinsed under running water for 4 hours and placed in 70, 80 and 95% ethyl alcohol successively, for 1 day. at the end of this period, the clearing procedure was completed by placing the samples in methyl salicylate (regent chemicals, mumbai)). at the end of the third day, complete transparency was achieved. india ink (united ink and varnishes pvt ltd, mumbai, india) was injected into the root canals of the transparent teeth using syringes with 27 gauge needle (shree uniya surgical, india). after the ink had dried, root canal morphology was examined by a magnifying glass of 5x magnification [figure 1], and the following observations were made: transparent specimen showing canal bifurcation number and type of root canals, presence and location of lateral canals and intercanal communications, location of apical foramina, andbifurcation of canals.canals were categorized into the first five types of vertucci's classification as follows: type i: a single canal is present from the pulp chamber to the apex.type ii: two separate canals leave the pulp chamber, but join to form one canal to the site of exiting.type iii: one canal leaves the pulp chamber, divides into two within the root, and then merges to exit in one canal.type iv: two separate and distinct canals are present from the pulp chamber to the apex.type v: single canal leaving the pulp chamber but dividing into two separate canals with two separate apical foramina. number and type of root canals, presence and location of lateral canals and intercanal communications, location of apical foramina, and bifurcation of canals. canals were categorized into the first five types of vertucci's classification as follows: type i: a single canal is present from the pulp chamber to the apex.type ii: two separate canals leave the pulp chamber, but join to form one canal to the site of exiting.type iii: one canal leaves the pulp chamber, divides into two within the root, and then merges to exit in one canal.type iv: two separate and distinct canals are present from the pulp chamber to the apex.type v: single canal leaving the pulp chamber but dividing into two separate canals with two separate apical foramina. type i: a single canal is present from the pulp chamber to the apex. type ii: two separate canals leave the pulp chamber, but join to form one canal to the site of exiting. type iii: one canal leaves the pulp chamber, divides into two within the root, and then merges to exit in one canal. type iv: two separate and distinct canals are present from the pulp chamber to the apex. type v: single canal leaving the pulp chamber but dividing into two separate canals with two separate apical foramina. results of this investigation indicate that one third of the teeth exhibit two canal system (36%). of the teeth with two canals, type iii configuration was most common followed by type ii and type v [figure 2]. although two canals were found in 36% of teeth, only 6.25% of canals exited in two separate foramina (type v) [table 1]. out of all the canals showing two canal configuration, around 83% joined and exited in single foramen (type ii and iii) and remaining 17% exited in two separate foramina (type v) [table 1].the apical foramen was found to coincide with the apical root tip in 47.2% of teeth [table 2]. in the present study, lateral canals were observed in around 13% of the cases [figure 3] [table 3]. anastomosis were found only in type iii canals which accounts for 2.5% of all the teeth [table 4]. apical ramifications were seen in around 7.42% of the teeth, out of which 75.7% were with two rami, 24.3%with three rami, and none with four rami [table 4] [figure 4]. in teeth with two canals, bifurcations were seen maximum in middle third (64%) followed by in cervical third 23.3% and in apical third 1.25% [table 5]. intercanal communications were observed in 28.4% of all teeth and in 70.2% of teeth with two canals [table 6]. canal configuration (from left to right) types i, ii, iii and v number and percentage of canal system types in mandibular incisors (n=480) in the study distribution of apical foramen in mandibular incisors (n=480) in the study curvatures in type i canal configurations- straight, s shaped and j shaped distribution of lateral canals in mandibular incisors in the study distribution of apical ramification in mandibular incisors in the study type i variations- apical ramification, reticular structure and lateral canals distribution of position of canal bifurcation in mandibular incisors in the study distribution of intercanal communications in mandibular incisors in the study various methods have been used to study root canal morphology including radiographic examination, root sectioning, staining and clearing techniques, direct observation with microscope, sectioning and macroscopic observation, stereo microscope, spiral computed tomography, and cone beam computed tomography. vertucci used the clearing technique to study the root canal morphology of extracted mandibular anterior teeth. it has been reported that fine details of the root canal system can be visualized by staining and clearing[figure 1]. this technique also makes canal negotiation with instruments unnecessary, thereby maintaining the original form and relation of canals, and provides a three-dimensional view of root canal. the process of changing the tooth into a transparent object involves many physical and chemical changes. the inorganic constituents of the tooth are first dissolved by decalcification, and further water, air, and lipid components are removed by dehydration and by subsequent immersion in the clearing agents, th and this method was used in the present study as well. the literature on mandibular incisors reveals that 1168% of mandibular incisors possess two canals, although in many of these cases, the canals merge into one in the apical 13 mm of the root. vertucci examined the root canal morphology of 300 mandibular anterior teeth and reported a second canal in 27.5% of mandibular incisors. miyashita et al., reported that 12.4% of mandibular incisors contained two canals; however, only 3% had two foramina. sert et al., noted that two canals were present in 68% of mandibular central incisors. mauger et al., evaluated the canal morphology at different root levels in one hundred mandibular incisors, and reported that 98100% of the teeth had one canal in the area 13 mm from the apex. the differences between these morphology studies may be related to variations of examination methods, classification systems, sample sizes and ethnic background of tooth sources. in a study in jordanian population, it was found that 73.8% of the mandibular incisors possessed a single root canal, and 26.2% of teeth were with two canals. the results of the present study indicate that one third of the teeth exhibit two canal system (36%) [table 2] [figure 2]. it was found that 63.75% of mandibular incisors possessed a single root canal (type i) with straight, j and s shaped curvatures [figure 3] as well as frequent apical ramifications, lateral canals and reticular structures [figure 4]. only 6.25% of canals exited in two separate foramina (type v). of the teeth with two canals, type iii configuration was most common followed by type ii and type v. therefore, the frequency of two canals in the present study was within the range of previous reports. this is due to failure of the dentist to recognize the presence of the second canal, and the need for access cavities to have appropriate inciso-gingival extension to facilitate the location of lingual canals. none of the teeth were seen with type iv canal system, which may be due to smaller number of samples examined in the present study and any conclusion drawn needs to be based on study of a larger population. the apical foramen was found to coincide with the apical root tip in 47.2% of teeth [table 2]. this is higher than reported in previous studies that demonstrated that the apical foramen coincided with the anatomical apex in 1746% of cases. in the present study, total apical foramen count stands at 510 (n=480), which is because of type v canal configuration and apical ramifications this finding may be of significance in working length determination which often depends on the average position of the apical constriction relative to root apex. in the present study, lateral canals were observed in around 13% of teeth and were found most frequently in the middle of the canal [table 3] [figure 4]. lateral canals in the apical third account for 2.94%.this is consistent with the findings of miyashita et al.; however, much lower than that reported by vertucci. anastomoses were found only in type iii canals which accounts for 2.5% of all the teeth. apical ramifications were seen in around 7.42% of the teeth out of which 75.7% were with two rami, and 24.3% with three rami, and none with four rami [table 4]. in teeth with two canals, bifurcations were seen maximum in middle third (64%) followed by the cervical third 23.3%; and, the apical third 1.25% [table 5].this requires an individualized procedure for preparation and filling in each of these conditions to obtain the most desirable results. pulp space anatomy of mandibular incisors in an indian population show high incidence of complexity which includes variations in canal configuration, number of canals and presence of isthmus. intercanal communications were observed in 28.4% of all teeth, and in 70.2% of teeth with two canals [table 6]. the high percentage of intercanal communications in teeth with two canals may be of clinical significance, because it may be difficult to debride and fill these communications adequately. within the limitations of the present study, it can be concluded that overall, 36% of mandibular incisors in this north-east indian population had two canals. in the teeth with two canals, the type iii canal system was the most prevalent followed by type ii.type v was the least prevalent.

aim: to aim of this study is to investigate the root canal characteristics of mandibular incisors in a north east indian population using a canal staining and tooth-clearing technique. materials and methods: four hundred and eighty extracted mandibular incisors, collected from dental clinics within north east india were selected for this study. following pulp tissue removal, the teeth were decalcified with 5% nitric acid, dehydrated with ascending concentrations of alcohol and rendered clear by immersion in methyl salicylate. after staining of the canal systems with india ink, cleared teeth were examined under 5x magnification and the following features were evaluated: (i) number and type of root canals; (ii) presence and location of lateral canals and intercanal communications; (iii) location of apical foramina; and, (iv) bifurcation of canals. results:the majority of mandibular incisors had a single canal (63.75% of teeth possessed a type i canal system). although 36.25% of the roots possessed two canals, only 6.25% had two separate apical foramina. conclusions:the prevalence of two canals in this group (of north east indians) of mandibular incisors was 36.25% and is within the range of previous studies performed on populations of different racial origin.

PMC3227278

pubmed-915

according to the new report from world health organization (who) published in september 2011, cardiovascular diseases (cvds) remain the leading cause of death and disability in the world. noncommunicable diseases accounted for more than 36 million deaths in 2008 with cvds responsible for 48% of these deaths, cancers 21%, chronic respiratory diseases 12%, and diabetes mellitus 3%. over 80% of cvd deaths occur in low- and middle-income countries. although, a large proportion of cvds is preventable, they continue to rise mainly because preventive measures are inadequate and it has been projected that by year 2030, almost 23.6 million people will die from cvds. similarly, majority of cvd deaths in low income countries occur in individuals less than 60 years of age [1, 2]. interestingly, while actions to reduce blood pressure and cholesterol are having an impact on overall cvd mortality in high-income countries, there is worsening of cvd risk profiles in most developing regions of the world. in sub-saharan africa, the prevalence of cvds has reached near epidemic proportions with sh being the main driver of cardiovascular complications. whereas sh was said to be rare in africans in the first half of the twentieth century, it is the commonest cause of heart failure, stroke, and kidney disease from many studies in africa. this upsurge in the incidence of sh and its complications in sub-saharan africa with high burden of infectious diseases and poverty had greatly reduced the life expectancy in this part of the world. although there are indications that sh plays a major role in cardiovascular outcomes, there is paucity of data particularly in the northeast nigeria on mortality profile of hypertension-related admissions into the medical wards, hence this study. this is a retrospective study which reviewed the frequency and outcome of hypertension-related admissions at the atbuth, bauchi north east nigeria. the hospital is a 650-bedded tertiary health care facility established in 2009 with 130 beds dedicated to medical admissions. the health facility serves as a referral centre for the residents of bauchi state with estimated population of 4.7 million (2006 nigerian population census) and neighbouring states of gombe, yobe, and adamawa. diagnoses and management of all patients admitted into the emergency unit are usually discussed the next day at the morning review with all consultants in attendance. this is to ensure accurate diagnosis of cases and to provide quality care to the patients. blood pressure was measured in the emergency department using a standard mercury sphygmomanometer (accoson) every 30 minutes until it was stable. hypertension was defined as admission blood pressure of 140/90 mmhg or antihypertension medication usage. records of all patients admitted between 1st november 2010 and 31st october 2011 were studied. although we independently reassessed patients ' diagnoses from their medical records, the final diagnosis used in this study was that of the managing consultants. this was because there was high correlation between reviewed diagnoses and the initial one by the managing team. case files of those admitted for various complications of sh were retrieved using both nurses and medical records admission books. necessary information was extracted from the case files and entered into a proforma designed for the study. patients who were admitted for hypertensive heart failure, stroke, or transient ischemic attack (tia) due to sh, hypertensive nephrosclerosis and hypertensive emergencies or urgencies were included. excluded from the study were cases with blood pressure less than 140/90 mmhg at presentation and who were not on antihypertensive medication and no documented stigmata of long standing sh. information obtained from the case notes was patient's age and gender, complications of sh, awareness of hypertension, duration of hypertension, drug compliance, alcohol use, and cigarettes smoking. others included patient's weight, height, body mass index, sbp, dbp, duration on admission and outcome. total number of admissions and hypertension-related admissions were noted, and the percentage of latter was calculated. a simple frequency distribution of sh complications was generated. student t-test was used to compare means of continuous variables while chi-square test was used to compare means of proportions. three thousand one hundred and eight patients consisting of 1603 (51.6%) males and 1505 (48.4%) females were admitted at the medical wards of atbuth during the period of the study. three hundred and fifty-two deaths occurred in males and 236 in females with percentage mortality being higher (p=0.0001) in males (21%) than in females (15.7%). of the total 3108 admissions, 735 (23.7%) were due to hypertension-related complications, with mean age of 51.9 17.5 years. diabetes complications with sh as comorbidity were seen in 96 (3.1%) patients, peripartal cardiomyopathy in 51 (1.64%), and stroke in the young not related to sh was diagnosed in 25 (0.8%) patients. others included dilated cardiomyopathy in 18 (0.6%) patients and coronary artery in 2 (0.06%) patients. a total of 1220 (39.3%) patients were admitted for cholera which ravaged bauchi state during the period of the study. when cholera patients were excluded from analysis three main occupations among the patients admitted included housewife (33.5%), trading (22.2%), and farming 21.4%). although hypertension-related admissions were 23.7% of total admissions, there was an excess of mortality from sh complications (42.9%). stroke/tia was the commonest complication of sh in the patients, and it accounted for 44.4% of cases. this was followed by hypertensive heart failure (27.8%), hypertensive emergencies (16.7%), and chronic kidney disease (11.2%). mean sbp and dbp were 167.4 18.2 and 98.6 13.5 at presentation. although 498 (67.8%) patients were aware of their sh status at presentation, only 269 (36.4%) were compliant with their antihypertensive medications. comparison of baseline characteristics of survivors and nonsurvivors of sh admissions is presented in table 2. nonsurvivors were older (p=0.001) and had longer duration of hypertension (p=0.041) and higher body mass index than survivors. similarly, pulse rate, sbp, and dbp were significantly higher in nonsurvivors than survivors (p=0.001, 0.001 and 0.001, resp.). the mean duration of hospital stay was significantly shorter in nonsurvivors (6.6 7.8 days) in comparison to survivors (11.7 13.6 days), p=0.001. the percentages for awareness of sh, compliance with antihypertensive medications, alcohol use, and cigarette smoking were similar between survivors and non-survivors. stroke had the highest mortality (39.3%), followed by chronic kidney disease (36.6%) hypertensive emergencies (30.9%) and hypertensive heart failure had the lowest intrahospital mortality (27.5%). while duration of sh and pulse rate at presentation was higher in males than in females, the mean body mass index was higher in females. on the other hand, mean age, duration on admission, sbp, and dbp were similar between the two groups. although mortality profile due to stroke, hypertensive heart failure, and chronic kidney disease as well as hypertensive emergencies was higher in males than females, the differences did not reach statistical significance. the probability of death increased with age (odds ratio=1.024, p=0.02), pulse rate at admission (odds ratio=1.02, p=0.001), and duration on admission (odds ratio=0.957, p=0.002). the findings from this study showed that hypertension-related admissions accounted for sizeable proportion (23.7%) of medical admissions among adult patients from a tertiary health institution in northeastern nigeria. this result is comparable to 20.2% from another tertiary hospital in southsouth and 18.4% from southeastern nigeria. similarly ndjeka et al. reported 19% from a rural hospital in south africa. interestingly, when cases of acute cholera admissions were excluded from the data because cholera was epidemic during the study period, the proportion of hypertension-related admissions rose to 38.9%. the three commonest complications of sh were stroke (44.4%), hypertensive heart failure (27.8%), and hypertensive emergencies (16.7%), respectively. although hypertension-related complications accounted for 23.7% of medical admissions, mortality related to this condition was 42.9%. this mortality profile is high and may be a reflection of severity of sh complications in the patients. casefatality of hypertensive-related admissions was 34.3%, and this was significantly influenced by age, duration of sh, body mass index, and admission pulse rate. factors that were predictive of mortality in this study were patients ' age, admission pulse rate, and duration of admission. some of these factors have also been identified in a study among congolese. the overall mean length of hospital stay was 10.1 12.7 days (median 7 days). non-survivors had a median duration of 4 days at death compared to survivors with median stay of 8 days, and most deaths occurred within the first week of admission. mean duration of hospital stay has been documented in some studies to be associated with mortality. one important finding of this study is the fact that two-thirds of the patients had been diagnosed as being sh prior to admission. among those who were aware of their sh status, drug compliance rate was 54% this proportion is similar to what had been reported in other studies in africa [11, 13] and may be one of the reasons for high mortality in the region. high level of illiteracy along with poverty and side effects of medications was among several reasons given for poor compliance. unlike the study from congo where death was associated with younger age, report of this study showed that hypertension-related death was more in older people. the tendency of older people having more cardiovascular comorbid conditions could partly explain this observation. high pulse rate was another factor that predicted risk of death from complication of sh in this study. several published data have demonstrated a positive association between pulse rate and all-cause mortality in hypertensive individuals [14, 15]. this has an important implication for choice of medication especially in the presence of complications. although smoking and alcohol abuse had been associated with poor outcomes in hypertensive patients, findings from this study did not show any significant difference amongst survivors and non-survivor in this regard. in europe and america, hypertensive cardiovascular complications are commonly seen in older age group [16, 17]; however, relatively younger individuals were affected in our study. majority of our patients were not covered by the national health insurance scheme which has just started in nigeria and thus had to pay for drugs and services at the point of care which increase out-of-pocket expenses. in addition, a sizeable number of our patients were housewives and students with little or no sources of income. these reduce their financial capability to purchase antihypertensive medications and compliance with their treatment. stroke was the commonest complication of sh amongst this group of nigerians, and this agrees with previous studies from other regions in nigeria [8, 9, 17]. recent community and hospital-based data have shown increasing cases of stroke in africa [18, 19]. on the other hand reports from high income countries seem to suggest a decline in hypertension-related complications. part of the reasons for this is availability of potent antihypertensive medications with minimal side effects which are associated with greater compliance. mortality among males (21%) was significantly higher than in females (15.7%). gender-related difference in mortality among medical admissions had been reported from a hospital-based study in nigeria. although females tend to use hospital more frequently, male patients often present with complications. part of the limitations of this study includes the fact that it was a retrospective study and being hospital based. it might be difficult to generalize our result to the entire community since study was from a tertiary hospital which serves as a referral centre to the neighbouring states. low postmortem rate to confirm or refute diagnoses was another limitation of the study. in conclusion, sh complications are common among medical admissions in bauchi nigeria and occur at a relatively young age. hypertension-related admissions are associated with excess mortality, and stroke is the commonest. community intervention to health-educate the populace on the need for early detection, dangers of sh, and other cardiovascular risk factors is urgently needed. women empowerment programs and provision of gainful employment by the government will reduce poverty and improve medication compliance among hypertensive individuals.

cardiovascular disease has reached near epidemic proportion in sub-saharan africa, and systemic hypertension (sh) remains the driver of cardiovascular complications. we studied hypertension-related admissions and their outcome at the abubaker tafawa balewa university teaching hospital (atbuth) bauchi, northeast nigeria. records of all patients admitted into the medical wards between 1st november 2010 and 31st october 2011 were studied, and case files of those managed for sh complications were selected for detailed examination. of the total 3108 admissions, 735 (23.7%) were hypertension related. mean systolic blood pressure (sbp) and diastolic blood pressure (dbp) were 167.4 18.2 and 98.6 13.5, respectively, at presentation. although, hypertension-related admissions were 23.7% of total admissions, there was an excess of mortality associated with sh complications (42.9%). stroke was the commonest, and it accounted for 44.4% of cases. stroke had the highest mortality (39.3%), followed by chronic kidney disease (36.6%); hypertensive emergencies (30.9%) and hypertensive heart failure had the lowest intrahospital mortality (27.5%). in conclusion, sh-related admissions are common among medical admissions in bauchi nigeria and are associated with high mortality. community interventions that promote early diagnosis and reduction of cardiovascular risk profiles are urgently needed to reduce sh deaths.

PMC3388339

pubmed-916

the prevalence of child obesity is increasing rapidly worldwide. based on the data of the world health organization (who), over 30% of the people in the middle east are overweight. a review of some studies from iran reveals that the prevalence of obesity among iranian children has been doubled between 1993 and 1999, which could be attributed to the changes in lifestyle.1, 3, 4 obesity in adults is not easy to treat and is often correlated with obesity during childhood. in the last decade, consumption of soft drinks and fast foods together with less activity and exercise contributed to the increasing number of overweight people worldwide. along with increasing caries risk, increased consumption of sugar-sweetened beverages and snack foods also has been linked to obesity. health risks associated with childhood overweight and obesity are strong indicators for predisposition to adult morbidity and mortality and include type 2 diabetes, cardiovascular disease, fatty liver disease, early maturation, endometrial, colon, breast and other cancers, psychological stress, as well as orthopedic problems, general poor health, and hepatic problems.7-10 an iranian study detected a high prevalence of cardiovascular risk factors in overweight and obese children aged 7-12 years. obesity is also related to several aspects of oral health, such as caries, periodontitis and xerostomia. fruit drinks and other sweetened beverages represent high sugar sources, which may contribute to cariogenic potential. oral health is strongly influenced by the daily intake of food and can also play a significant role in nutritional intake and general status of health, particularly in the elderly. who recommends limiting added sugars to 10% of total calories on the grounds that added sugars contributed to overall caloric density of the diet. a study in scotland showed that among 165 children aged 3-11 years, whom with the worst decay were significantly thinner also severe dental decay was associated with underweight. one brazilian study concluded that there was no statistically significant association between dental caries and obesity in adolescents aged 12 to 15 years. among children and teenagers aged 2-20 years, body fat amount changes as the body grows and is different for boys and girls. unlike body mass index (bmi) assessments for adults, assessments for children and teenagers take these growth and gender specific differences into account. these child-specific bmi values are referred to as bmi-for-age. categories describing amount of body fat for children and teenagers is also different from the categories describing amount of body fat in adults. among adults, bmi categories include underweight, normal, overweight, and obese. among children and teenagers, bmi-for-age categories include underweight, since the frequent consumption of high caloric and cariogenic substances are two of the many factors associated with caries and obesity, and to the best of our knowledge there are no studies that have investigated the association between bmi-for-age and dental caries in iran to date, the purpose of this study was to determine whether bmi-for-age, might be associated with dental caries in children in isfahan, iran. this cross-sectional study was conducted from june to august 2007 in isfahan, iran. using a random cluster sampling, a total of 1003 pupils aged 6-11 years from six private and state elementary schools with different social backgrounds the children thoroughly chewed one disclosing tablet (svenska dentorama ab, stockholm, sweden) and rinsed their mouth with water, according to the manufacturer s instructions. the children who had 70% or fewer teeth surfaces with plaque were invited for further evaluations. the sample size was estimated allowing for caries prevalence of 60%, and significance level of 0.05. all of the children participated in the clinical examination were iranians and permanent residents of the city. body weight was recorded to the nearest 100-gram using a standard beam balance scale with the subject barefoot and wearing light dresses. the balance was calibrated at the beginning of each working day and at frequent intervals throughout the day. body height was recorded to the nearest 0.5 cm according to the following protocol: no shoes, heels together and head touching the ruler with line of sight aligned horizontally. to avoid subjective errors, bmi-for-age percentiles, representing eating habits in children and teenagers, were used. bmi-for-age [(weight in kilograms)/(height in meters)] percentiles are dependent on gender and age-specific weight for height curves for those aged 2-20 years. according to these curves, underweight is defined as bmi-for-age<5th percentile, normal is defined as 5th percentile<bmi-for age<85th percentile, at risk of overweight is defined as 85th percentile<bmi-for-age<95th percentile, and overweight is defined as bmi-for-age>95th percentile. since the number of samples in underweight group was insufficient (three samples), this group was excluded from the study. the remaining 633 children (317 boys and 316 girls) enrolled in the study. all selected children were clinically examined for dental caries by a specialist utilizing the who criteria for diagnosis of dental caries. the examination was carried out using a cycle and cow horn two-headed dental explorer (aesculap ag, tuttlingen, germany), a plane mouth mirror (aesculap ag) and cotton rolls to remove any plaque or debris where necessary, and recorded on special charts. teeth were considered as decayed when in addition to showing clinical signs such as a color change, wedging and catching of an explorer tip during the examination of occlusal surface encountered some degree of resistance., a surface is diagnosed as decayed if the explorer is retained. dressed and restored teeth that had recurrent caries teeth filled with temporary materials were considered as filled, and not as decayed; no radiographs were taken. missing teeth were not marked correspondingly, since no definite statement could be made without a proper anamnesis whether the tooth really existed, or if an early extraction had taken place. to assess the caries frequency the dft index for the permanent dentition and the dft index for primary dentition were used, since it gives a good insight into the state of decay in the patient. if both the deciduous and permanent teeth were present, only the permanent teeth were evaluated. t-test was used to analyze the mean decayed and filled permanent/primary teeth (dft/dft) and the difference between groups, chi square test for evaluation of association between bmi-for-age and gender, and multiple linear regression for evaluation of association between bmi-for-age and dft/dft indices. the authors certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research. the findings showed that 16% of the children were within normal weight range, 16.9% at risk of overweight, and 67.1% overweight. there was no statistically significant association between bmi-for-age and gender (p=0.2) (table 1). the mean se of dft in the normal weight, at risk of overweight and overweight groups were 0.34 0.08, 1.23 0.13 and 0.73 0.05, respectively; and mean se of dft were 2.01 0.19, 2.76 0.18 and 2.59 0.13, respectively. multiple linear regression showed that there was no statistically significant association between bmi-for-age and dft (r=0.06; ci: 0.01 to 0.09), and bmi-for-age and dft (r=0.07; ci: 0.003 to 0.046). by taking the bmi-for-age into account, 27.7% of the children with normal weight were caries free, whereas only 14% of children at risk of overweight and 37.2% of the overweight children were caries free. there was statistically significant association between bmi-for-age and being caries free (p=0.0001). given the causative relation between refined carbohydrates and dental caries, it is appropriate to hypothesize that overweight might also be a marker for dental caries in children and teenagers. although bmi is widely used to screen adults for obesity, its use in adolescents is controversial. bmi is a commonly used measure of adiposity, because it is easy to calculate, quick to measure, and noninvasive. unfortunately, it is a fairly poor index in children unless age and gender are taken into consideration. therefore, bmi-for-age categories were used in this study which might produce controversial results compared to the other studies. results of the present study showed the high prevalence of overweight and at risk of overweight in the 6-11-year-old children in isfahan. a study showed that prevalence of overweight was 20.8% in 3-9 year-old children according to bmi. palmer implicated that the relationship between obesity and caries in children needs further exploration; it is clear that there are common denominators that both diseases share. negative changes in eating and activity patterns, increased frequency of snacking and increased consumption of fermentable carbohydrates are common in both obesity and caries in children. the mean dft/dft indices in the normal weight children were less than children at risk of overweight and the overweight. although it was hypothesized that bmi-for-age would be associated with increased dental caries prevalence, there was no association between bmi-for-age and dft/dft indices (r 0.07). the relationship between nutrition and dental caries is complex because it is a multifactorial disease; oral hygiene, available nutrients, saliva, and oral flora influence dental caries. chen et al investigated bmi index and dft score in three-year-old children. they concluded that there were no significant differences in the dft score of carious children among different bmi groups and there is no relationship between carious deciduous teeth and weight status. kantovitz et al in a systematic review on the relationship between obesity in childhood found that only one study with high level of evidence showed direct association between obesity and dental caries. macek&mitola concluded that there is no statistically significant association between bmi-for-age and dental caries prevalence for children in either dentition and overweight children with a positive history of dental caries in the permanent dentition exhibit fewer dmft than do their normal weight peers. the authors found that overweight children aged 6-17 years had a significantly lower dental caries severity than did children of normal bmi-for-age. hilgers et al found that the mean caries average for permanent molars significantly increased with increased bmi, even after adjusting for age and gender. they concluded that elevated bmi is associated with an increased incidence of permanent molar interproximal caries. burt&pai reported that children of low birth weight subsequently developed more caries in the primary dentition than did children with normal to high birth weight. they concluded that this may be related to social deprivation factors during the development of the primary dentition. willerhausen et al indicated a strong association of obesity and caries in 1290 children of elementary schools in a city in germany. another study found that there was a significant correlation between the bmi and caries frequency even after adjustment for the age of the children. one finnish study followed 516 children from birth to age 12 and used weight to predict caries experience (dmft/dmft). the investigators reported that obesity alone was not a very good predictor of dental decay. the present study demonstrated a significant association between bmi-for-age and being caries free (p=0.0001). this is in line with the results of the study of willerhausen et al that showed number of healthy teeth decreased with age and bmi.a swedish study examined the relationship between dental caries and risk factors for atherosclerosis and reported that children with a dmft score greater than 9 had significantly higher bmi-for-age values than caries-free children. glick noted that, while the consequences of obesity will have an indirect effect on oral conditions, this alone is not justification to get involved. rather, there needs to be a stronger desire to have an impact on the patients general health. these findings might also show that further investigation should be conducted to determine if an association exists between dental caries and bmi-for-age and to address what factors specific to overweight in children might be protective against dental caries especially in permanent dentition. the future preventive programs must include strategies for nutrition control to avoid obesity as well as dental caries. there is a high prevalence of at risk of overweight and overweight in 6-11-year-old children in isfahan. at risk of overweight and overweight children had higher dft/dft score than did normal children, but there was no association between bmi-for-age and dft/dft indices.

background and aims childhood obesity has been associated with high refined sugar intake. the relationship between sucrose intake and development of dental caries has been established. the aim of this study was to determine the association between age-specific body mass index (bmi-for-age) and dental caries in children. materials and methods in this cross-sectional study a total of 1003 children were screened. weight, height, bmi-for-age and dental caries of 633 (317 boys, 316 girls) 6-11-year-old children were clinically examined for dental caries using the world health organization criteria by a dentist on a dental chair. decayed and filled teeth (dft/dft) indices were used. bmi-for-age and dental caries were analyzed with multiple linear regression, chi-square, and t-tests using spss computer software. results the findings showed that 16% of the children had a normal weight, 16.9% were at risk of overweight, and 67.1% were overweight. in the normal weight, at risk of overweight, and overweight groups, the mean se for dft were 0.34 0.08, 1.23 0.13 and 0.73 0.05, respectively; and 2.01 0.19, 2.76 0.18 and 2.59 0.13 respectively, for dft. multiple linear regression showed that there was no statistically significant association between bmi-for-age and dft (r=0.06) and dft (r=0.07) indices. 27.7%, 14% and 37.2% of children with normal weight, at risk of overweight and with overweight were caries free, respectively. there was statistically significant association between bmi-for-age and being caries free (p=0.0001). conclusion there was no association between bmi-for-age and dft/dft indices. in addition, a high prevalence of at risk of overweight and overweight was seen among 6-11-year-old children in isafahan, iran.

PMC3529887

pubmed-917

rheumatoid arthritis (ra) is an obstinate, systemic inflammatory disease which has a negative impact on the quality of life.1 anti-tumor necrosis factor (tnf) therapy has proved to be beneficial to ra patients because it can suppress inflammation and joint damage;2 therefore, the percentage of ra patients treated with ant-tnf agents is steadily increasing. adalimumab (ada), a fully human anti-tnf monoclonal antibody, exhibits excellent effectiveness in ra; however, its use has been reported to cause many adverse events in ra patients. tnf is an important cytokine involved in initiating a protective immune response; therefore, patients receiving this therapy may be at a high risk of infection. legionella pneumophila is a fastidious intracellular gram-negative bacillus that requires special microbiological culture media., however, it seems that the risk of l. pneumophila pneumonia may be increasing in ra patients receiving tnf antagonist therapy.3 we report a rare case of pneumonia caused by l. pneumophila in an ra patient after only two ada injections. this report suggests a possible association between the use of ada and the incidence of pneumonia, which is a severe and often fatal infection. a seventy-eight-year-old japanese woman with seropositive ra, diagnosed in 1998 based on the acr 1987 criteria, underwent sulphasalazine (sasp) therapy before she visited our institute. in 2008 she was started on methotrexate (mtx) and low-dose prednisolone (psl) therapies. despite administration of low-dose therapies of mtx (6 mg/week), sasp (500 mg/day), and psl (2.5 mg/day), because of her age and history of drug intolerance, her ra disease activity remained high [tender joint count, 4/28, and swollen joint count, 11/28; patient global assessment score, 72 mm/100 mm; c-reactive protein (crp), 2.55 mg/dl; erythrocyte sedimentation rate (esr), 52 mm/hour; matrix metalloproteinase- 3, 71.7 ng/ml; and disease activity score 28-esr, 5.82]. furthermore, she had pulmonary emphysema and slight fibrosis in her bilateral lower lungs, and her sister had previously suffered from tuberculosis. however, the patient had no other lung diseases, including tuberculosis (negative result on the tuberculin test quanti-feron), no medical history of any viral infection, was not a hepatitis b virus carrier, and showed normal serum kl-6 and beta-d-glucan levels (410 u/ml and<2.84 pg/ml, respectively). therefore, we decided to include anti-tnf therapy along with her current therapy after giving isoniazid (inh) 300 mg/day for three months. ada at a dose of 40 mg was introduced in addition to mtx (6 mg/week), sasp (500 mg/day), psl (2.5 mg/day), and folic acid (5 mg/week) in june 2011. she experienced excellent pain relief in her joints after her first subcutaneous ada injection, with no immediate adverse effects; her second injection was therefore safely administered two weeks after her first injection. eight days after her second ada injection, she had fever (38.6 c), fatigue, and bloody sputum for two consecutive days; thereafter, she was admitted to our hospital. at that time, we first heard that she regularly visited a public bath and had continued to do so after ada treatment. on admission (day 0, 8 days after her second ada injection), her body temperature was 38.6 c and she was slightly tachycardic (96 beats/minute) with a blood pressure of 142/68 mmhg. her heart sounds were normal, and she had neither chest pain nor visible rash. however, coarse crackles were audible in her right lower lung, and oxygen monitoring showed hypoxemia (spo2, 92%). her joints were not swollen, painful, or warm, so we did not consider this state as a flare of ra. on the other hand, laboratory data showed marked acute inflammation (crp, 27.05 mg/dl; white blood cell count, 20,600/mm) and a positive urine l. pneumophila antigen test (binax, portland, or, usa). however, her serum beta-d-glucan levels were normal (< 3.30 pg/ml). in addition, her expectoration culture, collected two days later, was weakly positive for haemophilus influenzae (blnar). chest x-ray showed a permeation shadow in her right lower lung, and chest computed tomography (ct) showed pleural thickening, light ground glass opacities (ggos) right under the pleura, and consolidation with an air bronchogram in the right s6, s9, and s10 segments without fibrotic change from her baseline lung (fig. the patient was clinically diagnosed with pneumonia caused by l. pneumophila affecting her originally fibrotic ra lungs. she was then admitted and given intravenous pazufloxacin (pzfx) 1000 mg/day (after an initial administration of 2 g twice daily at days 0 and 1) for 10 consecutive days. she showed remarkable recovery after being injected with pzfx and was discharged on day 10. after discharge from our hospital, we confirmed her seroconversion of l. pneumophila and complete healing of pneumonia on chest ct. mtx (6 mg/week) and psl (5 mg/day) therapies were then restarted without ada because of an increase in her ra disease activity at day 20. however, pneumonia relapsed at day 55 along with loss of appetite, fatigue, and coughing. laboratory data showed acute inflammation (crp, 13.09 mg/dl; white blood cell count, 10,500/mm), and her urine l. pneumophila antigen test (binax) was again positive. in addition, both her serum kl-6 and beta-d-glucan levels were high (815 u/ml and 29.56 pg/ml, respectively) unlike before. blood gas data showed severe hypoxemia (po2, 47.5 mmhg; pco2, 29.5 mmhg). chest ct findings showed multifocal consolidation with ggo extending to the bilateral lower aggravated fibrotic lungs (fig. 2a and b) unlike pneumocystis pneumonia (pcp), which mostly showed diffuse ggo of the bilateral lungs. furthermore, neither dna of pneumocystis jiroveci from bronchoalveolar lavage fluid (balf) nor serum cryptococcus and aspergillus antigens were positive. we could not detect any bacteria from her balf culture. on the other hand, serum candida antigen and culture of candida albicans were positive. on the basis of these findings, we diagnosed a relapse of l. pneumophila pneumonia with deep mycosis (eg, candida infection) and interstitial pneumonia (ip). the patient s lungs were previously affected by ra, but not pcp. unfortunately, although we tried every possible treatment, the patient eventually died of aggravated ip on day 79 (kl-6, 5030 u/ml; beta-d-glucan, 8.05 pg/ml at day 72). ada, a fully human anti-tnf monoclonal antibody, has been used in japan since june 2008 for the treatment of ra and is administered at 40 mg once every 2 weeks or at 80 mg for cases of high disease activity which do not respond to 40 mg use. it is known to be very effective in decreasing inflammation, such as that in ra. however, it is also known that anti-tnf therapy may be a risk factor for a number of infections; in particular, ada treatment is considered a risk factor for reactivation of latent tuberculosis.46 anti-tnf agents including ada are said to be risk factors for bacterial pneumonia.7,8 in addition, ra disease severity in itself is known to be one of the strongest risk predictors of infection.9 as mentioned above, patients treated with anti-tnf agents are generally believed to be at an increased risk of bacterial infections.10 conversely, another study found that the severity of serious infections was not increased in anti-tnf-treated patients compared with a disease-modifying antirheumatic drugs (dmard)-treated cohort.11 considering the risks associated with the use of anti-tnf agents, we restrict their administration and occasionally exclude immunocompromised patients with co-morbidities, such as those with diabetes mellitus, heart disease, viral hepatitis, and lung disease, or elderly patients from these treatments. our patient was a 78-year-old woman with ra, who had a long-term smoking habit and had bilateral fibrotic lungs; these were possibly be risk factors for legionella infection in patient as well as other infections such as pcp. legionellosis presents as two types of diseases; one is legionnaires disease, the more severe form, and the other is pontiac fever, the milder form, which shows minimal heat and muscular pain, and has a rapid complete recovery.12 legionellos is also often presents as pneumonia and is mainly caused by the l. pneumophila serogroup 1 (sg1), a ubiquitous, opportunistic, gram-negative intracellular pathogen.3 tnf induces differentiation of monocytes into macrophages, which are essential in the induction of granuloma, and is important for maintaining the integrity of granuloma.13 in addition, tnf plays an important role in host resistance against infectious agents, especially those multiplying intracellularly.14 thus, treatment with tnf antagonists is associated with an increased risk of infection, particularly infections caused by intracellular microorganisms such as l. pneumophila. in contrast, in japanese post-marketing surveillance with tnf antagonists for ra, both fatal and surviving cases of legionella pneumonia were reported to be very rare (infliximab, 1 and 0 in 7522 cases; etanercept, 0 and 0 in 13894 cases; ada, 3 and 2 in 3084 cases, respectively). legionella pneumonia tends to be a severe and fatal form of community-acquired pneumonia very similar to pneumococcal pneumonia. nowadays, however, l. pneumophila pneumonia is considered to be a curable disease if appropriate antimicrobial therapy is administered at an early stage. usually, legionellosis is considered to be caused by inhalation of aerosols containing l. pneumophila and bathing in hot springs and circulation-type bathtubs available in public bathing facilities that are infected with l. pneumophila. but further investigations showed that her case was an isolated infection and was not part of a mass outbreak, suggesting that l. pneumophila infection in our patient was more likely caused after ada treatment. nowadays in japan, both the introduction of the urinary legionella antigen test and improvements in the infectious disease law have led to an increase in the number of reports of l. pneumophila. accordingly, milder cases of legionella pneumonia have been reported. in the past, many cases of legionellosis might have been overlooked, including milder cases of legionella pneumonia or minor pontiac fever. winthrop-university hospital criteria are said to be very useful for discriminating fairly well between l. pneumophila pneumonia and bacteremic pneumococcal pneumonia, at the time of hospitalization, for community-acquired pneumonia.15 chest ct findings of legionella pneumonia are said to be bilateral or unilateral with single and multifocal consolidation and ggo,16 while those of mild legionella pneumonia are said to be bilateral, with multiple affected segments and peripheral lung consolidation with ggo.17l. pneumophila includes 16 types of serogroups, and only sg1 can be detected by the urine l. pneumophila antigen test (binax). the percentage of l. pneumophila sg1 may be high in japan, as it is in a lot of countries, but we are unable to detect all l. pneumophila by this antigen test. urine l. pneumophila antigen test may be positive for a few weeks after treatment of legionella pneumonia as seen in this case; however, we diagnosed this case as a recurrence of l. pneumophila pneumonia with deep mycosis (eg, candida infection) and ip on the basis of data from serum, culture, balf analysis, and ct scans, which showed multifocal consolidation with ggo extending to the lower parts of her fibrotic lungs bilaterally at recurrence (fig. the risk of l. pneumophila pneumonia is reported to increase in patients receiving tnf antagonists, with a relative risk of 16.521, compared with that in the overall population in france.3 the use of corticosteroids and mtx might also have played a role in the emergence of this infection, but the patient had been treated with these medications for a long time. legionella tends to colonize the respiratory tract of immune-compromised patients and causes severe health problems once their immune system is further affected.18 in our case, because the patient did not visit any public bath after discharge from our hospital, it is possible that although she initially recovered from l. pneumophila pneumonia and was discharged from the hospital, the organism was still present in her respiratory tract, causing pneumonia to recur and ultimately prove fatal. in such cases, it is important to prevent tuberculosis by inh, pcp by trimethoprim sulfamethoxazole, and pneumococcal pneumonia by vaccination, in all immune-compromised ra patients before the first treatment with anti-tnf agents; additionally, it is important to carefully monitor ra patients who have been given anti-tnf agents in the past, not only in the early phase when they are first given tnf inhibitors, but also afterwards, especially after they have recovered from infections such as that described in our case. to our knowledge, this is the first reported fatality triggered by relapsing pneumonia caused by l. pneumophila with deep mycosis and ip in an ra patient treated with ada. the patient eventually died of aggravated ip following a relapse of pneumonia caused by l. pneumophila. to avoid exacerbation of a patient s illness and to prevent fatality due to severe infection, it is important for us to be aware of the fact that the use of tnf-inhibitors can lead to infections such as that described here. because a delay in controlling some infections worsens the patient s ra disease activity during that period, it is important to control any infection rapidly and completely. recently, ra treatment has been rapidly and aggressively adopted based on the concept of treat-to-target; therefore, we need both to accumulate evidence from case studies to promote safe and effective treatments for ra, and to avoid therapeutic errors, which will result in the maximum possible survival of patients.

we present a rare fatal case of relapsing pneumonia caused by legionella pneumophila in a patient with rheumatoid arthritis after only two injections of adalimumab. a 78-year-old japanese woman with a 14-year history of rheumatoid arthritis was prescribed adalimumab because her disease activity remained high. however, 8 days after her second injection of adalimumab, she was admitted to our hospital and diagnosed with pneumonia caused by l. pneumophila. following intravenous antibiotic therapy, she recovered completely from pneumonia and was discharged on day 10, but pneumonia relapsed, resulting in death 79 days after the first episode of pneumonia. l. pneumophila can lead to recurrence of pneumonia that can ultimately prove fatal, similar to the present case. a review of the pertinent literature is also presented.

PMC3700963

pubmed-918

the aryl hydrocarbon receptor (ahr) is a transcription factor activated by numerous environmental ligands such as dioxins and polycyclic aromatic hydrocarbons (pahs). its endogenous ligand has not yet been described, but some endogenous compounds, notably oxidative derivatives of tryptophan, are already described as efficient activators. following ligand binding, ahr translocates to the nucleus, dimerizes with its partner the aryl hydrocarbon receptor nuclear translocator (arnt), and binds to xenobiotic responsive elements (xre) in target genes. ahr is known to be a key regulator of some xenobiotic degradation enzymes, notably cytochromes p450 belonging to the cyp1 family, which are involved in the bioactivation of various environmental procarcinogens including pah and arylamines. the ahr-mediated pathway is commonly viewed as an adaptive response toward these xenobiotic agents. recent data demonstrated that ahr mediates diverse endogenous functions in our close vertebrate relatives as well as our distant invertebrate ancestors, including cell proliferation, adhesion and migration, and inflammation [2, 3]. accidental exposure to dioxins, which are prototypes of environmental ahr ligands, leads to a broad spectrum of pathologies, ranging from cancers to cardiovascular diseases and type 2 diabetes [46], all of which involve an inflammatory process. using a triple-null mouse model that lacks the two receptors for tnf and tnf and the receptor for the il-1 and il-1 cytokines, it was demonstrated that il1-like cytokines play a central role in dioxin-induced inflammatory effects. we have shown in intestine that pah-induced ahr activation upregulates the expression of some inflammation target proteins, including proinflammatory cytokines such as il-1 and tnf [8, 9]. similar data have been observed in other cells and tissues, ranging from macrophages and breast cells to skin and lung [1013]. moreover, hollingshead et al. showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (tcdd) treatment in combination with il-1 or phorbol 12-myristate 13-acetate (pma) results in a marked synergistic induction of il-6 levels over what is seen without ahr activation. since tcdd induces il-6 expression through the ahr pathway, this synergistic effect could be partly explained by an inflammation-induced increase in ahr expression. the aim of this study on caco-2 cells was to investigate the effect of signals known to be proinflammatory on ahr expression and to describe the molecular mechanisms involved. phorbol 12-myristate 13-acetate (pma) was sourced from sigma (france), il-1 from peprotech (france), anti-il1 antibody (ab2105) from abcam (france), and proteasome inhibitor set i from calbiochem (france). caco-2 human colonic adenocarcinoma cells and thp1 human monocytic cells were cultured as previously described [8, 14]. at confluence, cells were starved for 12 h without fbs (replaced by 0.2% bsa) and treated for 1 h to 24 h with either 100 nm pma or 200 nm il-1. in some experiments, caco-2 or thp-1 cells were treated with conditioned media. to obtain the conditioned media, cells were treated for 2 h with 100 nm pma, washed 3 times with pbs, and further cultured in 0.2% bsa medium. media samples were collected after 224 h incubation, and a new caco-2 batch was treated for 8 h with these conditioned media. total rna was isolated using a nucleospin rnaii kit (macherey-nagel, france) and reverse-transcribed at 42c for 1 h using gibcobrl m-mlv reverse-transcriptase (life technologies, france) and random primers. expression levels of target genes (ahr, il1-, il-8, tnf, and tgf) were determined using a lightcycler 480 system (roche, france). pcr was performed with 0.5 m of each primer using the lightcycler with mastermix plus for sybr green i no rox. cycling conditions were 10 min denaturation at 95c, followed by 40 cycles of 30 s denaturation at 95c, 30 s primer annealing at 60c, and 30 s fragment elongation at 72c. ahr, il-1, il-8, tnf, and tgf mrna expressions were normalized to 2-actin expression, and data were quantified by the 2 method. the 2.7 kb of the human ahr gene 5-flanking region (the 2103/+637 region of the ahr gene) was subcloned into the pgl3-enhancer luciferase vector (promega, france) as previously described to obtain the p3.48 construct. transient transfections were performed by lipofection (lipofectin, life technologies) in a serum-free and antibiotic-free medium containing 2% l-glutamine, with 0.5 g of p3.48. after 48 h treatment with 100 nm pma, luciferase activity was evaluated using the luciferase assay system from promega. ahr promoter analysis with mathinspector software (genomatix software, germany) revealed the presence of 3 ap1 and 3 nfb putative binding sites. these sites were mutated using the quickchange site-directed mutagenesis kit (stratagene, france). cells were transfected with 0.5 g of the mutated vectors, and after a 48 h treatment with 100 nm pma, luciferase activity was evaluated as described above. statistical analysis was performed using a mann-whitney test on graphpad prism (graphpad software). in order to evaluate the effect of proinflammatory conditions on ahr mrna levels, caco-2 cells were treated with pma or with il-1. the maximal (4.9-fold) induction of ahr mrna was observed after 8 h of treatment with pma (figure 1(a)). we also evaluated the expression of various cytokines after exposure to pma (figure 2). peak tnf, il-1, and tgf upregulation (10-, 53-, and 286-fold, resp.) occurred after 8 h of exposure. treatment of caco-2 cells with the proinflammatory cytokine il-1 was also associated with an increase in ahr mrna that was maximal (6.5-fold) after 8 h of treatment (figure 1(b)). taken together, these results showed that enhancement of ahr expression was associated with signals involved in proinflammatory processes. to see whether ahr induction (mrna) in response to pma was associated with an increase in ahr transcription, reporter gene expression was analyzed using the p3.48 construct in which luciferase expression was driven by the ahr promoter. treating wt p3.48-transfected caco-2 cells with 100 nm pma led to a 2.3-fold increase in luciferase expression (figure 3), showing that increased ahr expression in response to pma was mainly of transcriptional origin. pma is well known to potentialize inflammation-related processes through ap-1 and nfb pathways. ahr promoter analysis using matinspector software revealed the putative presence of 3 ap-1 and 3 nfb binding sites. the mutation of ap-1 sites did not modify ahr induction by pma (data not shown), suggesting that only the nfb pathway was involved. the effects of mutations of the three nfb binding sites found in the ahr promoter are summarized in figure 3. the mutation of one of the three sites did not significantly modify luciferase induction, whereas mutation of the first site proved most efficient. double mutation of sites 2 and 3 reduced the induction of luciferase expression by 45%, while mutation of all three sites totally abrogated this induction. taken together, these data strongly suggest that ahr induction involves the nfb pathway. in order to gain stronger confirmation of the role of nfb in ahr expression, we reduced nfb activation by inhibiting ib degradation using a supplier-specified proteasome inhibitors cocktail that includes proteasome inhibitor i, lactacystin, and mg-132. as shown in figure 4, using the proteasome inhibitor cocktail led to a 65% reduction in ahr induction by 100 nm pma, along with an 86% decrease in il-1 enhancement, demonstrating that the proteasome inhibitor cocktail was able to prevent the il-1 induction triggered by the nfb transduction pathway. caco-2 cells are able to produce cytokines, notably tnf and il-1, in response to proinflammatory signals. therefore, treating caco-2 cells with conditioned media from pma-treated caco-2 cells should result in ahr induction. media collected from 6 to 24 h after treating caco-2 cells with pma significantly upregulated ahr mrna. maximal activity (4.2-fold increase) was obtained with the 24 h conditioned medium (figure 5(a)). pretreating the cells with an il-1 neutralizing antibody (dilution 1/100) 4 h before exposure to 24 h pma-conditioned medium inhibited the induction of ahr expression, while pretreatment with rabbit isotype igg had no effect (figure 5(b)). in another experiment, caco-2 cells were treated with conditioned media from pma-treated thp-1 cells, and similarly we observed an induction of ahr mrna (4.4-fold increase) (figure 5(c)). our data therefore point to the involvement of a signalization loop which could lead to an enhancement of inflammatory processes. this study suggested the induction of an inflammation loop resulting from an initial ahr activation in the colon. indeed, this tissue through diet is effectively chronically exposed to various ahr ligands such as pah or food residues like dioxins or polychlorobiphenyls. our results obtained in caco-2 cells clearly demonstrated that both pma- and il1- enhance ahr transcript expression. this phenomenon was associated with an increase of ahr promoter activity. as inflammation-related processes mainly involve nfb and ap-1 transduction pathways, we carried out site-directed mutagenesis of ap-1 or nfb binding sites. mutagenesis of ap-1 was unable to decrease the induction of ahr promoter activity, whereas mutation of the 3 putative nfb binding sites abrogated the increase in ahr promoter activity. we also pretreated cells with a proteasome inhibitor cocktail in order to prevent degradation of the ib subunit and therefore inhibit nfb activation. this pretreatment inhibited both the induction of ahr expression after pma exposure and the increase of il-1 expression, which is known to be mainly regulated by nfb. taken together, these results demonstrated that proinflammatory conditions induce ahr expression at least partly through the nfb pathway. caco-2 cells express a number of cytokine receptors on their cellular membrane and are also able to secrete proinflammatory cytokines in response to initial inflammatory signals. our results demonstrated that treating caco-2 cells with a conditioned media derived from pma-treated cells also leads to an increase of ahr expression, and that this induction involved il-1 signaling. these results are consistent with our results from site-directed mutagenesis experiments and the treatments of cells with proteasome inhibitors. moreover, these data suggested that an autocrine loop could occur and probably generate and amplify a proinflammatory signal. indeed, environmental exposure to ahr agonists like pahs has been demonstrated to induce the expression of proinflammatory cytokines such as il-1 [8, 9] and to activate nfb. our data demonstrated that proinflammatory conditions induced and sustained through ahr expression could, therefore, increase cell susceptibility to pah-induced inflammation. pahs are potent ahr ligands that are present in tobacco smoke as well as diet, notably grilled meats. cigarette smoking is emerging as a strong risk factor in the otherwise unknown etiology of chronic inflammatory diseases. there are reports of a dose-response relationship between exposure to tobacco smoke and inflammatory bowel disease (ibd). the exact mechanisms by which smoking influences the development of ibd are poorly understood, but nicotine does not appear to play a critical role. interestingly, a recent study in dextran sulfate sodium-induced colitis mice reported that the attenuation of ahr expression resulted in a protective effect. moreover, ahr and its downstream targets, such as il-8, were significantly upregulated in ibd patients versus controls. the authors concluded that abnormal ahr pathway activation in the intestinal mucosa of ibd patients may promote chronic inflammation, and our results support this hypothesis. figure 6 proposes a possible explanation of the link between the ahr pathway and ibd. pahs such as benzo(a)pyrene, are bioactivated by cyp1 family enzymes into diolepoxides, such as benzo(a)pyrene diol epoxide, which activate nfb. the activation of nfb promotes an inflammatory loop via il-1 expression and induces ahr expression. the upregulation of ahr would, in response to pah exposure, enhance both cyp1 inducibility and pah-inflammatory properties. nfb-controlled pathways were classically divided into two branches: the classical pathway involving rela subunit and ikk and the alternative pathway involving relb subunit and ikk. physical interactions between ahr and each one of the nfb subunits were reported to induce distinct effects via specific sequences. interaction of ahr with rela induced a downregulation of gene expression controlled by rela as in the case of cyp1a1 or il-6 gene. in opposite, interaction of ahr with relb enhances dre-reporter gene activity of cyp1a1 and transcription of some nfb target genes such as il-8 and other chemokines through binding on specific relb/ahre sequences [23, 24]. furthermore, nfb-binding sites that are preferentially recognized by relb/p52 are spontaneous targets for relb/ahr complexes (i.e., independently of addition of any exogenous ligand). relb/ahr complexes are also found to bind on xre, as well as nfb consensus elements, and relb drastically increases the tcdd-induced xre-luc reporter activity. vogel and matsumura propose that ahr assists the function of relb not only to mediate chronic inflammation but also to promote relb's function in resolution of inflammation via negative feedback mechanisms, whereas ahr antagonizes the action of rela to moderate acute cellular inflammation and/or protect cells from unwanted side effects of full activation of inflammatory effects of rela. our data showed that in vitro, proinflammatory conditions enhance ahr expression through nfb pathway, therefore, it would be of interest to evaluate if the enhanced expression of ahr was also associated with an increase of relb/ahr complex formation and if such an interaction promotes in vivo either inflammation or its resolution. in conclusion, we demonstrated for the first time that compounds inducing proinflammatory cytokine expression enhance ahr expression in intestinal epithelial caco-2 cells through the nfb transduction pathway. several pieces of evidence point to ahr as a potential new target in the management of ibd and suggest that the modulation of ahr signaling pathway via diet, smoking cessation, or the consumption of ahr antagonists such as resveratrol could be a viable new strategy for the prevention and treatment of ibd.

recent data suggest that apart from its well-known role in the regulation of xenobiotic metabolizing enzymes, ahr is also involved in inflammation. however, the influence of inflammation on ahr expression remains unknown. here, we demonstrated that proinflammatory conditions induced by either pma or il-1 enhance ahr expression in caco-2 cells. this was associated with an increase in ahr promoter activity. by means of directed mutagenesis experiments and the use of proteasome inhibitors, we demonstrated that inflammation-induced ahr expression involved the nfb pathway but not ap-1. moreover, conditioned media from pma-treated caco-2 cells were also able to induce ahr expression, and this induction was repressed by anti-il-1 blocking antibodies. similar results were obtained with conditioned media from pma-treated thp-1 cells. taken together, these data suggest that ahr could be involved in vivo in an inflammatory loop. ahr was recently suspected to be implicated in inflammatory bowel disease. our results support this hypothesis and suggest that ahr could be a new target for inflammatory bowel disease patient management.

PMC3818893

pubmed-919

compared to many other clinical settings, pain and distress are common complaints in dental clinical practice. closely linked is that, such complaint of pain or distress among dental patients can be indicative of either a physical or psychological pathology. although, psychological distress is a non-specific term for negative mood states that encompasses sadness, frustration and anxiety among others; however, it is a valuable indicator of emotional ill-health. this is because psychological distress often refers to both the symptoms of psychiatric disorders and emotional responses to adversity and it is sometimes used as a screening for measure of probable psychiatric morbidity. despite being contiguous with psychological distress, emotional pain (psychological pain or psychache) is more indicative of a sustained state of inner turmoil, a perception of negative changes in the self and its functions that are accompanied by negative feelings. this pain is deeper and more vicious than depression, although depression may be present as well. for instance, it is known that patients who experience emotional pain or bodily ache without identifiable and adequate physical causes may be symbolically experiencing an intra-psychic conflict or psychological disorder. dental patients may suffer from both physical diseases and mental disorders presenting with psychological symptoms like mild anxiety and depression. the diagnosis and treatment of mental disorders, therefore, should be especially relevant to dental practitioners. as it is, dental specialists often come across patients, who present with complaints of pain, abnormal sensation and movement around the orofacial region, and hypersalivation, which are manifestations of underlying emotional disturbance and not due to a clearly identifiable physical cause. for instance, existing literature suggest that recognizable psychopathology is seen in about 30% of patients attending dental clinics with complaints of distress or pain, and this often goes undetected and untreated. early and appropriate recognition of such psychological distress would benefit both the individual patient and health care providers. moreover, unattended emotional ill-health often delays presentations, compromises treatment adherence and impairs quality of life among dental patients. importantly, the knowledge of psychological distress and emotional pain among dental patients would directly provide a framework for collaboration between dental practitioners and psychiatrists, and indirectly lead to a better understanding of psychiatric disorders by dental specialists. unfortunately, there are no controlled study on the prevalence of psychological distress and emotional pain in adult nigerian dental patients to the best of our knowledge following an extensive literature search. therefore, the aim of this study was to compare, with a matched control, the prevalence of psychological distress and emotional pain among patients attending an outpatient dental clinic in nigeria. we hypothesized that compared to the controls, dental patients would experience significant burden of emotional pain with psychological distress; and some demographic factors would constitute identifiable correlates. a total of 201 participants were recruited over a period of six months into the study. of these, 101 were dental participants who were attendees of oral and maxillofacial unit of the study hospital. they presented with jaw pain and were to have oral surgery (test group) and were matched with 100 controls (non-patients; normal relatives of dental patients). specifically, the test subjects consisted of consecutive patients attending the maxillofacial surgical outpatient/exodontia clinic of lagos university teaching hospital. the control participants were selected from non-patients of relatives of dental patients and matched for age and sex with the test group. selection criteria included: being a patient registered at the dental outpatient clinic, informed consent, aged between 18 and 60 years and no current or past history of psychiatric illness. the study protocol was sent to the health and research ethics committee of lagos university teaching hospital and approval was obtained before commencement of the research. written informed consent was obtained from each of the participants to take part in the study and assurance was given to participants that they could decline participation at any point without any negative consequences. all data were treated with confidentiality and those with significant distress were counseled and referred for the indicated care. all participants completed a pre-designed socio-demographic questionnaire to elicit variables like age, sex, employment status, and educational status among others. psychological distress was measured with the general health questionnaire version 12 (ghq-12), which has been widely used in nigeria. lastly, the psyche ache assessment schedule (pas) was used to measure emotional pain. examples of question items include: i feel psychological pain, my psychological pain seems worse than any physical pain, i hurt because i feel empty, i ca nt take my pain any more, my pain is making me fall apart, and my pain is making me fall apart among others. the pas has been used locally in the study of emotional pain and a score of 28 and above is indicative of emotional pain. all questionnaires were filled in private with the help of the researchers before the dental procedures were carried out. data analyses were done with the statistical package of social sciences for windows version 16 (spss-16). normally distributed data were summarized using mean (sd), while categorical data were represented as proportions. the mean age of study group and control group was 33 (12) years and 36 (13) years respectively (p=0.180). most of them were above 20 years of age for both the test and control groups. most of them were males with 60.4% and 66.0% in the study and control groups respectively (p=0.41). they were mostly employed 70.3% and 61.6%, with about one third being unemployed (students) in both groups (p=0.17). majority had at least primary school education (study group=100%) and (control=95.9%). table 2 shows the comparison of psychological distress and emotional pain between dental patients and the controls. on the ghq-12, the mean score for the test and control group was 0.721.2 and 0.370.6 respectively (p=0.011). in the test group, 21.8% (n=22) of the subject had ghq-12 scores of 3 and above (suggestive of psychological distress) while only 7% of the control group had ghq suggestive of psychological distress. the mean score among the test groups was 19.37.1 and 17.87.8 among the control (p=0.0146). figure 1 shows the proportion of subjects with positive scores for ghq and pas in both groups. a positive score suggestive of emotional pain (psych ache) was found in over a third of the dental patients (37.6%, n=38), while on the other hand, only 13% of the controls experienced psych ache (p<0.001). (table 2). of the dental patients with positive pas scores, 50% had scores suggestive of psychological distress on the ghq-12. however, more than half (86.4%) of those with ghq positive scores did experience psych ache (p<0.001). the experience of psychological distress was almost the same in both gender groups, with 16.5% in males and 14% in females (p<0.05). while for psych ache a similar 25.2% for males and 25.6% of females (p<0.05) (table 2). there was no difference in the number of subjects who currently live with a partner in both groups. however, more than one-sixth of those that experienced psyche ache (60.4%) were not currently living with a partner. the complaint of pain is common in dental practice setting, and may often largely assume to be solely due to physical causes. however, recent evidence has implicated other causes including psychological problems. in this respect, while dental anxiety as a psychological experience faced by dental patients has been extensively examined, particularly by western researchers, other common psychological experiences among dental patients like emotional pain, dental fear or depression have been scantily researched, especially in resource-restricted countries like nigeria. therefore, findings in this study constitute an important contribution to knowledge on the emotional concerns of dental patients in order to improve the quality of care in dental setting. in this study, the burden of psychological distress among dental patients (21.8%) was about three folds higher than what is found in the control group (7%). this high prevalence of distress among dental patients, as observed in this study, is not only in keep with what has been fielded in earlier works, but can be attributed to the experience of varieties of psychopathologies by dental patients. for example, the presence of dental anxiety has been implicated, especially because anxiety is a form of psychological distress and may be detectable by the ghq-12. however, studies have shown a world-wide variation in the prevalence of dental anxiety with estimates as high as 11% prevalence among people with dental fear. the disparity in the burden of emotional distress in the cited studies and our study can be adjudged to several factors like methodological issues, study population and setting, clinical factors, personality traits and the extent of comorbidity of mental health disorders among others. to further buttress the importance of the roles of psychosocial factors on emotional well-being among dental patients, improved understanding of dental procedures and expectations have been associated with reduction in dental fear, while a female gender, low engagement in treatment and depressed mood tend to increase the occurrence of dental fear. as expected, the experience of emotional pain as reflected by the burden of psyche ache is high among these study participants. that said, it is interesting to note that psych ache, which is often a feature of depressive illness, was found to exist among more than one-third of the test subjects, out of which only a half had scores suggestive of psychological distress on ghq-12. this is postulated to suggest that while ghq may be useful in screening for all forms of psychological distress, the pas is more specific to chronic pain. a further explanation is that most patients in this environment tend to show up at the dental clinics late, and more often these patients have not experienced chronic dental pain, but they only show up when the pain has become unbearable. as it is, the diagnosis and treatment of mental health illnesses including depression should be relevant to dental practitioners because they are the primary care providers who treat a large cross-section of the community. although mental problems are common in the community, they are particularly evident in treatment settings. for instance, more than 20% of patients seen in primary care clinics report had clinically significant depressive symptoms. by identifying the symptoms of depressed people, that can include complaint of distress, and referring them for treatment, dentists can provide another important contribution to the health care of their patients. some researchers have proposed the collaboration between dentists and psychotherapeutic specialists in provision of modern dental treatment. a four stepped approach for addressing dental anxiety has been proposed by pawlicki, which includes assessment, categorization, relaxation training and referral when necessary. in spite of the potential benefits of this study in the expansion of scanty data on the emotional experience of dental patients in resource-limiting setting like nigeria, it is limited in certain respect. for instance, this study did not explore in details the past dental experiences, which may have proven useful in understanding the finding of psychological distress. also a randomization was done as a result of the small population in the clinic, as well as physical pain was not measured using specific instruments. additional, it is a clinic-based study, thus extrapolation of its findings to the general population should be cautiously done. the findings in this study confirm the experience of many-fold of psychological distress and emotional pain by dental patients in comparison to normal population. thus, dentists should be empowered to go the extra-mile to carry out broad evaluation of pain symptoms, while deploring useful holistic distress-relieving techniques as well. such broad evaluation of pain symptoms that include psychological evaluation along with psychosocial support should be integrated into standard dental care protocol. again, the exigency to develop collaboration framework between dental and mental health services is buttressed in this study.

we set out to carry out a case-control evaluation of psychological distress and emotional pain among adult attendees of a nigerian dental clinic. a total of 201 subjects, made up of 101 dental patients (test group) matched with age and sex with 100 normal subjects (controls), was recruited into the study. all participants completed a designed socio-demographic questionnaire. general health question naire and psyche ache assessment schedule were also administered to assess psychological distress based on cut-off scores 3 and emotional pain based on cut-off scores 28 respectively. the mean ages of study and control group were 33 (12) and 36 (13) years respectively, and both study and control groups were not significantly different in all the assessed socio-demographic parameters. overall, 21.8% (n=22) of the subjects had psychological distress, while only 7% of the control group had psychological distress. this difference was statistically significant (p=0.003). similarly, there was significant difference in the experience of psyche ache (unbearable psychological pain) as over a third of the dental patients (37.6%, n=38) had emotional pain, while only 13% of the controls experienced psych ache (p<0.001). in this study, the burden of psychological distress and emotional pain was many-fold in dental patients when compared with the controls.

PMC4926031

pubmed-920

early life stressors that shape the stress response in offspring have profound effects on mood and cognition in adulthood (davidson&mcewen, 2012). chronic exposure to glucocorticoids contribute to the dysfunction of the inhibitory network and impairment of rhythmic oscillations, which are critical for the regulation of brain activity and complex cognitive processes (hu&et al, 2010). a dysfunctional gabaergic system is associated with the pathogenesis of neuropsychiatric diseases such as schizophrenia, anxiety and depression (hines&et al, 2012). during brain development, gabaergic synapses are formed prior to the formation of glutamatergic synapses and the activation of the gabaa receptor depolarizes immature neurons (ben-ari and et al, 2012, ben-ari, 2002, ben-ari and et al, 2007). excitatory gaba transmission plays important roles in various neurodevelopmental processes including; neuronal migration, cell proliferation, neurite outgrowth and generating synchronized network activity (cherubini&et al, 2011). cation chloride cotransporter (ccc) is the key controlling factor in controlling the switch of the gabaa receptor function. cccs control the reversal potential of the gabaa receptor-mediated current (egaba), which is important for the modulation of the gabaa receptor function. there are two main types of cccs; the outwardly directed potassium-chloride cotransporter 2 (kcc2), and the inwardly directed sodium potassium-chloride cotransporter 1 (nkcc1). in immature neurons, kcc2, on the other hand, reduces the chloride reversal potential thus it extrudes cl out of the cell and shifts the actions of the gaba from excitation to inhibition. although the other chloride regulators channels and transporters also take part in this sequence (blaesse and et al, 2009, medina and chudotvorova, 2006), the expression of kcc2 is thought to initiate the developmental switch of the gabaa receptor function from excitatory to inhibitory transmission (ben-ari, 2002). in addition to the expression of cation-chloride cotransporters, the gabaa receptor undergoes postnatal changes in its structure and function by the differential expression of different subunits ' composition (jacob et al., 2008). the presence of gabaa receptor 1 subunits mediates phasic inhibition by inducing a more rapid decay rate in gabaa-mediated synaptic currents (dunning&et al, 1999). in contrast, the gabaa receptor 5 subunits mediate tonic inhibition, which can be characterized by a slow decay rate of the synaptic current (jacob et al., 2008). the 1 subunits are located at the synaptic sites and mostly found in mature neurons, while the 5 subunits are located at the extrasynaptic sites and found mostly in immature neurons prior to the formation of the inhibitory synapse (jacob et al., 2008, thus, the maturation of the gabaergic function requires the precise expression of specific subunits of the gabaa receptor during postnatal brain development. the early expression of the gabaa receptor 5 subunit is required for the tonic inhibitory function of gaba, while the late expression of the 1 subunit is required for the phasic inhibition that indicates the maturation of the gabaa receptor function. it is well documented that stress increases glucocorticoid hormones and thereby potentiates excitotoxic damage in hippocampal gabaergic neurons (elliott and sapolsky, 1992, stein-behrens and sapolsky, 1992). early life stress exerts an effect on the hippocampal neurons and predisposes individuals to psychosis (stumpf and et al, 1989, tornello and et al, 1982, zhang and et al, 1990, scheller-gilkey and et al, 2003). the hippocampus exhibits subtle alterations subsequent to neuropsychiatric diseases such as schizophrenia and mania depressive disorder (benes, 1999). previous studies in postmortem brains from schizophrenia patients have shown a decrease in hippocampal gabaergic activity that could potentiate excitotoxic damage to hippocampal interneurons, consistent with abnormal oscillatory rhythms and increased basal metabolic activity (benes, 1999). it is still unclear how prenatal stress affects the development of gabaergic synapses in the hippocampus of the offspring. in this study we hypothesized that prenatal stress may affect the structural and functional maturation of the gabaa receptor in the hippocampus of rat pups. therefore, the purpose of this study was to examine the effect of maternal restraint stress on the levels of nkcc1 and kcc2, as well as gabaa receptors 1 and 5 subunits in the hippocampus of the offspring to provide insights about the involved mechanisms of maternal stress as a cause of dysregulation of gabaergic synapses that are known to be associated with the pathogenesis of psychiatric diseases at adulthood. pregnant rats were obtained from the national laboratory animal centre, mahidol university, salaya, thailand. they were housed in a single housing condition with a temperature and humidity controlled environment and maintained on a 12 h light/dark cycle with free access to food and water. each pregnant female rat was weighed on gestation day (gd) 721. in the morning of gd 21, each pregnant rat received nesting material, and thereafter, the cage was checked twice daily for the appearance of a litter. the day a litter gets discovered becomes designated as postnatal day 0 (p0), and the length of gestation was noted. all experiments were conducted according to the guidelines for care and use of the laboratory animals and approved by the experimental animal ethics committee of the institute of molecular biosciences, mahidol university, thailand (coa.mb-acuc 2015/003). pregnant rats were divided into two groups; 1) control group, 2) maternal restraint stress group (n=4/group). for the restraint stress, each pregnant rat was put into a small plexiglas cylindrical cage in which the length can be adjusted to accommodate the size of each animal. the restraint stress was performed during gd14-20, at four hours daily intervals during the light phase of the cycle as previously described (surakul et al., 2011, gestation days 1420 were selected because they represent the most sensitive period for the behavioral teratogenic effect of prenatal stress (fride&weinstock, 1984). whole hippocampal tissues were collected from rat pups at different postnatal days (p) from p7, p14, p21, p28 and p40, with n=4/group. brain tissues were then suspended in a lysis buffer composed of 50 mm tris ph 7.4, 150 mm nacl, 1 mm edta, 0.5% sodium deoxycholate, 1% sds, 1 mm pmsf, 1% triton-x-100 and supplemented with a complete protease and phosphatase inhibitor cocktail set (calbiochem, germany), then homogenized twice with a sonicator for 10 s each. the homogenized samples were centrifuged at 14,000 rpm, 4 c for 15 min. cell lysates were mixed with a sodium dodecyl sulfate (sds) sample buffer and boiled. equal amounts (20 g) of extracted protein samples were resolved in 10% sds-page and electrophoresis at 100 v for 150 min. the protein bands were then transferred to the pvdf membrane (amersham, usa) at 100v for 135 min. the membranes were then incubated in a blocking solution containing 3% skimmed milk for nkcc1, kcc2 and gabaa receptor 1 and 5 subunits, and 5% skimmed milk for actin at room temperature for 60 min. then the membranes were incubated with the following specific primary antibodies purchased from the available commercial sources. polyclonal goat anti-nkcc1 (sc-21545; 1:500), polyclonal goat anti-kcc2 (sc-19420; 1:500), polyclonal goat anti-gabaa receptor 1 subunit (sc-7348; 1:500), polyclonal goat anti-gabaa receptor 5 subunit (sc-7357; 1:500), and monoclonal mouse anti-actin (sc-69879; 1:5000), all antibodies were purchased from santa cruz biotechnology, usa. the membranes were then thoroughly washed 3 times using 0.1% tween-tbs (ttbs) for 5 min each, and then incubated with an appropriate hrp-conjugated secondary antibody. after that, the membranes were washed 3 times using 0.1% ttbs for 5 min each, and then the signals were detected by an enhanced chemiluminescence system (ecl prime, amersham biosciences, usa) and film exposure. the intensities of the band were quantified using densitometry software (image j, national institutes of health, usa). the immunoblot data were corrected for corresponding product of the -actin extracted from the same tissue which serve as an internal control. quantitative results were expressed as mean sem, calculated from the duplicate experiments. the statistical significance of difference between means was evaluated using student's t-test (unpaired, unless otherwise stated). changes produced by prenatal stress were analyzed at different postnatal ages using a two way anova with the prenatal stress and postnatal ages as independent variables and the protein levels as dependent variables; followed by a tukey's post hoc multiple comparison test. the probability level of p 0.05 was considered to have a statistically significant difference between the two sets of data. we examined the effects of maternal restraint stress, during the gestation day (gd) 1420, on the levels of nkcc1 and kcc2 in the hippocampus of rat pups and compared between the groups at different postnatal ages. the results showed that maternal restraint stress caused a transient but significant increase in the level of nkcc1 in the hippocampus at p14 (p<0.05) but no significant difference when observed at the other periods (fig. 1). for kcc2, the results show that maternal restraint stress caused a transient but significant increase in the kcc2 level in the hippocampus of rat pups during the weaning period (p21) (p<0.01) and this was followed by a transient but significant decrease during the preadolescence period (p28) (p<0.05) (fig. 2). however, there was no difference in the level of kcc2 when compare between groups during the adolescence period (p40). we found that maternal restraint stress significantly increases the nkcc1/kcc2 ratio in the pup's hippocampus at p14 and p28 (p<0.01). during this period, the nkcc1/kcc2 ratios in the hippocampus of prenatally stress pups exhibited more fluctuations than those observed in the control group (fig. the developmental expressions of gabaa receptor 5 and 1 subunits in the hippocampus appear in the opposite way. the 5 subunit was highly expressed during p7p14, and then declined during p21p40 (fig. 4, white bar) while the 1 subunit was expressed at a very low level during p7p14, then continually increased during p21p28 (fig. maternal restraint stress caused a transient but significant decrease in the level of the gabaa receptor 5 subunit at p14 (p<0.05) and followed by a long term increase at p21 (p<0.01), p28 (p<0.05) and p40 (p<0.05) as compared to the control (fig. 4). in contrast, maternal restraint stress caused a transient but significant increase in the level of the gabaa 1 subunit at p14 (p<0.05) followed by a significant decrease at p21 (p<0.01) and p28 (p<0.01) (fig. we found no significant difference in the level of the 1 subunit when comparing between the groups at p40. when the ratios of the 5/1 subunit of the gabaa receptor were calculated and compared across the different postnatal ages, we found that maternal restraint stress causes a significant decrease in the ratio of the 5/1 subunits during p7p14 (p<0.01), but a significant increase in the ratios of the 5/1 subunits during p21p28 (p<0.05) (fig. 6). in fact, we found small increase in the 5/1 ratios at p40; however, there was no statistically significant difference when compared with the control group. gabaa receptor depolarization maintained by nkcc1 is important for proper brain development since it is a key factor in the control of several ca2-dependent developmental phenomena, including neuronal proliferation, migration and targeting (rivera&et al, 1999). kcc2, in contrast, shifts the gabaa receptor activity from depolarization to hyperpolarization in mature neurons. in a developing hippocampus, the level of nkcc1 continuously increases starting from p21 to adulthood (yan et al., 2001) while the hyperpolarizing gaba is completed by the second postnatal week due to the progressive reduction of nkcc1 activity in parallel with the enhanced activity of kcc2 (rivera and et al, 1999, emri and et al, 2001, gulyas and et al, 2001, stein and et al, 2004). previous studies reported that kcc2 expression significantly increases during the second postnatal week, which is the co-incidence time point when the developmental switch of gabaa receptor activity is observed (rivera and et al, 1999, clayton and et al, 1998) and continually increased until p28 (lu et al., 1999). in the hippocampus, nkcc1 and kcc2 expressions show relatively similar developmental patterns indicating that both are required for the reciprocal regulation of cl homeostasis, which is important for the functional maturation of the gabaa receptor. in this study, maternal restraint stress induced a transient increase in the level of nkcc1 in the hippocampus of rat pups only at p14, while there was a kcc2 increase at p21 and then a decrease at p28. previous studies reported that the alteration in nkcc1 under a stress response has no effect on the gabaa receptor function. in contrast, the stress-altered kcc2 level has a profound effect on the modulation of intracellular cl concentrations (hewitt&et al, 2009). our finding, that maternal restraint stress increases the kcc2 level in the pup's hippocampus at p21, indicates the protective mechanism that counteracts the higher level of nkcc1 at p14. maternal restraint stress, which increases kcc2 level at p21 and then decreases it at p28, indicates the dysregulation of intracellular cl concentrations and the gabaa receptor mediated current during the preadolescence period. in the mature pyramidal neurons, kcc2 inhibition positively shifts the gabaa receptor reversal potential, thus, gabaa receptor activation causes depolarization (rivera&et al, 1999). our results indicate that maternal restraint stress may induce prolonged depolarizing of gaba in the hippocampus of rat pups until the pre-adolescence period, while in the control pups, hyperpolarizing gaba was completed by the early postnatal week. different brain regions may exhibit differential vulnerability to the effects of stress on the level of kcc2 and its activity. for examples, in the rat hypothalamic paraventricular nuclei, acute restraint stress has no effect on the level of kcc2, but attenuates the kcc2 activity (hewitt&et al, 2009). maternal restraint stress has no effect on the level of kcc2 in the amygdala of male pups at p14 and p22 (laloux&et al, 2012). in contrast, prenatal stress causes a significant decrease in the kcc2 level and its activity in the hippocampus, as measured by phosphorylation of kcc2 on ser 940 residue (sarkar&et al, 2011). these findings, together with our results, indicate that maternal restraint stress might alter gabaa transmission in the hippocampus of prenatal stress pups during the post-weaning (p21) to pre-adolescence period (p28) and this mechanism might be due to the alteration in the levels of kcc2. the underlying mechanism by which prenatal stress alters kcc2 levels remains unclear. studies have shown that brain a derived neurotrophic factor (bdnf) regulates the expression of kcc2 in both the young and adult brains (aguado and et al, 2003, rivera and et al, 2002). bdnf is seen to down-regulate kcc2 expression in the adult hippocampal slices via the activation of tyrosine receptor kinase b (trkb) (rivera&et al, 2002). in addition, bdnf promotes kcc2 expression in the developing mice forebrain (aguado&et al, 2003). therefore, bdnf regulation of kcc2 expression varies depending on the developmental stages and brain regions. prenatal stress has been reported to decrease bdnf levels in the rat hippocampus at p21 (van den hove&et al, 2006). taken together, the results suggest that an alteration in bdnf levels caused by prenatal stress might affect the kcc2 level in the pup's hippocampus. although it has been noted that bdnf-induced alteration in kcc2 expression was not caused by neuronal excitability and network activity (aguado&et al, 2003), the endogenous action that is regulating the changes is still elusive. it is assumed that maternal restraint stress altering kcc2 level in the rat pups hippocampus at preadolescence period could affect the excitatory glutamatergic synapses as well. it has been reported that kcc2 has an important roles in the modulation of the dendritic spines and ampa receptor diffusion by interacting with sub-membranous actin cytoskeleton (gauvain&et al, 2011), therefore indicating kcc2 is also require for the production of long-term potentiation (ltp) in the hippocampus of young animals. prenatal stress has been linked to an increased risk of psychiatric disorders such as schizophrenia and depression (charil&et al, 2010). recent studies show that the kcc2 level is significantly decreased in the hippocampus of schizophrenia patients, while there was no change in the nkcc1 level (hyde&et al, 2011). thus, an increase in the nkcc1/kcc2 ratio indicates the delayed maturation of cation-chloride cotransporters in the patient's brain, which may underlie the pathology of neuropsychiatric diseases. recently, it was demonstrated the significant increase in the level of oxsr1 (oxidative stress response kinase1) and wnk3 (with no k [lysine] protein kinase3) in the post-mortem brain of schizophrenia patients (arion&lewis, 2011). consequently, changes in the level of oxsr1 and wnk3 can shift the balance of chloride transport and leading to an abnormal gabaergic transmission in the prefrontal cortex, thereby contributing to the impaired neural network synchrony and cognitive dysfunction in affected individuals (arion&lewis, 2011). for the development of gabaa receptor subunits, our results are consistent with those that have been previously reported (ramos and et al, 2004, laurie et al., 1992). we found the 5 subunit is highly expressed during the early postnatal period and declined to the adult level around the 3rd postnatal week, while the 1 subunit was initially expressed at a small level during the 1st and the 2nd postnatal week and gradually increased until reaching its peak around the 3rd postnatal week. prenatal stress delays the developmental shift of the gabaa receptor 1 and 5 subunits that normally occur around p21 in the control pups. this was clearly observed in the control pups that were seen to have manifested developmental increments in the 1 subunit at p21, but not in the prenatal stress pups, at least until p40. on the contrary, the control pups show a developmental decrease in the 5 subunit at p21, but not in the prenatal stress pups that were seen to maintain the expression of the 5 subunit at least until p40. as a result, the ratio of the 5/1 subunits in the prenatal stress pups exhibits a significant increase during p7 and p14, but shows a significant decrease when observed at p21 and p28, as compared to the control group. the underlying mechanism of prenatal stress that induces a prolonged increase of 5 subunits in the rat pup's hippocampus at preadolescence is still elusive. a prolonged increase in the 5 subunit in the pup's brain may create an unpredictable effect on gaba inhibitory transmission; especially during puberty (shen&et al, 2007). in the hippocampal ca1 and neocortical pyramidal neurons, the extrasynaptic 5 subunit of the gabaa receptor mediates tonic inhibition and plays an important role in memory and learning (rudolph&mohler, 2014). an increased expression of the gabaa receptors ' 5 subunit is associated with memory loss (wang&et al, 2012) while the antagonist of the receptor can enhance learning and memory processes (rudolph and mohler, 2014, ling and et al, 2015). thus, our results suggest that prenatal stress induces an increase in the gabaa receptor's 5 subunit expression and the 5/1 ratios in the hippocampus at the preadolescence period may underlie the long term effects of prenatal stress on learning and memory impairment in the offspring at adulthood. for the gabaa receptors 1 subunit, our findings are consistent with previous report that reveal exposure to stress in juvenile rats can induce biphasic changes in their behavior, including hyperactivity at juveniles which in adulthood becomes hypoactivity accompanied by behavioral anxiety that are associated with the decrease of 1 subunits in the hippocampus and amygdala (jacobson-pick&richter-levin, 2012). taken together the results are in agreement, the juvenile period is a sensitive time and is more vulnerable to stress than other periods and this supports the hypothesis that prenatal stress is a predisposing factor for various neuropsychiatric diseases and memory impairment at later life. in this study, we added further information that the developmental expression of the gabaa receptor 1 subunit is similar to the developmental pattern of the kcc2. both the gabaa 1 subunit and kcc2 reach their peak around p21p28 and this indicates that the gabaa 1 subunit and kcc2 might coordinate in enhancing the gabaa receptor mediated synaptic inhibition that occurs around this period. interestingly, we found that prenatal stress induces changes in the levels of the gabaa 1 subunit and the kcc2 in a similar way. our results correspond to what has been previously documented in that kcc2 could modulate the expression level of the gabaa receptor 1 subunit via an alteration in intracellular [cl] and the decay rate of gaba-mediated inhibitory transmission (houston&et al, 2009). indeed, lower intracellular [cl] resulted in a faster decay rate of the gaba transmission (moroni&et al, 2011). it has been shown that kcc2 can manipulate the expression of gabaa receptor subunits, i.e., overexpression of kcc2 results in the reduction of intracellular [cl] and leads to an increase in the level of 1 and subunits (succol&et al, 2012). taken together, these results are in accordance with the hypothesis that prenatal stress reduces the kcc2 levels, which might lower the intracellular [cl], that acts as the intracellular signal and induces a faster decay rate of the gabaa receptor gating and, thus, decreases the expression of the 1 subunit of the gabaa receptor (succol&et al, 2012). these changes indicate a delayed maturation of the gabaergic function in the hippocampus of prenatal stress pups, especially during the preadolescence period. additionally, it was reported that stress disrupts the gabaergic function in the brain in many ways. stress induces dysfunction of the inhibitory network and impairs rhythmic oscillations leading to cognitive deficits commonly found in psychiatric disorders (hu&et al, 2010). prenatal stress disturbs the distribution of gabaergic interneurons in the cortical plate, reflecting the changes occurring in tangential migration and radial integration in the developing cortex (stevens&et al, 2013). prenatal stress causes a significant decrease in the frequency of spontaneous ipscs in the immature hippocampal neurons (grigoryan&segal, 2013) and increasing the vulnerability to stressful situations in the offspring during adulthood accompanied by a reduction of benzodiazepine binding in the hippocampus (fride&et al, 1985). in summary, this study has shown that maternal restraint stress has the ability to differentially alter the levels of nkcc1, kcc2, and gabaa receptor 1 and 5 subunits in the hippocampus of rat pups. consequently, these changes can lead to an imbalance of inhibitory transmission that may delineate the linkage between prenatal stress and neuropsychiatric disorders in later life. our findings reveal that there is a strong connection between early life stress exposures with an increased risk of developing psychiatric disorders at adulthood. furthermore, a reduction of kcc2 levels has been linked to the cause of epilepsy, which is considered as a risk factor for schizophrenia and autism. moreover, a prolonged increase in the 5 subunits in the hippocampus of rat pups during adolescence indicates a prolongation of the slow decay rate of inhibitory transmission in the pup's hippocampus and predisposes it for neuropsychiatric diseases and memory impairment in adulthood. experiencing adverse events during pregnancy has a negative impact on brain development and may increase vulnerability to developing neurological and psychiatric disorders later in life. as we demonstrated, fetal exposure to maternal stress hormones delays structural and functional development of gaba transmission in the rat pup's hippocampus during the preadolescence period. these changes may lead to the dis-regulation of gaba inhibitory transmission in the developing hippocampus. similar patterns of changes in the kcc2 and gabaa receptor 1 subunits were observed in response to early life stress, accompanied by supporting evidence that indicates changes in kcc2 levels may underlie the effect of maternal stress on the alterations in the 1 subunit of the gabaa receptors. moreover, prenatal stress also increases gabaa receptor 5 subunit expression throughout the preadolescence period, which may underlie the learning and memory impairments in the offspring at adulthood. in summary, we have provided an explanation of certain prenatal factors mediating structural and functional development of the gabaergic synapse that may be the link between prenatal stress and the emergence of neuro-psychiatric disorders at adulthood.

the gabaergic synapse undergoes structural and functional maturation during early brain development. maternal stress alters gabaergic synapses in the pup's brain that are associated with the pathophysiology of neuropsychiatric disorders in adults; however, the mechanism for this is still unclear. in this study, we examined the effects of maternal restraint stress on the development of cation-chloride cotransporters (cccs) and the gabaa receptor 1 and 5 subunits in the hippocampus of rat pups at different postnatal ages. our results demonstrate that maternal restraint stress induces a transient but significant increase in the level of nkcc1 (sodium potassium chloride cotransporter 1) only at p14, followed by a brief, yet significant increase in the level of kcc2 (potassium-chloride cotransporter 2) at p21, which then decreases from p28 until p40. thus, maternal stress alters nkcc1 and kcc2 ratio in the hippocampus of rat pups, especially during p14 to p28. maternal restraint stress also caused biphasic changes in the level of gabaa receptor subunits in the pup's hippocampus. gabaa receptor 1 subunit gradually increased at p14 then decreased thereafter. on the contrary, gabaa receptor 5 subunit showed a transient decrease followed by a long-term increase from p21 until p40. altogether, our study suggested that the maternal restraint stress might delay maturation of the gabaergic system by altering the expression of nkcc1, kcc2 and gabaa receptor 1 and 5 subunits in the hippocampus of rat pups. these changes demonstrate the dysregulation of inhibitory neurotransmission during early life, which may underlie the pathogenesis of psychiatric diseases at adolescence.

PMC4730793

pubmed-921

obesity is associated with numerous chronic diseases and, currently, its prevalence is 34% in us adults. excess fat storage is the result of greater energy intake than expenditure for a period of time; thus, increasing energy expenditure is an important strategy to treat obesity. physical activity energy expenditure (paee) can be further divided into energy expended during structured exercise and during activities of daily living other than structured exercise. there has been extensive research investigating the effects of various exercise and/or caloric restriction interventions for inducing weight loss. in general, these programs are successful at inducing weight loss; however, there is some evidence showing that increases in total daily energy expenditure during prescribed exercise interventions are less than expected given the amount of energy expended during the prescribed exercise sessions [38]. in support of this, one study showed that accelerometer counts from physical activities outside of structured exercise decreased by 8% after a 12-week training period. this suggests that paee outside of the structured exercise may decrease as a result of the exercise treatment. all of these prior studies examined the chronic or longer-term effects of exercise interventions on paee. however, it is also important to determine whether there are potential changes in paee acutely on days in which the exercise is performed. yet, to date, only one small study examined this acute effect of exercise on daily paee. it reported that accelerometer counts from nonexercise activities were lower on days with, than without, structured exercise during a 12-week exercise program. thus, the purpose of the present study was to determine whether performing a single exercise bout impacts daily paee in postmenopausal women and to determine whether the intensity of the exercise bout plays a role in any potential changes in daily paee. women in this study are a subset of those enrolled in a randomized clinical trial that was designed to determine whether intensity of aerobic exercise affects the loss of abdominal adipose tissue and improvement in cardiovascular disease risk factors in postmenopausal women with abdominal obesity (clinicaltrials.gov: nct00664729). briefly, they were: (1) older postmenopausal (age: 5070 yr), (2) overweight or obese (bmi: 2540 kgm and waist circumference>88 cm), (3) nonsmoking, (4) not on hormone therapy, and (5) sedentary (< 15 minutes of exercise, two times per wk) in the past 6 months before enrollment. the study was approved by the wake forest university institutional review board, and all women signed an informed consent form to participate in the study according to the guidelines for human research. data used for the current analyses are from women who were randomized to caloric restriction plus moderate-intensity aerobic exercise (moderate-intensity) or caloric restriction plus vigorous-intensity aerobic exercise (vigorous-intensity) and completed the study interventions. there were 18 women in the moderate-intensity and 18 women in the vigorous-intensity groups who had paee data available from before the intervention and from days with and without center-based exercise in the last month of intervention. both the moderate-intensity and vigorous-intensity interventions were 5 months, and the energy deficit was designed to be approximately 2100 kcalwk from caloric restriction and 700 kcalwk from center-based exercise. individual energy needs for weight maintenance were calculated from each woman's resting metabolic rate (indirect calorimetry after an overnight fast by using a medgraphics ccm/d cart and breeze 6.2 software, medgraphics, st. paul, mn) and an activity factor based on self-reported daily activity (1.2-1.3 for sedentary lifestyle). individual diets were developed by a registered dietitian according to each woman's choices from a menu designed by a registered dietitian. throughout the course of the 5-month intervention, all women were provided with daily lunch, dinner, and snacks prepared by the general clinical research center (gcrc) metabolic kitchen. they were asked to eat only the food that was given to them or that was approved from the breakfast menu. energy make-up of the diet was approximately 25% from fat, 15% from protein, and 60% from carbohydrate. women were allowed to consume as many noncaloric, noncaffeinated beverages as they liked. they were also allowed 2 free days per month during which they were not provided food but were given guidelines for diet intake at their prescribed energy level. all women were provided with daily calcium supplements (500 mg, 2 timesd). they were asked to keep a log of all foods consumed, and the records were monitored by the dietitian to verify compliance. the exercise interventions were center-based walking on treadmills (lifefitness 9500hr, life fitness co., il) on 3 dwk under the supervision of an exercise physiologist. exercise progressed from 2025 min the first week to 55 minutes by the end of the sixth week for the moderate-intensity (4550% of maximum oxygen consumption, vo2max) group and it progressed from 1015 minutes the first week to 30 min by the end of the sixth week for the vigorous-intensity (7075% of vo2max) group. the target exercise intensity was determined based on each woman's target heart rate calculated from the karvonen equation [(hrr intensity)+resting heart rate], where hrr is the maximal heart rate, obtained from each woman's maximum exercise test, minus resting heart rate. treadmill speed and grade were adjusted on an individual basis to ensure women exercised at their prescribed exercise intensity. heart rate readings (by polar heart rate monitors; polar electro inc, lake success, ny) were taken before, at least 2 times during (to monitor compliance to the prescribed exercise intensity), and after the exercise. it is about the size of a pager and is worn by clipping onto the waist. it collects 3-dimensional data at one minute intervals and stores such data for 7 days. activity energy expenditure was computed using the manufacturer's software from the integrated acceleration and body mass with formula developed by the manufacturer. daily paee was calculated using the average calories expended per minute times 1440 minutes a day. women were asked to wear the accelerometer before and each month during the intervention, for 57 days including week days and weekend days. women were instructed not to change their activities while wearing the accelerometer at all times, except while sleeping and bathing. data collected from rt3 monitors were included only when there were valid data from both before the intervention and during the last month of intervention. during the last month of intervention, rt3 data were considered valid when data were collected from at least 2 days with center-based exercise and at least 2 days without center-based exercise. the average paee from days with and without center-based exercise were used for the current analyses. of note, the average daily paee from days with center-based exercise included the energy expended during the exercise sessions, and none of the women performed structured exercise at baseline. treadmill readings during the exercise sessions were recorded for each woman as a measure of energy expended during center-based exercise. height and weight were measured before and after the 5-month intervention with shoes and jackets or outer garments removed. all analyses were performed using sas software, version 9.1 (sas institute, cary, nc). paired t-tests were used to compare values within the same group at different measurement points. as shown in table 1, there were no differences in baseline characteristics such as age, racial distribution, body weight, and body mass index between the moderate-intensity and vigorous-intensity groups. the total amount of weight loss during intervention was similar between the moderate-intensity and vigorous-intensity groups (12.9 4.2 kg or 14.6 4.8% and 12.6 5.1 kg or 13.5 4.6%, resp.). during the last month of intervention, paee on days with center-based exercise was significantly higher in women performing moderate-intensity exercise than in women performing vigorous-intensity exercise (p=.052). in contrast, paee on days without exercise was not statistically different between the two groups (p=.135). in the moderate-intensity group, 13 of the 18 women had higher paee on days with than without center-based exercise (figure 1), and the average paee on days with exercise (577.7 219.7 kcald) was higher than on days without exercise (450.7 140.5 kcald, p=.011) (table 1). yet, the difference (127.0 188.1 kcald) was much smaller than the energy expended during exercise (325.0 79.6 kcald) (figure 2), suggesting that, during the 5th month of exercise training, women expended less energy on activities outside of the structured exercise when they exercised during the day. in support of this, paee on days with center-based exercise was not different from baseline paee (520.8 206.5 kcald; p>.05 for both) in women performing moderate-intensity exercise even though energy expended during exercise was included in the daily paee on days with exercise. on the other hand, in the vigorous-intensity group, 12 of the 18 women had lower paee on days with than without center-based exercise during the last month of the intervention (figure 1). the average daily paee on days with exercise (450.6 153.6 kcald) was lower than on days without exercise (519.2 127.4 kcald) (difference=68.6 136.1 kcald, p=.047) again even though energy expended during exercise (296.8 93.0 kcald) was included in daily paee on days with exercise (figure 2). this indicates that, during the 5th month of exercise training, women performing vigorous-intensity exercise were expending more total calories on nonexercise days than on exercise days. in addition, paee on days without exercise was not different from baseline daily paee (543.2 164.0 kcald; p>.05); however, paee on days with exercise (which included energy expended during center-based exercise) was significantly lower than baseline paee (p=.020). this study adds information to the literature regarding how acute exercise sessions affect daily paee and whether the intensity of exercise influences the effects. we found that, during the last month of a 5-month moderate-intensity exercise training intervention, the daily paee during days with center-based exercise was higher than days without exercise by an amount much smaller than the exercise energy expenditure. during the 5th month of a vigorous-intensity exercise training intervention, daily paee during days with center-based exercise was lower than days without center-based exercise sessions, even with energy expended during the exercise sessions included in paee. therefore, there was a reduction in paee outside of the center-based exercise sessions in both intervention groups, and this reduction appeared to differ based on the intensity level of the center-based exercise because it was greater in women performing vigorous-intensity, compared to moderate-intensity, exercise. our findings are in line with those of meijer et al., who found that accelerometer counts of total physical activity were similar between days with and without training, and that after energy expenditure during the training session was subtracted out, accelerometer counts were significantly lower on training days. in their study, the training program included one aerobic exercise of 60 minutes and one cardio- and weight-stack machine exercise of 90 minutes each week for 12 weeks in men and women of 55 years and older. we can not directly compare the magnitude of the paee responses in our study to their study because the intensity of the exercises was not specified and only accelerometer counts were reported in their study. we also showed that exercise at vigorous intensity induced greater compensation in paee than moderate-intensity exercise. of note, both exercise programs in our study are consistent with the current physical activity guidelines for adults [11, 12]. the frequency of the exercise sessions was the same for the moderate-intensity and vigorous-intensity exercise groups, and the volume of exercise was also similar. however, this does not exclude the possibility that volume or frequency of exercise of an exercise program may affect the chronic response in paee to the program. further studies are needed to address these questions because these are important factors to consider when designing exercise programs to better meet an individual's goal for participating exercise. in this study, thus, women were relatively trained so that paee responses to acute exercise may be somewhat different from those if women were untrained. however, we suspect that the compensation in paee is likely lower in the trained state. in other words, for a person who does not participate in regular exercise, an acute session of exercise may induce greater compensation in paee. on the other hand, the information found in this study may be more important because with the epidemic of obesity, many individuals participating in exercise programs may think that would satisfy the goal of weight control. thus, we should educate and encourage them to maintain higher daily activities while participating in exercise programs at the same time. the results of this study should be interpreted in light of a few considerations. the daily paee during days with and without center-based exercises was the average of at least two days. although this provides a good measure of activity energy expenditure, it would be better if data from more days were available. second, all exercise sessions were during the week. for paee during days without exercise, we did not have enough data to determine whether there was a difference in paee between those weekdays and weekend days. these are not accurate measures of energy expenditure; however, we believe this will not affect our conclusion given the big difference shown between exercise energy expenditure and the difference between paee during days with and without exercise sessions (figure 2). in summary, the main finding of this study is that women expended more energy during physical activities outside of prescribed exercise sessions on days they did not perform center-based exercise, especially if the prescribed exercise was of a higher intensity. more research is needed to determine what exercise prescription can minimize this compensation. this phenomenon may have biological and behavioral reasons, and future research investigating the underlying mechanisms is warranted. thus, health professionals should encourage individuals who are participating in exercise programs to maintain levels of activity in addition to the program, so that greater weight loss can be achieved .

this study determined whether performing a single moderate- or vigorous-intensity exercise bout impacts daily physical activity energy expenditure (paee, by accelerometer). overweight/obese postmenopausal women underwent a 5-month caloric restriction and moderate- (n=18) or vigorous-intensity (n=18) center-based aerobic exercise intervention. during the last month of intervention, in women performing moderate-intensity exercise, paee on days with exercise (577.7 219.7 kcald1) was higher (p=.011) than on days without exercise (450.7 140.5 kcald1); however, the difference (127.0 188.1 kcald1) was much lower than the energy expended during exercise. in women performing vigorous-intensity exercise, paee on days with exercise (450.6 153.6 kcald1) was lower (p=.047) than on days without exercise (519.2 127.4 kcald1). thus, women expended more energy on physical activities outside of prescribed exercise on days they did not perform center-based exercise, especially if the prescribed exercise was of a higher intensity .

PMC2931398

pubmed-922

screw clamping force via application of torque is an essential maintenance mechanism of implant prostheses.1 unstable screw joints induce complications such as screw loosening under continuous cyclic loading. the occurrence of screw loosening is reported to be 12.7% in single tooth restoration and 6.7% in partial fixed prostheses. 23 jemt et al.4 observed that most of the screw loosening occurred within a year and the frequency of screw loosening was reduced over time. henry et al.5 and khraisat et al.6 stated that screw loosening is more frequent in external type implants. in internal type connections, the stability of the prosthesis is obtained from the clamping force of the screw joint and the frictional force created by contact between the conical mating parts of the implant-abutment assembly. because of this connection, internal type implants have more favourable stress distribution, better stability, and superior resistance to lateral load.7 however, tsuge and hagiwara8 presented no significant difference between internal and external implant-abutment connections concerning their effect on the abutment screw loosening. theoharidou et al.9 also reported that the frequency of screw loosening in single tooth restoration was 2.7% in external type implants and 2.4% in internal type implants; this difference was not statistically significant. the frequency of screw loosening is reduced over time, but still occurs in spite of recent advances in the design and screw materials. screw loosening produces discomfort for patients; therefore, methods to prevent screw loosening are needed. one method for preventing screw loosening that is easily applicable to clinical practice is retightening. gradual functional loading causes sliding of the screw thread and relief of screw extension induced by preload. gradual reduction of preload below a critical point causes screw turning. applying a greater preload on the screw in the first phase of screw loosening siamos et al.11 reported that in order to minimize the loss of preload, it is necessary to retighten the screw 10 minutes after the first screw tightening. however, the retightening procedure may change the shape of the abutment screw and the inner screw thread of the implant; hence, stability during function may be affected.12 in addition, conflicting results on the effect of retightening have been reported. according to tzenakis et al.,13 if the abutment screw is repeatedly retightened, a higher preload can be obtained because of surface wear that can lower the coefficient of friction. however, weiss et al.14 reported that repeated tightening/removal procedures decreased the reverse torque value (rtv) of screws. riccardi-copped et al.15 reported that repeated tightening and removal of titanium screws caused the rtv to decrease gradually. the effect of screw retightening remains controversial and on the timing and frequency of retightening are lacking. the aim of this study was to assess the effect of dynamic loading and screw retightening on rtv in external and internal connection type implants. twenty external type implants (diameter, 4 mm; length, 10 mm; sola, shinhung, seoul, korea) and 20 internal type implants (diameter, 4 mm; length, 10 mm; luna, shinhung, seoul, korea) were used (fig. 1). abutments for cementretained prostheses (diameter, 5 mm; gingival height, 5.5 mm; esthetic abutment for external implant and duo abutment for internal implant, shinhung, seoul, korea) were connected to each implant (fig. each implant and abutment assembly was held in a customized jig in all testing procedures. using a digital torque controller (mgt 12, mark-10 co., new york, ny, usa) after 10 minutes, the assemblies were retightened with 30 ncm torque to compensate for embedment relaxation.11 after tightening the screws twice, an initial measurement of rtv was made. the implant/abutment assemblies were divided into 4 groups (10 assemblies per group) in order to evaluate the effect of intermittent retightening: 1) ext-n, external type implant with no retightening; 2) ext-rt, external type implant with retightening; 3) int-n, internal type implant with no retightening; 4) int-rt, internal type implant with retightening. the implant/abutment assemblies were fitted to the mounting base of a universal testing machine (electroplus e 3000, instron, washington dc, wa, usa) with the long axis of the implant fixed at 30 degrees relative to the vertical axis (fig. the testing device delivered sine curved cyclic loading between 20 and 250 n at 14 hz for 100,000 cycles. in the groups with no retightening (ext-n and int-n), the rtv was measured after 100,000 cycles of loading, while rtv was measured after 3, 10, and 100 cycles as well as every 20,000 cycles in the retightening groups (fig. 3). for all statistical evaluations, spss version 20.0, spss inc., one-way analysis of variance (anova) was used for comparing rtv depending on the implant system and retightening. when the rtv was measured repeatedly after various loading cycles, tukey's test was used for post hoc comparisons, and the significance level was set at =0.05. the initial rtvs were 27.8 1.3 ncm in external implants and 25.1 1.9 ncm in internal implants. both implant systems showed decreased rtvs when compared with the tightening torque value of 30 ncm. the initial rtv of external implants was significantly higher than that of internal implants (p<.05). the rtvs of all implant/abutment assemblies were significantly reduced after cyclic loading (p<.05, table 1, table 2). the rtvs of the groups without retightening were 25.4 1.2 ncm in the ext-n group and 15.8 1.8 ncm in the int-n group. the ext-n group showed a significantly higher rtv when compared with the int-n group (p<.05). the rtvs of the groups with retightening were 23.8 1.8 ncm in the ext-rt group and 19.9 3.3 ncm in the int-rt group. the ext-rn group showed a significantly higher rtv than the int-rn group (p<.05). in external implants on the other hand, retightened internal implant/abutment assemblies showed superior rtvs when compared to internal implants with no retightening (table 1, table 2). screw loosening is the most frequently occurring mechanical complication of implant restorations.516 abutment screw loosening has been reported in a large number of studies with an incidence ranging from 2% to 15% of abutments. 2359 screw loosening is caused by inadequate tightening torque, settling of implant components, inappropriate implant position, inadequate occlusal scheme or crown anatomy, poorly fitting frameworks, improper screw design/material, and heavy occlusal forces.171819 to overcome screw loosening and joint instability, many technical solutions have been suggested. for example, new abutment screw designs and materials for maximizing preload,120 mechanical torque-applying instruments for optimizing tightening torque,21 precise implant components for antirotation, and internal conical connection implants with no micromotion or microgaps have been proposed. in the current study, the initial rtv was always smaller than the tightening torque (30 ncm) in external and internal type implants. haack et al.22 reported that most of the tightening torque is used to overcome the friction of the surface and only 10% of the tightening torque is used to generate preload. they suggested that 75-80% of tightening torque remained in titanium or gold screws.22 kim et al.23 reported that the amount of remaining torque was affected by the screw material (77% remained in titanium alloy and 66% remained in gold alloy). for standardization of the results, the same titanium alloy was used for both internal and external abutment screws in the present study. the results showed that the remaining torque was 90.9% in external implants and 83.3% in internal implants. these relatively high remaining initial rtvs might have been caused by advanced milling technique and/or the screw design/material. in the current experiments, initial rtvs and post-cyclic loading rtvs were higher in external implants than in internal implants. this is a result of the difference in connection type between the implants. in external implants, joint stability is obtained by the tension of the screw, while it is achieved mainly by friction between the abutment and the implant in internal type implants.24 most of the tightening torque is used to produce preload in external implants,25 while in internal implants, tightening torque is distributed between friction with the abutment and preload on the screw. moreover, internal implants are susceptible to the wedge effect, which arises when tightening torque and mechanical load are applied, leading to axial displacement of the abutment, so that the tensile force of the screw is lost and preload is decreased.232425 according to lee et al.24 and kim et al.,25 axial displacement of the abutment is greater in internal implants, while rtv is greater in external implants. although screws from the same manufacturer were used for standardisation, the screws for external implants have longer threads than those of the screws used for internal implants (fig. 1). the thread-engaging surface may affect the preload in screw-tightening procedures. to our knowledge, no internal/external implant system manufactured by the same manufacturer offers either similar or identical screw shapes owing to the different inner structures of internal/external implants. although the 2 implant systems had different screw sizes and dimensions, the lengths of the engaging or the mating threads were similar in both.24 the upper part of the thread in the screw of the external implant was not in contact and just passed through the inner surface of the implant. comparison of the groups with and without retightening after cyclic loading revealed no significant differences in rtvs in external implants. however, in internal implants, retightening resulted in higher rtvs than those found in implants without retightening. these results can be explained by the difference in connection type between the implants and the number of times the implants were retightened. in internal type implants, less settling effect occurs during tightening and loosening procedures, because tightening torque generates less preload due to dissipation of force via friction between the abutment and the implant. therefore, a greater number of retightening procedures allows more settling effect and making an adequate surface of the screw. cardoso et al.26 found that rtvs decreased as the number of insertion/removal cycles increased in external type implants. in contrast, tzenakis et al.13 reported an increase of preload in internal type implants. therefore, retightening of screws is strongly recommended for joint stability, especially in internal type implants. the appropriate number of retightening procedures is not clear from the results of previous studies. tzenakis et al.13 reported a gradual increase in gold screw preload from tightening for the first time to retightening 5 and 10 times in internal type implants. in the current study it should be noted that retightening more than 5 times is not recommended for the maintenance of preload. in the present study, this phenomenon was observed in both external and internal type implants. according to delben et al.,27 rtvs were maintained constantly by retightening after every 100,000 cycles of loading in external type implants. according to binon and mchugh,28 the average daily number of mastication is about 2,700 and so 100,000 cycles corresponds to around one month. in the current study, the load cycle was decreased to 20,000 cycles (corresponding to one week) in order to infer the timing of retightening, because previous studies found no significant differences with 100,000 load cycles. in the current experiments, rtvs decreased only 10 rounds of cyclic loading in external and internal type implants. in our previous study,24 thus, axial displacement of the abutment screw can affect joint stability, especially in internal implants. to increase preload and secure joint stability, it is recommended that retightening be performed after 10 loading cycles for both external and internal implants. moreover, retightening after 1 week is also profitable for internal type implants. this study evaluated rtv according to retightening and cyclic loading in external and internal type implants. initial rtv and postcyclic loading rtv were higher in external implants than in internal implants. in external type implants, retightening did not produce a significant difference in rtv when compared with the non-retightening group after 100,000 rounds of cyclic loading. in internal type implants, the retightening group showed significantly increased rtvs when compared with the non-retightening group after cyclic loading. a decrease in rtv occurred after only 10 rounds of cyclic loading, which represents the early stage of cyclic loading. after 20,000 cycles of loading, rtvs were maintained constantly in both external and internal type implants.

purposethe aim of this study was to evaluate the effect of cyclic loading and screw retightening on reverse torque value (rtv) in external and internal type implants. materials and methodscement-retained abutments were connected with 30 ncm torque to external and internal type implants. experimental groups were classified according to implant connection type and retightening/loading protocol. in groups with no retightening, rtv was evaluated after cyclic loading for 100,000 cycles. in groups with retightening, rtv was measured after 3, 10, 100 cycles as well as every 20,000 cycles until 100,000 cycles of loading. resultsevery group showed decreased rtv after cyclic loading. before and after cyclic loading, external type implants had significantly higher rtvs than internal type implants. in external type implants, retightening did not affect the decrease in rtv. in contrast, retightening 5 times and retightening after 10 cycles of dynamic loading was effective for maintaining rtv in internal type implants. conclusionretightening of screws is more effective in internal type implants than external type implants. retightening of screws is recommended in the early stage of functional loading.

PMC4551784

pubmed-923

osteoporosis most commonly affects postmenopausal women, placing them at a significant risk of fractures. in particular, hip fractures are an important cause of mortality and morbidity among postmenopausal women. approximately 20% of patients with hip fractures die within a year, most of the deaths occurring within the first 6 months after a fracture (cumming et al 1997). among the survivors, recent reports have shown that the rate of hip fractures is falling in ontario and finland (jaglal et al 2005; kannus et al 2006), probably because of recent indications that clinical attention to osteoporosis management is increasing and that specific programs of selective screening and aggressive treatment to prevent bone loss may really be able to reduce the fracture rate among the population most at risk (melton et al 2005). however, there is no indication that the use of effective osteoporosis interventions is increasing dramatically in regions including asia where the growth in the elderly population is most rapid. because elevated bone turnover markers and low bone mineral density (bmd) are independent predictors of hip fracture risk, and the risk is multiplied when both are present (garnero et al 1996), bone turnover and bmd are important factors in decreasing the risk of hip fractures. both reduction in bone turnover and increase in hip bmd may be necessary to decrease the fracture risk of the hip, which is primarily composed of cortical bone and may require greater proportionate changes than trabecular bone (epstein 2007). in fact, drugs with a smaller effect on bone turnover reduce the risk of only vertebral fractures, whereas those with a greater effect reduce the risk of both vertebral and nonvertebral fractures including hip fractures (delmas 2002). furthermore, only those drugs associated with relatively large bmd increases together with greater reductions in bone turnover were effective for reducing the risk of nonvertebral fractures including hip fractures (hochberg et al 2002). treatment with potent anti-resorptive drugs is a strategy for preventing hip fractures in postmenopausal women. several therapeutic agents are currently available to treat or prevent postmenopausal osteoporosis (iwamoto et al 2006, 2007). among commercially available drugs, however, only alendronate, risedronate, intravenous zoledronate, strontium ranelate, and hormone replacement therapy (hrt) have shown anti-fracture efficacy against hip fractures in post-menopausal women (black et al 1996, 2007; mcclung et al 2001; rossouw et al 2002; reginster et al 2005). alendronate and risedronate, which are the second- and third-generation bisphosphonates, respectively, have been used as first-line drugs in the treatment of osteoporosis for many years. in particular, evidence to explain the efficacy of alendronate against hip fractures has been accumulated for postmenopausal osteoporosis. thus, this paper discusses, based on a review of the literature, the efficacy of alendronate against hip fractures and the mechanism for this anti-fracture efficacy in postmenopausal women with osteoporosis. treatment of osteoporosis is conducted in accordance with the principles of evidence-based medicine (ebm). ebm takes into account information derived from the highest-quality investigations with clinical judgment and patient values, to allow optimal clinical management. all available clinical evidence is classified hierarchically into various levels; strictly conducted systematic reviews with homogeneity represent the highest level, followed by strictly conducted randomized controlled trials (rcts) with narrow confidence interval or meta-analyses with homogeneity of rcts, which have long been considered as the gold standard in the context of clinical investigations (centre for evidence based medicine). the important points that must be considered in the choice of drugs for the treatment of osteoporosis are the effects of the drugs in reducing the incidence of fractures, the consistency of the results of rcts of the drugs, and the long-term efficacy and safety of the drugs. a meta-analysis conducted by papapoulos et al (2005) demonstrated the efficacy of alendronate against hip fractures in postmenopausal women with osteoporosis. in total, 6,804 women with bmd t-score of 2.5 or less (age range: 3991 years) from 6 rcts were analyzed. the rates of hip fractures were 29 per 10,000 person-years at risk (pyr) in the alendronate group and 62 pyr in the control group. the overall risk reduction rate was 55% with the consistent results of rcts (figure 1). the efficacy of alendronate against hip fractures in postmenopausal women with osteoporosis is supported by a report of the world health organization (who) scientific group (who scientific group 2003). rcts have also demonstrated a similar incidence of gastrointestinal tract adverse events in patients treated with alendronate and a placebo, with no severe adverse events seen in the alendronate group (liberman et al 1995; black et al 1996; cummings et al 1998). the long-term (10-year) safety of alendronate has been confirmed (bone et al 2004; black et al 2006); reductions in bone turnover remain stable throughout 10 years of alendronate treatment and are associated with continued gains in lumbar spine and hip bmd. thus, alendronate could be a candidate for postmenopausal women with osteoporosis who are at a higher risk of hip fractures. vitamin d and calcium supplementation would also be needed in patients with vitamin d insufficiency/deficiency and low calcium intake. according to the analyses of the fracture intervention trial, postmenopausal women treated with alendronate with the greatest percentage reduction in bone-specific alkaline phosphatase (bsap) have the lowest risk of hip fractures, while those with the smallest reduction in bsap have the highest risk of hip fractures (figure 2) (bauer et al 2004). each 1 standard deviation reduction in a 1-year change in bsap is associated with 39% fewer hip fractures in alendronate-treated postmenopausal women (bauer et al 2004). alendronate-treated postmenopausal women with at least 30% reduction in bsap (56% of alendronate-treated subjects) have a 74% lower risk of hip fractures relative to those with less than 30% (bauer et al 2004). alendronate is effective in reducing the risk of hip fractures across a spectrum of ages (figure 3) (hochberg et al 2005). the effectiveness appears to be greater in patients with a femoral neck bmd t score 2.5 than in patients with a femoral neck bmd t score 2.0 (hochberg et al 2005). a retrospective analysis of a large population of new users of alendronate with diagnosed osteoporosis has confirmed that both compliance and persistence, in actual practice, is low and inadequate (rabenda et al 2008). siris et al (2006) analyzed the fracture probability across the full range of possible compliance values expressed as a medication possession ratio (mpr) (full mpr range: 0.01.0 or 0%100%), which is defined as the number of days supply of a medication with bisphosphonates during a certain period (sclar et al 1991). at an mpr from 0% to 50%, the probability of fracture during a period of 24 months remains consistent at about 11% and declines progressively once a threshold value of 50% is achieved (siris et al 2006). less than half the women were found to be compliant with bisphosphonate therapy (mpr 0.8) and approximately 40% of women persisted with treatment for 12 months without a substantial gap in therapy (rabenda et al 2008). poor adherence to alendronate treatment is associated with an increased risk of hip fractures (rabenda et al 2008). thus, adherence (compliance and persistence) with treatment has represented a major challenge in patients receiving alendronate. once-weekly alendronate, which is the preferred dosing regimen, has been reported to be theoretically equivalent to daily dosing, providing postmenopausal osteoporotic women with a more convenient dosing option that enhances their adherence to treatment (kendler et al 2004; rossini et al 2006). once-weekly alendronate has safety and tolerance profiles similar to daily alendronate in postmenopausal women with osteoporosis (schnitzer et al 2000; rizzoli et al 2002; simon et al 2002). rabenda et al (2008) have reported that the mpr is a significant predictor of the occurrence of hip fractures in postmenopausal women treated with alendronate. the logistic regression model has estimated that for each decrease in the mpr of 1%, the adjusted risk of hip fractures increases by 0.4%. as shown in figure 4 (rabenda et al 2008), there is a negative estimated linear relationship between the probability of hip fractures and the value of the mpr. the treatment regimen is also an important determinant of hip fracture occurrence: postmenopausal women who receive weekly alendronate are 16.4% less likely to suffer from hip fractures than those on the daily regimen (rabenda et al 2008). rabenda et al (2008) have also shown that persistence to alendronate treatment is a significant predictor of incurring hip fractures. the relative risk reduction for hip fractures is 60% for persistent patients compared with nonpersistent patients (hazard ratio: 0.404; 95% ci: 0.357, 0.457; p<0.0001). thus, both high compliance and persistence with prescribed osteoporosis medication are significantly associated with reduced hip fracture risk. the once-weekly alendronate regimen provides benefit in terms of better adherence (compliance and persistence) of the patients to the treatment regimen than the once-daily dosing regimen, leading to greater efficacy against hip fractures. osteoporosis is defined as a skeletal disorder characterized by compromised bone strength, predisposing a person to an increased risk of fracture (nih consensus development 2001). properties related to bone strength include rate of bone turnover, bmd, geometry, microarchitecture, and mean degree of mineralization (epstein 2005). theoretically, microdamage might weaken bone, but no relationship to bone strength or fracture risk has been established (epstein 2005). the proximal femur of the hip is primarily composed of cortical bone. in cortical bone, high bone turnover, as observed after the menopause, is associated with decreased bmd, increased cortical porosity due to increased bone remodeling in the haversian canal, decreased cortical thickness due to increased endocortical bone remodeling, and decreased mean degree of mineralization (hedlund et al 1989; bousson et al 2000; boivin et al 2000; szulc et al 2006). increased cortical thinning, increased porosity, and decreased mean degree of mineralization of bone (mdmb) are observed in the cortex in cases of hip fractures (loveridge et al 2004; bell et al 1999a; bell et al 1999b; bell 1999c), which might be associated with increased indices of haversian remodeling. these changes caused by high bone remodeling in terms of high bone turnover translate to alterations in bone strength and risk of fractures. alendronate inhibits osteoclastic bone resorption, strongly reduces bone turnover, increases hip bmd, decreases cortical porosity, and produces more uniform mineralization (ie, increases the mdmb) in cortical bone, possibly contributing to a reduction in the risk of hip fractures (liberman et al 1996; black et al 1996; cummings et al 1998; boivin et al 2000; roschger et al 2001). in alendronate-treated postmenopausal women, the distribution of the degree of mineralization in cortical bone shows a clear shift toward the highest mineralization values and a decrease in the number of bone structure units having low values of mineralization (boivin et al 2000). the mdmb augmentation probably accounts for most of the increase in bmd seen with alendronate (boivin et al 2000). according to the hypothesis proposed by boivin et al (2000), the reduction in the activation frequency caused by the anti-resorptive effect of alendronate is followed by a prolonged secondary mineralization that increases the percentage of bone structure units having reached a maximum degree of secondary mineralization and, though this mechanism, augmentation of the mdmb (table 1). recently, it has been possible to extract cross-sectional geometry from dual-energy x-ray absorptiometry (dxa) scan imaging. hip structure analysis (has) is a computer program designed to perform this function with dxa scans of the hip. greenspan et al (2005) have demonstrated that alendronate improves parameters of hip structure geometry as evaluated by the has, such as cortical thickness, cross-sectional area, section modulus, and buckling ratio in the narrow neck, intertrochanteric region, and femoral shaft (figure 5) (greenspan et al 2005). these data provide additional information on a potential mechanism for hip fracture reduction with alendronate. in addition, improvements in the above parameters in the narrow neck and intertrochanteric region are greater in combination therapy with hrt (figure 5) (greenspan et al 2005). this result supports the concept that anti-resorptive therapies that produce larger decreases in bone turnover markers together with larger increases in bmd are associated with greater reductions in hip fracture risk, especially at sites primarily composed of cortical bone (hochberg et al 2002). osteocyte viability has been observed to be an indicator of bone strength, with viability as the result of maintaining physiological levels of loading and osteocyte apoptosis as the result of a decrease in loading (epstein 2007). osteocyte apoptosis and decrease are major factors in the bone loss and fractures associated with aging (epstein 2007). a recent animal study demonstrated that low doses of risedronate or alendronate suppressed osteocyte apoptosis induced by fatigue loading of the ulna in rats (follet 2007). however, the effect of alendronate on osteocyte apoptosis remains to be established in clinical studies. evidence derived from the literature, stratified and based on strict ebm guidelines, suggests the efficacy of alendronate against hip fractures in postmenopausal women with osteoporosis with an overall risk reduction rate of 55%. according to the analyses of the fracture intervention trial, each 1 standard deviation reduction in a 1-year change in bsap is associated with 39% fewer hip fractures in alendronate-treated postmenopausal women, and those with at least a 30% reduction in bsap have a 74% lower risk of hip fractures relative to those with less than 30%. alendronate is effective in reducing the risk of hip fractures across a spectrum of ages. the mechanism for this anti-fracture efficacy has been clarified; alendronate suppresses bone turnover and subsequently increases hip bmd, decreases cortical porosity, improves parameters of hip structure geometry (cortical thickness, cross-sectional area, section modulus, and buckling ratio), and produces more uniform mineralization (increases the mdmb) in cortical bone. a once-weekly alendronate regimen provides better adherence (compliance and persistence) of the patients to the treatment than the once-daily dosing regimen, leading to greater efficacy against hip fractures. thus, the efficacy of alendronate against hip fractures has been confirmed in postmenopausal women with osteoporosis, especially with once-weekly dosing regimen.

osteoporosis most commonly affects postmenopausal women, placing them at a significant risk of fractures. in particular, hip fractures are an important cause of mortality and morbidity among postmenopausal women. anti-resorptive therapies that produce greater decreases in bone turnover markers together with greater increases in bone mineral density (bmd) are associated with greater reductions in fracture risk, especially at sites primarily composed of cortical bone such as the hip. thus, treatment with potent anti-resorptive drugs like alendronate is a strategy for preventing hip fractures in postmenopausal women with osteoporosis. the purpose of this paper is to discuss the efficacy of alendronate against hip fractures and the mechanism for this anti-fracture efficacy in postmenopausal women with osteoporosis. a meta-analysis of randomized controlled trials has shown that alendronate reduces the risk of hip fractures by 55% in postmenopausal women with osteoporosis. according to the analyses of the fracture intervention trial, each 1 standard deviation reduction in a 1-year change in bone-specific alkaline phosphatase (bsap) is associated with 39% fewer hip fractures in alendronate-treated postmenopausal women, and those with at least 30% reduction in bsap have a 74% lower risk of hip fractures relative to those with less than 30%. alendronate is effective in reducing the risk of hip fractures across a spectrum of ages. the mechanism for this anti-fracture efficacy has been clarified; alendronate strongly suppresses bone turnover and subsequently increases hip bmd, decreases cortical porosity, improves parameters of hip structure geometry (cortical thickness, cross-sectional area, section modulus, and buckling ratio), and produces more uniform mineralization (increases the mean degree of mineralization of bone) in cortical bone. a once-weekly regimen of alendronate administration provides better patient compliance and persistence with the treatment than the once-daily dosing regimen, leading to greater efficacy against hip fractures. thus, the efficacy of alendronate against hip fractures has been confirmed in postmenopausal women with osteoporosis, especially with a once-weekly dosing regimen.

PMC2682380

pubmed-924

stressful events activate the sympathetic nervous system and hypothalamic-pituitary-adrenal axis, which lead to the release of biochemical mediators of stress, such as cortisol, catecholamines, and neuropeptides [1, 2]. these stress mediators trigger a variety of physiological changes meant to improve the performance of the organism, such as increasing blood pressure and heart rate and enhancing the immune response. thus, a short, acute stress has been shown to exert various beneficial effects. however, when stress becomes chronic, the prolonged exposure to the same stress mediators, which were beneficial in acute stress, often triggers pathological processes and contributes to the development or exacerbation of various diseases, including cancer. chronic stress has been implicated in the stimulation of tumor development and progression by both clinical and animal studies [46]. initially, stress-induced suppression of the immune response was suggested as the major mechanism of this phenomenon. as opposed to acute stress, which enhances immunity and has been shown to increase resistance to cancer, chronic stress impairs immune responses and in this way facilitates tumor growth [8, 9]. however, there is also growing evidence indicating that stress mediators, such as glucocorticoids and sympathetic neurotransmitters, can directly affect tumor cell proliferation and survival as well as tumor angiogenesis. the direct effects on tumor cells vary significantly between different stress mediators and types of tumors [1013]. in contrast, their actions on tumor vascularization involve interactions with common angiogenic factors, such as vascular endothelial growth factor (vegf), and seem to be universal between different tumor types [5, 1416]. thus, stress mediators and their receptors can become novel targets in antiangiogenic tumor therapy. norepinephrine (ne) and epinephrine (e) belong to a family of catecholamines and are one of the best characterized stress neurohormones. ne is released primarily from the sympathetic nerves, while e is mainly secreted from the adrenal medulla. as the sympatho-adrenomedullary system is responsible for the body's fight-or-flight stress response, circulating levels of both catecholamines ne and e activate the same and adrenoreceptors (ar), which are widely distributed in all tissues. recently, ne and e have been implicated in stress-induced augmentation of tumor growth and progression. in an orthotopic model of ovarian carcinoma, the growth-promoting effect of stress was mimicked by a -ar agonist, isoproternol, and blocked by its antagonist, propranolol [5, 6]. similarly, activation of -ar resulted in an increase in metastases in animal models of lung and breast cancer [18, 19]. in all of the above models, the growth-promoting effects of stress, as well as direct activation of -ars, was associated with a significant increase in tumor vascularization, while -ar blockers reduced vessel density [5, 6]. moreover, tumors derived from stressed animals had elevated levels of vegf and other angiogenic factors, and the growth promoting actions of -ar activation was reduced by blocking the vegf pathway. thus, an increase in angiogenesis appears to be the main mechanism of growth-promoting effects of ne and e. indeed, in various cancer cell types, such as ovarian cancer, colon cancer, melanoma, pharyngeal carcinoma, and multiple myeloma, activation of -ars present on tumor cells led to a dramatic increase in synthesis and release of angiogenic factors vegf, il-8, and il-6 [5, 16, 2023]. these effects were mediated primarily via a -ar-dependent increase in camp levels, which resulted in the activation of protein kinase a (pka) and src [5, 22]. adrenergic stimulation has also been shown to increase the secretion of metalloproteases, mmp-2 and mmp-9, which further augment angiogenic and metastatic processes. interestingly, catecholamine-induced release of angiogenic factors from tumor cells can be further enhanced by its secretion from stromal cells, such as -ar-positive tumor-associated macrophages [24, 25]. although the stimulatory effects of ne and e on the release of angiogenic factors seem to be the major mechanism of their tumor-promoting actions, these neurotransmitters can also exert direct trophic effects on endothelial cells (ecs) through -ars. phenylepinephrine, a non-vasoconstrictive -ar agonist, has been shown to induce ec proliferation and migration as well as promote capillary formation. since tissue ischemia is known to stimulate ne release from the sympathetic nerves, the direct angiogenic effect of ne can be significantly enhanced in hypoxic areas of tumors. thus far, the results of experimental studies have confirmed that ar agonists exert strong stimulatory effects on tumor growth and agree that the release of angiogenic factors is the main mechanism of these actions. these discoveries open new possibilities of treatment with well-known drugs, such as antagonists of ars. some clinical data indicating decreased incidence of prostate cancer among cardiovascular patients treated with -blockers corroborated the above findings [28, 29]. however, it is important to remember that the indirect, pro-angiogenic effect of ar agonists mediated by other angiogenic factors depends on the presence of these receptors on tumor cells, thus it can be tumor-specific. moreover, the angiogenic actions of ne and e can be further modified by their direct effect on tumor cell proliferation and invasiveness, which in turn may differ among various tumors. in many cancer cell types, such as colon, ovarian, and prostate ,. however, adrenergic stimulation can also inhibit proliferation of some tumor cells, as shown in melanoma and neuroblastoma [30, 31]. in breast cancer, the adrenergic agonists seem to increase motility of cancer cells but at the same time inhibit their proliferation [13, 32]. in agreement with these data, another clinical study indicated no effect of treatment with -blockers on the risk of breast cancer among cardiovascular patients [33, 34]. thus, the success of potential cancer therapy targeting ars will depend on the type of tumor, its receptor expression pattern, and environmental factors, such as stress, which augment ne and e effects. dopamine (da) is not only a precursor of ne and e but is also an important neurotransmitter in the brain acting via two types of receptors d1 and d2. in the periphery, da is synthesized in mesenteric organs as well as released from sympathetic neurons and adrenal medulla. levels of da are elevated during stress, but rather than mediating the fight-or-flight response, as ne and e do, its role involves coping with stress. it has been shown that administration of da inhibits the growth of various tumors, such as stomach, breast, and colon cancers [14, 36]. consistently, in mice lacking the da transporter, which is normally responsible for uptake of this neurotransmitter, the elevated da levels were associated with reduced growth of lewis lung carcinoma. in gastric cancer, the endogenous levels of da were significantly lower than those in surrounding healthy tissue, indicating that the neurotransmitter acts as an endogenous tumor suppressant that needs to be inactivated to allow tumor progression. the main mechanism of these growth-inhibitory actions of da involves its direct antiangiogenic effect on ecs. in all animal models, treatment with da led to a significant reduction in tumor vascularization [14, 36, 37]. da has also been shown to block vegf-induced ec proliferation, migration, and vascular permeability. further studies revealed that da, acting through its d2 receptors, enhances endocytosis of vegf-r2 and decreases its membrane expression. this activity of da interferes with vegf signaling by reducing vegf-induced phosphorylation of its vegf-r2 and preventing the activation of downstream kinases fak and p42/44 mapk [38, 39]. in addition to its effect on mature ecs, da has also been shown to block vegf signaling in endothelial progenitor cells (epcs). as a consequence, da not only inhibits trophic functions of vegf in these cells but also blocks their recruitment from bone marrow. it has been shown that da levels are decreased in the bone marrow of tumor-bearing mice, which facilitates epc mobilization. since recent data strongly support a role for epcs in the tumor vascularization, da effect on epc function may significantly contribute to its growth-inhibitory effect. the role of da in stress-induced changes in tumor growth and progression has not been characterized. it seems that da is an endogenous inhibitory factor which requires inactivation for tumor growth, rather than sympathetic activation. however, in contrast to ne and e acting on specific tumors, da effects appear to be more universal, influencing various tumor types, via its direct actions on ecs and epcs. neuropeptide y (npy) is a 36-amino-acid peptide coreleased with ne from sympathetic nerves. npy is mainly known due to its anxiolytic effect in the brain and central regulation of food intake. in the periphery, npy inhibits the release of ne after sympathetic stimulation and acts as a vasoconstrictor. there is also a growing number of evidences that npy is a growth factor for variety of cells. the peptide has been shown to stimulate proliferation of vascular smooth muscle cells and neuronal precursors, while the trophic effect of npy on ecs revealed its angiogenic properties [4247]. the peptide stimulates proliferation and migration of ecs and promotes capillary tube formation, while in vivo, endogenous npy facilitates vascularization of ischemic tissues [43, 46, 47]. these actions are dependent on endothelial nitric oxide synthase (enos) activation and, partially, on the vegf pathway. the angiogenic activities of npy are mediated mainly by its y2rs, since npy-induced angiogenesis is severely impaired in y2r/ mice [48, 49]. due to its angiogenic properties, npy has been implicated in various pathological conditions associated with a deregulation of tissue vascularization, such as retinopathy, wound healing, atherosclerosis, and obesity [48, 5052]. recently, its role in tumor angiogenesis has also been shown. in malignancies originating from neuroendocrine tissues, such as neuroblastoma and ewing's sarcoma, npy released from tumor cells seems to be an essential factor involved in their vascularization. antagonists to npy receptors blocked the effect of both neuroblastoma and ewing's sarcoma-conditioned media on ec proliferation. consequently, treatment with exogenous npy significantly increased vascularization of subcutaneous xenografts derived from both tumor cell types. as in the case of ne and e, the angiogenesis-related growth-stimulatory actions of npy are further modified by its direct effect on tumor cell growth and survival. for example, in neuroblastoma, the peptide stimulates proliferation of tumor cells via the same angiogenic y2rs, thereby further augmenting the growth of neuroblastoma xenografts. in contrast, in ewing's sarcoma, npy induces tumor cell apoptosis via y1 and y5rs. as a result, exogenous npy inhibits growth of ewing's sarcoma xenografts in vivo, despite increase in their vascularization. although neuroendocrine tumors, which synthesize and release endogenous npy, seem the most susceptible to tumor growth regulation by this peptide, npy and its receptors have also been implicated in nonneuronal types of tumors. for example, peptide yy (pyy), which belongs to the same family of peptides and acts through the same receptors as npy, has been shown to inhibit proliferation of breast and prostate cancer cells via y4rs and pancreatic cancer cells via y2rs [5356]. thus, these direct effects on tumor cell proliferation and survival are an important aspect of npy actions in tumors and are often potent enough to overcome its angiogenesis-mediated growth-promoting effect. thus far, most of the studies addressing the role of stress in promoting cancer growth focus on the best known stress mediators catecholamines and glucocorticoids. there are no studies directly linking npy with stress-induced tumor growth and progression. however, systemic npy levels are also upregulated during stress, particularly those intensive and prolonged in nature. hence, once stimulated, the elevated levels of npy persist for a longer period of time. the physiological role of npy is to help cope with stress due to its central, anxiolytic effects [58, 59]. however, it has been shown that elevated peripheral circulating levels of npy induced by intensive chronic stress can result in significant deleterious effects, such as enhanced atherosclerosis and diet-induced obesity, both of which are diseases associated with intensive tissue growth and upregulated angiogenesis [52, 60]. thus, while high levels of npy in the brain improve stress coping, chronically elevated levels of the peptide in the circulation can result in a variety of side effects. as summarized above, the discoveries of recent years provided a significant body of evidence confirming an important role of sympathetic neurotransmitters and, consequently, chronic stress in regulating of tumor vascularization (figure 1). this research opens new avenues for developing novel therapeutics, as well as using already existing and well-characterized drugs, such as -blockers and da receptor agonists, in new clinical settings. this seems to be particularly important, since cancer diagnosis per se is usually a stressful event for the patient. however, careful consideration needs to be given to other actions of stress mediators, such as cancer-specific effects on tumor cells themselves, as well as changes in immune system, which can indirectly affect tumor development and progression. finally, since patterns of neuro-hormonal activation vary with different types of stress, tumor exposure to particular stress mediators would vary, too. thus, potential therapeutic value of modifying particular stress pathways may be dependent on a variety of factors.

recent evidence supports a longstanding hypothesis that chronic stress can influence tumor growth and progression. it has been shown that sympathetic neurotransmitters, such as catecholamines and neuropeptides, can affect both cancer cell growth and tumor vascularization. depending on neurotransmitter and type of tumor, these effects can be both stimulatory and inhibitory. norepinephrine (ne) and epinephrine (e) are potent stimulators of vascularization, acting both by inducing the release of angiogenic factors from tumor cells and directly on endothelial cell (ec) functions. as a result, activation of the adrenergic system increases growth of various types of tumors and has been shown to mediate stress-induced augmentation of tumor progression. dopamine (da), on the other hand, interferes with vegf signaling in endothelial cells, blocks its angiogenic functions and inhibits tumor growth. another sympathetic neurotransmitter coreleased with ne, neuropeptide y (npy), directly stimulates angiogenesis. however, proangiogenic actions of npy can be altered by its direct effect on tumor cell proliferation and survival. in consequence, npy can either stimulate or inhibit tumor growth, depending on tumor type. hence, sympathetic neurotransmitters are powerful modulators of tumor growth and can become new targets in cancer therapy.

PMC2874925

pubmed-925

bone is a dynamic tissue which undergoes continuous self-renewal, and bone homeostasis relies on functional equilibrium among three types of cells: osteoclasts essential for bone resorption, osteoblasts responsible for bone matrix formation, and osteocytes involved in the reception and transduction of mechanical stimuli and in the regulation of osteoclast/osteoblast differentiation and function. the balance between bone synthesis and resorption is finely tuned and any perturbations of this balance in adults trigger bone disease. osteopetrosis (osteo: bone and petros: stone) regroups a set of rare, heterogeneous, and inherited bone diseases characterized by increased bone mass. osteopetrosis is therefore an osteocondensing disease. in principle, two causes could give rise to this osteocondensing phenotype: increased bone formation or failure of resorption by osteoclasts. however, osteopetrosis is known to result from defective osteoclast differentiation or function [3, 4]. important progress has been made during the past decades in understanding the molecular mechanisms underlying the development of hereditary diseases characterized by increased bone mass [3, 5]. our objective in this review is not to give a detailed description of all the sclerosing bone diseases; such information can be found in other reviews [3, 4, 6, 7]. instead, we discuss recent findings regarding osteopetrosis and how the study of this disease has contributed to new understanding of functions associated with the skeleton [810]. osteoclasts are highly specialized cells responsible for the dissolution of bone mineral and for the degradation of organic matrix. osteoclasts are multinucleated cells (containing up to 50 nuclei), derived from the fusion of mononuclear cells belonging to the monocyte-macrophage lineage. under the influence of factors secreted by osteoblasts and/or stromal cells present in the bone microenvironment m-csf (macrophage colony stimulating factor) is expressed by osteoblasts and binds the c-fms receptor on osteoclast precursors, stimulating their proliferation and the expression of rank (receptor activator of nf-b) receptor. the interaction of rank-l, expressed and secreted by osteoblasts and stromal cells, with its receptor propels the fusion of osteoclast progenitors to form a giant multinucleated cell. a terminally differentiated osteoclast is able to degrade extracellular bone matrix by the action of specific proteins. to resorb bone matrix, osteoclasts must be perfectly polarized with a ruffle border and a sealing zone. these two features allow the creation of a resorption lacuna into which h ions are actively secreted in order to acidify it, leading to dissolution of bone matrix hydroxyapatite. type ii carbonic anhydrase (caii) hydrates co2 to form carbonic acid, which spontaneously dissociates into protons and hco3 ions. the protons are actively transported into the resorption lacuna by a vacuolar atpase proton pump located in the ruffled border domain [1, 11]. the hco3 ion is exchanged with cl by a bicarbonate/chloride antiport on the basolateral membrane of the cell. the chloride ion is translocated into the resorption lacuna through chloride channel 7 (clcn7), recently reclassified as chloride/proton antiport. the acidic environment promotes the dissolution of inorganic content and also exposes the organic matrix, which is then ready to be digested by secreted proteolytic enzymes [1, 11]. the collagenous bone matrix is dissolved by two groups of enzymes, the matrix metalloproteases and the lysosomal cathepsins. this enzyme is secreted into the resorption lacuna and degrades type i collagen in the acidic environment. the acquisition and maintenance of osteoclast membrane polarity require a complex system of vesicle trafficking and ongoing cytoskeletal renewal. one of the proteins involved in these processes is plekhm1 (pleckstrin homolog domain containing family m with run domain member 1). this protein plays a crucial role in acidification and trafficking of intracellular vesicles [13, 14]. a recently discovered protein important for osteoclast trafficking activity is snx10 (sorting nexin 10). snx10 belongs to a family of about 30 proteins sharing the px (phox homology) phospholipid binding domain and is involved in protein trafficking and osteoclast differentiation/function [15, 16]. osteopetrosis is a generic name for a group of rare genetic bone diseases characterized by osteoclast failure. the adult autosomal dominant type ii form or albert-schnberg disease classified as mild is sometimes associated with bone symptoms. in contrast, the infantile recessive osteopetroses are severe forms and usually lethal in childhood without treatment [3, 57, 17]. mutations in at least 8 genes (table 1) have been identified as being responsible for osteopetrosis pathogenesis in humans. autosomal recessive osteopetrosis is a severe disease diagnosed in the first months of life owing to a variety of problems. they also show bruising and frequent bleeding secondary to medullar hyperplasia caused by bony invasion of the medullar space. untreated children usually die during their decade from hemorrhage, pneumonia, anemia, or infection. hematopoietic stem cell transplantation (hsct) is the only treatment option currently far available [3, 5, 18]. the number of osteoclasts is normal or high, but their acidifying activity, compulsory for bone resorption, is impaired. several genes are known to be involved in this form of osteopetrosis (table 1, figure 1). about 50% of aro patients harbors loss-of-function mutations of tcirg1 which codes for the proton pump v-atpase 3 subunit [17, 19]. loss-of-function mutations of the clcn7 gene, coding for chloride channel 7, have also been described in ~10% of aro patients. mutations in ostm1 (osteopetrosis associated transmembrane protein 1), coding for a protein involved in transport of clcn7 to the ruffled border (and considered as a subunit of clcn7), have been described as causing severe osteopetrosis in ~5% of patients [2123]. primary neurological defects can also be present in patients bearing ostm1 or clcn7 mutations [20, 23]. deformity of the distal femora, bone pain, and chondrolysis of the left hip were described in one patient. recently, a mutation in the snx10 gene was found in 15 families in which the patients displayed a heterogeneous phenotype. mild growth retardation, hypocalcemia, hydrocephalus, severe hematological abnormalities, and visual impairment have been described in patients with loss of function mutations of snx10 [3, 15, 16, 24]. less than 4% of aro patients harbors loss-of-function mutations of tnfsf11, encoding rank-l, or of tnfrsf11a, encoding rank receptor, and constitute a distinct subgroup of recessive osteopetrosis. indeed, bone biopsies from these patients revealed a complete lack of osteoclasts [2527]. in addition, patients with tnfsf11 mutations exhibit some immune abnormalities and not palpable lymph nodes, but b and t lymphocyte numbers are normal. by contrast, most of the patients with tnfrsf11a mutations have a more severe immunological phenotype with a defect in memory b lymphocyte differentiation and a reduction in immunoglobulins levels [2527]. however, osteopetrosis caused by tnfsf11 mutations can not be treated by hsct, because an osteoblast defect is the basis of this pathology. in practice, a molecular genetic diagnosis should be made before transplantation to ensure that the pathology is not due to a rank-l mutation. in 1983, renal tubular acidosis can explain the hypotonia, apathy, and muscular weakness occurrence in some patients. by radiography caii deficiency resembles other forms of osteopetrosis, but brain calcifications can develop during childhood and osteosclerosis and bone modeling spontaneously decrease instead of increasing in the course of pathology evolution. metabolic acidosis occurs during the neonatal period, and renal tubular acidosis, both proximal and distal, has been described [29, 30]. caii is expressed in many different tissues including brain, kidney, red blood cells, cartilage, lung, and digestive mucosa. ado ii is commonly called benign osteopetrosis but presents with an extremely heterogeneous course from asymptomatic to rarely fatal. adoii clinical and radiological signs occur quite late in childhood or in the teens, although earlier occurring has sometimes been reported. adoii patients usually displayed osteosclerosis at the vertebral level (so-called sandwich vertebrae) and also a bone in bone aspect observed mainly in the iliac bones, but sometimes in other epiphyses. in addition, on radiography, alternating dense and light bands are often seen in iliac bones and at the extremities of long bones [7, 33]. bone fractures occur in 80% of patients, with a mean of 3 fractures per patient. the femur is the most fractured bone in this pathology, but fractures can occur on any long bones and even at the posterior arch of the vertebrae, which often leads to a spondylolisthesis. hip arthritis is frequent (in 50% of the cases) and could be due to excessive stiffness of the subchondral bone. auditory or visual impairment occurs in less than 5% of affected individuals [7, 33]. arthropathies are technically difficult and postsurgical complications, such as strengthening delay, infections, and pseudoarthritis are frequent (50% of cases) due to bone stiffness. for example, in 3 families in which most of the affected individuals expressed only a mild form of adoii, some members exhibited anemia and blindness caused by optical nerve compression. this phenotype has been called intermediate osteopetrosis because of its overlap with that of mild aro. about 70% of patients affected by adoii harbors heterozygous dominant negative mutations of the clcn7 gene (figure 1, table 1). in the remaining ~30% of cases, no mutations in clcn7 gene sequences were found, suggesting involvement of further genes in the pathogenesis of this form of osteopetrosis. osteopetrorickets is a bone disorder characterized by increase of bone mass with a defect of skeletal mineralization. schinke and coauthors performed histological analysis of undecalcified bone biopsies of 21 patients who received a diagnosis of osteopetrosis. in patients with loss-of-function mutations in the tcirg1 gene, the same pathological enrichment of osteoid was confirmed in oc/oc mice carrying a loss-of-function mutation of the tcirg1 gene, while no increase was revealed in osteopetrotic scr mice. the increase of osteoid volume was associated with hypocalcemia, due to a defect of intestinal calcium uptake. indeed it was shown that tcirg1 is also expressed in the fundus, a region of the stomach involved in gastric acidification, and loss-of-function mutations induce hypochlorhydria and reduced intestinal calcium uptake in both humans and mice. gastric acidification is a prerequisite for efficient intestinal calcium uptake; in hypochlorhydria, intestinal calcium uptake is lowered leading to parathyroin hormone (pth)-dependent activation of osteoclasts and an osteoporosis phenotype. in the case of loss-of-function mutation of tcirg1, intestinal calcium uptake is reduced and pth-dependent stimulation of bone resorption is blocked, resulting in an osteopetrorickets phenotype. performed histomorphometric analysis of bone biopsies of 9 osteopetrotic patients with loss-of-mutation in the tcirg1, clcn7, and tnfsf11a genes. in contrast, there was no sign of osteopetrorickets in patients with clcn7 and tnfsf11a gene mutations [3537]. osteocalcin can exist in two different forms, undercarboxylated and carboxylated on 3 glutamic acid residues [10, 38]. the carboxylated form has high affinity for the hydroxyapatite, facilitating its engraftment in the bone matrix. ferron et al. investigated whether acidic bone resorption lacuna promotes the decarboxylation of osteocalcin. indeed they observed that in oc/oc mice the levels of undercarboxylated osteocalcin were reduced by 30% compared to wild-type animals. similar features were observed in wild-type mice that received fetal liver hematopoietic stem cells (hscs) from oc/oc mice confirming the relevance of osteoclast function in osteocalcin-insulin signaling. moreover they observed that oc/oc mice were glucose intolerant, with reduced serum insulin levels, pancreas insulin content, and insulinexpression in the pancreas [40, 41]. interestingly, it was shown that osteopetrotic patients affected by autosomal dominant osteopetrosis with osteoclast acidification defects have lower levels of insulin and a lower undercarboxylated/carboxylated osteocalcin ratio but diabetes was not reported [40, 41]. osteocalcin is very important for the cross talk between bone and the systems responsible for male fertility [42, 43]. karsenty's group showed that osteocalcin is able to stimulate, in a camp response element binding (creb) protein-dependent manner, the production of testosterone by testes. this function is mediated by the interaction of osteocalcin with gprc6a, a g-coupled receptor expressed in leydig cells [42, 43]. in 1997 cohen et al. showed that op/op mice (which lack colony stimulating factor 1, csf-1) have reduced mating ability, low sperm numbers, and low serum testosterone levels due to decreased leydig cell steroidogenesis. the study also showed how csf-1 is essential for the development and function of the hypothalamic-pituitary-gonadal axis. further studies in osteopetrotic animal models will be important to confirm the interaction between bone and male fertility. it now well established that there is a tight correlation between bone and the immune system, which has led to a new discipline called osteoimmunology. this research area is just now expanding and we are beginning to better understand the relevance of this interplay in bone diseases. associated defects in b cell function were attributed to mutations in genes involved in osteoclast differentiation or function or to an abnormal medullary microenvironment. oc/oc mice display a block at the pro-b to pre-b cell transition, which is due to a defect of the bone microenvironment rather than to a cell autonomous defect of b cells, because in vitro experiments showed that b cell progenitors isolated from osteopetrotic mice were able to differentiate into immature b cells apart the alterations of b cell differentiation, kong and coauthors described a reduction in thymus size and a block of thymocyte development at the cd4cd8cd44cd25 stage. many studies have been published regarding the effects on osteoclast differentiation and function following alterations of immune cells. in particular, it was shown that animal overexpressing the transcription factor foxp3 (forkhead box p3) displayed osteopetrotic phenotype with increased bone mass and reduced osteoclast number and activity. moreover in vitro experiments suggested that treg cells could inhibit osteoclast differentiation and function by suppression of cytoskeletal reorganization. bone and bone marrow can be considered as two distinct compartments of the same functional unit, the bone-bone marrow organ. indeed it was shown that dysfunction of osteoclast activity results in aberrant formation of the hsc niche, leading to retention of hsc in the spleen. the frequency and absolute number of linnegsca1ckit (lsk cells) were decreased by 90% and 99.8%, respectively, in the bone marrow of oc/oc mice compared to controls. this alteration was associated with a defect of mesenchymal stem cells to differentiate into osteoblasts. the effect was revealed by a dramatic reduction in the expression of the osteoblast markers runx2, alp, osteocalcin and bsp and a reduced proportion of cells expressing cd51 and the integrin 5 (cd49e). the study showed that osteoclasts promote the formation of the hsc niche, regulating the osteoblast differentiation important for the niche. indeed the authors showed that the absence of osteoclast activity affects formation of the bone marrow hsc niche and impairs ability of mesenchymal stem/stromal cells to recruit hematopoietic progenitor cells. moreover the restoration of osteoclast function by treatment with cd45sca1 cells reestablishes normal levels of hematopoietic progenitors in the bone marrow [46, 53]. the relationship between bone and adipose tissue is an area of intensive investigations because molecules involved in bone-fat interactions could be used as pharmacological targets to prevent osteoporosis and bone fractures. in particular, ligands for ppar- include long-chain fatty acid and synthetic compounds such as thiazolidinedione. ppar- functions are associated with activation of the adipogenesis and inhibition of the osteoblastogenesis [56, 57]. these mice developed increased bone mass with a parallel reduction of bone marrow cavities and extramedullary hematopoiesis. indeed deletion of ppar- resulted in impaired osteoclast differentiation and activity, since it regulates c-fos expression involved in rankl signaling. demonstrated that the absence of ppar- in white adipose tissue led to lipodystrophy, increased bone mineral density, and extramedullary hematopoiesis in spleen. this interplay between bone and adipose tissue has clinical important implications, since a long-term treatment with the ppar- agonist rosiglitazone in patients affected by type 2 diabetes could result in osteoporosis and bone fractures [54, 58]. rare hereditary diseases inducing a bone condensation have shed new light on several aspects of bone cellular biology that were not well known. indeed the study of these diseases allowed the identification of new mechanisms of osteoclast differentiation and function and the discovery of new functions associated with the skeleton. much evidence suggests that the skeleton has a central role in bone physiology since bone disorders usually impact other organs [8, 9, 42, 43, 45, 53, 54, 60]. however, there are some features of these diseases that require further investigation. for example, as in other monogenic diseases, the genotype-phenotype correlation is not always clear and consistent. indeed, the same mutations can give rise to different phenotypes, as exemplified by the clcn7 gene heterozygous mutations. efforts to identify the mutations responsible for the remaining 30% are on-going. from a pathophysiological point of view, it is worth noting that the pathologies caused by reduced osteoclastic activity such as osteopetrosis lead to frequent fractures. this might be linked to a skeleton elasticity defect, but also to an inability to repair micro damage in bones because of a lower rate of bone turnover. this situation illustrates the well-known discrepancy between the bone quantity and its resistance to mechanical stress. in contrast, pathologies caused by an increase in bone formation due to increased activity of the wnt signaling pathway (striated osteopathy) or to tgf activating mutations (camurati engelmann disease) are not associated with an increased incidence of fractures. in conclusion, further study of osteopetrosis will allow us to better understand the physiology of bone and its impact on the whole body. moreover our challenge for the future will be to identify new therapeutic approaches for this disabling disease, particularly for those forms for which only palliative intervention is currently available.

osteopetrosis is a rare genetic disorder characterized by an increase of bone mass due to defective osteoclast function. patients typically displayed spontaneous fractures, anemia, and in the most severe forms hepatosplenomegaly and compression of cranial facial nerves leading to deafness and blindness. osteopetrosis comprises a heterogeneous group of diseases as several forms are known with different models of inheritance and severity from asymptomatic to lethal. this review summarizes the genetic and clinical features of osteopetrosis, emphasizing how recent studies of this disease have contributed to understanding the central role of the skeleton in the whole body physiology. in particular, the interplay of bone with the stomach, insulin metabolism, male fertility, the immune system, bone marrow, and fat is described.

PMC4385565

pubmed-926

it is widely believed that the rate of mortality associated with anesthesia is steadily decreasing, and that anesthesia today is very safe indeed. however, this assertion has been questioned by lagasse, who argues that the definitions of anesthetic mortality used in different studies over the years have not been consistent. perhaps the best data on anesthesia mortality come from australia, where the state-based anesthetic mortality review committees have followed a relatively consistent approach with clear definitions (table 1) for 20 years. case mix clearly makes a substantial difference to the risk of anesthesia, but the sequential triennial reports of these committees do suggest that anesthesia has become safer over time and that the risk of death definitely and entirely attributable to anesthesia today is close to 1 in 200,000 in patients who are essentially healthy and undergoing minor or moderate surgical procedures. it is disappointing, however, that even these data relate primarily to deaths within either 24 or 48 hours of anesthesia, whereas most people would probably be more interested in knowing their chances of actually going home from hospital and surviving for a reasonable period thereafter (30 days post-surgery, for example). there is very little information on this, although at least one european study suggests that these risks may be much higher than generally appreciated. this european study also confirms earlier evidence suggesting that anesthesia makes an important contribution to outcome after surgery. fundamental to any discussion of the safety of anesthesia is the matter of when mortality should be attributed to anesthesia. the australian mortality committees have provided definitions by which perioperative deaths can be attributed to one of eight categories, three of which relate to anesthesia (table 1). the critical words are: caused by the anesthesia or other factors under the control of the anesthetist. it is these other factors that have largely been overlooked in efforts to estimate the true incidence of mortality (let alone morbidity) attributable to anesthesia. perioperative myocardial ischemia is a case in point: clearly it may be attributable or amenable to factors under the control of the anesthetist; its consequences often manifest several days postoperatively so are easily overlooked, particularly in studies that focus on the first 24 or 48 hours after surgery; and it may contribute to death, although often only some time later. in a study by the study of perioperative ischemia research group, patients surviving a postoperative in-hospital myocardial infarction had a 28-fold increase in the rate of subsequent cardiac complications within 6 months following surgery, a 15-fold increase within 1 year, and a 14-fold increase within 2 years. are these extra deaths attributable to anesthesia? we would argue that they are. in the perioperative ischemic evaluation (poise) study, a multicentre prospective blinded controlled trial involving 8,351 patients, the incidence of perioperative myocardial infarction in patients randomized to receive metoprolol was 4.2% compared with 5.7% in those receiving placebo (confirming the value of beta-blockers in protecting the heart), but the overall mortality was 3.1% and 2.3%, respectively. in other words, the perioperative use of beta-adrenergic blocking drugs, a factor very much under the control of anesthetists, was associated with an excess mortality rate of 0.8%. the wider question of how and when to use beta-blockers during anesthesia, and of the other factors relevant to optimal management, is of course complex, which is exactly why anesthetists are rebranding themselves as experts in perioperative medicine. however, one can not have it both ways: the fact that the overall management of patients perioperatively can make a substantial difference to outcome is strong endorsement of the argument for a central role for well-resourced medically qualified practitioners in the provision of anesthesia, but it also makes a nonsense out of blanket claims that the risk of dying from an anesthetic is very low. there are many parts of the world in which anesthesia mortality rates are many multiples of those in high income countries such as the usa, europe, and australia [11-13]. clearly, many advances that have been made in the safety of anesthesia have not been realized everywhere. even worse, many patients do not receive desperately needed surgical and anesthetic services at all. the number of surgical operations undertaken around the world every year has only recently been estimated, and, at about 234 million, actually exceeds the number of births. unfortunately, these operations are very unevenly distributed, with only 3.5% being undertaken amongst those 34.8% of the global population who live in countries spending $100 or less per person on healthcare. it seems that about 11% of the global burden of disease measured in disability-adjusted life years arises from conditions amenable to surgery, so access to appropriate surgery is clearly essential, and obviously depends on equal access to safe anesthesia. even in high income countries there is substantial variance in access to services: more disturbingly, there is astonishing variance in practices over and above the variance attributable to resource differences. furthermore, there is ongoing evidence that even those patients who actually do receive appropriate healthcare (including surgery and anesthesia) are at unacceptable risk of harm from avoidable errors [17-20]. the safe surgery saves lives initiative of the world health organization has developed a surgical safety checklist to address some of these errors through a cost-effective tool applicable to surgery and anesthesia everywhere. in a study undertaken in eight pilot sites around the world, introduction of this checklist significantly and substantially reduced harm associated with surgery. the pilot study was not a randomized controlled trial, but it was prospective and large (data from almost 4,000 patients collected at baseline were compared with data from a similar number after the introduction of the checklist). it will not eliminate errors, but it is highly likely to reduce them and, through enhanced teamwork, to improve outcomes more generally. there is now a considerable onus on senior clinicians to promote the use of the checklist in a meaningful manner involving the engaged participation of all members of the operative team. there is also considerable onus on the organizations associated with anesthesia to continue to support initiatives to improve the training and resources available for anesthesia in resource-limited regions of the world. the world federation of societies of anaesthesiologists has provided strong leadership in this regard and in recent years the support for anesthesia from the world health organization has increased considerably. as with so many things, the sophistication and accuracy of the way in which we measure outcome has increased in parallel with other advances in anesthesia. this has the effect of highlighting risks of anesthesia that, in previous years, may not have been appreciated at all. so is anesthesia becoming safer? certainly-and this is self evident to anyone who has practiced for more than three decades. certainly not, especially in higher-risk patients, and in low-income regions of the world. we are making commendable progress, but there is a long way to go to achieve the goal that no patient shall be harmed by anaesthesia. the authors have financial interests in safer sleep llc, which produces a system to promote safety in anesthesia.

in well-resourced areas of the world anesthesia has become safer over the past decades, and anesthesia mortality does seem to be reducing. however, there is a lack of international agreement over definitions of anesthetic mortality and, therefore, difficulty in knowing exactly what the rate of anesthetic mortality is. avoidable harm from error is still a problem, and sophisticated analysis suggests that more deaths than generally appreciated may be attributable to factors under the control of anesthetists. mortality rates in low income areas of the world are unacceptably high. there is more to be done if anesthesia is to become truly safe for all patients.

PMC2948295

pubmed-927

severe sepsis is characterized by acute release of systemic inflammatory (systemic inflammatory response syndrome [sirs ]) and anti-inflammatory mediators (compensatory anti-inflammatory response syndrome [cars ]) caused by infection [1, 2]. the cytokines associated with sirs include interleukin- (il-) 1, il-6, il-12, il-17, and tumor necrosis factor- (tnf-). the cytokines associated with cars include il-4, il-10, and transforming growth factor- (tgf-) 1. this immune imbalance results in multiorgan dysfunctions and eventually death in patients with severe sepsis. dexamethasone inhibited lipopolysaccharides- (lps-) stimulated release of tnf-, il-6, il-8, and il-10 from whole blood of septic patients. similarly, hydrocortisone decreased il-1 and il-6 production from ex vivo lps-stimulated whole blood of septic shock patients. steroid might affect cytokine production from circulatory immune cells of septic patients. according to the surviving sepsis campaign (ssc) guidelines for the management of severe sepsis and septic shock (2008 and 2012), patients with septic shock can be prescribed low-dose steroids if adequate fluid resuscitation and vasopressor therapy are unable to restore hemodynamic stability [6, 7]. septic shock patients treated with low-dose steroids could reverse shock at an early stage, but final mortality rate was similar to those without low-dose steroids. the adjusted hospital mortality rate was significantly higher (odds ratio=1.18, p<0.001) in patients who received low-dose steroids compared with those who did not. however, a multicenter observation study found that steroid use was associated with low mortality rates in patients with severe sepsis. thus, the use of low-dose steroids in patients with severe sepsis is still controversial. serial increase in monocyte human leukocyte antigen-dr (hla-dr) expression and il-12 response in stimulated peripheral blood mononuclear cells (pbmcs) are associated with higher survival rate in patients with severe sepsis. thus, we analyzed our study database to explore the relationship of low-dose steroid treatment with hla-dr expression and cytokine responses in patients with severe sepsis by repeated detections. this study is a post hoc analysis using our previously published study database. from july 2008 to june 2009, 35 patients who were admitted to a 20-bed intensive care unit (icu) in a regional teaching referral hospital for severe sepsis were enrolled in this study. six nonsurvivors died within 7 days and they all received low-dose steroid therapy. the data of these six patients was excluded because of lack of repeated cytokine response results for analysis. the sirs was defined as two or more of the following criteria: (1) body temperature>38c or<36c; (2) respiratory rate>20 breaths/min; (3) heart rate>90 beats/min; and (4) white blood count>12000/l or<4000/l or>10% bands. sepsis was defined as sirs according to a confirmed infectious etiology. for validating experimental findings, 22 men and 8 women visiting our health evaluation center for examinations were enrolled as healthy controls with mean age of 60.8 1.9 years old. severe sepsis was defined according to the consensus criteria of sepsis with one or more organ dysfunctions such as shock, respiratory failure, acute renal failure, jaundice, and thrombocytopenia [14, 15]. septic shock was defined as sepsis-induced hypotension unresponsive to fluid resuscitation within 24 hr after admission to icu. acute renal failure was defined as a rapid increase in creatinine levels (> 0.5 mg/dl). jaundice was defined as hyperbilirubinemia (total bilirubin>2 mg/dl), whereas thrombocytopenia was defined as a platelet count of<150,000/l. disease severity was assessed by the acute physiology and chronic health evaluation (apache) ii score. standard treatment according to guidelines was provided to all patients [6, 8]. a course of low-dose steroid therapy could be prescribed in septic shock patient with 7 days of intravenous hydrocortisone 50 mg every 6 hours if shock developed within 24 hr after admission to icu. the institutional review board at chang gung memorial hospital approved our previous study (96-1465b) and the patients ' close family members provided informed consent. patients who survived longer than 28 days after icu admission were defined as survivors. whole blood (10 ml) was obtained from each patient at 08:30 a.m., within 48 h of admission to icu, and immediately mixed with heparin. pbmcs were isolated via differential centrifugation over ficoll-plaque (amersham biosciences, uppsala, sweden) from 8 ml of residual whole blood within 2 h of collection. 2.5 10 of pbmcs were suspended in 50 l of phosphate-buffered saline (pbs) and incubated in the dark for 15 min at room temperature with 20 l of hla-drpercp, cd11bpe, and cd14fitc antibodies (becton dickinson, ca, usa). the monocytes were detected by a three-color flow cytofluorimeter (beckman coulter, ca, usa) with positive controls for cd11bpe and cd14fitc. monocyte hla-dr measurements were expressed as percentages of hla-dr-positive monocytes and as means of fluorescence intensities (mfi) in relation to the entire monocyte population, thus reflecting the hla-dr density per cell. flow cytometry analysis was performed using kaluza software v1.1 (beckman coulter, ca, usa). the figure of analysis strategy for monocyte hla-dr expression was presented in our previously published paper. 5 10 pbmcs were plated in two wells of a flat-bottomed 24-well plate (nunclon, aarhus, denmark) in 1 ml of sterile rpmi 1640 tissue culture medium containing 5% heat-inactivated bovine serum, 1 mm of l-glutamine (gibco, grand island, usa), and 1 mm sodium pyruvate. the cells in second well were stimulated with 1 pg/l of lps (sigma, missouri, usa). the plate was incubated at 37c in 5% carbon dioxide for 24 h. supernatants of the culture wells were sampled and stored at 80c until use. cytokine levels of supernatants were measured with a human enzyme-linked immunosorbent assay (elisa) kit, according to the manufacturer's instructions. the elisa kit of il-10 was manufactured by pierce biotechnology, illinois, usa. the il-6, tgf-1, and il-17 were purchased from r&d systems, inc., the il-12, tnf-, and il-1 were purchased from becton dickinson, ca, usa. cytokine responses were defined as the difference in supernatant levels with and without lps stimulation. changes in cytokine responses were defined as the difference in cytokine response on day 7 minus the cytokine response on day 1. statistical analysis was performed using the statistical package for the social sciences (spss) software v17.0 for windows (spss inc., differences in continuous variables between two groups were analyzed using the mann-whitney test, whereas differences in categorical variables were analyzed using the chi-square test or fisher's exact test. differences in continuous variables in the same subjects were analyzed using the wilcoxon signed-rank test. generalized linear model analysis was used to determine the association between clinical characteristics and cytokine response. of the final 29 enrolled subjects with severe sepsis, 18 patients were prescribed low-dose steroids and 11 were not (table 1). in the low-dose steroid group, 12 patients survived for 28 days and six died (table 2). the apache ii score in low-dose steroid group was higher than no-steroid group, although the statistical analysis did not show difference. there were no significant differences in age, gender, histories, infection sources, and initial appropriateness of antibiotics between groups with and without low-dose steroid therapy. patients administered low-dose steroid therapy displayed higher percentages of septic shock (94% versus 36%), compared with no low-dose steroid group. the rates of gastrointestinal bleeding, acute renal failure, thrombocytopenia, jaundice, bacteremia, and mortality were similar between the two groups. tnf- response in the control group was significantly higher than in both patient groups (table 3). however, il-1 response in the control group was significantly lower than in both patient groups. il-12 and tnf- responses on days 1 and 7 in steroid group were significantly lower than in no-steroid group. there were no differences in il-1, il-6, il-10, il-17, and tgf-1 responses on days 1 and 7 between the two patient groups. il-1 and tgf-1 responses in patients administered low-dose steroid therapy decreased from day 7 to day 1 (table 3). compared to day 1, there were no changes in il-6, il-10, il-12, il-17, and tnf- responses on day 7 in patients who received low-dose steroid therapy. il-12 response in patients without low-dose steroid treatment significantly increased from day 7 to day 1 (figure 1). similarly, there were no changes in il-1, il-6, il-10, il-17, tgf-1, and tnf- production in patients treated without low-dose steroid therapy from day 7 to day 1. in survivors with severe sepsis, there were no changes in il-1, il-10, il-17, tgf-1, and tnf- production from day 7 to day 1 in survivors. all cytokine responses detected did not change from day 7 to day 1 in nonsurvivors (table 4). table 5 shows regression analysis results demonstrating the relationship between changes in il-1 levels and low-dose steroid therapy and other clinical characteristics. apache ii score levels were independently and positively associated with changes in il-1 response (b=3.110). patients who were severely ill produced more il-1 in pbmcs after 6 days compared with those who had milder illness. however, low-dose steroid therapy was not independently associated with the change in il-1 levels. low-dose steroid therapy did not influence il-1 production in pbmcs of severe septic patients. low-dose steroid therapy was also independently and negatively linked to the increase in il-12 after regression analysis (b=750.743; table 6). il-12 recovery from day 7 to day 1 was lower in patients with low-dose steroid therapy. the presence of septic shock did not independently correlate with the change in il-12 levels. il-12 recovery from day 7 to day 1 was high in male patients (b=447.838). although tgf-1 response in patients receiving low-dose steroidtherapy decreased from day 7 to day 1, the regression analysis did not find independent factors associated with the change in tgf-1 levels (table 7). there was no difference in monocyte percentage, positive hla-dr percentage in monocytes, and mfi of hla-dr between patients treated with or without low-dose steroids on day 1 or 7 (figure 2). in addition, there was no change in monocyte percentage, positive hla-dr percentage in monocytes, and mfi of hla-dr in patients treated with or without low-dose steroids from day 7 to day 1. previous studies often used in vitro cultures to test the effectiveness of different doses of steroids or repeatedly measured plasma levels between the patients receiving or not receiving steroids at specific times. these studies have a common fault; they indirectly detect the function or response of immune cells after a course of low-dose steroid therapy. since circulating cytokines can be produced by immune and nonimmune cells, such as smooth muscle cells and endothelial cells, circulating cytokine levels only reflect a relatively broad response. in this study, we demonstrated that a complete course therapy with low-dose steroid was associated with decreased il-12 production in pbmcs from patients with severe sepsis. in contrast to our results, a 6-day crossover study with serial detecting plasma cytokine levels in patients with septic shock reported that the inhibitory effect of hydrocortisone infusion did not decrease plasma il-12 level. the first possible reason for this result is that only 3 days of hydrocortisone infusion was administered to each group; therefore, the effect of low-dose steroid did not have a chance to develop. second, ficoll density gradient centrifugation for monocytes isolation resulted in lower cell function than by positive selection by magnetic microbeads. the function of il-12 production from pbmcs in this study might be influenced by ficoll solution. other reasons may be that there is increased il-12 production in dendritic or human b-lymphoblastoid cells, resulting in similar amounts of circulating il-12. in this study, we did not find an association between il-6 response and low-dose steroid therapy. they found that il-6 production in lps-stimulated diluted whole blood transiently fell from day 1 to 3 and returned after day 5 in a low-dose hydrocortisone therapy group. in terms of circulating cytokine levels, there were no significant differences in plasma il-6 levels between treatment groups of continuous 6 h infusion of endotoxin+hydrocortisone or endotoxin+saline in an animal study. furthermore, a double-blind, randomized, placebo-controlled study showed similar results. two doses of hydrocortisone (100 mg per 8 h) were administered after bilateral total knee replacement in the study group. il-6 levels were 40% lower in the study group after 10 h but returned to levels similar to that of the control group at 24 h. however, other studies have reported different results. stress dose of hydrocortisone infusion in patients with septic shock significantly decreased plasma il-6 levels on day 5 between the steroid and placebo groups. hydrocortisone infusion reduced plasma il-6 levels in a 6-day crossover study in patients with septic shock. generally, low-dose steroid therapy may influence plasma il-6 levels but not il-6 response in pbmcs, based on current evidence. il-10 production in stimulated pbmcs did not differ between patients receiving or not receiving low-dose steroid therapy on day 1 or 7. even after 6 days of therapy, il-10 response did not change in the low-dose steroid group. moreover, dexamethasone inhibited lps-stimulated release of il-10 from diluted whole blood of septic patients in a dose-dependent manner. after in vivo administration of high or low amounts of cortisol, high cortisol therapy further increased lps-induced il-10 expression in isolated monocytes from healthy participants, whereas low cortisol therapy decreased il-10 expression. more studies are needed to determine the role of low-dose steroid therapy on il-10 production in patients with severe sepsis. endogenous cortisol is one of the main anti-inflammatory mediators induced by our central nervous system during severe sepsis. it has been described that steroids have downregulating effects on monocyte hla-dr expression [2527]. high endogenous cortisol levels observed in septic shock patients may play a role in the loss of monocyte hla-dr expression via its effect on hla-dr transcription. however, monocyte hla-dr expression seems not to be influenced by low-dose steroid therapy. in this study, there was no change in monocyte percentage, positive hla-dr percentage in monocytes, and mfi of hla-dr in patients with low-dose steroid therapy from day 1 to 7. cortisol can influence the immune system and is crucial for the host for defense against pathogens. cytokines mediate a high glucocorticosteroid output with regulation from the neuroendocrine to the immune-endocrine system. as a result, high levels of adrenal glucocorticosteroid are vital in preventing an uncontrolled inflammatory response to cytokines. the main immunological function of il-12 is to enhance native t lymphocyte differentiation to type 1 help t (th1) cells. th1 cells secrete interferon- that regulates macrophage and natural killer (nk) cell activation, stimulates immunoglobulin secretion by b cells, and enhances th1 cell differentiation. in this work, this might explain why low-dose therapy was associated with an increase in adjusted hospital mortality in ssc database. decreased il-12 response in patients receiving low-dose steroid therapy may depress a protective effect by decreased cellular immunity and phagocytic functions. first, the percentage of septic shock was different between steroid and no-steroid groups. the reason why steroid group had higher percentage of shock is that patients with septic shock are appropriate for steroid treatment according to guidelines. in septic shock group, not in no-shock group, patients with low-dose steroid had significantly lower change of il-12 response than those without low-dose steroid. although generalized linear model analysis was used to exclude the confounding effect of shock, there was not a directly statistical analysis to demonstrate low-dose steroid affected il-12 response recovery in patients without septic shock. second, patients with low-dose steroid treatment have received steroid when their blood was drawn on day 1. although the time from onset of steroid treatment to blood sampling was short and not more than 48 hr, cytokine responses and hla-dr expression might be influenced. il-12 response observed in pbmcs increased from day 7 to day 1 in severe septic patients. we demonstrated that a course of low-dose steroid therapy influenced il-12 production from in vitro lps-stimulated pbmcs of severe septic patients. there was no correlation between low-dose steroid therapy and monocyte hla-dr expression.

background. sepsis-induced immunosuppression may result in higher mortality rates in patients. methods. we examined the relationship of cytokine responses from stimulated peripheral blood mononuclear cells (pbmcs) and monocyte human leukocyte antigen-dr (hla-dr) expression (days 1 and 7) with low-dose steroid therapy in 29 septic patients. patients were treated according to the guidelines. thirty healthy controls were enrolled for validation. results. eighteen patients were prescribed low-dose steroids and 11 were not. interleukin- (il-) 12 responses in patients without low-dose steroid therapy on days 1 and 7 were higher than those with low-dose steroid therapy. compared to day 1, il-12 responses significantly increased on day 7 in patients without low-dose steroid therapy. after regression analysis, the change in the il-12 response from day 7 to day 1 was found to be independently associated with the low-dose steroid therapy. there was no difference in monocyte hla-dr expression between patients treated with and without low-dose steroid on day 1 or 7. no change in monocyte hla-dr expression from day 7 to day 1 was observed in patients with or without low-dose steroid therapy. conclusion. decreased il-12 response was associated with the low-dose steroid therapy in pbmcs of septic patients.

PMC4983364

pubmed-928

congenital causes of intestinal obstruction include duodenal atresia, malrotation, duodenal obstruction from ladd bands, other congenital bands, superior mesentery artery syndrome, enteric duplications as well as several other entities. congenital anomalous bands resulting in proximal jejunal obstruction are a rare entity that should be considered in the differential in patients with clinical signs of obstruction and no prior history of abdominal surgery. we present a case of an 8 year-old girl with a nine-month history of intermittent vomiting and no history of prior surgery and review the embryology and radiologic findings of this entity using different imaging modalities. an 8 year-old girl with a past medical history of constipation presented with a history of vomiting for nine months. the vomiting was intermittent, non-bloody, at times bilious, and occasionally contained food particles. an abdominal ultrasound was performed which demonstrated debris within a dilated proximal duodenum. the superior mesenteric artery (sma) and superior mesenteric vein (smv) were visualized with the sma lying to the left of the smv with normal anatomic alignment [figure 1]. (a) ultrasound of mid abdomen shows superior mesentery vein (arrow) and superior mesentery artery to the left (b) the doppler flow study confirms arterial flow in the superior mesentery artery (arrowhead) and the venous flow in the uncompressed superior mesenteric vein (arrow). a standard barium single contrast upper gastrointestinal (ugi) series was performed with the contrast entering the antrum and duodenal bulb. the proximal duodenum and descending duodenum were markedly dilated in caliber with apparent tapering of the third part of the duodenum. there was an abrupt cutoff with no contrast extending beyond approximately the mid abdomen [figure 2]. delayed images were obtained while the patient was supine with contrast never extending beyond the third portion of the duodenum [figure 3]. single contrast upper gastrointestinal series shows on a single view plain radiograph the contrast abruptly cutting off approximately at the mid abdomen (arrow). single contrast upper gastrointestinal series shows on delayed images with patient laying supine, contrast never extending beyond third portion of duodenum with persistent cutoff (arrow). contrast is seen in antrum of stomach (star), proximal duodenum, and descending duodenum. patient was subsequently placed prone for several minutes with eventual slight transit of contrast [figure 4]. thirty minute delayed prone images demonstrated contrast in the proximal small bowel, however proximal duodenum remained markedly dilated and contrast/debris filled [figure 5]. additionally delayed overhead images demonstrated contrast extending to the distal small bowel after patient had been upright for greater than 30 minutes [figure 6]. markedly dilated proximal duodenum with abrupt cutoff in the third portion of the duodenum raised concern for a questionable mass like positionally dependent extrinsic compression of the third portion of the duodenum. delayed prone images demonstrated contrast in the proximal small bowel with persistent dilatation of the proximal duodenum. additional delayed image following several minutes of prone position demonstrates a small amount of contrast progressing to the jejunum (arrow). delayed abdominal radiograph following the upper gastrointestinal series after patient had been upright for greater than 30mins demonstrates further progression of contrast into the jejunum and ileum (star). distal most extent of contrast near the cecum (arrow). a subsequent computed tomography (ct) of the abdomen and pelvis the duodenum was distended with the third part of the duodenum tented downward [figure 7]. the duodenal/jejunal junction was severely narrowed and coursed distally anterior to the right common femoral artery below the bifurcation [figure 7]. computed tomography (ct) of the abdomen and pelvis shows dilated fluid filled duodenum (asterisk, a). duodenum tented medially (black arrow, b) and downward (white arrow, c, black arrow, e). duodenum severely narrowed as it crosses anterior to right femoral vein below the bifurcation (white arrow, d). the patient was taken for an exploratory laparotomy and found to have dense vascularized bands without intestinal malrotation. upon entering the abdomen ,. further exploration in the abdomen revealed the proximal jejunum tethered by dense vascularized bands running from approximately five inches distal to the ligament of treitz down into the pelvis. the cecum was incidentally noted to be folded back on itself and adherent to this tented portion of the jejunum. after these adhesions were ligated, the bowel was examined and no other anatomical abnormalities were discovered. the patient experienced a normal post-operative course with subsequent complete resolution of symptoms. the congenital causes of intestinal obstruction in infants and children are intestinal atresia and stenosis, congenital bands, remnants of embryologic structures such as vitelline arteries or veins, omphalomesenteric ducts or a mesourachus. in addition intestinal obstructions caused by other anomalous congenital bands have only been described sporadically in the literature and are otherwise rare.[57] an example of anomalous congenital bands is ladd bands. they arise from the cecum and extend to the sub-hepatic region, posterior peritoneum or abdominal wall. ladd bands result in compression of the 2 or 3 duodenal portions, which lead to intestinal obstruction. however, the location of these bands is different from embryonic remnants such as vitelline vessels or omphalomesenteric ducts. akgur et al., cited a few series of cases to suggest that these bands are the result of a mesenteric developmental anomaly. mesenteric anomalies have not been well documented aside from cases of malrotation and anomalous intestinal fixation. in our patient, upper gastrointestinal (ugi) barium examination showed a dilated duodenum near the ligament of treitz with sharp obstruction. most duodenal obstructions by external compression are caused by abnormal duodenal rotation with internal herniation or ladd bands. in our patient our case is particularly rare as abnormal dense vascularized bands were seen distal to the ligament of treitz, which is not characteristic of ladd bands. this resulted in dilatation of the proximal jejunum which was not readily identified on the ugi barium examination. though imaging demonstrated a high grade obstruction, the patient's symptoms were chronic and mild. furthermore prone positioning partially alleviated the degree of obstruction presumably by altering the position of the anomalous bands and relieving the obstruction. finding an anomalous band tethering the proximal jejunum to the pelvis and adhering the jejunum and cecum is unique and unlike previously described congenital peritoneal bands. as no previous operation had been performed on this patient and operative findings did not show any other abnormal post inflammatory adhesions between the small and large intestines, the etiology of these bands was determined to be congenital., discovered a band running from the antimesenteric wall of the proximal jejunum just distal to the treitz's ligament to the root of the mesentery. the anomalous congenital band in that case was considered to be the remnant of ventral mesentery that failed to resorb completely. in both our case and the case described by liu et al. external compression of the proximal jejunum by an anomalous band should be included in the differential diagnosis of obstruction of the distal duodenum or duodeno-jejunal junction for patients without previous history of surgery, especially when ugi barium examination shows normal duodenal rotation and duodeno-jejunal fixation.

congenital anomalous bands at the proximal jejunum resulting in obstruction have been described sporadically in the literature and are otherwise rare. we present a case of an 8 year-old girl with a nine-month history of intermittent vomiting and no history of prior surgery. the imaging workup includes an abdominal ultrasound, a single contrast upper gastrointestinal series, and a dual contrast computed tomography of the abdomen and pelvis. surgical intervention revealed the presence of dense bands at the proximal jejunum without evidence of malrotation. our report reviews the embryology and radiologic findings of this entity using different imaging modalities.

PMC3551515

pubmed-929

in generalized ligamentous laxity, the range of motion across various joints in an individual is increased compared to the mean range of motion in the general population (1). although this could occur as a result of genetic disorders affecting the connective tissue, in most individuals there is no genetic aberrancy and it exists in isolation (2). the prevalence of generalized ligamentous laxity varies among different races from 5% to 57% of the general population (3). it is well known that generalized ligamentous laxity is implicated in various musculoskeletal injuries, especially in the lower extremities, such as cruciate ligament injuries, patellar instability, and ankle injuries (4, 5). although generalized ligamentous laxity is a predisposing factor for shoulder instability (6), to the best of the authors knowledge no study has investigated the role of generalized ligamentous laxity in acute and chronic shoulder injuries in athletes. this study aimed to determine whether generalized ligamentous laxity can be a predisposing factor for acute and chronic shoulder injuries in athletes. this cross-sectional study was conducted from april to october 2015. to obtain a representative sample of athletes with shoulder injuries, we sought all athletes who had sustained shoulder injuries in hamadan province in iran over the previous three years according to the documents of the local branch of the iranian federation of sports medicine in hamadan province. all the relevant data were recorded, including demographic information, type of sport, duration of sporting activity, and the beighton score for generalized ligamentous laxity (table 1). our inclusion criteria were being 17-37 years old and having a history of sports activity for at least six months (if there was a shoulder injury, then the requirement was at least six months of sports activity before the injury). the exclusion criteria were having any deformity or disorder that interfered with the beighton score assessment and a lack of documents proving at least six months of regular sports activity. several athletes were excluded, mainly because of a lack of documentation proving sports participation. using a sampling formula for cross-sectional studies where =0.05, d=0.065, and p (ligament laxity prevalence according to other studies)=0.15, all the volunteers signed a consent form to confirm their participation in the study, and the study was approved by the ethics committee of hamadan university of medical sciences (no.: ir.umsha.rec.1395.101) we considered generalized ligamentous laxity as point 4 or more of the beighton score and divided all the participants into two groups: the participants in group a had ligamentous laxity (with ligamentous laxity) and those in group b did not (without ligamentous laxity). any acute or chronic shoulder injuries and functional issues among the participants were also assessed according to the standard farsi (contemporary persian) translation of the quickdash measure. a total of 118 volunteer athletes participated in our study, which included 32 athletes with shoulder injuries and 86 athletes without shoulder injuries. they were divided into two groups according to the beighton score: group a (with ligamentous laxity) consisted of 43 participants and group b (without ligamentous laxity) consisted of 75 participants. we then compared the following factors between the two groups: acute shoulder injury, chronic shoulder injury, shoulder instability, chronic shoulder pain, and dash scores (table 2). a statistical analysis was carried out using the statistical package for social sciences (spss) version 20 software (spss inc., chicago, il). the chi-squared test was used to analyze the effect of the generalized ligamentous laxity on the nominal scale values, namely chronic shoulder pain, and chronic and acute shoulder injuries. spearman s test was used to analyze the generalized ligamentous laxity effect on the last value, successful return to sports activities, for which the dash score was ordinal. first, we evaluated the relationship between ligamentous laxity and chronic shoulder pain (table 3). sixteen participants (37.2%) in group a and 13 participants (17.3%) in group b suffered from chronic shoulder pain (p=0.016). we then evaluated the number of chronic shoulder injuries in both groups (table 4). thirteen participants in group a (30.2%) and 11 participants in group b (14.6%) had chronic shoulder injuries, which was statistically significant (p=0.032). with respect to acute shoulder injuries, 11 participants in group a (25.5%) and 19 participants in group b (25.3%) had chronic shoulder injuries (table 5). we then assessed upper limb function using the quickdash measure (table 6) and found that there was a significant difference between the functional status of the two groups (p=0.030). finally, we compared the history of acute and chronic instability between the two groups based on dislocation documentation or a history of shoulder pain with at least two positive clinical tests for instability (table 7). eleven participants in group a (25.5%) and five participants in group b (6.6%) had instability, which was significant (p=0.004). the shoulder joint gets injured frequently in sports like volleyball, handball, swimming, basketball, and overhead sports, and an abnormal increase or decrease in range of motion is an important factor in sports injuries involving the shoulder (7). generalized ligamentous laxity refers to an increased range of joint motion compared to that of the general population. its prevalence is 5%-15% but varies among different race groups, with an incidence as high as 57.3% among africans (1, 6). although there is no standard for defining ligamentous laxity, there are several clinical scoring systems for measuring joint laxity in individuals and populations. the beighton score is considered an excellent method for screening generalized ligamentous laxity (8, 9). bin abd razak et al. evaluated generalized ligamentous laxity in the musculoskeletal injuries of 100 young patients in a primary care center for comparison with a healthy control group (1). they found that the individuals who had musculoskeletal injuries were 3.35 times more likely to have generalized ligamentous laxity compared to healthy people. several studies have demonstrated the relationship between generalized ligamentous laxity and sports injuries in the lower extremities, including cruciate ligament injuries (4) and patellar dislocation (5). found a significantly increased risk of knee joint injury among hypermobile participants who played contact sports (10). notwithstanding, little evidence exists regarding the relationship between generalized ligamentous laxity and shoulder injuries. studied generalized ligamentous laxity and increased external rotation of the shoulder as a predisposing factor for primary shoulder dislocation in young, healthy individuals. they found that men with shoulder dislocation were 6.8 times more likely to have generalized ligamentous laxity (6). to the best of our knowledge, no study has investigated a range of shoulder injuries in athletes with or without ligamentous laxity. hence, we evaluated the relationship between generalized ligamentous laxity and various shoulder injuries in athletes who had participated in sports activities for at least six months. the athletes with generalized ligamentous laxity had more chronic shoulder pain, chronic shoulder injuries, shoulder instability (acute and chronic), and less functionality according to their dash scores compared to the athletes without generalized ligamentous laxity. interestingly, there was no difference in the number of acute shoulder injuries between the two groups. this was because we considered fractures around the shoulder to be acute injuries in the study. if we had omitted fractures in both groups, the difference in acute injuries would have been significant. the strength of our study was the evaluation of all the volunteer professional athletes in our province who had participated in at least six months of regular sports activity, including professional athletes with shoulder injuries. since the treatment of injured athletes is the responsibility of the federation of sports medicine in iran, this federation had a complete list of all the injured athletes in the province. the limitation of our study was that we only evaluated athletes in one province of iran. although hamadan province is ethnically diverse and includes most iranian ethnic groups (azeri, kurd, lur, and fars), and parallel research results will thus be quite compatible with the current research findings, it is suggested that the same study be conducted in different provinces of iran with a greater number of athletes. a sports injury can be influenced by the athlete s level of professionalism. although we excluded athletes who had participated in professional sports activities for less than six months, it is not clear if their knowledge and ability to prevent sports injuries would be the same as those with more experience. this study highlights the importance of having screening programs in athletic clinics, sports clubs, and sports offices at high schools and universities to identify individuals with generalized ligamentous laxity, and emphasizes the need for physicians and surgeons to practice prophylactic measures. there may be a role for shoulder-specific proprioceptive and strength training protocols for shoulder injuries in individuals with generalized ligamentous laxity who participate in high-risk sports, especially contact sports.

backgroundgeneralized ligamentous laxity is defined as an increased range of joint motion compared to that of the general population. it is a predisposing factor for sports injuries, especially in the lower extremities. nevertheless, there is little evidence about the relationship between generalized ligamentous laxity and sports injuries in the upper extremities. objectivesto evaluate the relationship of generalized ligamentous laxity with acute and chronic shoulder injuries in athletes. patients and methodsour study comprised 118 volunteer athletes with a history of at least six months of sports activities and a shoulder injury in the three years prior to participation in our study. the athletes were divided into two groups: those with or without generalized ligamentous laxity. acute and chronic shoulder injuries, shoulder pain, shoulder instability, and functional status assessed via the quickdash measure were determined and compared between the two groups. a p value of less than 0.05 was considered significant. resultsgroup a (with ligamentous laxity) consisted of 43 participants (36.4%) and group b (without ligamentous laxity) consisted of 75 participants (63.6%). the athletes in group a had more shoulder pain (p=0.016), chronic shoulder injuries (p=0.032), and shoulder instability (p=0.004), and less functionality (p=0.030) than those in group b. if fracture were not considered an acute injury in both groups, the athletes with generalized ligamentous laxity would have had more acute shoulder injuries. conclusionsgeneralized ligamentous laxity is an important predisposing factor for acute and chronic shoulder injuries in athletes. prescreening programs for beginners and rehabilitation shoulder programs for sports athletes at high risk are strongly recommended.

PMC5010880

pubmed-930

data types and tools evolve, predictive approaches developed for such work can also be directed at a number of closely connected issues. some of these include taking advantage in motif discovery of increasing numbers of genomes, including low-coverage sequences; quantifying the contributions to motif discovery of different genomes or sets of genomes; improving the predictive reliability of motifs by genome-scale clustering and co-occurrence; determining a best minimal set of motif discovery methods, probably in a discovery approach that uses multiple methods (6); and using coexpression and other functional data types. in this report, we describe a new cisred database that contains predictions for whole-genome discovery of regulatory elements in mammals and other eukaryotes. we also describe the predictive system behind the database, which uses genome-scale approaches to predict deeply conserved ab initio motifs and identifies groups of similar motifs and co-occurring patterns of motifs. the system is designed to be readily maintained and extended in the context of rapidly evolving resources, data types and tools. the upstream section of the pipeline loads the database with significant discovered atomic motifs, and the section downstream of the database identifies groups of similar atomic motifs and co-occurring patterns of motifs. an atomic motif consists of a set of sequences, typically with a common length between 6 and 12 bp, members of which are present in a sequence region on the target species and in corresponding regions on other genomes. the system uses genome resources that are a combination of directly downloaded and processed sequences, annotations and relationships (e.g. orthology, coexpression and interactions). these are stored in an automatically updated local resource that holds a wide range of public and commercial databases. motif discovery was carried out in a search region based on a single major transcript for each gene. an input target sequence set consisted of a sequence from the target species, and corresponding sequences from homologous vertebrate genes. the following rules were used to assemble target sequence sets for cisred 1.2e. we identified homologous genes by combining data from compara (7), homologene (8), inparanoid (9) and kegg (10). for each target human gene and each of its orthologues, we took the major ensembl (7) transcript if its protein sequence was n-terminal complete. we further required that the human gene had an annotated ensembl 5-untranslated region (5-utr) that was at least 10 bp long. for each target gene's orthologue set, we required at least one of dog, mouse and rat, and at least one of chicken, frog, fugu, tetraodon and zebrafish. if the human 5-utr was<500 bp, we applied no utr requirements to orthologues; however, if the human 5-utr was>500 bp, we required that all orthologues had annotated ensembl 5-utrs that were at least 10 bp long. after this filtering, for any orthologous region that was missing from an input sequence set, we added a sequence region from the current ucsc (11,12) multiple sequence alignment. for a small subset of the target genes, corresponding regions from encode-specific species were then added from the current encode multiple sequence alignment (13). finally, regions were added from an internally processed version of the unannotated macaca mullata genome from. these rules delivered 7500 human target genes with sequence sets that contained a minimum of three and an average of six orthologous sequences from other genomes. sequence regions extended 1.5 kb upstream and 100 bp downstream of transcription start sites (tss), net of all types of repeats except ltr/erv1, ltr/ervl, ltr/malr and of coding sequences, which were masked. in addition to a target sequence set, we supplied each discovery program with a genomic background input file. the background input for each species consisted of 1000 concatenated search regions that were randomly selected from the genome's entire set of search regions. we used multiple discovery methods in parallel, running each method with a range of parameter settings; typically target motif width and motif occurrence model were varied. we used a compact but diverse base set of discovery methods that consisted of consensus (14), meme (15) and motifsampler (16). raw discovered motifs were post-processed, for example, to remove identical motifs reported by the same algorithm, and to merge strongly overlapped motifs. we assigned p-values to motifs discovered by multiple methods across large sets of target genes whose sequence sets varied in species composition, as follows. first, we identified a representative subset of target sequence sets that sampled the range of species compositions of the target sets. then, for each representative target sequence set, we created random sequence sets by retaining the original sequence from the target species and replacing each orthologous sequence with a synthetic sequence. the random synthetic sequence was generated from the target sequence by a tool we developed to simulate neutral evolution using published substitution rates and indel rates and lengths. we then submitted each target sequence set and all random sequence sets to identical motif discovery and post-processing procedures. we assigned method-independent (mi) scores to all motifs discovered in target and random sequence sets, using a trainable function that contained four non-negative parameters: score()=(1+1d)(1+2cremote)(1+4w)(1+3(1cclose))b, where coefficients took the following possible values: d, w r, b {0, 1} and c [0, 1]. in the above equation, d characterized the number of site sequences in a motif; w and b characterized the shape of a motif's information content profile; and c characterized motif sequence conservation. the score increased when the motif was conserved for species that are evolutionarily remote from the target, and decreased when the motif was not conserved for species that are close to the target (primates, in the case of human). we used the distribution of mi scores for motifs from a target gene's random sequences to transform the mi scores for the target gene's motifs into p-values. finally, we loaded the database with motifs whose p-values were below a threshold (which, for cisred 1.2e, was 0.05). a library of known transcription factor binding sites, split into mutually exclusive training and testing fractions, was used to optimize the scoring function and to characterize the performance of the system. the library contained 1000 sites for 300 human genes from transfac v9.1 (17), and 250 binding sites that we curated from the literature. we optimized the scoring function by simulated annealing, using a training fraction of known sites from randomly selected genes and two objective functions: the area under a receiver operating characteristic (roc) curve and the number of experimentally known motifs that were not predicted. we assessed the system's predictive performance with a test fraction of known sites, using observables like sensitivity, specificity and positive predictive value (ppv) (e.g.). although comparisons of method performance are constrained by many factors, current system performance compared favourably to results in a recent study (6). to identify groups of similar motifs, we defined two pairwise motif similarity metrics. for the first metric, we used a version of the levenstein edit distance between two sequences that was modified to permit no internal gaps (18). for each motif, m, and its reverse complement, rc(m), we scanned motif pairs relative to each other, and reported the overall minimum average mutual edit distance to motif k, i.e. min(d(m, k), d(rc(m),k)). the second metric was based on the maximum information content shared between position frequency matrices derived for each motif, and also treated a motif and its reverse complement as equivalent. we hierarchically clustered pairwise dissimilarity matrices with the local density-based optics algorithm (19). we extracted clusters from optics ' reachability output by applying an automatic cluster recognition method that identifies cluster boundaries as inflection points in the reachability plot (20), then traversing these hierarchical segmentation results with an algorithm that traced a deepest available path, constrained by a maximum preset depth. the large size of mammalian genomes makes it challenging to organize the computational hardware and software infrastructure required to address such issues routinely. we did the large-scale discovery, similarity and co-occurrence calculations on a beowulf-style, 400 cpu (pentium iii, xeon, opteron) oscar cluster () running red hat linux 9; and remotely on the beowulf 1700 cpu glacier cluster at westgrid (). we clustered motifs on a 12 dual-core cpu (ultrasparc iv) smp server with 96 gb of ram running solaris 9. the database infrastructure is designed to evolve to hold results from a range of mammals, as well as results from model organisms. because promoter regions are enriched sources of regulatory elements, motif discovery and the cisred design were both based on regions around tsss. cisred human v1.2 contains motifs from promoter regions of 7500 human genes using ensembl v30 (ncbi 35) data, as well as a pilot result set of 250 mouse genes. the current database design makes three types or levels of information available for regulatory elements: (i) atomic motifs, which are discovered independently in each target region sequence set; (ii) groups of similar motifs, each of which is a putative model for the binding site of a single transcription factor; and (iii) co-occurring patterns of models, which are putative regulatory modules. predicted regulatory elements can be viewed directly in cisred's web user interface. from this interface, motifs can be viewed in the ucsc genome browser, in the ensembl genome browser via a das server, or in the sockeye comparative genomics workspace (21). a user can filter the displayed motifs by criteria like the p-value threshold, the orthologous species present in a motif and the discovery method. the database contains a table of high-confidence globally coexpressed genes (22); genes coexpressed with each cisred target gene are listed on that gene's page. the database also contains a table of single nucleotide polymorphisms (snps) from dbsnp (8) that occur in predicted motifs. when a predicted human motif contains a snp, the cisred atomic motif page highlights this variation on the target sequence of a motif site sequence set, and the highlighted base hyperlinks to the snp's primary source information in dbsnp. a current schema diagram is available from the databases&methods page, and direct sql queries can be run on the mysql databases at. a user can download the data, with sql files, as well as a compressed file that contains all input fasta sequence sets. because older versions of the database may not be compatible with the current user interface, only recent versions for each species can be accessed through the web or via a mysql client. however, historical releases of cisred databases are archived and can be downloaded. certain parts of the system's software are available from a tab on the cisred home page. sockeye (21) permits, for example, a user to assess details of conserved regions relative to genomic annotations in multiple sequence alignments. the hitplotter visualizer displays large sets of discovered motifs from multiple-method discovery runs, and is available on request as a beta release. database contents will be extended to include large-scale results for human, mouse, rat, caenorhabditis elegans and drosophila melanogaster. the input sequence sets for this database were based on human tsss that were identified by considering ensembl (7) and refseq (23) annotations. to address the limitations of gene and transcript annotations for non-human species, corresponding vertebrate search regions were taken from ucsc (11,12) and encode multiple sequence alignments (13). we are extending our ability to take advantage of unannotated and low-coverage genome sequence data. we are continuing to improve motif post-processing and scoring. optimizing the scoring function depends on having a large library of known motif sites; we anticipate that a newly created web database for submitting and curating binding sites from the literature may help us to enlarge and improve this resource (; s. b. montgomery and o. l. griffith, manuscript in preparation). given a scalable clustering method, we continue to assess motif similarity metrics and how best to use the hierarchical information output from optics. given groups of similar motifs, we are applying group labels to atomic motifs, then identifying overrepresented co-occurring motif patterns using hypergeometric statistics, imposing separation constraints on neighbouring motifs, and searching in two stages for patterns larger than pairs (24,25). we are implementing methods for annotating discovered motifs as known or novel against known site resources. given annotated motifs, we will annotate overrepresented co-occurring patterns of human motif pairs as known versus novel using the transcompel resource (26). we are applying genome-scale motif clustering and co-occurrence as filters for predicted motifs that may improve the predictive reliability and the resulting catalogue of conserved regulatory elements. we have assembled a large multi-species coexpression resource that contains public microarray and sage data from diverse sources (22) (table 1). we have shown that combining global coexpression data from multiple platforms improves confidence in coexpression predictions when assessed against the gene ontology (go) (27). from this, we established go-based pearson correlation thresholds that identified high-confidence globally coexpressed gene pairs. the coexpression database makes results available from this global analysis and from two other recent analyses (28,29). although coexpressed genes can have similar regulatory elements, the system's predictive performance improved only marginally when inputs included coexpressed genes in addition to orthologous genes (data not shown). given these results, currently we are assessing an approach that includes no coexpressed genes in motif discovery inputs, but uses coexpression information to assess groups and co-occurring patterns identified in genome-scale sets of atomic motifs. we will extend the database user interface to offer more complex user filtering, as well as motif searches based on consensus strings or matrices. for work with classes of regulatory elements that are defined by wet lab data types based on, for example, chip or dnase i hypersensitivity (e.g. see encode tracks within the ucsc genome browser) (11,13), we have designed a new schema that is based on search regions rather than on genes, and will extend the user interface to support this. we will continue to assess the contributions to regulatory element predictions of different genomes and sets of genomes. we will integrate and assess new motif discovery methods, and will identify a best minimal set of methods on an ongoing basis. data processing system for high-throughput motif discovery, clustering, co-occurrence, annotation and performance assessment. database contents and high-level links, from a web user perspective, as of cisred human v1.2. groups are clusters of similar motifs that are identified by large-scale optics (19) clustering.

we describe cisred, a database for conserved regulatory elements that are identified and ranked by a genome-scale computational system (). the database and high-throughput predictive pipeline are designed to address diverse target genomes in the context of rapidly evolving data resources and tools. motifs are predicted in promoter regions using multiple discovery methods applied to sequence sets that include corresponding sequence regions from vertebrates. we estimate motif significance by applying discovery and post-processing methods to randomized sequence sets that are adaptively derived from target sequence sets, retain motifs with p-values below a threshold and identify groups of similar motifs and co-occurring motif patterns. the database offers information on atomic motifs, motif groups and patterns. it is web-accessible, and can be queried directly, downloaded or installed locally.

PMC1347438

pubmed-931

g. v. black was the first to describe a systematic method of cavity preparation and the ideal cavity form to restore carious lesions. classical cavity forms and principles remained appropriate and largely unchallenged for a period of 50 years using amalgam. amalgam has been the most widely used dental restorative material for the restoration of posterior teeth due to straightforward handling procedures, well tested material properties, and clinical success which has been documented for over a century despite esthetic shortcomings. low material price and rapid application however, amalgam cavities require precise procedures, usually resulting in uniform depths, particular wall forms, and specific marginal configurations with excessive tooth damage to ensure retention of amalgam. thus, patient demand for tooth colored restorations, public concerns related to mercury in dental amalgams, and the desire for a minimally invasive restorations, have made posterior composites an indispensable part of the restorative process instead of amalgam. the increased conservation of healthy dental structure with resin based composite restorations compared to the amalgam ones is the greatest advantage of the former. many clinicians have used this class of materials quite successfully during the last 5 to 10 years in posterior stress bearing areas.1 however, the inherited problems faced using resin based composites were inadequate wear resistance, marginal leakage, secondary caries and lack of appropriate contact.2 as manufacturers continue to search for tooth-colored resin-based composite materials that will have good physical properties, the introduction of new materials has taken dentistry a step closer to the goal. recently, a new posterior composite material, quixfil, was introduced into the dental market. the bimodal filler technology of quixfil shows particle distribution with two distinct peaks at 0.8 and 10 m and polymerization shrinkage is stated 1.7 vol.% by the manufacturer. a longitudinal randomized clinical assessment of stress bearing class i and ii restorations showed that quixfil exhibited good clinical results for 3 years.3 nanotechnology may offer unique solutions to improve the performance or handling characteristics of restorative dental materials. resin composite systems made by the use of nanotechnology can offer high translucency, high polish and polish retention similar to that of microfilled composites while maintaining physical properties and wear equivalent to several hybrid composites.4 grandio was used as one of the first resin composites with incorporated nanofillers beside conventional hybrid type fillers, being called nanohybrid composites. kramer et al5 investigated clinical performance and margin analysis of grandio in class ii cavities and stated that they were satisfactory after four years. as a large number of new improved resin brands are being released to the market, it is important for dentists to be aware of the probable longevity and likely modes of failure in posterior composite restorations. this information is best obtained from randomized controlled trials conducted clinically and in the laboratory for a definitive assessment of dental materials.6 hence, the purpose of this study was to evaluate 12 month clinical performance of a nanohybrid and a low-shrinkage posterior resin composite in class i and class ii restorations. thirty-one patients (10 male and 21 female) participated and provided written informed consent to participate in the study. detailed exclusion and inclusion criteria were as follows: inclusion criteria were: (1) permanent premolars and molars requiring class i and ii for treating primary carious lesions,(2) with at least one neighbouring tooth and in occlusion to antagonistic teeth, (3) good oral hygiene. exclusion criteria were: (1) patients with fewer than 20 teeth, (2) poor hygiene, (3) heavy bruxism habits, (4) periodontal problems and known allergic reactions against any components of the used materials, (5) pathologic pulpal diagnosis with pain (nonvital), (6) fractured or visibly cracked teeth, (7) defective restorations adjacent or opposing to the tooth, (8) rampant caries, (9) atypical extrinsic staining of teeth or staining of any existing tooth colored restorations. a total of 82 teeth (41 pairs) were restored with either a nanohybrid resin composite grandio (voco gmbh, germany) and its self-etch adhesive futurabond nr (voco, germany) or a low-shrinkage posterior composite quixfil (denstply, germany) and its self etch adhesive xenoiii (dentsply, germany) (table 1). the distribution of materials and tooth locations were randomized by tossing a coin (table 2). however, interference in the randomization procedure within patients was performed in order to equally distribute materials into some important variables such as tooth type and position, restoration class type in such a way that minimized the influence of those factors. the teeth were prepared using conventional instruments and adhesive conservative techniques, appropriate local anesthesia have been achieved preoperatively unless declined by the patient. the average facio-lingual width of the cavities was approximately one third of the intercuspal width. calcium hydroxide (dycal, dentsply caulk, germany) was placed where indicated for deep cavities. the location of the cervical margins was not recorded. for class ii restorations, the dentists used metal matrix bands (toefflemire, teledyne waterpik technologies, usa) and wooden wedges. placement of resin composites followed the incremental technique (2 mm-thick layers). the resin composite was adapted with a flat faced or elliptical condenser and light cured using a halogen light of 500 mw/mm intensity (hi-lux ultra, benlioglu, turkey). the light output of the curing unit was monitored with a light meter (curing radiometer model 100; demetron corp, usa) a post occlusal adjustment was performed with carbon paper and the quality of interproximal contacts and cervical adaptation was checked by means of dental floss and interproximal radiographs. the restorations were finished under water-cooling with fine and super fine diamond points (kg finishing kit, karensen ltd, brasil) and rubber polishing kits (eveflex polisher, eve ernst vetter gmbh, germany). all restorations were clinically evaluated after 1 week (baseline), 6 months and 12 months by 2 investigators using the modified usphs criteria as first described by cvar and ryge7 and adapted by wilson et al8 for retention, color matching, marginal discoloration, marginal adaptation, secondary caries, surface texture, anatomic form and postoperative sensitivity (table 3). the examiners were not involved in the placement of the fillings and were unaware of the materials used in this double-blind study. when disagreement arose during evaluation, the examiners had to reach a consensus. all evaluations were carried out under a dental operating light, using flat surfaced mouth mirrors and dental explorers. restorations were scored as follows: alpha represented the ideal clinical situation; bravo was clinically acceptable; charlie was clinically unacceptable situations where the restoration had to be replaced. for secondary caries detection bitewing radiographs statistical analysis was performed using pearson chi-square and fisher s exact test for assessing the difference between the restorative materials (p<.05). cochran s q test was also employed for evaluating the difference between examination recalls of the same restorative material. at the end of 12 months, all restorations (grandio or quixfil) were present and a total of 82 restorations were available for clinical evaluation in 31 patients (recall rate 100%). none of the restorations had shown any marginal discoloration and anatomic form loss until the end of the 12 months and no restorations exhibited post-operative sensitivity at any evaluation period. summary of clinical findings of ryge criteria with respect to color match, marginal adaptation, secondary caries and surface texture is shown in table 4. mainly, the difference between the restorative materials (grandio and quixfil) at the end of 12 months was not statistically significant and demonstrating acceptable clinical performance. at the 6-month recall all the restorations received alpha score with respect to each evaluation criteria. nevertheless, there were some statistically different issues regarding evaluation criteria of each material itself between the examination recalls. the percentages of alpha scores for color match were 95% (n=39) for grandio restorations and 100% (n=41) for quixfil restorations. two grandio restorations (5%) received bravo score (p=0.135) at 12-month recall. four grandio restorations (10%) received bravo ratings while 37 restorations (90%) received alpha ratings for marginal adaptation. this difference was found to be statistically significant (p=0.018) between baseline and 12 month recalls. quixfil restorations marginal adaptation score was alpha for 40 restorations (98%) and bravo for 1 restoration (2%) at the end of 12 months (p=0.368). the evaluation of secondary caries results revealed that 39 restorations (95%) received alpha scores among quixfil restorations where 2 restorations (5%) received charlie ratings (p=0.135). the alpha and bravo scores of grandio restorations for surface texture were 40 (98%) and 1 (2%), respectively (p=0.368). all the quixfil restorations received alpha scores in terms of surface texture at 12 months. however, these developments have been so rapid that long-term clinical data on specific products are rarely available because of regular introduction of improved versions. laboratory tests might provide useful information to the potential performance of a filling material and its handling, but such tests can not adequately evaluate the clinical performance of a material or clinical handling characteristics. besides, in vitro studies can not answer questions about in vivo longevity of these tooth colored restorations.9 long term results with some of these newly developed materials are lacking and remain controversial as studies report inconsistent clinical results.10,11 while usphs system has served well for clinical evaluation, there are some concerns about the sensitivity of the approach in short term clinical evaluations. the lack of sensitivity of the ryge system to record small early changes, combined with the continually evolving clinical designs and non standard investigator modifications of the categories, scales, and reporting methods, has created a body of literature that is extremely difficult to meaningfully interpret. in many cases, the relative insensitivity of the ryge methods during short and medium term clinical trials (< 3-5 years) may be misinterpreted.12 however, this system is still being used in the clinical researches to compare these finding with the previous ones that utilize the same system. the first 6 up to 24 months appear as the critical period for the development of deteriorations.13 mair14 evaluated posterior composite restorations over a 10-year period. inevitably, this study can be criticized that the duration of the study is insufficient to confirm long-term suitability of the tested materials; nevertheless these findings provide an indication of their initial clinical performance. in the present study, the bonding of the two restorative materials was sufficient to provide adequate retention over 12 months and none of the restoration was lost. the findings of this study were similar to the results of other clinical studies examining the resin restorations for the same evaluation period.15,16 however, 2 quixfil restorations failed after twelve months due to secondary caries and these restorations were replaced. many studies17,18 have indicated that up to 30% of the study populations have reported post-operative sensitivity following the placement of a posterior resin restoration. self-etch primers make the smear layer part of the hybrid layer, as it dissolves the smear layer, incorporating it into the mixture of collagen fibers and resin monomers. since the smear layer becomes an integral part of the hybrid layer, low sensitivity response may be the outcome, which was also seen in the present study.19 in regard to the clinical performance of self-etch systems, the literature contains contradictory findings, as the bonding effectiveness of these adhesives seems to be material dependent.20,21 a great variety of self-etch systems are available on the market. they differ in the number of bottles, steps, and acidity of the primer solution, among other factors. a closer analysis of the aforementioned clinical trials20,21 reveals that the self etching adhesive with good clinical performance did not belong to the group of strong self etching adhesives, but to the group of mild self etching adhesives. futurabond nr s and xeno iii s ph are both 1.4 belonging to the same group. the loss of marginal adaptation and the presence of secondary caries are predictors of the failure of posterior resin based composites and the reason for the replacement of the restoration.17 this study revealed that two quixfil restorations demonstrated secondary caries although the evaluation period was short. according to mjr22 and saleh,23 development of secondary caries is not only due to the material itself. clinical environment, caries experience of patients, criteria for replacements, different handling characteristics appeared to affect clinical results. additionally, bernardo et al24 reported that the overall risk of failure due to secondary caries was 3.5 times higher in composite restorations than in amalgam restorations. grandio restorations have already showed 10% bravo scores in terms of marginal adaptation, which is statistically significant between baseline and 12 months. similar to our results, kramer et al5 found that for marginal adaptation grandio showed 17% bravo scores after one year clinical evaluation period. however, previous researches demonstrated that evaluation of the composites during initial periods of evaluation depicted minor changes compared to the baseline.25,26 marginal adaptation is directly influenced by the type of composite resin used.27 altering the amount and quality of the filler particles can change the esthetics and mechanical properties of restorative composite resins. furthermore, lowering a material s viscosity by modifying the composition of the monomer system permits a higher filler load and at the same time improves the handling properties. 28 grandio has a filler degree of 87% w/w (71% volume) by combining spherical nano particles and none of the restorations had shown any marginal discoloration and anatomic form loss until the end of the 12 months and no restorations exhibited post-operative sensitivity at any evaluation period. quixfil has 86% by weight (66% volume) filler load, which is approximately the same. in a previous study, manhart et al29 evaluated the clinical performance of quixfil for 18 months and found significant increase in marginal discoloration with time. while, marginal defects were observed for both materials in our study, none of the restorations showed marginal discoloration. many of the these marginal defects appeared to result from the fracture of thin flashes of resin composite material extended on non-instrumented enamel surfaces adjacent to the cavity margins. the use of phosphoric acid etching30 and aggressive self-etch adhesives32 may reduce the occurrence of such defects, especially in high-stress-bearing areas, because of the improved enamel etching.30 in the present study, mild self-etch adhesive systems were used and marginal adaptation results for 12 months may be related to absence of acid-etching procedure. in consistent to our results, abdalla and garcia-godoy31 evaluated the clinical performance of futurabond nr in class v lesions and reported less deteriorations in regards to marginal adaptation and marginal discoloration when adhesive resin was applied following enamel etching. in the present study, both of the restorative materials demonstrated good color stability and surface texture. at the 1-year recall, the majority of scores were alfa, bravo scores were recorded for only two grandio restorations for color stability and one grandio restoration for surface texture. however, it has been reported that changes in surface texture and color stability of resin composite restorations could increase after one year.6,33 in our study, the greater range of shades was available for grandio and we expected better color matching ability for this material. although, quixfil was available in one universal shade, none of the restorations showed bravo scores at baseline. good color match results might be related to chameleon effect of quixfil, blending into the tooth structure around the restoration. in the present study, both of these restorative materials were used with their self-etch adhesive systems and demonstrated acceptable clinical performance after 12 months. these successful findings might be related to the relatively short evaluation period, which is consistent with many studies in which there were no significant differences between composite materials in early evaluation periods.15,16 it should be noted that the time frame for this study was not of such duration to indicate the long term suitability of the tested materials, but it may provide an indication for detecting material-related initial changes in color and surface topography regarding their future performance. it was concluded that nanohybrid (grandio) and low-shrinkage posterior composite (quixfil) demonstrated acceptable clinical performance after 12 months.

objectives: the purpose of this study was to evaluate and compare the 12 month clinical performances of two different posterior composites in class i and class ii restorations. methods:thirty-one patients (10 male, 21 female) were recruited into the study. a total of 82 class i and class ii cavities were restored with either a nanohybrid composite (grandio) or a low-shrinkage composite (quixfil), using their self etch adhesives (futura bond and xeno iii) according to manufacturers instructions. the restorations were clinically evaluated 1 week after placement as baseline, and after 6 and 12 months post-operatively using modified usphs criteria by two previously calibrated operators. statistical analysis were performed using pearson chi-square and fisher s exact test (p<.05). results: all patients attended the 12-month recall. lack of retention was not observed in any of the restorations. with respect to color match, marginal adaptation, secondary caries and surface texture, no significant differences were found between two restorative materials tested after 12 months (p>.05). none of the restorations had marginal discoloration and anatomic form loss on the 12 month follow-up. restorations did not exhibit post-operative sensitivity at any evaluation period. conclusions:clinical assessment of nanohybrid (grandio) and low-shrinkage posterior composite (quixfil) exhibited good clinical results with predominating alpha scores after 12 months. however; further evaluations are necessary for the long-term clinical performance of these materials.

PMC2798791

pubmed-932

we now appreciate that the immune system recognizes cancer and that patients with strong natural immune reactions have better survival compared with those with weak responses. this concept was first demonstrated in patients with colorectal cancer (crc) by pages et al. here they demonstrated that the immune contexture, defined by number, type and location of tumour immune infiltrates in primary tumours, are key prognostic factors for disease-free and overall survival (os) in crc patients. importantly, the immunoscore, an immune-based classification strategy derived from three aspects of the immune contexture, proved superior over the standard tumour node metastases classification in predicting outcomes in survival and relapse of crc patients and their response to therapy. collectively, a good immunoscore, defined as high densities of tumour-infiltrating lymphocytes particularly with a t helper type 1 (th1) and cytotoxic orientation, was associated with longer disease-free survival and/or improved os. this correlation was first documented in crc but has now been extended to a number of other cancers, including melanoma, head and neck, breast, ovarian and lung. importantly, recognition that cancer development and progression is influenced by the host immune system has led to the development of a conceptual framework called cancer immunoediting, ' which explains the dual host-protective and tumour-promoting roles of the immune system. furthermore, the demonstration that cancers actively evade immune destruction has led to its inclusion as an emerging hallmark of cancer in 2011. tumours can escape immune detection and destruction by a number of mechanisms that have been previously discussed. these can include: (i) downregulation of antigen and major histocompatibility complex class i (mhc i) expression, (ii) production of immunosuppressive mediators (for example, interleukin-10 (il-10), transforming growth factor-, adenosine, vascular endothelial growth factor-a (vegf-a), indoleamine 2,3-dioxygenase (ido)) to dampen effector cell activation and (iii) recruitment of immune cell subsets (t regulatory cells (tregs) and myeloid-derived suppressor cell (mdsc)) to suppress effector immune cell function and facilitate its growth and metastases. in addition to these pathways, we now understand that physiological negative feedback loops that curtail t-cell activation, to prevent autoimmunity, can be exploited by tumours to limit anti-tumour responses. for example, after activation, t cells upregulate checkpoint receptors such as cytotoxic t-lymphocyte antigen 4 (ctla-4), which bind to b7 ligands with higher affinity than the co-stimulatory receptor cd28, thus attenuating effector t-cell function. other t-cell checkpoint receptors that limit activated t-cell effector function include programmed cell death-1 (pd-1), b and t lymphocyte attenuator (btla), t-cell immunoglobulin mucin 3 (tim3) and lymphocyte activation gene-3 (lag-3). recently, it has been demonstrated in melanoma patients that tumours can also counter attack from activated t cells though another mechanism termed adaptive immune resistance. in this study, tumour-infiltrating lymphocytes producing inflammatory cytokines such as interferon- in the tumour microenvironment upregulated pd-l1/l2 expression on tumours, which delivers an inhibitory signal to pd-1-expressing t cells. over the past 5 years, the use of antibodies to target checkpoint receptors (also termed checkpoint blockade) has emerged as one of the most effective ways to overcome tumour-induced immunosuppression and re-activate an endogenous anti-tumour response. in 2011, anti-ctla-4 (ipilimumab) was the first immunomodulatory monoclonal antibody approved by the food and drug administration for the treatment of patients with advanced melanoma on the basis of improved survival benefits. recently, second-generation checkpoint inhibitors, anti-pd-1 and anti-pd-l1, have induced better anti-tumour efficacy (31% objective response rate) compared with ipilimumab (6%). with the recent observation of further increase in anti-tumour efficacy (53%) through the combination of anti-ctla-4 and anti-pd-1 clinical trials are currently in different stages of accrual and development to assess checkpoint receptor blockade in combination with (1) chemotherapies, (2) radiotherapy, (3) small-molecule inhibitors, (4) vaccines and (5) co-stimulatory receptors (for example, cd40, ox40 (also known as cd134), cd137, gitr (glucocorticoid-induced tumour necrosis factor (tnf) receptor-related protein) and cd27. the induction of iraes is an important factor to consider when attenuating co-stimulatory and co-inhibitory immune receptors for cancer immunotherapy. thought also has to be given as to how targeting of these receptors in combination or with other therapeutics may induce combination-specific iraes that may not be predicted or observed by monotherapies. iraes are thought to be related to therapy-associated cytokine release and t-cell-mediated organ infiltration and different from bona fide autoimmunity as they normally resolve following therapy or with corticosteroids or anti-tnf therapy. iraes can generally involve the gastrointestinal, liver, skin, nervous and endocrine systems (figure 1). in a pooled analysis of 325 patients treated with 10 mg kg of anti-ctla-4, iraes of any grade were observed in 72.3%. similar proportions were also observed in patients treated with anti-pd-1 alone, although with less severity. in most cases, iraes are mostly mild and generally manageable with appropriate treatment algorithms now having been developed. this includes dose interruption/discontinuation and the use of systemic high-dose corticosteroids. specifically, grade 3 or 4 iraes, which developed in each of these patients (14% for anti-pd-1 and 25% for anti-ctla-4) warranted attention, as in extreme cases these can be severe and life threatening. patients who resolve their grade 2 iraes can recommence treatment, although any grade 3 or 4 iraes (with the exception of grade 3 skin toxicity) is a contraindication to further therapy with ipilimumab. importantly, almost all patients (93%) developed iraes following concurrent anti-pd-1 and anti-ctla-4 monoclonal antibody therapy with an increase in grade 3 or 4 iraes (50%) being observed compared with those treated with monotherapy alone. moving forward, the better safety and clinical efficacy of targeting pd-1 (most likely due to its role in regulating t-cell activation in peripheral tissues) suggest that it will be the checkpoint receptor of choice to combine with other therapies for cancer treatment. nevertheless, one may predict that the act of combining immunotherapies increases the proportion of patients who may develop severe iraes. this may limit the number of patients who can fully benefit from combination cancer therapies due to discontinuation, as seen in 21% of patients treated with anti-ctla-4 and anti-pd-1. iraes generally manifest during the induction phase of treatment (initial 912 weeks of therapy) and organ-specific iraes appears to follow kinetics of appearance. in the case of ipilimumab where iraes of skin and mucosa develop after about 36 weeks, this is followed by diarrhoea/colitis around week 5 and liver toxicity and endocrinopathies around week 6. more recently, an update on patients treated with anti-pd-1 reported on 2 years of safety monitoring and found that similar to what has been reported for ipilimumab, most adverse events tended to occur within the first 6 months of therapy. hence, the goal of therapies targeting checkpoint receptors in combination with other approaches should aim to generate a strong therapeutic index over the induction period of therapy. questions that have to be answered include whether it is possible to release/activate anti-tumour immunity without development of iraes if the right combination, dosing and scheduling is utilized. alternatively, is the development of iraes a predictor of anti-tumour responses? in one study, it was reported that ipilimumab-treated patients who developed grade 3 or 4 iraes were more likely to achieve a clinical response compared with those who had no iraes. interestingly, in a retrospective analysis of 498 patients pooled from four phase ii clinical trials treated with different doses of ipilimumab, feng et al. reported that higher exposure (dose) was associated with better survival, albeit at a greater risk of developing iraes in the grade 3 or higher category. a caveat is that this correlation is observed simply because the longer the patients responding are treated, the greater the possibility of developing iraes. however, the study by feng et al. also suggested that patients who did not experience iraes during the induction period were not likely to experience it during maintenance therapy with ipilimumab. it would be interesting to perform retrospective analysis to determine whether these group of patients also did not benefit as much clinically. potentially these analyses may help answer the question of whether tumour reactive and autoreactive t cells are the same or distinct. it should be noted that, in some studies, there were patients, albeit at a low proportions, who developed durable responses with no associated iraes. analysis of these patient cohorts may provide clues as to the requirements necessary to release anti-tumour immunity without inducing iraes. for patients who discontinued combination therapy due to iraes, do they still benefit clinically and what is the minimum period of therapy required to allow immune activation and/or release of immune suppression? a recent report suggests this is likely, where patients with advanced melanoma treated with ipilimumab were found to survive for up to 10 years, confirming the durability of the overall survival (os) trend observed. these results, the largest analysis of os for 1861 patients, included 2 phase iii trials, 8 phase ii trials and 2 retrospective trials. their findings demonstrated that in 1015% of the total patients who responded, 22% of patients can achieve 310 years os and this os reaches a plateau at 3 years. this was observed regardless of dose (3 or 10 mg kg), previous treatment history and whether they had been kept on a maintenance therapy with ipilimumab. analysis of safety data, which were not included in these results, may shed light on the association of iraes ' severity with os as well as clinical benefits obtained following therapy cessation. in addition to checkpoint receptors, agonistic antibodies targeting co-stimulatory receptors belonging to the tnf receptor family (cd40, ox40, cd137, gitr and cd27) are also being assessed. these receptors, in general, act through activation of effector cell function and are currently being evaluated for their anti-tumour efficacy in various phase i/ii clinical trials. in particular, the data for agonistic antibodies targeting cd40 and cd137 (4-1bb) are most mature where some objective responses, as well as corresponding iraes, have been reported. in a number of phase i clinical trials, agonistic anti-cd40 monoclonal antibodies have been evaluated against solid tumours or b-cell non-hodgkin's lymphoma. objective responses were reported in a proportion of these patients, with the most common iraes being primarily grade 2 cytokine release syndrome, which manifests as transient chills, fever and rigors and mild elevations in liver enzymes and decreases in circulating platelet numbers. interestingly, iraes such as colitis or dermatitis, which are normally associated with checkpoint receptor blockade and can result in dose interruption/discontinuation, were not observed in targeting cd40, suggesting the comparative safety of anti-cd40. targeting of another co-stimulatory receptor cd134 (ox40) in one phase 1 clinical trial was also safe as no acute toxicities were reported at the dose tested. by contrast, grade 3/4 iraes (severe liver toxicity) were observed in patients treated with anti-cd137 (urelumab) particularly at high doses, resulting in the termination of a number of phase i trials. currently, lower doses of anti-cd137 are being evaluated for their safety in a phasei/ii clinical trial with reports suggesting anti-tumour activity can still be elicited without significant liver toxicity. the experience with ipilimumab and immunotherapies in general is their kinetics of response is much slower but more durable compared with those seen with cytotoxics. recently, a long-term follow-up study of 177 patients treated with ipilimumab reported a median time to achieve complete response of 30 months, consistent with slow kinetics of tumour regression. similarly, the kinetics of anti-pd-1 responses is atypical with some patients responding rapidly as early as 8 weeks into therapy, whereas other patients displayed new tumours or growth of existing tumours before responding. in addition, some patients continued to respond despite cessation of anti-pd-1 therapy. strikingly, a different kinetics of response was observed in patients treated with the maximum tolerated doses of anti-pd-1 and anti-ctla-4, given in combination. impressively, these patients all had tumour reduction of 80% (classified as deep response) or more at their first scheduled assessment at 12 weeks. this is in contrast to patients who were given anti-ctla-4 and anti-pd-1 in a sequenced regime. this suggests anti-tumour responses can be rapid if the right combination is given at optimal dosage and timing. the question now is whether (1) maintenance dosage is required for patients treated with combination therapies who responded rapidly so as to minimize their risk of further developing more iraes or (2) can patients who developed grade 3 or 4 iraes and ceased therapy still benefit and can one correlate the number of doses they received before cessation, with clinical benefit to determine what is the minimum period of therapy required? (3) if iraes are predictors of response, are there biomarkers that can be used to determine which patients will develop them and thus enable monitoring and prophylactic treatment to prevent their escalation into grade 3 or 4 toxicities and cessation of treatment? given that many patients with iraes treated with systemic corticosteroids and/or anti-tnf resolve their symptoms rapidly if treated when symptoms first appear, it would suggest serum tnf and other inflammatory cytokines like interleukin-6 could be measured as biomarkers and targeted. there seems to be a consensus that corticosteroids or anti-tnf administration do not impede anti-tumour immune responses following checkpoint blockade. given that most patients will develop some form of iraes following immunomodulatory therapies, particularly in combination, it would suggest concurrent administration of anti-tnf and/or corticosteroids with combination therapies is an option that can be investigated. the demonstration that targeting checkpoint receptors activates endogenous anti-tumour immunity and leads to significant clinical benefit in different cancer types now spurs the question of how their efficacy can be further improved through rationale combination approaches. in advanced melanoma, concurrent anti-ctla-4 and anti-pd-1 therapy resulted in clinical benefits in 50% of patients, which to date is the best result obtained with cancer immunotherapies. nevertheless, 50% of patients did not respond, and in some cancer types such as pancreas and prostate, checkpoint blockade does not appear to provide any significant clinical benefits. thus, early-phase clinical trials are exploring how checkpoint inhibitors combine with other agents, including (1) conventional therapies (radiotherapy, chemotherapy), (2) alternative immunotherapies (vaccines, cytokine, adoptive cellular therapy), (3) targeted therapies (braf or vascular endothelial growth factor inhibitors) or (4) immunomodulatory antibodies. with combination immunotherapies increasingly being tested in clinical trials, iraes will be an ongoing issue that has to be dealt with (as discussed above) as it may limit their usage. furthermore, even when both agents have regulatory approval and have distinct mechanisms of action and toxicity profile, unexpected specific iraes induced by the combination can occur. this was illustrated by a recent clinical trial that reported on the unexpected hepatotoxicity observed with melanoma patients after concurrent treatment with a braf inhibitor (vemurafenib) and ipilimumab, resulting in cessation of the trial. given the beauracracy, cost and time associated with conducting clinical trials, utilizing preclinical mouse models that can more accurately model tumour immunity and iraes may allow more informed assessment of which therapies can be combined to induce optimal therapeutic index (figures 2 and 3). tumour-associated autoimmune syndrome is very rare in patients with cancer, such as melanoma-associated retinopathy and vitiligo. however, new data suggest that it is not merely a side effect of robust anti-tumour immunity. the development of vitiligo is considered to be a benefit to the prognosis of the patient with malignant melanoma. in a preclinical study, mice with vitiligo generated more cd8 memory t cells, which protected them from melanoma expressing the same melanocyte antigens. in another study, inflammatory monocytes or dendritic cells accumulated in the skin were shown to be capable of killing melanoma cells as well as normal melanocytes. however, whether the relationship of tumour-associated autoimmune syndrome and tumour immunity is dependent on the innate immune system or adaptive immune system or both requires further investigation. in contrast, iraes are not true autoimmune diseases, as they typically resolve with cessation of therapy and appropriate immunosuppressive regimes. in addition, the experience with ipilimumab and immunotherapies in general is their kinetics of response is much slower but more durable compared with those seen with cytotoxics. thus utilizing spontaneous mouse tumour models may better mimic the response kinetics that is observed in the clinic and may potentially allow for the development of any therapy-induced toxicity. these models can be divided into those that are carcinogen-induced such as methylcholanthrene (mca)-induced fibrosarcomas and 7,12-dimethylbenz[]anthracene (dmba)/12-o-tetradecanoylphorbol-13-acetate (tpa)-induced skin papillomas or genetically engineered mouse tumour models, which have enforced expression of oncogenes and/or the loss of function of tumour suppressors, often in a tissue-specific and/or temporally controlled manner (figure 2). examples include the her2/neu or pymt transgenic mice to mimic breast cancer, the mt/ret model of spontaneous metastatic melanoma and braftyr-creerpten mice in which 4-hydroxytamoxifen (4-ht) induces de novo melanoma as well as the use of adenoviral vectors encoding cre recombinase to selectively introduce mutations in the oncogene kras and the tumour-suppressor gene tp53 in the pulmonary epithelia to induce autochthonous lung tumours. interestingly, it has been noted that carcinogen-induced models are very immunogenic, whereas germline mutation models are often not, most likely due to the former carrying more passenger mutations, thereby generating more neoantigens that can potentially be recognized by the immune system. this was elegantly illustrated in two studies investigating the role of t cells in selecting for non immunogenic sarcomas using either a mouse model of sarcomagenesis driven by cre recombinase-triggered activation of the kras oncogene and inactivation of the trp53 tumour-suppressor gene or a mca-induced mouse model of sarcoma. in the first model, sarcomas that developed did not appear to be recognized by adaptive immunity as these tumours grew equally well when transplanted into wild-type or rag-2-deficient mice. on the contrary, mca-induced sarcomas, which had many passenger mutations in addition to kras and trp53, were demonstrated to generate tumour-specific t cells, which could then selectively sculpt tumour immunogenicity. interestingly, mouse mca-induced sarcomas were also found to have qualitative and quantitative genomic mutation profiles to carcinogen-induced human cancers, such as smoker's lung cancer. thus carcinogen-induced mouse models of cancer may better mimic cancers that are immunogenic. in contrast, transplantable tumours, which grow more rapidly and are employed as an initial model to assess the therapeutic potential of combination therapies, may be less useful. as most transplantable tumours are grown subcutaneously on the flanks of mice, an alternative approach is to inoculate them orthotopically to better mimic the tumour environment from which they originated. indeed, immunotherapies that demonstrate efficacy against a renal tumour grown subcutaneously were not as effective when the same tumour was transplanted into the kidney. although checkpoint blockade in patients commonly induces iraes, such events have rarely been commented upon or observed in preclinical mouse models, even when 3 different pathways are targeted. it is possible that while treated mice appeared outwardly healthy, closer examination of animals may have revealed the presence of biochemical autoimmunity, which is frequently observed in patients even if they do not display clinical symptoms. in addition, observable iraes may not have manifested owing to the generally short nature of preclinical mouse models experiments or, perhaps, the strain of mice used may be more resistant to developing iraes compared with humans. this was elegantly shown in a recent study where repeated dosing of anti-ctla-4 in sjl/j mice over 5 weeks was able to induce hypophysitis, an irae that appears to be associated with ipilimumab treatment. testing checkpoint blockade in combination or with other therapies in mouse strains more prone to development of autoimmune symptoms (for example, nod, sjl/j) may be one strategy to assess the development of tumour immunity and autoimmunity. furthermore, it has also been shown that repeated dosing of agonistic antibodies to co-stimulatory receptors such as gitr induced anaphylaxis in tumour-bearing mice that was caused by serum antibodies, and dependent on cd4 t cells, b cells, basophils, platelet-activating factor and gitr. alternatively, to observe iraes in mice, a more dramatic alteration of the immune system such as the complete and systemic depletion of tregs may be required. intratumour treg depletion by antibodies such as anti-ctla-4 (now a recognized mechanism) does not seem sufficient to induce iraes in mice, as are observed in humans. in addition, heterogeneity in the kinetics of response is normally seen in patients after immunotherapy, such as those that were treated with anti-pd-1, and thus a tumour model that mimics what is observed in the clinic will be useful. using the de novo model of mca-induced fibrosarcoma, we observed that complete treg depletion in dereg mice bearing established mca-induced tumours displayed a similar heterogeneous range of tumour responses to that observed in the clinic. in our study, no overt autoimmunity with treg depletion was observed, but this may be explained by the potential compensatory effect of other nontransgenic regulatory cells in the dereg mice. comparative experiments in foxp3-dtr knock-in mice, where autoimmunity is more easily generated upon treg depletion, are currently underway in our laboratories. in clinical trials, concurrent blockade of ctla-4 and pd-1 checkpoint receptors induced deep and rapid anti-tumour responses in contrast to the slower response kinetics seen in patients given anti-ctla-4 and anti-pd-1 in a sequenced-regime. this suggests that anti-tumour responses can be rapid if the right combination is given at optimal dosage and timing. it was not made clear if patients who had the rapid deep objective response had high/low grade iraes. indeed, we recently reported the use of the poorly immunogenic b16f10 melanoma model to characterize a very heterogeneous anti-tumour effect of the immune response induced by complete treg depletion in dereg mice. strikingly, the duration of the tumour immune system interaction induced in individual treg-depleted mice positively correlated with their propensity to develop vitiligo. a rapid complete tumour rejection was not associated with the development of autoimmunity; however, a proportion of mice that suppressed but did not effectively clear b16f10 melanoma did develop vitiligo. we would postulate that approaches that combine with treg depletion to rapidly reject tumours may also diminish iraes. currently, cancer patients who have chronic autoimmunity are generally precluded from clinical trials with checkpoint blockade. however, patients with certain autoimmune diseases (pernicious anaemia, crohn's disease, ulcerative colitis, systemic lupus erythematosis and psoriasis) are generally thought to be at higher risk of cancer due to the underlying dysregulation of their immune system, as well as a consequence of their treatment regime. thus, it is imperative we understand how and if toxicities induced by combination therapies in this cohort of patients differ to cancer patients who do not have underlying autoimmunity, that is, whether the therapies will be more toxic. given that many tumour-associated antigens are self-antigens, and that self-reactive t/b cells are increased in patients with autoimmunity, should a case be made that cancer patients with stable underlying autoimmunity be allowed treatment with immunotherapies as they may respond better? of course, these patients have to be monitored stringently owing to their increased risk of developing iraes or have their autoimmune symptoms exacerbated. nevertheless, studies in this cohort of patients may potentially help in answering the question of how much overlap they are between tumour reactive and autoreactive t cells as well as their relative importance in different cancers. the foxp3-dtr mouse represents one model where we can evaluate how combination immunotherapies attenuate tumour immunity/iraes. in this setting, tumour-bearing foxp3-dtr mice can be conditionally depleted of their tregs to mimic the maximum release of suppression on all immune cells and can then be treated with different immunotherapies/agents. potentially, this may allow us to assess how different co-inhibitory/co-stimulatory receptors combine with each other or with other therapies to attenuate anti-tumour immunity/iraes. although clinical observations suggest that autoimmune effectors are intricately involved in tumour cell killing, evidence using antigen-specific preclinical mouse models indicates that, in some cases, the immune system can selectively target tumour tissue while sparing self. indeed how self antigen-specific t cells target tumour versus healthy tissues, and how they are regulated in different microenvironments, is still unclear. to address this issue, miska et al. evaluated the ability of specific t effector cells to kill tumour versus healthy cells expressing the same self antigen in the same animal. their studies concluded that tumours suppressed autoimmune t effector cells locally without distal impairment, suggesting that higher levels of suppression exist in tumours compared with healthy tissues expressing the same self antigen. their studies also highlighted an increased potency of regulatory mechanisms such as treg and mdscs in tumours, although the molecular mechanisms responsible for this increased suppression remain to be elucidated. thus antigen-specific cd4 or cd8 t cells that are unable to eliminate tumours can still mediate destruction of healthy tissues, suggesting that the threshold of inducing autoimmunity is lower than tumour immunity. this implies that systemic administration of immunotherapies that result in t-cell activation will almost always induce autoimmunity unless a substantial antigenic difference is identified between tumour target and healthy tissue. thus strategies that can alleviate immune suppression specifically at the tumour site will be the way forward for next-generation combination therapy approaches. another issue for consideration is that many of the cancer patients may have underlying metabolic syndrome such as obesity and diabetes, which have repeatedly been associated with increased incidence for some common cancers. how would combination therapies impact on this cohort of patients in terms of tumour immunity/iraes? this question may be assessed by testing immunotherapies with the obese diabetic mouse (ob/ob) model. the durable clinical benefits obtained with immune checkpoint blockade have reinvigorated the field of cancer immunotherapy. moving forward, checkpoint blockade looks set to be explored in combination with other therapies to further improve their therapeutic efficacy and their impact on more cancer types. however, the proportion of patients developing severe iraes following combination immunotherapies most likely will increase. utilizing relevant preclinical mouse models of cancer that better model tumour

the current excitement surrounding cancer immunotherapy stems particularly from clinical data involving agents mediating immune checkpoint receptor blockade, which have induced unprecedented efficacy against a range of tumours compared with previous immunotherapeutic approaches. however, an important consideration in targeting checkpoint receptors has been the emergence of associated toxicities termed immune-related adverse events (iraes). in light of the clinical benefits observed after co-blockade of checkpoint receptors and data from preclinical mouse models, there is now a strong rationale to combine different checkpoint receptors together, with other immunotherapies or more conventional therapies to assess if clinical benefits to cancer patients can be further improved. however, one may predict the frequency and severity of iraes will increase with combinations, which may result in premature therapy cessation, thus limiting the realization of such an approach. in addition, there is a limit to how many different combination therapies that can be tested in a timely manner given the legal, regulatory and budgetary issues associated with conducting clinical trials. thus, there is a need to develop preclinical mouse models that more accurately inform us as to which immunotherapies might combine best to provide the optimal therapeutic index (maximal anti-tumour efficacy and low level iraes) in different cancer settings. in this review we will discuss the iraes observed in patients after checkpoint blockade and discuss which mouse models of cancer can be appropriate to assess the development of tumour immunity and iraes following combination cancer immunotherapies.

PMC4232074

pubmed-933

type 1 autoimmune hepatitis (aih) is a progressive liver disorder predominantly affecting women that is characterized by the presence of circulating autoantibodies [anti-nuclear antibody (ana) and/or anti-smooth muscle antibody (asma)], hypergammaglobulinemia, and a favorable response to immunosuppressive therapy (1-5). individuals affected with aih often have concurrent non-hepatic autoimmune disorders, such as chronic thyroiditis (9.2%), sjgren's syndrome (7.2%), and rheumatoid arthritis (2.8%), but associations with immune thrombocytopenic purpura (itp) are rare (1.4%) (5). in the present report, we describe the clinical outcome of a patient with aih associated with itp who was successfully treated with prednisolone (psl). a 75-year-old japanese woman was admitted to the national hospital organization shinshu ueda medical center, japan, in july 2013 for further investigation of elevated serum aminotransferase levels and thrombocytopenia. the laboratory data from her referring institution were aspartate aminotransferase (ast) 935 iu/l, alanine aminotransferase (alt) 913 iu/l, total bilirubin 7.7 mg/dl, and platelet count 2.810/l. she had been diagnosed with essential hypertension three years prior and was taking a candesartan cilexetil/amlodipine besilate compound product. her family history was negative for liver or autoimmune disease, and she denied exposure to agents relevant to hepatitis, such as blood transfusion or herbal (kampo) medicine. the laboratory data on admission at our facility (table 1, 2) showed severe thrombocytopenia and increased levels of hepato-biliary enzymes and gamma-globulin. platelet-binding igg (pbigg) was negative, but platelet-associated igg (paigg) was 894 ng/10 cells (nl: 47). her thyroid-stimulating hormone and free-thyroxin levels were within normal ranges, although anti-thyroglobulin (tg) and anti-thyroperoxidase (tpo) antibodies were positive. the diagnostic criteria for disseminated intravascular coagulation (dic) proposed by the japanese ministry of health, labor and welfare were not satisfied in this patient (score: 4). tests for the hepatitis a, b, c, and e viruses, epstein-barr virus, cytomegalovirus, and herpes simplex virus were all negative. her human leukocyte antigen (hla) a bone marrow examination uncovered normoplastic marrow with platelet depletion and a megakaryocyte count of 30/l (fig. ret: reticulocyte, fdp: fibrin/fibrinogen degradation products laboratory findings on admission (2). ana: anti-nuclear antibody, asma: anti-smooth muscle antibody, ama: anti-mitochondria antibody, anti-lkm1 ab: anti-liver/kidney microsome type 1 antibody, paigg: platelet-associated igg, pbigg: platelet-binding igg, tg antibody: anti-thyroglobulin antibody, tpo antibody: anti-thyroperoxidase antibody, h. pylori ab: helicobacter pylori antibody, ebv: epstein-barr virus, cmv: cytomegalovirus, hsv: herpes simplex virus, hla: human leukocyte antigen the histological findings of a bone marrow examination. magnification: (a) 40, hematoxylin and eosin (h&e) staining; (b) 400, h&e staining. a diagnosis of itp was made based on the above clinical and laboratory findings. according to the criteria of the international autoimmune hepatitis scoring system (6), the patient's pre-treatment clinical score without histology was 16 (female:+2, alp (alkaline phosphatase)/alt ratio:+2, igg level:+2, ana titer:+2, anti-mitochondrial antibody (ama): 0, viral markers:+3, drugs:+1, alcohol:+2 and immune disease:+2), and indicative of definite aih. the patient promptly began treatment with 30 mg/day (0.6 mg/kg/day) of psl on day 5 of admission, which quickly restored her liver enzyme levels to within normal limits (fig. 3a and c), infiltration of lymphocytic and plasma cells, and mild interface hepatitis around the portal vein (fig. (d) the specimen showed infiltration of inflammatory cells consisting of lymphocytes and plasma cells, with mild interface hepatitis in the portal zone. magnification: (a) 40, hematoxylin and eosin (h&e) staining; (b) 40, azan-mallory staining; (c) and (d) 400, h&e staining. following an uneventful treatment course, the patient's serum transaminase and igg levels became elevated without thrombocytopenia due to a recurrence of aih when psl was tapered to 5 mg/day. restoring the steroid dose to 20 mg/day normalized her laboratory findings, and the serum transaminase and igg levels and platelet count have since remained normal for over 19 months with a maintenance dose of 10 mg/day psl (fig. aih is defined as a chronic liver disease with unknown etiological factors that is associated with aberrant auto-reactivity and a genetic predisposition (1-5). in contrast, itp is an acquired autoimmune disorder mediated by antibodies against platelet membrane glycoprotein (gp) ib or iib/iiia complexes and thrombopoietin receptor (8-10). the precise mechanism underlying itp remains elusive (11), and itp complicating aih is rare and of uncertain pathogenesis (12-14). in japan, given that type 1 aih represents the overwhelming majority (15) of aih cases, all reported cases of aih associated with itp have been type 1. summarized 16 cases of aih associated with itp appearing in the japanese literature (12). while aih preceded itp in 5 cases, the disorders manifested simultaneously in the others. in agreement with these findings, wada et al. reported on a patient with a six-year history of aih who developed itp (13), and yamaike et al. relapses of aih and/or itp were not found in any case. in our patient, either aih and itp developed simultaneously or aih shortly preceded itp, because liver histology (biopsied 9 days after commencing steroid therapy) showed very mild fibrosis (fig. 3b). the first-line therapy for both aih and itp is corticosteroids (1-5). roughly 24-50% of aih patients experience relapse during steroid tapering or withdrawal (1,15). approximately 75% of patients with itp respond to corticosteroids, but only 5-30% achieve a sustained remission (16). to our knowledge, the present case is the first of its kind, showing relapsing aih and non-relapsing itp during steroid tapering. we suspected that the response thresholds of the two diseases to psl were different in our subject. hla class ii molecules on antigen-presenting cells play a crucial role in triggering the immune response, which begins with the recognition of peptide antigens in the hla class ii groove by the t-cell receptor of cd4 t cells through direct contact of both molecules. we previously reported that the most influential gene associated with type 1 aih susceptibility was hla-drb1*04:05 (17,18), while drb1*15:01 conferred disease resistance (19). strong associations between anti-gp autoantibodies and hla class ii genes have been identified, as follows: anti-gpiib-iiia antibody association with drb1*04:05 and dqb*04:01; and anti-gpib-ix antibody with drb1*08:03 and dqb1*06:01 (20). the hla typing pattern in the present case was drb1*04:05 and drb1*15:01. thus, the drb1*04:05 allele may be linked to aih associated with itp. however, it is difficult to explain the low frequency of aih with itp, although the immunologically different mechanisms of the two disorders, which remain unresolved, may be involved. since itp and aih manifest as autoimmune-mediated disorders, the treatment for both conditions is psl. here, aih recurred after psl tapering but itp did not. this differential immunosuppressive effect is of clinical interest and may provide insights into the mechanisms and interplay of autoimmune diseases.

although autoimmune hepatitis (aih) is frequently complicated with chronic thyroiditis or other autoimmune disorders, reports on its association with immune thrombocytopenic purpura (itp) are scarce. we herein describe a case of aih associated with itp. a 75-year-old japanese woman was admitted to our hospital due to increased aminotransferase levels and severe thrombocytopenia. elevated serum immunoglobulin g (igg) was detected, and tests for platelet-associated igg and anti-nuclear antibody were positive. following the diagnosis of aih-associated itp, prednisolone treatment of 0.6 mg/kg/day resulted in a decrease in the aminotransferase levels and an increased platelet count.

PMC5337457

pubmed-934

deep brain stimulation (dbs) is a neurosurgical technique aimed at improving functional neurologic disorders. to facilitate intraoperative neurophysiologic microelectrode recording (mer) and neurocognitive testing of the eloquent parts of the brain, dbs is commonly performed via an awake approach under a local anesthetic and conscious sedation. while a deeper plane of anesthesia can be used during the portion of the procedure not involved in testing, an awake and cooperative patient with fully preserved brain function is essen-tial during mer to ensure accurate lead placement 1,2. our case involved a developmentally delayed 24-year old, 80-kg male with traumatic brain injury (tbi) as an infant with resultant post-traumatic stroke, hydrocephalus, and right upper extremity hemidystonia, causing a prominent dystonic flexor position at the elbow. the surgical plan included implantation of intracranial dbs leads in the globus pallidus internus in an attempt to relieve the hemidystonia. a peripheral iv was placed and midazolam 2 mg was administered en route to the or. standard asa monitors were applied along with 2 l/min of oxygen via nasal cannula, while the neurosurgical team prepared for head frame placement. a dexmedetomidine intravenous infusion at 0.5 g/kg/h was started, and a total of 1.2 g/kg of fentanyl was administered incrementally for local anesthetic infiltration at the head frame pin sites. during this period, the patient maintained spontaneous ventilation and did not react to local infiltration, pin insertion, or head frame manipulation. a radial arterial line and second peripheral iv were placed under local and propofol infusion at 100 g/kg/min. before mer and neurocognitive testing, propofol was turned off in order to transition to the awake segment of the procedure. once mer was complete, the propofol infusion was restarted to supplement the dexmedetomidine infusion during surgical site closure. the patient's postoperative course was uneventful, and he was discharged home 2 days after the procedure. at patient's 6-month follow up clinic visit, he had a dramatic reduction in his right upper extremity tremor and had a significant improvement in his ability to perform daily activities. dbs involves the placement of wire electrodes into deep brain nuclei. for movement disorders, these deeper brain structures include the subthalamic nucleus, internal segment of the globus pallidus, and thalamic nucleus ventralis intermedius. electrical impulses are delivered to these structures with the objective of altering specific neural networks, resulting in a therapeutic effect. the electrodes are then connected to an implanted pacemaker that produces high-frequency stimulations that help ameliorate the clinical manifestations of functional neurologic disorders, while having the added qualities of titratability and reversibility 3. targeting specific deep brain structures in dbs surgery is based on stereotactic principles that use advanced brain imaging and atlases. the surgical trajectory is guided by stereotactic platforms, which may be framed or frameless. the deep brain nuclei targets are verified by their electrophysiological signature using intraoperative neurophysiologic mapping. this presents several unique challenges to the anesthesiologist: providing patient reassurance and comfort, especially during head frame placement and burr hole drilling, and maintaining this level of supportive care over a potentially long surgery; ensuring proper positioning to allow for safe and optimal surgical conditions; and maintaining access to the patient's airway while anticipating emergency airway interventions in the event ventilatory compromise occurs under sedation. this is especially important as the head frame system (fig.1) can impair access to the airway, making mask ventilation problematic. patient cooperation, providing an anesthetic technique that will minimally interfere with mer, and maintaining spontaneous ventilation and airway patency are crucial to a successful outcome in these cases. finally, an appropriate blood pressure should be maintained in order to minimize the risk of intracranial bleeding, a well-known complication in dbs procedures 4. the anesthetic management of an awake craniotomy has changed over the past 20 years in an attempt to address the aforementioned challenges. currently, the most popular technique is the asleep-awake-asleep technique. this employs a heavier sedation or general anesthesia during the initial stages, alternating with consciousness using rapid-onset medications with a short duration of action during brain mapping and neurocognitive testing 2. after mapping is complete, deep sedation or general anesthesia can be reestablished for surgical closure and removal of the pins. propofol, remifentanil, and dexmedetomidine are some of the anesthetics that have been used for awake craniotomy (table1). while propofol and remifentanil are short-acting, propofol can attenuate mer and remifentanil may cause muscle rigidity. both drugs can result in respiratory depression, so they must be titrated carefully. while high doses of dexmedetomidine can abolish mer, low dose infusion rates (0.30.6 g/kg/h) usually preserve mer integrity, while also providing analgesia and sedation without respiratory depression 1. the -2a receptors in the locus ceruleus are responsible for the sedative, analgesic, and sympatholytic effects of dexmedetomidine. this specific effect over -2a receptors may allow cortical neurons to continue functioning upon stimulation in contrast to propofol or even barbiturates that produce generalized neuronal hyperpolarization. commonly used anesthetic agents in awake craniotomies and dbs in a recent case study, 6 pediatric patients with severe dystonia successfully underwent dbs procedures with the use of low-dose dexmedetomidine as part of the anesthetic technique with unimpaired neuroelectrophysiological signals 5. since it does not directly affect the activity of subthalamic neurons, dexmedetomidine may be an ideal sedative medication for neurophysiologic monitoring 6. low-dose dexmedetomidine (0.10.4 g/kg/h) infusion has been shown to produce mer from subthalamic neurons similar to mer during the awake state 7. additionally, dexmedetomidine use has decreased the intraoperative need for antihypertensive medications used to prevent intracranial hemorrhage 4. as seen in the presented case, the surgeon was able to perform successful cortical mapping during low-dose dexmedetomidine infusion allowing the patient to follow simple verbal commands. there was no evidence of respiratory depression, and dexmedetomidine also likely decreased the amount of narcotic needed for this stimulating procedure. it has been well-documented in the literature that dexmedetomidine decreases the need for narcotic administration in various surgical procedures. despite the patient's severe dystonia and developmental delay dexmedetomidine helped facilitate this by decreasing the patient's anxiety and restlessness during the procedure and allowing for an easy transition from sedation to responsiveness. while dbs is most commonly performed for generalized dystonias, there are few reports in the literature describing dbs for tbi-induced dystonias. here, we have presented a case of dbs in an awake patient with post-traumatic hemidystonia. although various anesthetic techniques and combinations of drugs have been described for an awake craniotomy, dexmedetomidine appears to be useful in procedures, involving dbs and neurophysiologic monitoring. as the indications for dbs in the pediatric patient population are expanding and the demand for this procedure increases, the creation and implementation of standardized protocols guiding the complex anesthetic management for these cases would be of value.

key clinical messagedeep brain stimulation in an awake patient presents several unique challenges to the anesthesiologist. it is important to understand the various stages of the procedure and the complexities of anesthetic management in order to have a successful surgical outcome and provide a safe environment for the patient.

PMC4498869

pubmed-935

allergic rhinitis and asthma are both chronic heterogeneous disorders, with overlapping epidemiology of prevalence, health care costs and in quality of life (2). these two clinical entities are observed over time as separate diseases. epidemiological and clinical evidence, as well as experimental observations have suggested a link between rhinitis and asthma leading to a definition of allergic rhino-bronchitis or united airways diseases and the concept of one airway one disease. most important of them are pollens, dust mites, and animal products (4). house dust mites dermatophagoides pteronyssinus (df p) and dermatophagoides farinae are the most common indoor aeroallergens all over the word. other aeroallergens animal origin, which are often described as a cause of respiratory allergies are cockroach, feathers and animal hair (5). clinical epidemiological and pathophysiological studies suggest a strong functional and immunological relationship between the nose and bronchi and survey results report that pollens, especially grass pollens are the major cause of respiratory allergies worldwide (6, 7). the aim of this study was to determine hypersensitivity to the most common aeroallergens among our patients with nasobronchial allergy and to determine whether there are differences in hypersensitivity between rhinitis and asthma. the study is a retrospective, population analysis of the results of skin prick tests to aeroallergens in patients with nasobronchial allergy. the study included 2254 patients, aged from late adolescence (from 16 years of age) to adults, who were tested at the department of dermatovenerology, clinical centre of banja luka, during ten years period. ambulatory patients, who were referred by family doctors, otolaryngologist, dermatologists, or pulmonologists were subjects of testing. patients were tested by skin prick test, which is used worldwide as an assured and rapid method for allergy screening. in the first series there were seven allergens: df p, weed pollen mixture, grass pollen mixture, tree pollen mixture, feathers, animal dander and cockroach (blatella germanica). patients who tested positive for the grass pollen mixture, were further tested for: cocksfoot (dactilys glomerata), meadow grass (poa pratensis), rye-grass (lolium perenne), timothy (phleum pratense), cultivated wheat (triticum aestivum) and corn (zea mays). patients who tested positive for the weed pollen mixture, were further tested for: ragweed (ambrosia elatior), mugwort (artemisia vulgaris), sorrel (rumex acetosella), plantain (plantago lanceolata) and wall pellitory (parietaria officinalis). patients who tested positive for the tree pollen mixture were further tested for: birch (betula verrucosa), hazel (corylus avellana), elder (sambucus nigra), linden (tilia cordata) and ash (fraxinus americana). according to the diagnosis in order to perform the necessary statistical tests we used the statistical software package spss for windows (version 13). for the analysis of the data, descriptive and inferential statistics methods where used. from descriptive statistical parameters the mean value and measures of variation were used, which describe the main characteristics of the data in a quantitative sense. from inferential statistical methods student s t-test, person the limit value of the existence of a statistically significant difference was set at p<0.05. the total sample of our study included 2254 patients with nasobronhial allergy, 1376 (61%) women, and 848(39%) men. the difference between number of female and male patients was statistically significant (t test; p<0.01). almost three times more patients, had allergic rhinitis than asthma (t test; p<0.01). in the rhinitis group were 957 (58.6%) were female and 677 (41.4%) were male patients, mean age was 45.7216.1. in the asthma group 419 (67.6%) were women and 201 (32.4%) were men, mean age 45.6816.0. in both group of all the patients, highest recorded number of positive prick tests was df p (27.5%) and weed pollens (21.9%), followed by grass (18.3%) and tree pollens (10.1%). although there were more female than male patients in our total sample (p<0.001), results show that the number of positive prick tests, according to all allergens was larger in male patients (2 test, p<0.05). the patients with allergic rhinitis have a higher number of positive skin prick tests to df p, and all pollen mixtures, than patients with asthma (2 test, p<0.05). for cockroaches, feathers and animal hair, determined by a small number of positive skin prick tests, there were no statistically significant differences between patients with rhinitis and asthma (table 1). positive skin prick tests in the total sample, by the sex of patients and type of allergic disease.*dermatophagoides pteronyssinus results of skin prick test for individual weed pollens in the total sample and by group of disease show that they were mostly positive to ragweed (28.9%), then followed by mugwort (11%) and plantain (8.1%). more patients with allergic rhinitis than with asthma had positive skin prick test to ragweed and sheep sorrel (2 test, p<0.05). other weed pollens showed no statistically significant difference between the two groups of patients (table 2). positive skin prick tests to weed pollens in the total sample tested to six individual, grass pollens showed highest number of positive tests to cocksfoot (14.4%) and meadow grass (14.3%). this was followed by rue-grass (12%) and wheat (10.2%). patients with rhinitis had more positive tests for cocksfoot (15.3%), but the patients with asthma for meadow grass (12.6%). there was no statistically significant difference for grass pollens between the two groups of patients, except for timothy grass (2 test, p<0.032), in rhinitis group. positive skin prick tests to grass pollens in the total sample of patients, the highest number of positive tests, was to birch pollen (7.3%), then hazel (5.7%), while for the other tree pollens, there were a small number of positive skin reactions. both groups of patient had the highest positive tests for birch, but without statistically significant difference, as for the other tree pollens (table 4). main findings of this study are that our patients with nasobronchial allergy more often have allergic rhinitis than asthma, and that df p is the most common cause of allergy. although in the study there were more women than man, allergy was more often found in men. it is consistent with the results of similar studies which included more women than men, and with a higher incidence of adult non-atopic rhinitis in women. however, there are studies that found no difference in the gender distribution (3). the fact that a significantly greater number of patients had allergic rhinitis rather than asthma coincides with epidemiological data on the incidence and prevalence of these diseases in adolescence and adulthood (1, 7). finding that df p is the common allergen in both groups of patients in our study, especially in the rhinitis group, is consistent with the results of a large number of similar studies (3, 8). when it comes to pollens, we found that the greatest number of positive skin prick tests was to weed pollens, followed by grass pollens and the smallest number for tree pollens. aerobiological and allergic studies show that the pollen map of europe is changing and that depends of cultural factors, major international moving and climate changes (9, 10). a number of studies provide data that grass pollens are the main source of respiratory allergies worldwide (11, 12, 13, 14). in the same studies, tree pollens are generally found as well as in our study, according to the frequency in second place behind the grass and weed pollens. however, in recent years, weed pollens allergy has been increasing in certain parts of france, italy, austria, croatia and bulgaria (15). our research, as the majority of studies in europe, shows that ragweed pollen is the leading weed pollen (6). in hungary, at least 60% of the patients who have an allergy to pollen are allergic to ragweed pollen (16). in croatia, which is a geographically close region, nearly half the patients with seasonal rhinitis and asthma have had sensitization to ragweed (17). in our study, after ragweed, the second most common weed pollen is mugwort. ragweed and mugwort have nearly identical flowering season, and clinical and serological studies in europe suggest that hypersensitivity to this two pollens are frequently associated (18). plantain, third weed pollen in our study, is cited as a significant cause of pollinosis in temperate regions of north america, australia, europe as well as in japan (19). the most common grass pollens in our study is cocksfoot and meadow grass. in similar studies meadow, cocksfoot and rye-grass are stated as the most common allergens (20, 21). our result show that the birch is most usually allergenic tree pollen which is consistent with results from other studies that identified it as one of the most common causes of allergic rhinitis in europe (5). in the literature, numbers of adults which are allergic to birch vary depending on geographic region (22). authors from the united kingdom were recommended that birch pollen is one of the seven allergens which are sufficient for identification of hypersensitive individuals in epidemiological studies (23). this pollen is often referred to the cause of pollen allergy in central and northern europe (24, 25). our results show that a small number of patients have positive skin prick tests for cockroach, animal hair and feathers. distribution of these allergens varies depending on the geographic region, climate and housing conditions, and the literature data are different. in recent years, a number of studies have investigated that cockroach exposure is a major risk factor for the development of asthma (26, 27). however, the results for cockroach allergy are similar to ours, as was also confirmed by croatian authors (28). some studies show that one third of patients with respiratory allergy have an allergy to cat allergens, while multicenter study from china noted significant number of respondents have dog hair allergy (29). our study, like the others, showed a very low percentage allergy for animal hair (30). more patients with nasobronchal allergy have rhinitis than asthma. although majority of patients were female, allergy is more common in men, than in women. dermatophagoides pteronyssinus and weed pollens are the most common aeroallergens for both groups of patients. the rhinitis group of patients had the greatest number of positive skin prick tests for house dust mite and all pollens, than the asthma group. cocksfoot is the most common grass pollen in rhinitis group, and meadow and in asthma group of patients.

abstractbackground: aeroallergens are the most common causes of allergy.aim:the aim of this study was to determine hypersensitivity to aeroallergens in patients with nasobronchial allergy. methods: this retrospective population study included 2254 patients with nasobronchial allergy, from late adolescents to adults. their response to aeroallergens was assessed by skin prick tests. results:more patients had rhinitis (72.7%), than asthma (27.6%). although majority of patients were female, allergy is more common in men than in women (p<0.05). both groups of patients had the greatest number of positive skin prick tests for dermatophagoides pteronyssinus (27.5%) and weed pollens (21.9%), followed by grass (18.3%) and tree pollens (10.1%). ragweed is the most common positive weed pollen in both groups, more in patients with rhinitis (p=0.022). the cocksfoot is the most common grass pollen in rhinitis group (15.3%), but meadow grass (12.6%) in asthma patients. birch is the most common tree allergen in the both groups. conclusions:more patients with nasobronchial allergy have rhinitis than asthma. skin prick tests are usually positive for dermatophagoides pteronyssinus and weed pollens, followed by grass and tree pollens, and they are more common positive in patients with rhinitis than asthma.

PMC4272505

pubmed-936

in 1935, rous and beard were the first to describe the progression of virus-induced papillomas to carcinomas in rabbits 1. during subsequent decades, it has been widely accepted that the development of human cancer follows a multi-stage process involving tumor initiation, promotion and progression 2. these stages are paralleled by an accumulation of several mutations in genes regulating critical cellular pathways, which provide a growth advantage for individual tumor cells. in this regard, only a few genetic modifications enable the clonal expansion of normal cells during tumor initiation. additional mutations further support tumor growth during promotion, and tumor cells finally develop a malignant phenotype including invasive growth and metastasis during progression 3, 4. concerning colorectal cancer (crc), it has been known for several decades that carcinomas mostly develop from adenomas. in 1988, vogelstein et al. described four specific mutations that accumulate during the progression of adenomas to carcinomas 5. these mutations have subsequently been shown to involve so-called care- and gatekeeper genes, which enable genetic or epigenetic instability and support tumor growth respectively. although several other mutations involved in crc development have been added during recent years, most sporadic crcs are believed to develop as a consequence of the mutations initially described by vogelstein et al. various factors have been shown to be responsible for the accumulation of mutations in crc including inheritance and environmental factors (e.g. composition of diet, obesity, diabetes mellitus, smoking, alcohol consumption) 6. of note, also chronic inflammation is regarded as an important risk factor for the development of cancer. this is especially apparent in patients with inflammatory bowel diseases (ibd), which have an increased risk for the development of colitis-associated crc depending on the duration and severity of intestinal inflammation 7. whereas the contribution of chronic inflammation to tumor development has been widely attributed to its ability to induce mutations (e.g. through reactive oxygen or nitrogen species) 8, recent data propose a direct effect of inflammation on tumor growth. several pro-inflammatory cytokines released by innate and adaptive immune cells have been shown to regulate cancer cell growth and thereby contribute to tumor promotion and progression. among these, interleukin-6 (il-6) seems to take a center stage in human cancer development. an increased expression of il-6 has been detected and associated with an unfavourable prognosis in patients with various types of cancer including both sporadic and colitis-associated crc. experimental studies found an activation of important oncogenic pathways in cancer cells through il-6. in this article, we review the role of il-6 during sporadic and inflammation-associated crc development. besides data from human crc, molecular mechanisms of il-6 signaling in experimental models of crc will be discussed with an outlook on future therapeutic implications. following its initial description as a b cell differentiation factor in 1986 9, a versatile role has been attributed to il-6 for the regulation of innate and adaptive immunity. in fact, il-6 is involved in the regulation of the acute phase response through the induction of acute phase proteins in hepatocytes, the differentiation of monocytes to macrophages, the proliferation and resistance against apoptosis of t cells and th2 cytokine production 9-11. importantly, recent data suggest a critical role for il-6 during chronic inflammation, since il-6 is required for the induction of effector th17 cells and inhibits the differentiation of regulatory t cells. il-6 is produced by various cell types including monocytes, macrophages, fibroblasts, keratinocytes, endothelial cells, b cells, t cells, and also several tumor cells 12. however, monocytes and macrophages seem to be the predominant producers of il-6 during acute and t cells during chronic inflammation 13. in these cells, il-6 expression is regulated through the activation of several transcription factors such as nf-kb, c/ebpbeta (caat/enhancer-binding protein beta) or ap-1 (activator protein 1). the regulation of il-6 expression through these transcription factors enables a rather unspecific upregulation of this cytokine during nearly every type of inflammation. il-6 binds to the membrane-bound il-6 receptor alpha (mil-6r, cd126) subunit of the il-6 receptor on target cells. this complex then associates with a homodimer of the second receptor subunit, glycoprotein 130 (gp130, cd130), and thereby enables the activation of subsequent downstream signaling. this so-called classic signaling is restricted to cells expressing both the mil-6r subunit and gp130. although gp130 is widely expressed, mil-6r expression is limited to hepatocytes and some leukocytes 14. importantly, a soluble form of il-6r (sil-6r) enables il-6 signaling in cells that do not express mil-6r through trans-signaling. sil-6r is either produced by limited proteolysis of mil-6r through the metalloproteinase adam17 or translation from a splice variant of il-6r mrna 14. sil-6r binds il-6 with a similar affinity as mil-6r and the complex of sil-6r and il-6 can interact with gp130 on target cells that do n't express mil-6r. as signal transduction during both classic and trans-signaling these include the activation of janus kinases (jaks) with a subsequent activation of the transcription factor signal transducer and activator of transcription 3 (stat3) through phosphorylation. stat3 activation has been shown to be an important step for promotion and progression through the induction of various target genes. these target genes are involved in tumor cell survival (e.g. bcl-2, survivin, mcl-1), proliferation (e.g. c-myc, cyclin d1, cyclin b), angiogenesis (e.g. hif1alpha, vegf), metastasis (e.g. mmp2, mmp9), cell adhesion (e.g. icam-1, twist1), inflammation (e.g. il-6, il-17, il-23, cox2) and others (for review see 15, 16). among these, suppressor of cytokine signaling 3 (socs3) is a direct inhibitor of stat3 signaling. although the influence of these pathways on immune cells has been known for several years, only recent data provide a molecular insight on the importance of il-6 signaling during tumor development. concerning the role of il-6 in b cell differentiation, it's not surprising that multiple myeloma was among the first types of cancer that have been shown to be influenced by il-6. in fact, il-6 acts as an auto- and paracrine growth factor for myeloma cells and antibodies against il-6 inhibit myeloma cell growth in vitro and in vivo 17. today, il-6 is regarded as an important tumor promoting factor in various types of human cancer including glioma, lymphoma, melanoma as well as breast, ovarian, pancreatic, prostate, renal and, of course, colorectal cancer. various studies have found an increased expression of il-6 in patients with crc, where il-6 levels are elevated in the serum of patients and in tumor tissue itself 18-20. according to a review article by knpfer and preiss, il-6 expression can be associated with tumor stage, size, metastasis and survival of patients with crc 21. although data on il-6 expression in sporadic crc are well proven, there is an on-going debate about the source of il-6 expression in non-inflammation-associated cancer. for instance, belluco et al. described an association of a polymorphism of the il-6 promoter with serum levels of this cytokine in patients with crc 22. an additional mechanism could be an amplification of the il-6 gene, as reported in patients with glioblastoma, although this mechanism has not been shown for crc so far 23. another explanation for increased il-6 levels could be an infiltration of tumors with il-6 secreting inflammatory cells as seen in colitis-associated cancer (cac). as mentioned above, patients with inflammatory bowel diseases such as crohn's disease (cd) and ulcerative colitis (uc) have an increased risk for the development of cac. for instance, the cumulative risk for the development of crc in patients with uc is about 17.8% after 30 years of disease 24. in patients with large bowel involvement of cd, there is a 8.3% risk for crc over a period of 30 years 25. in fact, various studies have shown that il-6 is an important regulator of ibd pathogenesis, mainly through its effect on immune cell function 11. for instance, il-6 trans-signaling has been shown to activate t cells in the lamina propria of patients with ibd and induces resistance of these cells against apoptosis through upregulation of anti-apoptotic factors such as bcl-2 and bcl-xl 27. due to the correlation of il-6 expression with crc prognosis and the increased expression of il-6 in patients with ibd, il-6 is thought to act as a link between chronic inflammation and tumor development. importantly, corvinus et al. could show an increased phosphorylation of stat3 in crc cells, but not in normal intestinal epithelial cells 28. these data propose a functional relevance for il-6 directly acting on tumor cells in sporadic crc. this direct effect of il-6 on colorectal cancer cells is likely mediated through trans-signaling, as intestinal epithelial cells usually do not express mil-6r 14. in fact, evidence for this hypothesis comes from various experimental studies in mouse models of colitis-associated cancer. in 1992, lahm et al. were among the first to describe a growth-promoting effect of il-6 on colorectal cancer cell lines in vitro 29. however, it was not until 2004 that becker et al. were able to show that il-6, secreted by lamina propria t cells and macrophages, is also important for the development of cac in vivo 30. in the widely used aom+dss mouse model of cac, the authors found an il-6 dependent growth of intestinal tumors that was dependent on il-6 trans-signaling in intestinal epithelial cells, possibly with downstream activation of stat3. the tumor promoting effect of il-6 could be inhibited through treatment with anti-il-6r antibodies or sgp130fc, a designer variant of soluble gp130 that specifically blocks trans-signaling. similarly, grivennikov et al. found reduced tumor development in il-6 -/- mice exposed to the aom+dss model 31. in this work, the authors underlined the importance of an il-6 dependent stat3 in tumor cells as critical for proliferation and the inhibition of apoptosis. these data were complemented by bollrath et al. showing increased cac development following aom+dss treatment in gp130y757f mice, which have stat3 hyperactivation, and attenuated tumor development in conditional knockout mice with a specific deletion of stat3 in intestinal epithelial cells 32. the effect of stat3 on tumor cells was mediated through the expression of various regulators of g1/s and g2/m cell cycle progression. interestingly, in both studies defective il-6/stat3 signaling was impairing intestinal inflammation in aom+dss treated animals. in this regards, il-6 and stat3 were proposed as important regulators of intestinal homeostasis. in addition to il-6 and stat3, in a study by rigby et al. the specific deletion of socs3 in intestinal epithelial cells of mice treated with aom+dss was associated with increased tumor development 33. in contrast, overexpression of socs3 in vitro was able to reduce proliferation of crc cell lines. whereas most effects of stat3 have been attributed to a direct regulation of cell cycle progression through this transcription factor,, we were able to show that il-6 induces the expression of vegfr2 (vascular endothelial growth factor receptor 2) in intestinal epithelial cells and enables an auto-/paracrine feedback loop, which promotes proliferation of tumor cells in the aom+dss model of cac through vegfr2-dependent stat3 activation 34. despite substantial progress of crc treatment during recent years, crc still belongs to the leading causes of cancer related death in industrialized countries. therefore, new therapeutics, especially for patients with advanced disease, are desperately required. according to the growing evidence supporting a critical role for il-6/stat3 signaling during various aspects of both sporadic and inflammation-associated crc development, therapeutics targeting this pathway could be promising options for affected patients. in fact, several therapeutics inhibiting the il-6/stat3 pathway have been developed for the treatment of human disease. these include anti-il-6 or anti-il-6r antibodies, soluble gp130fc (sgp130fc) and selective small molecule jak inhibitors 35 (table 1). anti-il-6 antibodies were among the first therapeutics targeting the il-6/stat3 pathway to be used in clinical studies on human cancer during the 1990s. these include the treatment of multiple myeloma or aids-associated kaposi's sarcoma 36, 37. however, these treatments only produced a limited response, and, as the antibody used (be-8) was from murine origin, an immune response against this therapeutic was induced in treated patients 38. as a consequence, the chimeric, murine-human monoclonal anti-il-6 antibody siltuximab was generated during subsequent years. although there are currently no data on the use of siltuximab in patients with crc, a study analyzing the effect of siltuximab in various types of solid cancer including crc has just been finished and the results are awaited eagerly (clinicaltrials.gov identifier: nct00841191). however, initial clinical studies using siltuximab in patients with metastatic renal cancer, ovarian cancer or prostate cancer provided mixed results 39-41. a possible explanation might come from the fact that antibody-associated il-6 is not cleared from the circulation and thus increases systemic il-6 concentrations 35. so far, several clinical studies have shown a promising effect of tocilizumab in chronic inflammatory diseases and led to the approval of this antibody for the treatment of rheumatoid arthritis and juvenile idiopathic arthritis by the fda 43. although there are preclinical data on a therapeutic effect of tocilizumab in cancer, clinical trials in human cancer are missing so far. another strategy to inhibit il-6/stat3 for instance, the small molecule jak1 and 2 inhibitor ruxolitinib has shown promising results in a clinical trial in patients with post myeloproliferative neoplasms and acute myeloid leukemia 44. currently, there are no clinical data on the use of ruxolitinib in crc. however, cep-33779, another jak inhibitor, could successfully reduce tumor growth in experimental cac 45 all of the treatments mentioned above inhibit both classical and trans-signaling, and therefore also block physiological functions of il-6. in contrast, a specific inhibition of trans-signaling could be achieved by sgp130fc. sgp130fc is a designer cytokine that specifically binds il-6/sil-6r complexes and therefore only blocks trans-signaling. in fact, sgp130fc has been shown to be effective for the treatment of experimental cac in the study by becker et al. the substance will soon enter clinical development and it will be interesting to see its effect on human cancer 46. il-6 has been shown to be an important tumor promoting cytokine that enforces proliferation and anti-apoptotic effects in tumor cells. clinical and experimental data strongly propose a contribution of il-6 signaling to the development of both sporadic and colitis-associated colorectal cancer development. in this regard, several components of the il-6 signaling pathway such as il-6, il-6r, jak have been proposed a promising targets for crc therapy. as initial clinical studies using anti-il-6 therapy are on their way, it will be interesting to see, if preclinical data will live up to their promise.

growing evidence proposes an important role for pro-inflammatory cytokines during tumor development. several experimental and clinical studies have linked the pleiotropic cytokine interleukin-6 (il-6) to the pathogenesis of sporadic and inflammation-associated colorectal cancer (crc). increased il-6 expression has been related to advanced stage of disease and decreased survival in crc patients. according to experimental studies, these effects are mediated through il-6 trans-signaling promoting tumor cell proliferation and inhibiting apoptosis through gp130 activation on tumor cells with subsequent signaling through janus kinases (jaks) and signal transducer and activator of transcription 3 (stat3).during recent years, several therapeutics targeting the il-6/stat3 pathway have been developed and pose a promising strategy for the treatment of crc. this review discusses the molecular mechanisms and possible therapeutic targets involved in il-6 signaling in crc.

PMC3491448

pubmed-937

reinforced surveillance systems aimed at monitoring the introduction of chikv have been implemented in 6 departments in southeastern france, including the var department, where ae. albopictus has spread since its introduction in 2004, presumably from northern italy (4). on august 29, 2010, a 7-year-old girl (patient 1) with acute febrile syndrome, headache, and abdominal pain sought treatment in the city of frjus (var) 1 day after she had returned from rajasthan, india. continuous chikv circulation in northern india districts has been reported during 20092010 (www.promedmail.org). three weeks after the notification of patient 1, another young girl (patient 2) experienced clinical symptoms that began on september 18 with fever, arthralgia, backache, headache, and retro-orbital pain. patient 2 s physician reported that a young girl (patient 3), a close friend of her patient, showed clinical symptoms compatible with chikv infection at the same time. patient 3, who lives near patient 1, had invited patient 2 to spend the night of september 15 at her home. a serum sample from patient 3 was collected 1 week after onset of fever and monoclonal antibody capture elisa detected high titers of specific anti-chikv immunoglobulin m. the serum sample also showed a weak rt-pcr signal for chikv. given that patients 2 and 3 did not report any recent travel to areas endemic for chikv no complications were recorded, but all 3 patients had persistent weakness and joint pain 3 months after the acute phase. intensive mosquito control measures, including spraying for adult mosquitoes and destroying breeding sites, were undertaken around the patients residences and areas visited by confirmed case-patients. no further cases were found by the active case finding system (a local physician and laboratories network) implemented for 45 days after the declaration of the last autochthonous case. a molecular study of france/2010 chikv strains isolated in frjus obtained from patients 1 (imported case) and 2 (autochthonous case) was performed. viral genomic rna was extracted from chikv grown once in mosquito c6/36 cells and then subjected to rt-pcr amplification by using a set of primers targeting the structural genes of chikv (7). paired sequence analysis of the e26k e1 junction showed that the 2 france/2010 chikv strains display a divergence rate<0.05% at the nucleotide level, whereas 100% identity was observed at the amino acid level. phylogenetic analysis demonstrated that these viral strains belong to a cluster that is closely related to strains from india within the ecsa lineage (figure). the france/2010 chikv isolate from patient 2 might be derived from an indian strain introduced by patient 1 (index case). genotypes e2-211 t, e2-312 m, e2-386a, 6k-8i, and e1-284e that are found in the currently circulating strains belonging to the ecsa lineage were identified in france/2010 chikv isolates (2,3,7). these isolates also display the genotype e1-211e specifically shared by viral strains belonging to the asian phylogenetic group (table). the residue ala at position e2-264 has not been previously described in any chikv strains. phylogenetic relationships among chikungunya virus isolates from cases of chikungunya fever in france, based on complete e2-6k-e1 nucleotide sequence (2,771 nt) analysis. phylogenetic analysis was inferred by using the maximum-likelihood method as implemented in mega version 5 software (www.megasoftware.net). the sequence of the strains from france described in this study has been deposited in genbank (accession number pending); other sequences were retrieved from genbank.*molecular signatures were based on the analysis of complete amino acid sequence e2-6k-e1 (923 aa). the numbering of amino acid positions refers to the african isolate s27 (genbank access no. letters in parentheses after strain names refer to east/central/south africa (a), asia (b) and west africa (c) phylogroups. recent attention has focused on the predominant role of e1 and e2 proteins in successful chikv infection of the anthropophilic ae. vector competence experiments with la runion/2006 chikv isolates demonstrated the importance of the newly acquired e1-ala226val substitution for efficient transmission by ae. albopictus from northern italy and from southeastern france showed disseminated infection rates ranging from 75%90% for chikv strains with e1-226v (10). the 2 france/2010 chikv strains isolated in frjus have ala at position e1-226 (table). the presence of an asp residue at position e2-60, found in most of the ecsa chikv strains, may in part counterbalance the less favorable transmission of e1-226a strain in ae. albopictus (table). the thr residue at position e2-211 potentiates the infectivity of chikv in ae. the presence of e2-211 t in chikv isolates from france underlines the risk for emergence of a fully adapted viral variant if the e1-226v genotype was selected during continuous transmission within ae. the efficient chikv transmission in italy and southeastern france sheds new light on its dissemination potential in europe from 1 index case, regardless of the viral genetic background and mosquito species in the region of origin of the imported chikv (1,10,11). in emerging regions, such as italy and runion island, where the seroprevalence in the population was<50% however,>2 years passed since the end of the epidemic in runion island before a local transmission of chikv was again detected. in europe where sylvatic cycles are absent, vertical transmission may participate in the maintenance and/or cyclic reemergences of chikv. albopictus that may have more efficient vertical transmission than mosquito populations in eastern italy and tropical regions (11,12). in 2010, southeastern france faced the concomitant emergences of dengue virus (denv) and chikv (13). for each of these viruses, only 2 autochthonous infections were confirmed, which suggests that rapid detection and control measures implemented around imported and autochthonous cases have been efficient. a recent report mentioned the dual emergence of chikv and denv in southeastern france and urged the implementation of specific surveillance and response measures to reduce the risk for arbovirus emergence (14). since 2006, a specific chikungunya/dengue national preparation and response plan based on rapid detection and investigation of imported and suspected autochthonous cases, mosquito control measures, and efficiency evaluation in the treated areas has been activated from may through november and then modified after annual debriefing meetings involving all partners. in 2010, this model proved to be well adapted to the early detection and control of chikv and denv. albopictus mosquitoes and the successful emergence of chikv in italy and france, reinforced surveillance and response to chikv and denv dissemination should become a higher priority in europe (15).

in september 2010, autochthonous transmission of chikungunya virus was recorded in southeastern france, where the aedes albopictus mosquito vector is present. sequence analysis of the viral genomes of imported and autochthonous isolates indicated new features for the potential emergence and spread of the virus in europe.

PMC3321794

pubmed-938

cd47 (originally named integrin-associated protein (iap)) is a cell surface protein of the immunoglobulin (ig) superfamily, which is heavily glycosylated and expressed by virtually all cells in the body. cd47 was first recognized as a 50 kda protein associated and copurified with the v 3 integrin in placenta and neutrophil granulocytes and later shown to have the capacity to regulate integrin function and the responsiveness of leukocytes to rgd-containing extracellular matrix proteins [14]. soon after this integrin-associated protein was cloned, it was shown to be identical to the erythrocyte cell surface antigen cd47. the fact that cd47 is also expressed by cells like erythrocytes, that do not express integrins, indicates that it can be more appropriate to refer to this protein as cd47 than using its original name integrin-associated protein (iap). the protein is fairly well conserved between species and has about 6070% similarity in the amino acid sequence when comparing human cd47 with that of mouse, rat, and bovine cd47. cd47 has also been shown to be identical to the oa-3/ovtl3 antigen highly expressed on most ovarian carcinomas [6, 7]. it also shows homology to a protein family of variola and vaccinia viruses [1, 5, 6], the significance of which is still unclear. cd47 consists of an extracellular igv domain, a five times transmembrane-spanning domain, and a short alternatively spliced cytoplasmic tail. in both humans and mice, the cytoplasmic tail can be found as four different splice isoforms ranging from 4 to 36 amino acids, showing different tissue expression patterns. the 16 amino acid form 2, which is by far the predominant isoform, is expressed in all cells of hematopoietic origin, as well as in endothelial and epithelial cells. in contrast, the 36 amino acid form 4 is expressed primarily in neurons, intestine, and testis. expression of the 4 amino acid form 1 is found in epithelial and endothelial cells, while the expression pattern of the 23 amino acid form 3 resembles that of form 4. despite a study showing that cd47 form 3 and 4 could be associated with memory retention in rats, and that form 2 is predominating in astrocytes, little is known as to what these splice variants mean in terms of possible difference in the functionality of the protein. a new twist in the understanding of cd47 comes from a recent finding that cd47 can be expressed as a proteoglycan with a molecular weight of>250 kda, having both heparin and chondroitin sulfate glycosaminoglycan (gag) chains. this form of cd47 was found to be expressed in both the human jurkat t cell line and in murine primary t cells, as well as in human umbilical vein endothelial cells (huvecs), murine lung endothelial cells, and in human smooth muscle cells. functionally, the gag chains at cd47 ser were found to be crucial to inhibit t cell receptor signaling following the ligation of cd47 by its ligand thrombospondin-1. early studies of cd47 were based on the use of monoclonal antibodies (mabs) raised against the cd47 protein purified from placenta [2, 11], showing a role of cd47 in mediating an enhanced igg-mediated phagocytosis response in the presence of rgd-containing ligands, such as fibronectin, fibrinogen, vitronectin, or collagen type iv [2, 11]. the same mabs were also found to block neutrophil transendothelial migration stimulated by interleukin 8 (il-8) or the bacterial peptide n-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) and to inhibit neutrophil migration across tumor-necrosis-factor-- (tnf-) stimulated endothelial cells, where cd47 on both the neutrophils and the endothelial cells was found to be important. generation of other anti-cd47mabs, raised against epithelial membrane preparations, showed that cd47 is present at the basolateral membrane of epithelial cell monolayers, that mabs blocking cd47 on either neutrophils or the epithelial cells delay neutrophil trans-epithelial migration, and that efficient neutrophil chemotaxis correlates with an increased neutrophil cell surface expression of cd47. the basement membrane protein entactin, which contains an rgd sequence, was also found to stimulate neutrophil adhesion and chemotaxis in a cd47-dependent manner in vitro. generation of cd47-deficient mice further proved the importance of this protein in regulating neutrophil inflammatory responses, by showing an increased sensitivity to bacterial infection due to a delayed neutrophil accumulation in bacterial peritonitis. cd47-deficient neutrophils also show a strongly impaired rgd-stimulated neutrophil adhesion, phagocytosis, and respiratory burst. for v 3 integrin-mediated cellular responses to the extracellular matrix protein vitronectin, cd47 was found to be required for v 3-mediated binding to vitronectin-coated beads, but not v 3-mediated adhesion to vitronectin-coated surfaces. in addition to its original association with v 3 integrins, cd47 has also been shown to interact with and regulate the integrins 2 1 and iib 3 on platelets [16, 17], the 2 1 integrin on smooth muscle cells, the 4 1 integrin on sickle red blood cells and b lymphocytes [19, 20], the 6 1 integrin in microglia, and the 5 integrin in chondrocytes (figure 1). thrombospondin-1 (tsp-1) is the prototypic member of the thrombospondin family of extracellular matrix glycoproteins, which are implicated in regulating cell motility, proliferation, and differentiation. the extracellular igv domain of cd47 was found to be a receptor for the c-terminal cell-binding domain (cbd) of tsp-1, since the expression of cd47 in otherwise cd47-deficient cells promotes adhesion to tsp-1 or its cbd, and a functional blocking mab against cd47 can block endothelial cell chemotaxis against tsp-1 or the cd47 binding cbd-peptide 4n1k. it was later shown that tsp-1, its cbd, or the 4n1k peptide stimulates v 3 integrin-mediated cell spreading on vitronectin in a cd47-dependent manner. in platelets, tsp-1, the tsp-1 cbd, or 4n1k activates the platelet iib 3 integrin and induces platelet spreading on fibrinogen, platelet aggregation, and increased focal adhesion kinase (fak) tyrosine phosphorylation, which are all dependent on interaction between cd47 and integrin iib 3. furthermore, cd47 was found to mediate a synergistic effect of soluble type i collagen and tsp-1 or 4n1k, which enhance 2 1 integrin-mediated platelet activation or vascular smooth muscle cell chemotaxis [17, 18]. early experiments also suggested that cd47 regulates tsp-1-induced cell spreading or platelet activation by affecting signal transduction in a pertussis toxin-sensitive way via a heterotrimeric gi protein [16, 24]. cd47 was later shown to functionally associate with heterotrimeric gi, to suppress camp levels, and mediate the inhibition of erk in platelets and smooth muscle cells. more recently, cd47 ligated by tsp-1 was found to inhibit nitric oxide (no) signaling in vascular cells and to oppose no/cgmp-mediated inhibition of integrin activation to facilitate platelet aggregation. in addition to signaling through gi proteins, cd47 has been shown to signal via the 3 integrin cytoplasmic tail. although it is not entirely clear how much these two signaling pathways overlap, accumulation of cd47/ 3-integrin-complexes in cholesterol-rich lipid rafts, which appears to depend on both the cd47 igv domain, the multiple transmembrane-spanning domain, and a long range disulfide bond between cys in the igv domain and cys in the transmembrane domain, engage in gi signaling. cd47 has also been shown to be involved in the regulation of intracellular ca ([ ca]i), exemplified by its regulation of an integrin-dependent increase in [ca]i in endothelial cells and tumor cells, and that cd47 synergizes with t cell receptor-stimulated elevations of [ca]i in t lymphocytes [33, 34]. the cytoplasmic tails of the form 2 and form 4 splice variants of cd47 were found to bind to the cytosolic ubiquitin-related proteins plic-1 and plic-2 (plic, proteins linking iap to cytoskeleton). in b lymphocytes, a role of cdc42 and rac has also been suggested in cd47-dependent regulation of neuronal development and neurite formation [36, 37]. adhesion of intestinal epithelial cells to collagen i induces the association of cd47 with 2 integrins, and cd47 is necessary for collagen i-induced cyclooxygenase-2 (cox-2) expression and epithelial cell migration, which is mediated by g i3 (figure 1). sirp proteins belong to the ig family of cell surface glycoproteins, where the first member identified was sirp (also known as shps-1, cd172a, bit, mfr, or p84) [3944]. sirp is highly expressed in myeloid cells and neurons, but also in endothelial cells and fibroblasts, and has three extracellular ig-like domains, one distal igv-like domain, and two membrane proximal igc-like domains [41, 42]. in addition, an alternatively spliced form having only one igv domain has also been reported. in its intracellular tail, sirp has two immunoreceptor tyrosine-based inhibitory motifs (itims), which when tyrosine phosphorylated can bind the src homology 2 (sh2) domain-containing protein-tyrosine phosphatases shp-1 and shp-2. additional cytoplasmic binding partners for sirp are the adaptor molecules src kinase-associated protein of 55 kda homolog/skap2 (skap55hom/r), fyn-binding protein/slp-76-associated phosphoprotein of 130 kda (fyb/slap-130), and the tyrosine kinase pyk2. sirp is also a substrate for the kinase activity of the insulin, egf, and bpdgf receptors, and the overexpression of sirp in fibroblasts decreases proliferation and other downstream events in response to insulin, egf, and bpdgf. since sirp is also constitutively associated with the m-csf receptor c-fms, sirp overexpression partially reverses the v-fms phenotype. two other family members have also been identified, sirp (also known as cd172b) [42, 47] and sirp (also known as cd172 g or sirp2), whose extracellular ig-like domains are similar to that of sirp. however, the cytoplasmatic regions of sirp and sirp are different from that of sirp. sirp has a very short cytoplasmatic tail with no signaling motifs. instead, the transmembrane region contains a positively charged lysine residue, which can bind the immunoreceptor-tyrosine-based-activating-motif- (itam-) carrying adaptor protein dnax activation protein 12 (dap12/karap) [49, 50]. sirp has no recognizable signaling motif or capability to interact with cytoplasmic signaling molecules and is therefore unlikely to generate intracellular signals. cd47 has been shown to be a ligand for sirp [52, 53] and sirp [54, 55], but does not bind sirp. the cd47/sirp interaction regulates not only a multitude of intercellular interactions in many body systems, such as the immune system where it regulates lymphocyte homeostasis [56, 57], dendritic cell (dc) maturation and activation, proper localization of certain dc subsets in secondary lymphoid organs [5961], and cellular transmigration [62, 63], but also regulates cells of the nervous system (reviewed in [64, 65]). an interaction between these two proteins also plays an important role in bone remodeling [66, 67]. cellular responses regulated by the cd47/sirp interaction are many times dependent on a bidirectional signaling through both receptors [51, 64, 65] (figure 1). the finding that cd47 on host cells can function as a marker of self and regulate phagocytosis by binding to sirp will be further described in a subsequent section. the interaction between cd47 and sirp has proven to be very specific species, as shown by the relatively weak binding of cd47 from mouse, rat, or cow to human sirp [69, 70]. in addition, the glycosylation of cd47 or sirp does not seem to be necessary for their interaction, but the level of n-glycosylation of sirp has, however, an impact on the interaction such that over glycosylation reduces the binding of cd47. the long range disulfide bond between cys in the cd47 igv domain and cys in the transmembrane domain is also important to establish an orientation of the cd47 igv domain that enhances its binding to sirp. ligation of cd47 by anti-cd47 mabs was found to induce apoptosis in a number of different cell types. this phenomenon was first described in jurkat t cells, in anti-cd3 activated but not in resting primary t cells, and in b-cell chronic lymphocytic leukemia (b-cll) cells [72, 73]. cd47-induced apoptosis can be induced by several different mabs; however, while some of these (e.g., ad22, 1f7, or mabl) show potent apoptosis induction in suspension [7376], others (e.g., b6h12 and 2d3) need to be immobilized to a surface to promote cell death [72, 77]. of the two sirp-family members known to bind the cd47 igv domain (sirp and sirp), sirp as a soluble fc-fusion protein does not induce cd47-dependent apoptosis, while sirp or sirp bound onto the surface of beads induces apoptosis through cd47 in jurkat t cells and the myelomonocytic cell line u937. in addition, tsp-1 or the cd47-binding tsp-1 cbd-peptide 4n1k also induces cd47-dependent apoptosis [74, 75, 78, 79]. indeed, mice deficient in cd47 or tsp-1 sustain oxazolone-induced inflammation significantly longer than wild-type mice due to a deficiency in t cell apoptosis. this form of cell death was initially described to be characterized by cell shrinkage, reduction in mitochondrial transmembrane potential, and exposure of phosphatidylserine (ps) on the cell surface, but to be independent of fas (cd95) or tnf receptor signaling. classical apoptosis, and it is independent of cysteinyl aspartate protease (caspase) activation [72, 73] (figure 2). furthermore, inhibitors of actin polymerization or mitochondrial electron transfer prevent cd47-induced ps exposure. in support of a role for the actin cytoskeleton, peripheral blood mononuclear cells (pbmcs) from wiskott-aldrich syndrome (was) patients, where mutations in the was protein (wasp) results in defective cdc42-induced regulation of the actin cytoskeleton, are resistant to cd47-induced apoptosis. although the mitochondrial transmembrane potential is affected in cd47-induced apoptosis, it does not involve the mitochondrial release of cytochrome c or apoptosis-inducing factor (aif), but does involve the production of reactive oxygen species (ros). in jurkat t cells, it was shown that the inhibition of gi signaling with pertussis toxin can counteract cd47-induced apoptosis, that ligation of cd47 reduces intracellular camp levels, and that camp elevating agents prevents apoptosis by cd47 ligands. this signaling pathway, which likely also involves reduced signaling through protein kinase a (pka), is not only described in t cells, but also in several breast cancer cell lines. in the latter cell type, it was shown that epidermal growth factor can inhibit the cd47 death pathway via protein kinase c (pkc). a yeast two-hybrid screen, where cd47 was used as bait, identified the pro-apoptotic bcl-2 family member bcl-2-homology-3- (bh3-) only protein 19 kda interacting protein-3 (bnip3). in t cells, bnip3 was found to physically associate with cd47, which prevents its degradation in proteasomes and sensitizes t cells to cd47-induced apoptosis [78, 79]. ligation of cd47 induces the translocation of bnip3 to mitochondria, and attenuation of bnip3 activity inhibits cd47-induced apoptosis, which together suggests that bnip3 is crucial as a mediator of this cell death pathway. moreover, bnip3 gene expression was found to be increased and regulated by hypoxia-inducible factor-1 (hif-1) following the ligation of cd47 by single-chain fragments of an anti-cd47 mab which kills b-cll cell lines both in vitro and in vivo, where the knockdown of hif-1 represses cd47-induced cell death (figure 2). the finding that a jurkat t cell clone lacking cd47 is resistant to fas- (cd95-) induced apoptosis, but that expression of cd47 restores the sensitivity to fas-ligation, suggested that cd47 can augment fas-induced apoptosis via a mechanism that requires neither cd47 signaling nor its association with lipid rafts. in fact, the lack of cd47 impairs important proapoptotic events downstream of fas, such as loss of mitochondrial membrane potential, cytochrome c release, caspase activation, poly(adp-ribose) polymerase (parp) cleavage, and dna fragmentation. this function of cd47 is likely also important in primary cells, since t cells from cd47-deficient mice are protected from fas-induced apoptosis. in hematopoietic cells, cd47-induced apoptosis has been described in hematopoietic tumor cells [54, 7274, 7678, 81, 82, 84, 85] and in activated primary t or b cells [73, 74, 77, 79]. however, whether apoptosis can be induced through cd47 in nonactivated leukocytes is still somewhat unclear. the only situation where nonactivated t or b cells have been found to undergo cd47-induced apoptosis is when immobilized anti-cd47 mab has been used, but not when using soluble cd47 ligands or mabs known to induce apoptosis in activated cells or tumor cells [73, 74, 79]. surprisingly, although cd34 hematopoietic progenitors express cd47, they are resistant to cd47-induced apoptosis by either immobilized or soluble mab [76, 77]. in addition, immature human monocyte-derived dendritic cells (idcs) were described as resistant to cd47-induced apoptosis, following incubation with an immobilized cd47 mab for 18 hours. however, another study showed that freshly isolated human monocytes or human monocyte-derived idc undergoes a rapid (within 60 min) cell death in response to the cd47-ligand 4n1k. this cell death, which was described to occur in a subset of cells and where monocytes or idc not affected by 4n1k remain viable in culture, is associated with cellular features previously described for cd47-induced apoptosis, such as not only ps exposure, increased plasma membrane permeability, reduced mitochondrial membrane potential, caspase independence, but also included dna fragmentation. thus, these findings suggest that although a subset of idc may undergo cd47-induced cell death at an early time point, this may not be detectable at later time points. however, it raises the question if specific subsets of monocytes or idcs are sensitive to this form of rapid cell death whereas others are resistant and maintain their viability in culture. in addition to hematopoietic cells, overexpression of cd47 can induces cell death of cultured cerebral cortical neurons, which is enhanced by the coexpression of sirp and prevented by brain-derived neurotrophic factor (bdnf) when cd47 and sirp are coexpressed. apoptosis in neurons overexpressing cd47, however, is dependent on caspases and apoptotic cells have condensed apoptotic nuclei with fragmented dna. it has also been shown that endothelial cells incubated under static conditions in the absence of flow increase their expression of tsp-1, which uses the cd47/ v 3 integrin as a receptor to trigger endothelial cell apoptosis. this mechanism also appears to be involved in endothelial cell apoptosis during proatherogenic turbulent flow conditions and in mechanosensitive induction of apoptosis in fibroblasts. tsp-1-mediated apoptosis, mapped to the type-3 repeat/c-terminal domain of tsp-1, in promyelocytic leukemia cells (nb4-lr1) has also been suggested to depend on the engagement of both cd47 and the v 3 integrin. in contrast to the proapoptotic effects of cd47 described above, it was reported that the tsp-1-derived peptide 4n1 could abolish c2-ceramide-induced apoptosis in primary porcine thyroid cells by preventing reduction in intracellular camp levels, an effect blocked by the functional blocking antihuman cd47 mab b6h12. a similar effect of 4n1 peptide was also found to inhibit the cytotoxic effects of the anticancer drugs camptothecin and doxorubicin in thyroid carcinoma cells. although it is unclear how these effects of cd47-ligation can be explained in relation to the proapoptotic effects of this molecule, it is interesting to note that tumor cell tsp-1 overexpression has been linked to disease recurrence and decreased survival [9496], and it was suggested that this pathway could be one explanation behind drug resistance in thyroid cancers. mature erythrocytes express high levels of cd47, but do not express integrins, which early indicated that other important functions of cd47 could be expected in these cells. the fact that individuals with the rhnull phenotype, which do not express any of the proteins of the rh protein complex, only express about 25% of normal levels of cd47 suggested a close relation between cd47 and erythrocyte rh proteins [5, 97]. in the erythrocyte cell membrane, rh polypeptides associate in a complex with many other proteins (e.g., rh associated glycoprotein (rhag), glycophorin b, lw, and cd47). another erythrocyte membrane protein complex is formed by the band 3 anion exchanger and several other proteins (e.g., glycophorin a, protein 4.2, and ankyrin). the latter multiprotein complex mediates the anchorage of the erythrocyte membrane to the spectrin cytoskeleton [99, 100]. in addition, it has been suggested that the rh complex and the band 3 complex may in fact be associated in the erythrocyte membrane. mutations in band 3, or complete band 3 deficiency, in human erythrocytes results in reduced expression of rh polypeptides and rhag and results in a virtual lack of cd47. moreover, human erythrocytes deficient in protein 4.2 also show a marked deficiency of cd47 as well as an altered glycosylation of rhag. when combining these finding, a hypothesis was put forward, which suggests that cd47 of the rh complex may indeed form a link to the band 3 complex by binding to protein 4.2 (figure 3). studies of protein mobility within the erythrocyte membrane have shown that cd47 is associated with the erythrocyte cytoskeleton as being a part of the rh complex, but that cd47 is also present as a noncytoskeleton anchored pool which is more mobile in the erythrocyte membrane. thus, cd47 in erythrocytes may serve several different functions depending on its grade of mobility in the plasma membrane. it has been suggested that the freely mobile cd47 pool may be of importance to accumulate cd47 in specific membrane areas upon cell-cell contacts, for example, in order to efficiently interact with sirp on other cell types such as phagocytic cells. importantly, data from studies of murine erythrocytes have shown remarkable differences as compared with human erythrocytes, and the link between cd47, the rh complex, and protein 4.2 in murine erythrocytes is not well understood. first, there are little or no rh polypeptides in erythrocytes from band 3-deficient mice, while the expression of cd47 is only slightly reduced. second, erythrocytes from cd47-deficient mice were found to contain normal amounts of murine rh and rhag polypeptides. taken together, the total picture suggests that cd47 seems to interact with rh polypeptides and protein 4.2 in human erythrocytes, whereas the interaction between cd47, rh polypeptides, and proteins of the band 3 complex in murine erythrocytes is still unclear. a progressive decrease in the cd47 expression level has been observed on human erythrocytes stored under blood bank conditions at+4c for more than 14 days [107109] and in a mouse model of erythrocyte storage which tried to mimic human blood bank conditions. however, the magnitude of reduction in cd47 expression was rather different, from a modest reduction of up to 6% at day 42 of storage, around 30% reduction on day 28 of storage, to a more than 50% reduction on day 14 of storage. furthermore, a recent study indicated that the cd47 levels of erythrocytes stored for 35 days were not different from that of fresh cells. the discrepancy between these findings may have several explanations, such as the exact storage conditions or methods for the quantification of erythrocyte cd47 expression levels. in addition, the loss of cd47 may also be sensitive to leukocytes remaining in the erythrocyte concentrates. indeed, a gradual loss of cd47 from erythrocytes during storage was observed in stored erythrocytes irrespective of whether the buffy coat was removed or not before storage, although buffy coat removal resulted in an increased expression level of cd47 at all time points tested, showing a significant correlation between the number of remaining leukocytes and erythrocyte cd47 levels. a study of cryopreserved leukoreduced erythrocytes was unable to detect any effects on erythrocyte cd47 expression levels. microparticles released in the blood from blood cells or endothelial cells have recently gained interest due to their possible role in regulating a variety of normal or pathological biological functions. such microparticles are also released from erythrocytes during cryopreservation or storage at+4c, and these microparticles do among other erythrocyte membrane proteins also carry cd47 [112, 114]. although the presence of cd47 on these microparticles suggests a mechanism for the observed loss of cd47 from stored erythrocytes, soluble cd47 has also been detected in the supernatants of erythrocytes stored at+4c. in addition to a possible loss of cd47 on stored erythrocytes, a recent study also indicated that storage as well as experimental aging in vitro results in a conformational change of the cd47 protein. this modification can be detected as a selectively increased binding of the anti-cd47 mab 2d3, which recognizes an epitope different from the epitope involved in the binding of tsp-1 or sirp and recognized by mab b6h12. however, the use of mouse models has been informative to further understand if the level of cd47 is changing during erythrocyte aging. in these models, biotin is injected intravenously to label all blood cells at one or two specific time points [115, 116]. analyses of circulating erythrocytes at later time points will then allow for discrimination between older biotin-positive and younger biotin-negative circulating erythrocytes [115, 116]. using this approach, it has been found that cd47 is gradually lost from the surface of circulating murine erythrocytes, where the oldest erythrocytes may have up to 30% lower cd47 expression levels as compared with younger erythrocytes [115, 117]. sickle cell disease, caused by a mutation in the hemoglobin chain and a formation of insoluble intracellular aggregates of mutated hemoglobin, resulting in characteristic sickle shaped erythrocytes, is associated with severe vasoocclusive crisis [118, 119]. one important mechanism behind this pathology is the enhanced adhesion of sickle erythrocytes to vascular endothelium [118, 119]. cd47 was shown to play a role in the pathologic sequestration of sickle erythrocytes to vascular endothelium under shear stress by binding to tsp. it has been found that the reticulocyte-enriched fractions of sickle erythrocytes are most efficiently binding to endothelial cells, that this function requires heterotrimeric g proteins and tyrosine kinase activity, and is mediated by 4 1 integrins on the reticulocytes. interestingly, sickle erythrocytes also show an increased expression of the cd47 epitope recognized by mab 2d3. in gaucher's disease, a sphingolipidosis caused by glucocerebrosidase deficiency, macrophages accumulate glucosylceramide following the excess phagocytosis of erythrocytes, which converts splenic macrophages to pathogenic gaucher cells. erythrocytes from patients with untreated gaucher's disease have been found to have reduced levels of cd47, which can be reversed upon enzyme-replacement therapy. it has been suggested that the anemia associated with the untreated disease can in part be explained by a combination of reduced cd47 levels together with other morphological erythrocyte abnormalities observed in this disease. the distinction between self and non-self is central to the maintenance of integrity in a multicellular organism and allows for a powerful and successful elimination of potentially dangerous pathogens, while carefully preserving healthy host cells and tissues. this distinction has been well studied in the adaptive immune response, which is specialized in the recognition of foreign peptides by virtue of small modifications to major histocompatibility complex (mhc) molecules made by these peptides. for the innate immune system, recognition is thought to be based on a large extent on the recognition of specific microbial structures, pathogen-associated molecular patterns (pamps). the innate immune system of the host organism has through the evolution developed a group of receptors, pattern recognition receptors (prrs), which serves to specifically recognize pamps. in this way, the host may utilize a certain number of receptors, encoded in the genome, for the recognition of various evolutionary stable molecular pathogen-associated structures. while some of the prrs (like the mannose receptor (mr)) recognize pamps directly, others (like complement receptors (crs)) are specialized to detect products generated secondary to pamp recognition. however, a system based on specific recognition of foreign is clearly flawed as it prevents the recognition of anything that is also present on the organism's own cells. this can be very elegantly circumvented by a defense system that has very broad recognition, when it is combined with specific molecules that mark host cells and tissues as self. thus, recognition of self will inhibit the activation of innate immune cells, whereas the recognition of missing self allows for an immune response to proceed. in other words, a system where the own cells express a unique marker of self, not present on foreign cells, would make the distinction between self and foreign very simple. in that way, it would not matter if the organism's own cells express substances or ligands that are similar to those found on foreign cells/particles. innate immune cells such as macrophages would only look for the presence of the self marker on the recognized particle, where the presence of self would release the recognized particle but the absence of self would allow for activation and destruction. this would significantly simplify the recognition process, as there would only be a need for a few broad specific recognition receptors instead of a countless number of foreign-specific receptors. it would also fulfill the criteria for a defense system that effectively recognize and destroy foreign objects but at the same time reduces the risk of damaging the organism's own structures. such a system for macrophage activation would be analogous to the well-established missing-self hypothesis for natural killer (nk) cell activation. nk cells recognize target cells by a range of activating receptors which also recognize ligands on many normal cells. however, in the nk cell system expression of self-mhc class i will protect the recognized cell via the ligation of nk cell inhibitory receptors specific for it is, however, important to note, that nk cell inhibitory receptors are now described, which seem to have ligands other than mhc class i. upon the binding of self-mhc class i, ligated nk cell inhibitory receptors will recruit and activate the phosphatases shp-1/shp-2 that mediate the inhibition of nk cell activation. nk cells thus spare cells whenever they express markers of normal self, and eliminate them when these markers are absent or inadequately expressed. other leukocytes also express molecules related to nk cell inhibitory receptors, further suggesting that similar mechanisms are operative also, for example, in macrophage activation. many of these inhibitory receptors recognize mhc class i, but the marker of self could in principle be any ubiquitously expressed surface molecule. a new chapter in the understanding of cd47 and its functions started when cd47 was found to bind sirp [52, 53]. at the time, sirp was regarded as one of several immunoreceptors with cytoplasmic itim motifs, generally suggested to be involved in negative regulation of cellular functions, mediated by the recruitment of the tyrosine phosphatases shp-1 and/or shp-2 or the inositol phosphatase ship to the tyrosine phosphorylated itims. in addition, sirp was found to be highly expressed in primary macrophages and macrophage cell lines, as well as in other myeloid cells such as monocytes, granulocytes, and dendritic cells. in macrophages, sirp was found to negatively regulate signaling through tyrosine kinase-dependent signaling pathways (e.g., fcri). although both shp-1 and shp-2 can bind to the phosphorylated sirp itims, only shp-1 has been associated with the inhibitory function of sirp in macrophages, similar to that of the nk cell inhibitory receptors, whereas shp-2 associated with sirp leads to a phosphatase-dependent enhancement of the signal in many situations. when studying cd47-deficient mice, we were struck by the fact that cd47-deficient bone marrow can not reconstitute and engraft in lethally irradiated syngeneic wild-type recipient mice, while it engrafts normally in cd47-deficient recipient mice. our work on trying to understand this controversy first involved experiments where different leukocyte populations were transferred from cd47-deficient mice into wild-type recipient mice. however, we later simplified the system by studying transfusion of erythrocytes instead of leukocytes. the reason for this is that erythrocytes in contrast to many leukocyte populations have a long half-life in circulation, they do not divide, do not express mhc class i, and they do not home to extravascular tissues or organs. these studies showed that fresh erythrocytes isolated from the blood of cd47-deficient mice, labeled with a fluorescent cell tracker dye and transfused into wild-type recipient mice, have markedly reduced survival, whereas their half-life is normal in cd47-deficient recipients. the clearance rate of such freshly isolated cd47-deficient erythrocytes is remarkably fast with complete clearance within 24 hours. to put this in perspective, the average lifespan of murine erythrocytes in circulation is somewhere between 45 and 60 days, depending on the mouse strain investigated [135, 136]. the rapid clearance of cd47-deficient erythrocytes from the circulation of wild-type recipient mice does not require complement, since cd47-deficient erythrocytes are also cleared from the circulation of complement factor 3- (c3-) deficient mice. neither is there a requirement for lymphocytes or antibodies to enable the clearance of cd47-deficient erythrocytes from the circulation, since clearance is normal in rag1-deficient mice, which lack mature t and b lymphocytes. rather, the transfused cd47-deficient erythrocytes that are eliminated from the circulation of wild-type mice are recognized and cleared by splenic red pulp macrophages, and the removal of these macrophages by splenectomy or by treatment with macrophage-toxic clodronate liposomes abrogates the elimination of cd47-deficient erythrocytes. furthermore, macrophage sirp is tyrosine phosphorylated upon contact with cd47 on erythrocytes, and when sirp on isolated splenic macrophages is blocked, it increases the level of phagocytosis of wild-type erythrocytes to that seen with cd47-deficient erythrocytes, whereas phagocytosis of the cd47-deficient erythrocytes is unaffected by the antibody treatment. in addition, studies in sirp-mutant mice, where the cytoplasmic signaling domain of the receptor is deleted, have shown a shorter half-life of normal cd47-expressing erythrocytes in these mice, which also present with mild anemia. such spontaneous anemia is, however, not seen in cd47-deficient mice, suggesting that cd47 could also be needed on the macrophages to facilitate the clearance of erythrocytes in the spleen. however, lack of cd47 on platelets also results in very rapid clearance when transfused into wild-type recipients, and both cd47-deficient mice and sirp-mutant mice have a mild spontaneous antibody-independent thrombocytopenia [139, 140]. altogether, these findings indicated that all erythrocytes can be phagocytosed by splenic red pulp macrophages when sirp is blocked or cd47 is missing, and that these macrophages must have a receptor for erythrocytes. indeed, we have identified ldl receptor-related protein (lrp-1), which by recognizing calreticulin on the surface of normal erythrocytes can mediate phagocytosis of untreated cd47-deficient erythrocytes [141, 142]. by showing that macrophages in the splenic red pulp can recognize normal circulating erythrocytes, but that these macrophages do not phagocytose erythrocytes as long as they display cd47 on their surface, these findings were the first to prove that macrophages are perfectly capable of recognizing normal host cells and rely on self recognition for proper function. in addition, it also demonstrated major similarities between mechanisms for self recognition in nk cells and macrophages. based on the findings that unopsonized erythrocytes lacking cd47 can be phagocytosed by splenic macrophages, and that the cd47/sirp interaction can potently inhibit the prophagocytic mechanism operating in that situation, we next hypothesized that the cd47/sirp interaction might as well be able to negatively regulate phagocytosis of opsonized erythrocytes, and that for the cells of the innate immune systems front line defense (i.e., macrophages and dc) there would be a balance between signals from activating receptors (e.g., fcr or cr) and the inhibitory signal from sirp ligated by target cell cd47. indeed, the cd47/sirp interaction can also regulate phagocytosis of igg-opsonized or complement opsonized erythrocytes as well as other opsonized host cells, making cd47-deficient cells severely sensitive to being phagocytosed [139, 143145]. in addition, cd47-deficient mice are severely sensitive to experimental autoimmune hemolytic anemia (aiha) and experimental thrombocytopenia [139, 146]. on an autoimmune background prone to develop spontaneous aiha, lack of cd47 results in a more rapid and lethal aiha. using motheathen viable (me/me) mice, which only have about 20% of normal levels of shp-1, but normal levels of shp-2 [147149], we could pinpoint the role of these phosphatases in mediating the phagocytosis inhibitory effect of the cd47/sirp interaction in vivo. when transfused into me/me mice, igg-opsonized wild-type erythrocytes are cleared with the same rapid kinetics as seen with equally opsonized cd47-deficient erythrocytes, showing that at that particular signaling strength through prophagocytic fc receptors, the level of cd47 on normal erythrocytes was not enough to prevent phagocytosis if the shp-1 level was reduced by about 80%. in contrast, the prophagocytic signaling mediating phagocytosis of unopsonized cd47-deficient erythrocytes (presumably mediated by lrp-1) is likely much weaker, since unopsonized wild-type erythrocytes are not cleared with the same rapid kinetics as cd47-deficient cells in me/me mice. however, while unopsonized cd47 heterozygous erythrocytes (expressing about 50% of the cd47 found on wild-type erythrocytes) show normal half-life when transfused into wild-type recipients, they were found to be cleared more rapidly when transfused into me/me mice. these findings thus proposed that a reduction of the cd47 level to 50% of normal, in combination with a strongly reduced level of shp-1 in the macrophages, together weakened the inhibitory signaling through sirp such that clearance of unopsonized erythrocytes was allowed. furthermore, the rate of phagocytosis of igg-opsonized erythrocytes is distinctly regulated by the amount of cd47 present on the surface of the erythrocytes both in vivo and in vitro. thus, activation of phagocytosis in a macrophage in contact with a target erythrocyte (or any other host cell) can be viewed as a balance between signals from activating receptors (i.e., fcr, cr, or lrp-1) and the inhibitory signal from sirp ligated by target cell cd47. in the macrophage, neither signal appears to be dominant, but rather the decision to phagocytose a target host cell is based on an integration of positive prophagocytic signals and inhibitory cd47/sirp signaling (figure 4(a)). the same functional regulation also seems to be operating in dcs and in microglia. in hemophagocytic lymphohistiocytosis, hematopoietic stem cells are phagocytosed by bone marrow macrophages as a result of systemic inflammation. in this disease, hematopoietic stem cells were found to express reduced levels of cd47, which shows that pathological conditions may occur where a combination of inflammatory macrophage activation and reduced expression of cd47 results in a severe loss of critical cell types. however, not all prophagocytic receptors seem to be regulated by the cd47/sirp interaction. although fc receptor-mediated phagocytosis of igg-opsonized oxidatively damaged erythrocytes is strongly inhibited by the cd47/sirp interaction, scavenger receptor-mediated uptake of unopsonized oxidized erythrocytes turned out to be insensitive to this inhibitory mechanism. the mechanism whereby recruitment of shp-1 to sirp can inhibit phagocytosis has been suggested to involve the tyrosine kinase syk and phosphoinositide 3 kinase (pi3 kinase). more recently, it was suggested that shp-1 mediates dephosphorylation of nonmuscular myosin iia at the phagocytic synapse between the phagocyte and a host cell, as a result of the interaction between macrophage sirp and cd47 on the host cell, which brings further insight into the mechanism behind phagocytosis inhibition by the cd47/sirp interaction. based on the seemingly important interaction between cd47 and sirp to prevent phagocytosis of host cells, and the fact that cd47-deficient cells are rapidly phagocytosed when transfused into wild-type mice, it is clearly a puzzle why cd47-deficient mice do not present with a more severe phenotype where the macrophages phagocytose a large fraction of the cd47-deficient cells in those mice. however, this phenomenon is rather similar to what is described in 2 microglobulin deficient mice, which lack expression of mhc class i but where the nk cells still do not attack and destroy host cells and also show hyporeactivity to cells carrying ligands for activating nk cell receptors, cells which are efficiently killed by wild-type nk cells. one explanation proposed for this function in nk cells is that inhibitory nk cell receptors need to interact with licenced and able to become activated and kill target cells lacking or expressing reduced levels of self- mhc class i. by investigating the ability to phagocytose cd47-deficient cells in mice where cd47 was expressed by hematopoietic cells, nonhematopoietic cells, or both, the phagocytic tolerance to cells lacking cd47 in cd47-deficient mice it was found that macrophages developing in an environment where nonhematopoietic cells lack cd47 become tolerant to cells lacking cd47, which allowed cd47-defcient leukocytes to avoid clearance. curiously, this did not involve erythrocytes, which were cleared by splenic macrophages also in the bone marrow chimeras where nonhematopoietic cells lacked cd47 and macrophages had become the latter phenomenon has so far not been explained, but may suggest that phagocytosis of erythrocytes is regulated differently from that of leukocytes. although cd47-deficient macrophages express normal amounts of sirp, which can also become tyrosine phosphorylated upon contact with cd47 expressing cells (oldenborg et al., unpublished observations and), it is possible that the tolerance to cd47-deficient cells that develop in cd47-deficient mice has to do with tuning of intracellular signaling pathways. in favor of such a hypothesis is the observation that transfused igg-opsonized wild-type erythrocytes are cleared from the circulation at a significantly slower rate in cd47-deficient mice, as compared with that seen in wild-type recipient mice (oldenborg et al. this is unlikely due to a different expression of fc receptors in cd47-deficient mice, since cd47-deficient igg-opsonized erythrocytes are cleared with the same kinetics in both wild-type and cd47-deficient recipient mice (oldenborg et al. apoptosis is a physiological process of programmed cell death which is important for embryologic development, maintenance of homeostasis, and elimination of damaged cells. an important event related to this process is the rapid uptake of apoptotic cells or apoptotic bodies by phagocytic cells. the efficacy of this process in the body is shown by the fact that apoptotic cells are apparently removed with such extremely high efficiency that apoptotic cells are very hard to detect in tissues under normal physiological conditions. it has even been suggested that if apoptotic cells are in fact detected in vivo, this may indicate a possibility of defects in their clearance or the presence of a large overload of apoptotic cells [159162]. the largest part of apoptotic cell phagocytosis is mediated not only by professional phagocytes like macrophages and dcs, but also to some extent by nonprofessional phagocytes such as fibroblasts; epithelial cells and stromal cells are equipped with this function. one example of the latter phenomenon is the phagocytosis of apoptotic mammary epithelial cells by bystander epithelial cells during mammary gland involution [163, 164]. when cd47 was identified as a cell surface protein on host cells that can negatively regulate their phagocytosis through sirp, one important question was to understand if this signaling pathway is altered during apoptosis to facilitate uptake of apoptotic cells. one hypothesis would be that apoptotic cells downregulate the amount of cd47 on their surface, which together with an increased amount of prophagocytic ligands exposed during apoptosis would result in phagocytosis of the dead cells. indeed we found a reduced expression of cd47 on apoptotic fibroblasts and neutrophils, but curiously not on apoptotic jurkat t cells or apoptotic murine thymocytes. this issue became even more confusing when apoptotic murine t cells lacking cd47, in contrast to the solid data showing that cd47 on host cells inhibits phagocytosis by macrophages, were found to be phagocytosed less efficiently by macrophages than equally apoptotic cd47 wild-type t cells [143, 165]. in apoptotic murine t cells, cd47 was rather found to be important in mediating tethering of the apoptotic cells to the phagocyte [143, 165]. in addition, cd47 is redistributed into patches on the apoptotic cell surface, areas of the plasma membrane which are different than those harbouring clusters of proteins that function as ligands for prophagocytic receptors [141, 143]. thus, one possibility would be that the segregation of cd47 away from these phagocytosis promoting ligands could allow for tethering mediated by the cd47/sirp interaction, but that sirp would be too far away from the prophagocytic receptors to be involved in negative regulation (figure 4(b)). another very interesting hypothesis was recently suggested, which shed new light on this process. as described above in section 4.2 experimentally oxidized erythrocytes, as well as erythrocytes stored in blood bank conditions, showed enhanced expression of the cd47 epitope recognized by mab 2d3, suggesting a conformational change in the cd47 igv domain. importantly, this is associated with an increased binding of tsp-1 to cd47, which together could generate a new binding site for sirp that induces a prophagocytic signal instead of the normal inhibitory cd47/sirp signal. however, this very interesting hypothesis still has to also be investigated in the context of macrophage phagocytosis of apoptotic nucleated cells such as neutrophils or t cells. thus, cd47 may operate in several different ways during cellular senescence in order to switch its normal phagocytosis-preventing role to rather facilitate and promote the phagocytosis of senescent cells. lrp-1 is a multifunctional scavenger receptor, shown to be involved in mediating uptake of apoptotic cells [141, 166, 167], and as already mentioned it can also bind to calreticulin on viable erythrocytes to induce phagocytosis if the inhibitory cd47/sirp interaction is not strong enough [141, 142] (figure 4(b)). glucocorticoids are powerful in treatment of inflammatory conditions, which can be attributed to their ability to downregulate production of and cellular responses to proinflammatory cytokines and their ability to inhibit the recruitment of inflammatory cells [168, 169]. however, another mechanism whereby glucocorticoids may also reduce inflammation is to stimulate phagocytosis of apoptotic cells [170, 171]. interestingly, glucocorticoid treatment of macrophages results in increased macrophage expression of lrp-1 and increased phagocytosis of apoptotic cells or viable cd47-deficient erythrocytes. since cd47 can inhibit phagocytosis of host cells, and the amount of cd47 on the cell surface is clearly important in determining the phagocytosis efficiency of macrophages, this also raised the question on whether cells such as tumor cells can also increase their cd47 expression levels in order to escape macrophage elimination. indeed, early studies showed that ovarian carcinoma cell lines express increased levels of cd47 [6, 7], the functional significance of which was unclear at that time. more recently, an enhanced expression of cd47 has been reported for murine myeloid leukemias, as well as human normal and leukemic hematopoietic stem cells and many human solid tumors [172174], and that increased cd47 expression is associated with reduced patient survival in aml and solid tumors [173, 174]. to challenge the hypothesis that cd47 on the leukemic cells protects from phagocytosis by macrophages and supports tumor cell growth, a xenogenic mouse model was used to study a particular human aml clone (molm-13) with very low endogenous cd47 expression. expression of murine cd47 in molm-13 cells and transplantation of molm-13 clones expressing low or high levels of cd47 showed an enrichment and spreading of the cd47-high expressing clones to many bones of recipient mice, while the cd47-low clones showed minimal engraftment. depletion of macrophages in recipient mice allows for engraftment of cd47-low clones, and macrophage phagocytosis of aml cells both in vitro and in vivo is selective for tumor cells expressing low amounts of cd47. importantly, by blocking sirp on macrophages, phagocytosis of cd47-high aml cells is increased to that seen with cd47-low clones. thus, these findings strongly suggest that an increased cd47 expression level on tumor cells could serve to avoid macrophage clearance and promote dissemination of tumor cells (figure 4(c)). it was also recently shown that treatment serving to block the cd47/sirp interaction can result in elimination of aml stem cells in a xenogenic model. furthermore, inline with our original finding that the cd47/sirp pathway also potently inhibits fc receptor-mediated phagocytosis of igg-opsonized host cells [139, 145, 146, 150], it has been shown that antibody-mediated blocking of cd47/sirp signaling promotes phagocytosis of non-hodgkin lymphoma cells treated with rituximab and antibody-dependent cellular cytotoxicity (adcc) against her2/neu-positive breast cancer cells treated with traztusumab. thus, blocking the cd47/sirp interaction may prove to be a powerful tool in the treatment of various tumors. while organ transplantation is an important procedure to treat end-stage organ failure, it is hampered by a shortage of organ donors. to solve this problem, it has been suggested that pigs could serve as donors of organs, tissues, or cells to be transplanted into humans (i.e., xenotransplantation) [158, 178]. both the innate and adaptive immune system participates in the rejection of xenogenic transplants, the exact details of which is outside the scope of the present paper. however, of particular interest here are the findings suggesting that even in the absence of a functional adaptive immune system and in the absence of nk cells (both of which react to cell surface determinants of xenogenic cells recognized as non-self), rejection of xenogenic cells and tissues is seen, which suggests that macrophages could play an important role in this process. indeed, in studies of transplantation of pancreatic islets [179, 180] or hematopoietic cells from pigs to mice, macrophage depletion significantly enhances engraftment. the ability of macrophage sirp to inhibit phagocytosis by binding to cd47 on a target cell, and the fact that this interaction is rather species specific suggested that this interaction could play a role in macrophage-mediated clearance of xenogenic cells. indeed, it was found that porcine cd47 can not bind to murine sirp and induce tyrosine phosphorylation of the sirp itims, which could explain the rapid clearance of porcine hematopoietic cells by macrophages both in vitro and in vivo [181, 182]. however, expression of murine cd47 in porcine cells markedly inhibits macrophage-mediated phagocytosis of these cells in vitro and prolongs their survival in vivo. although a human sirp fusion protein was found to bind to porcine cd47, porcine cd47 does not activate human sirp. as a result, porcine hematopoietic cells are rapidly phagocytosed by human macrophages, but expression of human cd47 in porcine cells results in attenuated phagocytosis by human macrophages. these findings indicate that transgenic pigs expressing human cd47 could be an important possibility to further help reducing the rejection of porcine cells in xenotransplantation. the finding that cd47, by binding to sirp, could regulate rat alveolar macrophage fusion and formation of multinucleated giant cells in vitro suggested a function also in regulating formation of bone-resorbing osteoclasts and regulation of bone homeostasis. using functional blocking monoclonal antibodies against either cd47 or sirp, the formation of tartrate resistant acid phosphatase (trap) multinucleated osteoclasts is strongly inhibited in murine cell culture systems in vitro. this finding can also be confirmed in vivo, since cd47-deficient mice have reduced numbers of osteoclasts in bone and are protected from tumor metastasis and subsequent bone resorption. there are a few hypotheses presented to explain the mechanism whereby the interaction between cd47 and sirp promotes osteoclastogenesis. one suggests that binding of cd47 results in sirp tyrosine phosphorylation and subsequent recruitment of shp-1 to the phosphorylated itims, which next would mediate dephosphorylation of nonmuscle cell myosin iia to promote fusion and formation of osteoclasts. this hypothesis is contrasted by a study of sirp mutant mice, expressing a truncated nonsignaling sirp cytoplasmic domain, where cultures of bone marrow cells generated the same number of osteoclasts of the same size, as found in wild-type bone marrow cultures. the important observation made in the latter study was that osteoclasts generated in sirp mutant mice had an increased bone resorbing activity, suggesting that sirp negatively regulates this function in osteoclasts. the contradictory findings presented regarding a possible role of signaling through either cd47 or sirp during fusion of preosteoclasts and formation of multinucleated osteoclasts must also be put in context of the originally presented hypothesis that the interaction between cd47 and sirp may mainly facilitate cell fusion by promoting cell-cell adhesion and bringing the plasma membranes of two cells close enough to facilitate cell fusion. here it has also been highlighted that cd47 may interact with the shorter sirp isoform having only one extracellular igv domain, which would further reduce the distance between two opposing cellular membranes where cell fusion could take place. cd47 can regulate many important physiological cellular mechanisms by interacting with integrins, tsp-1, or sirp. as outlined above, much knowledge has been collected over the past two decades, but much is probably still to be discovered regarding the functions of this protein. there are several areas where one can assume that further progress may result in new novel ways to interfere with biological mechanisms in pathological conditions. although cd47 is expressed by virtually all cells in the body, in healthy as well as pathological cells, the data so far indicates that cd47-induced apoptosis may still be a way to kill, for example, tumor cells, since it appears that tumor cells and activated cells are much more sensitive to this cell death mechanism than nonactivated nave cells or hematopoietic stem cells. the ability of cd47 to inhibit phagocytosis is clearly an important mechanism whereby innate cells as macrophages or dendritic cells can discriminate between self and non-self, and maintain tolerance to host cells. in the field of xenotransplantation, further development and research to create xenotransplants carrying human cd47 may prove important to obtain even better conditions where xenogenic cell, tissues, or organs can be tolerated when transplanted into humans. in the field of cancer research, where tumor cells may express higher levels of cd47 as a way of avoiding phagocytosis and clearance by phagocytic cells, and where cd47-blocking antibodies have been shown to promote clearance of and to reduce the dissemination of tumor cells, brings hope that careful development of reagents that can block the cd47/sirp interaction may indeed be useful to treat many forms of cancer without having too much of a negative side effect in terms of inducing clearance of host cells. also in the field of bone research, one may suggest that manipulation of the interaction between cd47 and sirp may be a way to modulate bone resorption and to prevent osteoporosis. however, data showing that these two proteins may also be involved in regulating bone formation indicate that manipulation of this signaling system in bone tissue may be more complicated.

interactions between cells and their surroundings are important for proper function and homeostasis in a multicellular organism. these interactions can either be established between the cells and molecules in their extracellular milieu, but also involve interactions between cells. in all these situations, proteins in the plasma membranes are critically involved to relay information obtained from the exterior of the cell. the cell surface glycoprotein cd47 (integrin-associated protein (iap)) was first identified as an important regulator of integrin function, but later also was shown to function in ways that do not necessarily involve integrins. ligation of cd47 can induce intracellular signaling resulting in cell activation or cell death depending on the exact context. by binding to another cell surface glycoprotein, signal regulatory protein alpha (sirp), cd47 can regulate the function of cells in the monocyte/macrophage lineage. in this spotlight paper, several functions of cd47 will be reviewed, although some functions may be more briefly mentioned. focus will be on the ways cd47 regulates hematopoietic cells and functions such as cd47 signaling, induction of apoptosis, and regulation of phagocytosis or cell-cell fusion.

PMC3564380

pubmed-939

the demand for food of animal origin increases each year. to satisfy this demand, livestock production within the european union (eu) in 2011 was approximately 10 million heads of goats, 80 million heads of sheep, 80 million heads of cattle, and 150 million heads of pigs. to produce safe and nutritional food products, the animals need to be in good health. even though veterinary medicines contribute to improving and maintaining animal health, administration of these medicines by the farmer is carried out under licence via a veterinarian. the amount of drugs employed in food production estimated by kools et al. was 6051 t, with antibiotics the most frequently used class of drug (5393 t). the groups of tetracyclines and -lactams were used in high amounts and antiparasitic agents (194 t) were the second most frequently used class of drug. intensively produced animals are often fed with concentrated feed, a mixture of various materials (oats, wheat, barley, rye, cottonseed, and crambe) and additives. antibiotic sulphonamides, tetracyclines, and -lactam are the most frequently used antibiotics, and coccidiostats and ivermectin are the most frequently used antiparasitic agents. coccidiostats and histomonostats are a group of antiparasitic agents that have been shown to be persistent during the manufacture of feed and carry-over of this type of drugs has been demonstrated. the eu introduced maximum levels for these substances in nontarget feed in 2009; this regulation was later modified for some coccidiostats by the regulation ec/574/2011. cross contamination between medicated and nonmedicated feed could occur with any type of drug added to the feed, not only to coccidiostats, particularly when the cleaning process between batches is inefficient. recently, a study conducted by stolker et al. in the netherlands confirmed that nonmedicated feed batches were contaminated with antibiotic residues such as tetracyclines, penicillins, and sulfonamides. from 140 samples the fact that antibiotics could be present as contaminants in feed without the farmers ' knowledge implies that withdrawal times will not be considered and antibiotic residues could remain in animal products (meat, eggs, milk, honey, seafood, and fish), in addition to the development of antibiotic resistant bacteria. due to the problems related to food safety, the authorities involved regularly monitor the presence of veterinary drugs in food of animal origin (eggs, milk, muscle, and liver). controls on the water and food consumed by the animals have also been implemented, but the analysis conducted only evaluated the presence of substances such as pesticides [7, 8], nitrofurans, and mycotoxins [10, 11]. methods based on hplc-ms/ms detection are considered confirmatory methods because these types of methods provide full or complementary information, enabling substances to be unequivocally identified and if necessary quantified at the level of interest, according to the european commission decision 2002/657/ec. therefore, it is recommended to use confirmatory methods to detect the presence of antibiotics in nontarget feed. van poucke et al. reported a method for the analysis of zinc bacitracin, spiramycin, tylosin, and virginiamycin with quantification limits below 500 g/kg. published a multiclass method for the analysis of 33 analytes for 14 groups of antibiotics (including tetracyclines, quinolones, penicillins, ionophore coccidiostats, macrolides, and sulphonamides) with quantification limits of 3.865.0 g/kg. of the eight forms of commercially available tetracyclines, four are frequently used in food animal production (chlortetracycline, doxycycline, oxytetracycline, and tetracycline). maximum residue limits (mrl) for these four tetracyclines and their three epimers have been introduced for various foods of animal origin, including eggs, muscle, and milk. as maximum levels (ml) for these substances in feed samples concentrations of tetracyclines in medicated feed are variable and depend on the target animal, with dosage rates between 25 and 700 mg/kg. therefore, carry-over should be expected in the batch of feed manufactured after a medicated feed. reported that 100% of samples from the first batch of feed produced after the manufacture of medicated feed were contaminated with tetracyclines, with concentrations of 0.5154 mg/kg. based on these results, carry-over contamination is also expected for the other two commonly used tetracyclines (chlortetracycline and tetracycline) as they have similar chemical properties [16, 17]. based on the common use of tetracyclines in food animal production and the absence of confirmatory methods for the presence of the four tetracyclines in animal feed, the aim of this work is to present an hplc-ms/ms method for the analysis of tetracyclines in nonmedicated feed samples at levels of g/kg. disodium hydrogen phosphate dehydrate, anhydrous citric acid, ethylenediaminetetraacetic acid disodium salt (edta), trichloroacetic acid (tca), and formic acid (purity>99% by analysis) were purchased from sigma-aldrich (mo, usa). tetracycline, chlortetracycline, doxycycline, and oxytetracycline (purity>98%) and demeclocycline, used as the internal standard (is), were supplied by sigma-aldrich (mo, usa). organic solvents, methanol and ethyl acetate, hplc or analytical grade, were purchased from scharlau chemie (barcelona, spain) and demineralised water (resistivity 18 mu cm) was prepared in-house with a milli-q water system (millipore, bedford, ma, usa). mobile phase a consisted of milli-q water acidified to 0.04% with formic acid and mobile phase b consisted of methanol, acidified to 0.1% with formic acid. to prepare the individual stock solution of tetracycline, 20 mg of tetracycline was dissolved in 20 ml of methanol and stored at 20c for up to six months. the intermediate solution, a mixture of tetracyclines, was prepared by diluting the stock solution of each tetracycline to a final concentration of 50 g/ml and stored at 20c for up to one month. a standard working solution of tetracycline was prepared freshly each day by diluting the intermediate stock solution to a final concentration of 1 g/ml. for the internal standard stock solution, mcilvaine buffer was prepared with 10.8 g of citric acid, 10.93 g of disodium hydrogen phosphate, and 33.62 g of ethylenediaminetetraacetic acid disodium salt dihydrate (c10h14n2na2o8). once the three reagents were completely dissolved they were mixed and the volume was made up to 1 l and the ph adjusted to ph 4. hplc-ms/ms determination of tetracyclines was performed according to a previously reported method. the hplc-ms/ms consisted of an hplc alliance 2795 and a ms quattro premier xe triple quadrupole (waters, manchester, uk) controlled by the software masslynx 4.1 (waters, manchester, uk). the chromatographic analyses were performed by injecting 25 l of extract into a sunfire c18 column (150 2.1 mm i.d., 5.0 mm) (waters, manchester, uk). mobile phases a and b were mixed on a gradient mode and with a flow rate of 0.25 ml/min. an electrospray ionisation (esi) probe was set up on the triple quadrupole ms to evaporate the mobile phase coming from the hplc and to ionise the tetracyclines. analytes were detected in positive-ion mode and under the following conditions: capillary voltage 3 kv, source temperature 120c, desolvation temperature 350c, cone gas flow 49 l h, and desolvation gas flow 650 l h. as indicated in the decision 2002/657/ec, tetracyclines were identified on the basis of their selected reaction monitoring (srm) transition and their retention time (rt). different volumes of the tetracycline working standard solution were added to 2 g feed samples (exempt of tetracycline) and shaken in the dark for 30 min. concentrations of tetracycline in the matrix matched feed samples were 0, 400, 800, 1200, 1600, and 1600 g/kg. to extract the tetracyclines from the feed samples, 2 g of grounded feed, 8 ml of mcilvaine buffer, 300 l of tca, and 0,1 ml of is working solution were added to a 50 ml polypropylene tube. after shaking the sample in the dark for 10 min, 6 ml of ethyl acetate was added and the samples were shaken in an orbital shaker at 200 rpm for 20 min. after 15 of centrifugation at 4500 rpm, in a model 5415d centrifuge (eppendorf, hamburg, germany), 2 ml of the supernatant was transferred to a 10 ml amber conical tube and evaporated to dryness, in a turboevaporator model turbo vap ii de zyrmark (hopkinton, ma, usa). the final residue was dissolved in 0.5 ml of a mixture of mobile phase components (90a:10b) and vortexed. to filtrate the final extract ultrafree-mc centrifugal filter (millipore, ma, us) was employed and centrifuged at 9000 rpm for 10 min. the filtrate was transferred into an hplc vial which contained a 0.3 ml microinsert and stored at 20c; analyses were conducted within 3 days. the guidelines used to validate the method and to interpret the results were those established in the commission decision 2002/657/ec. the decision establishes criteria and procedures for the validation of analytical methods to ensure the quality and comparability of analytical results generated by official laboratories. aspects such us trueness/recovery, precision (under repeatability and reproducibility conditions), specificity, and applicability/ruggedness/stability of the method were investigated. trueness and the other validation parameters were assessed through recovery of additions of known amounts of tetracyclines in blank feed samples (except tetracyclines) as no certified reference material exists for this type of analysis and following the recommendation included in the decision 2002/657/ec. for validation one batch of matrix-matched samples was prepared with 21 samples fortified with tetracyclines at six concentrations (0, 400, 800, 1200, 1600, and 4000 g/kg). for concentrations 400, 800, and 1200 g/kg six replicated samples were prepared with only one sample for the remaining concentrations (0, 1600, and 4000 g/kg). additionally, two reagent samples were prepared for control, a blank reagent (containing only the reagents) and fortified reagent (containing 1200 g/kg of tetracycline and the reagents). to conduct the validation three batches of matrix-matched samples were prepared; each batch consisted of 21 samples fortified with tetracyclines at 0, 400, 800, 1200, 1600, and 4000 g/kg. while for levels of 400, 800, and 1200 g/kg six replicated samples were prepared, only one sample was used for levels of 0, 1600, and 4000 g/kg. after fortification, and prior to extraction, samples were shaken on an orbital shaker at 200 rpm for 10 min. after the samples extraction procedure explained above was applied. as well as the 21 matrix-matched samples, with each batch of samples two additional samples were prepared with reagents (no feed); one was spiked with tetracycline at 1200 g/kg (fortified reagents) and the other was not spiked with tetracyclines (blank reagent). additionally, 20 feed samples were analysed to determine selectivity/specificity. ten were spiked with tetracyclines at a validation level (800 g/kg) and with 400 g/kg of three antimicrobial drugs commonly used in food animal production (sulfadiazine, sulfamethoxazol, and trimethoprim). the other ten samples were analysed without adding any veterinary drugs (decision 2002/657/ec). the decision limit (cc) and detection capability (cc) were determined as described by freitas et al., following the decision 2002/657/ec requirements and applying the validation level of 800 g/kg. nonmedicated feed samples were collected from 50 milk farms located in galicia, spain, to investigate the presence of tetracycline residues in feed that are being consumed by cows that are producing milk daily. additionally, samples were supplied by feed manufacturers to investigate carry-over levels of tetracyclines after making medicated feed. the sampling procedures in both cases were conducted following the requirement of the regulation 691/2013. feed samples were stored at room temperature and in the dark with the objective of using similar store conditions compared to that in the industry and farms. the main raw materials of the feed samples according to the labels were corn genetically modified (between 36 and 15%), soy flour produced from soya been genetically modified (present in some samples, between 4 and 38%), colza flour (present in some samples, between 3 and 47%), and barley (present in some samples, between 6 and 18%); each feed sample had a particular composition which normally depends on the manufacture. on the other proximate composition the feed samples were crude protein (between 18 and 26%), crude fibre (between 4 and 10%), oil and fat crude (between 2.5 and 6%), crude ash (between 6 and 10%), and sodium (between 0.7 and 4%). tetracyclines have more than three hydroxyl groups that are easily ionisable by esi to enhance their detection. therefore, esi is commonly used for the ionisation of tetracyclines when analysis is conducted by hplc-ms/ms, independently of the matrix type [14, 2024]. to optimise the ms parameters for a high ms signal response, standard solutions of 1 g/ml of individual tetracyclines were infused directly into the ms. during this procedure, one precursor ion and two product ions were selected for each tetracycline to conduct srm analysis (table 1). with the hplc-ms/ms operating on srm mode, two transitions and the retention time were used to achieve four identification points for each of the analytes, as required in the decision 2002/657/ec. it should be highlighted that with other detection methods, such as diode array or a fluorescence detector, only one identification point is achieved and more steps are required. the use of precursor and product ions identified in this research for tetracyclines was also reported by boscher et al. 2010 for the analysis of these analytes in feed, royal jelly, and muscle [14, 20]. the separation gradient could be run at ambient temperature; however, it was observed that an increase in mobile pressure can cause the system to collapse; therefore a temperature of 35c is recommended. based on previously reported methods for tetracycline analysis in food and feed matrices [14, 2527] different extraction protocols were tested. firstly, simple extractions with ethyl acetate, hexane, acetonitrile, methanol, and dichloromethane were tested. however, recoveries were low, due to the tendency of tetracyclines to form chelation complexes with different cations. therefore, initial extraction with mcilvaine/edta buffer was employed and gave satisfactory results, as in previously reported methods [7, 21, 28, 29]. tetracyclines dissolved in the mcilvaine/edta solution were then extracted with ethyl acetate, as these two solutions are immiscible, and separation could be easily conducted as the organic phase stays on the top layer. other authors employ spe cartridges such as oasis for dispersive spe instead of ethyl acetate in order to purify the extract. the use of spe was avoided to reduce time and cost of the analysis as satisfactory results were achieved with the presented method. animal feed is derived from a multitude of raw materials from plant and animal origin, as well as pharmaceutical and industrial sources. as feed ingredients vary depending upon the animal, that is, poultry, swine, and cattle, analysis of tetracyclines in different feed types could be more complicated, particularly as the fat content will vary. the method used in this paper has been tested in feed for cattle, laying birds and chickens, rabbits, and dairy cows, and satisfactory results were obtained in all cases. these matrices were tested by preparing matrix-matched calibration curves with each type of feed, depending on the animal that was going to consume the feed. calibration curves to quantify concentrations of tetracyclines were obtained by spiking feed samples with the analytes at different concentrations. if linear regression coefficients (r) were below 0.98 the extraction procedure was repeated. even if a signal to noise ratio (s/n) higher than 10 was achieved at 300 g/kg, validation was conducted at 800 g/kg to provide acceptable results at 0.5, 1, and 1.5 times the validation level (800 g/kg) recommended by the decision 2002/657/ec. figure 1 shows chromatograms of a blank sample, figure 2 shows a blank sample spiked with all the tetracyclines at 400 g/kg, and figure 3 shows the srm transition employed for each tetracycline in one of the samples spiked at 400 g/kg. reference materials were not available; therefore trueness of the method was calculated in terms of recoveries. however, the advantage of the presented extraction protocol is that it does not require solid phase extraction and the four main tetracyclines can be identified and quantified simultaneously. results for repeatability, calculated as the mean rds of the rsd (n=6) for each concentration on each day of the validation, were below 17% for chlortetracycline, doxycycline, oxytetracycline, and tetracycline (table 2). results for reproducibility, calculated as the rds of 21 samples at the same concentration, were below 23% for all tetracyclines (table 2). to determine selectivity/specificity, 10 blank samples and the same samples spiked with the four tetracyclines at 800 g/kg were analysed. the successful quantification of tetracyclines and the absence of interfering peaks at the retention times of each analyte demonstrated the selectivity/specificity of the method. the limit of detection (lod) and limit of quantification (loq) of the method were calculated and verified with feed samples spiked with the tetracyclines at different concentrations. based on s/n above 3 for lod and above 10 for loq in matrix-matched samples, the lod and loq of the method were set at 35 and 47 g/kg for chlortetracycline, 40 and 60 g/kg for oxytetracycline, 24 and 40 g/kg for tetracycline, and 100 and 150 g/kg for doxycycline. cc and cc were determined using the conditions for substances for which no permitted limit has been established. cc and cc were higher than lod and loq for all the compounds, meaning that tetracyclines detected at a higher level than the cc will be positive and levels of tetracyclines were quantifiable, without doubt. cc and cc for chlortetracycline, doxycycline, oxytetracycline, and tetracycline were below 400 g/kg (table 2). validation results already published have shown, in some cases, higher repeatability, reproducibility, and lower loq, such as the work conducted by boscher et al. who reported rsd lower than 12% and loq of 20 g/kg. similarly, the method reported by stolker et al. achieved a loq of 0.1 g/kg for doxycycline and oxytetracycline. however, it should be highlighted that none of the methods reported, based on the authors knowledge, have been validated according to the decision 2002/657/ec. from 75 feed samples investigated oxytetracycline was the tetracycline more frequently detected present in 8% of the samples (n=6); its concentration range was between 90 and 400 mg/kg. each tetracycline was detected in individual samples and their concentrations were 150 and 110 mg/kg. chlortetracycline was the pharmaceutical detected at the highest concentration in this study, 15.14 mg/kg in feed samples for calves. it is important to highlight that the consumption of contaminated feed at level such as 15.14 mg/kg could cause food safety problems giving positive food samples. carry-over during feed manufacture has been proved for veterinary drugs such as coccidiostats and a similar case can be considered for tetracyclines, a group of antimicrobial agents commonly used in food animal production, due to its low cost. the research work presents a simple and fast method for the analysis of the four tetracyclines regulated in the production of food of animal origin (chlortetracycline, doxycycline, oxytetracycline, and tetracycline). the method was validated according to the european guideline and successfully applied to 75 nonmedicated feed samples. results showed the presence of tetracyclines in 15% of the samples, indicating that cross contamination occurs and maximum levels for tetracyclines may be required in the future.

the commission regulation 574/2011/ec set up maximum levels of coccidiostats and histomonostats in nonmedicated feed as a consequence of carry-over during manufacturing. carry-over takes place from medicated to nonmedicated feed during feed production. similar contamination could also occur for other pharmaceuticals such as tetracyclines, a group of antibiotics commonly employed in food production animal. the objective of this work is to present a simple and fast method for the simultaneous detection of four tetracyclines (chlortetracycline, doxycycline, oxytetracycline, and tetracycline) in nontarget feed at a g/kg level. validation of the method was performed according to the guideline included in the commission decision 2002/657/ec for official method. the validated method was successfully applied to 50 feed samples collected from different milk farms and 25 samples obtained from feed manufacturers. while oxytetracycline was the tetracycline most frequently detected, chlortetracycline was the analyte measured at the highest concentration 15.14 mg/kg. from 75 nonmedicated feed analysed 15% resulted to be positive for the presence of one tetracycline.

PMC4993920

pubmed-940

alterations in the influence of inhibitory gabaergic circuits can have a profound impact on the excitability of neural network function, and have been associated with hyperexcitable conditions such as epilepsy [1, 2]. recent work has identified what may be one of the most important processes in ensuring that networks maintain appropriate activity levels; homeostatic plasticity is thought to maintain network spiking activity levels within a physiologically relevant range through compensatory adjustments in intrinsic cellular excitability, as well as excitatory and inhibitory synaptic strength [38]. this phenomenon has been identified in several systems, at different developmental stages, in vitro and to a lesser extent in vivo. when activity levels of cultured neuronal networks (cortical, hippocampal, spinal) are altered for days, cellular excitability and synaptic strength within the network are adjusted in a direction that appears to oppose the alteration in activity [914]. for instance, when spiking activity is blocked for 2 days, ampaergic synaptic strength increases and gabaergic synaptic strength decreases in excitatory neurons. when network spiking activity is increased, ampaergic synaptic strength decreases. in each case the amplitude of miniature postsynaptic currents (mpscs) changed in a direction to compensate for the perturbation. several reviews have examined homeostatic plasticity, typically focusing on excitatory components within networks [36]. in this paper we will instead concentrate on the findings of homeostatic plasticity within gabaergic neurons and at gabaergic synapses onto excitatory neurons. based on previous work studying homeostatic plasticity in the glutamatergic system, we make the simplistic prediction that inhibition would be reduced following network activity blockade and increased following increases in network activity. therefore, following chronic reductions in network activity (figure 1 left), we would expect compensatory weakening of both gabaergic synapses on excitatory neurons and glutamatergic synapses on inhibitory neurons and to see reductions in the intrinsic cellular excitability of inhibitory neurons. if network activity is increased for days (figure 1 right), we would expect compensatory strengthening of both gabaergic synapses on excitatory neurons and glutamatergic synapses on inhibitory neurons and to observe increases in the intrinsic cellular excitability of inhibitory neurons. the most studied aspect of inhibition in homeostatic plasticity has examined the inhibitory gabaergic inputs to excitatory neurons (figure 1(a)). immunocytochemical studies gave the first indication that gabaergic circuits experienced homeostatic plasticity, as reduced visual input led to decreased cortical expression of gabaa receptors, gaba, and gad [15, 16]. compensatory changes in the amplitude of gabaergic mpscs have now been demonstrated in excitatory neurons following network activity perturbations in several different studies [10, 1720]. these changes in gabaergic mpsc amplitude are often mediated by changes in the number of synaptic gabaa receptors, and this is typically shown by quantitative immunocytochemistry [10, 20, 21]. in addition, compensatory changes in the vesicular inhibitory amino acid transporter, viaat, have been observed suggesting there are coordinated presynaptic contributions to homeostatic changes in mipsc amplitude [18, 20, 21]. while these studies have shown that mipsc amplitude is reduced following chronic activity blockade or increased following increased network activity, two studies suggest the opposite can occur. one study demonstrates that a subset of gabaergic inputs to hippocampal pyramidal cells are strengthened following activity block; however, the overall population of mipscs homeostatically scale downward. another study shows that in vivo application of ttx for 2 days resulted in an increase in mipsc amplitude in pyramidal cells recorded from cortical slices, which also does not fit the simple homeostatic model. these studies highlight the need to carry out more homeostatic studies in vivo, as perturbations in living networks are likely to be more complicated in terms of network homeostasis, but crucial in elucidating the goals of homeostatic plasticity. in a separate study where spiking activity was blocked in vivo for 2 days in the embryonic spinal cord these changes in gabaergic currents were compensatory because gaba was depolarizing and excitatory at this developmental stage. interestingly, homeostatic increases in gabaergic mpscs occurred through increased chloride accumulation, thus depolarizing the gabaergic reversal potential (egaba) and enhancing the driving force for these currents. similarly, another study indirectly demonstrated that homeostatic changes in gabaergic currents could be produced by a shift in egaba. in this study, activity was perturbed in hippocampal organotypic cultures and compensatory changes in gabaergic currents were observed in pyramidal cells at a stage when gaba was no longer excitatory. these findings are important for understanding the maturation of gabaergic synaptic strength but also may have implications for neuronal injury in mature circuits where the same depolarizing shifts in chloride reversal potential are observed following spinal cord injury, peripheral nerve injury, and traumatic brain injury [2638]. it is tempting to speculate that following injury, homeostatic mechanisms may be engaged that produce the maladaptive increases in excitability associated with neuronal injury. consistent with this idea, work in a model of febrile seizure suggests the possibility that compensatory increases in gabaergic strength appear to promote hyperexcitability by triggering the hyperpolarization-activated current, ih [39, 40]. other studies in cultured networks demonstrated that homeostatic changes in mipsc amplitude were not due to changes in egaba [10, 41]. however, these studies used whole-cell recordings to measure egaba, which may dialyze intracellular cl and mask the experimenter's ability to observe changes in gabaergic driving force. future studies assessing homeostatic changes in mipscs could use perforated patch recordings or chloride indicators to resolve this issue. although not as common as homeostatic changes in mipsc amplitude, homeostatic changes in mipsc frequency have been reported. increases or decreases in network activity have been shown to increase and decrease mipsc frequency, respectively, in excitatory neurons [10, 20]. this appears to be mediated by changes in the number of gabaergic inputs to excitatory pyramidal cells. to a large extent, gabaergic mpsc amplitude and frequency in excitatory neurons follow the homeostatic model, strengthening after chronic increases in activity and weakening after activity blockade. our homeostatic model predicts that ampaergic synaptic inputs to gabaergic neurons will strengthen following increases in activity and weaken following activity blockade (figure 1(b)). using hippocampal cultures, it was shown that parvalbumin-expressing inhibitory interneurons (pv ins) increased mepsc amplitude following chronic enhancement of activity levels and reduced mepsc amplitude following activity blockade. the changes in mepsc amplitude were mediated by changes in the number of an ampa receptor subunit, glua4, which was regulated homeostatically by neuronal activity-regulated pentraxin (narp). similarly, chronic increases in activity induced a strengthening of excitatory inputs to inhibitory interneurons in neocortical cultures, expressed presynaptically as an increase in the vesicular glutamate transporter, vglut2. consistent with these findings, another study demonstrated that increasing bdnf levels, as occurs with increased network activity, led to an increase in mepsc amplitude in inhibitory bipolar interneurons. in this study, the increase in mepsc amplitude was mediated by an increase in the sensitivity of the postsynaptic cell to glutamate, consistent with an increase in synaptic glutamate receptors. however, when spiking activity was blocked for days in several different cultured cortical networks, mepsc amplitude was unaltered in multiple classes of inhibitory interneuron [8, 41, 44]. thus far, the results are consistent with the idea that increased network activity levels triggered homeostatic increases in mepsc amplitude in interneurons, but that mepsc amplitude was typically unaltered by reductions in network activity. in none of these studies were changes in mepsc frequency observed. finally, we know of no homeostatic studies examining mipscs in inhibitory interneurons following activity perturbations. changes in interneuronal intrinsic cellular excitability (figure 1(c)) following activity block have been described in 2 different cortical cultures. in both studies, intrinsic cellular excitability was increased following activity blockade in 3 different classes of inhibitory interneuron [44, 45]. one of the studies suggested that the increased excitability was the result of an increase in input resistance. from a simplistic network perspective, increasing the excitability of an inhibitory neuron in an activity-blocked network is not what our homeostatic model would predict (figure 1(c) left). the enhanced inhibition may be offset by the observation that pyramidal cells also have increased intrinsic excitability following activity blockade, but the finding underlines the complexity of the homeostatic process [22, 25, 44, 45]. it is possible that activity perturbations result in changes in synaptic strength that are homeostatic for the network, while changes in intrinsic cellular excitability are homeostatic from the perspective of the individual cell. we have focused on mpscs because they provide a nice measure of a standard unit of synaptic strength. however, another potentially useful measure of synaptic strength is provided by looking at the functional connections between 2 components of the circuitry. the strength of the connections between inhibitory and excitatory neurons can be assessed through paired recordings, stimulating an inhibitory or excitatory neuron and recording a response in the other. the results of these studies have been somewhat mixed. when retinal activity is reduced in vivo by ttx infusion or lid suture, input to pyramidal cells in the visual cortex from inhibitory interneurons was homeostatically reduced in certain cases [46, 47]. in other cases reductions of visual input to cortical neurons resulted in a strengthening of both inhibitory inputs to pyramidal cells and pyramidal input to inhibitory neurons [46, 48]; from a network perspective, these results are opposite to that predicted by our model of homeostatic plasticity. one complication in these studies is that when visual input is perturbed in vivo, it is not always clear how this affects the activity of the visual cortical circuitry that is being studied; for instance, different results have been described when retinal activity is altered by lid suture versus ttx infusion. however, in one study in neocortical organotypic cultures, where network activity was clearly blocked, changes in the strength of connections between excitatory and inhibitory neurons were not simplistically homeostatic. brain-derived neurotrophic factor (bdnf) has been implicated in the signaling pathway for homeostatic plasticity of both glutamatergic and gabaergic systems. bdnf exerts its influence through changes in intrinsic cellular excitability, mepsc and mipsc amplitude and frequency. from these studies a pattern is beginning to emerge; when bdnf signaling is reduced, as occurs during activity blockade, there is an increase in the influence of excitatory neurons; when bdnf signaling is increased, as occurs during chronic increases in network activity, there is an increase in the influence of inhibitory neurons (figure 2). when activity is blocked in cortical cultures using ttx, pyramidal cells become more excitable through increases in mepsc amplitude, decreases in mipsc or spontaneous ipsc amplitude [21, 49], and increases in the intrinsic cellular excitability of these cells. all three of these compensatory changes appear to be mediated by reduced bdnf signaling because they are prevented by coapplication of bdnf and ttx and recapitulated by blocking bdnf signaling through its receptor, trkb. on the other hand, increases in bdnf signaling that would be associated with overly active networks enhanced the influence of inhibitory interneurons through increases in interneuronal projections (increased mipsc frequency), or through increased mepsc amplitude onto inhibitory interneurons [20, 41]. while the model shown in figure 2 is well supported by most of the experimental evidence, one exception to the homeostatic model is the observation that activity block triggers a bdnf-dependent increase in inhibitory interneuron intrinsic excitability in cortical cultures. the sensors of activity that trigger homeostatic plasticity changes are a major focus in the field but are poorly understood. activity sensors triggering changes in inhibitory circuitry are even less well understood. in the vast majority of homeostatic studies, network activity is reduced by ttx or glutamate receptor blockers or increased by gabaa receptor antagonists. all of these treatments alter activity levels, as well as neurotransmission, throughout the network. therefore, it is possible that changes in network spiking activity, cellular spiking activity, or synaptic transmission trigger homeostatic changes in mipscs. one recent study increased spiking activity in an individual cell in an otherwise unperturbed network. the finding is consistent with the idea that increases in individual cellular spiking activity trigger homeostatic compensations of gabaergic inputs. however, when activity was blocked in individual hippocampal pyramidal cells by transfecting them with a k channel or a mutant voltage-gated na channel, no change in mipsc amplitude was observed. the finding indicated that reductions in the activity of individual excitatory neurons did not trigger homeostatic changes in mipsc amplitude, but suggested that reductions in network-wide activity or neurotransmission may be required to induce this plasticity. consistent with the possibility that sensors assess neurotransmission, we have determined that in vivo blockade of depolarizing gabaa transmission in the embryonic spinal network triggered compensatory increases in excitatory gabaergic mpsc amplitude and cellular excitability in motoneurons [50, 51]; these forms of compensatory plasticity were not dependent on alterations in spiking activity, suggesting that the network could sense reduced spiking activity levels through reduced gabaa transmission, essentially using gaba as a proxy for activity levels. a better understanding of the sensors that trigger compensatory changes in inhibitory neurotransmission will require more extensive work than the current studies, but it will be important to consider the possibility that neurotransmission is involved in the process. gabaergic inputs to excitatory neurons in several different networks are strengthened following increases in activity and weakened following activity block; increases in activity lead to increased mepsc amplitude in inhibitory neurons; increases in bdnf signaling (associated with increases in activity) increase the excitability of inhibitory interneurons, while decreases in bdnf signaling (associated with decreased activity) increase the excitability of excitatory neurons. however, there are several clear examples that do not fit into any simplistic homeostatic model (interneuron intrinsic excitability, mepsc amplitude in interneurons following activity block, evoked responses between excitatory and inhibitory neurons). it will be important to identify common mechanisms of homeostatic plasticity, but it is likely that different preparations (e.g., in vitro versus in vivo) and different neural circuits use different homeostatic mechanisms. further, compensatory mechanisms will be experienced in different elements of the circuitry at different developmental stages. in addition, the methods of altering network activity are likely to trigger different homeostatic mechanisms, for instance, increasing versus decreasing activity. in some cases, particularly in vivo studies, assumptions are made about alterations in cellular or network activity, but are not directly tested, leaving open the possibility that apparent antihomeostatic responses are actually homeostatic, or vice versa. it is also possible that in some cases absolute levels of spiking activity are not the homeostatic goal, but rather some more sophisticated pattern of activity, for instance, synchrony of the output neurons, which could be achieved through more complicated changes in gabaergic interneurons [53, 54]. in the end, it is important to recognize that changes in gabaergic synaptic strength or cellular excitability in inhibitory neurons are being tested in isolation, but they occur within complex networks where it is difficult to know the functional consequences of these changes. as the field matures it will be important to take these complexities into consideration. because network-wide activity is clearly maintained across many neural circuits, there are likely to be strong homeostatic mechanisms maintaining global network activity; it will be important to differentiate these homeostatic mechanisms from those that maintain individual cellular activity or individual synaptic activity, each potentially being triggered by different sensors.

homeostatic plasticity ensures that appropriate levels of activity are maintained through compensatory adjustments in synaptic strength and cellular excitability. for instance, excitatory glutamatergic synapses are strengthened following activity blockade and weakened following increases in spiking activity. this form of plasticity has been described in a wide array of networks at several different stages of development, but most work and reviews have focussed on the excitatory inputs of excitatory neurons. here we review homeostatic plasticity of gabaergic neurons and their synaptic connections. we propose a simplistic model for homeostatic plasticity of gabaergic components of the circuitry (gabaergic synapses onto excitatory neurons, excitatory connections onto gabaergic neurons, cellular excitability of gabaergic neurons): following chronic activity blockade there is a weakening of gabaergic inhibition, and following chronic increases in network activity there is a strengthening of gabaergic inhibition. previous work on gabaergic homeostatic plasticity supports certain aspects of the model, but it is clear that the model can not fully account for some results which do not appear to fit any simplistic rule. we consider potential reasons for these discrepancies.

PMC3159121

pubmed-941

the lake malawi cichlid fauna comprises over 800 species offering a spectacular example of adaptive radiation with virtually all niches in the lake being filled by members of this family [2, 3]. with a few exceptions, all lake malawi cichlids form a monophyletic group as supported by mitochondrial [46] and nuclear ([ 79] but see) markers as well as allozymes [11, 12]. the phylogenetic reconstruction of lake malawi cichlid fauna has recovered six main mitochondrial dna (mtdna) lineages [5, 6, 10, 13]. the remaining cichlid fauna has been traditionally divided into two groups: one containing predominantly the rock-dwelling species commonly called mbuna and the second containing the remaining lake malawi cichlids. however, phylogenetic reconstructions have shown that both groups are artificial [5, 10, 13, 14]. several lethrinops, aulonocara, and alticorpus species (ecologically and morphologically typically assigned to the non-mbuna) cluster within the mbuna clade. furthermore, the non-mbuna genus copadichromis has been shown to have representatives belonging to both the non-mbuna clade, as well as to a separate lineage. the genus copadichromis, together with the genus nyassachromis and the newly erected genus mchenga, constitute the utaka, a species assemblage of midwater-feeding zooplanktivorous cichlid species. not recovering mtdna monophyly within this assemblage: m. eucinostomus and c. borleyi were placed within the non-mbuna clade, while c. mloto (reidentified as copadichromis sp. virginalis kajose, j. snoeks pers. and some other individuals of the copadichromis virginalis complex seemed to represent a different, well-diverged lineage. therefore, currently available mtdna phylogenies are inconclusive as to whether the utaka are genetically associated with the non-mbuna clade, whether they constitute an originally separate ancestral lineage, or whether only one or a few species or specimens cluster in a separate lineage. if specimens of a species cluster in genetically distant lineages, this may be a result of the retention of ancestral polymorphism, the existence of a cryptic species, or traces of a past hybridisation/introgression event. support for these alternative hypotheses may be gained by using a multilocus approach (e.g., [10, 1416]). if the nuclear genetic signature is concordant with the mtdna in subdividing a species into genetically separated units, this may point towards a cryptic species. on the other hand, if a mtdna split within a species is not supported by the nuclear genetic data, this may be an indication of introgression of genetic material from another species, or of shared ancestral polymorphism. whereas the resolution of the specific interrelationships within the major clades remains problematic, the six main mtdna clades of the malawi cichlid flock are clearly delineated [5, 6, 13]. shared polymorphism within taxa might result from incomplete lineage sorting, taxonomic inaccuracies, and/or hybridisation. while the other possibilities can not be completely ruled out, there is a growing number of studies acknowledging the important role of hybridisation in the evolutionary history of adaptive radiations (e.g., [1719]). in this study we aim at elucidating the phylogenetic position of the utaka within the malawian cichlid radiation and shed light on the causes for its taxonomic and molecular assignment inconsistency. we examined individuals of five utaka species (copadichromis sp. virginalis kajose, c. quadrimaculatus, m. eucinostomus, c. chrysonotus, and c. borleyi) from twelve localities throughout lake malawi and one locality in lake malombe (figure 1). voucher specimens were fixed in 10% formalin and are curated at the royal museum for central africa in tervuren, belgium. we included additional copadichromis species that were sampled during the sadc/gef project and previously published mtdna control region (complete d-loop) sequences of lake malawi cichlids, which we obtained from genbank. whole genomic dna was extracted from ethanol-preserved fin clips using proteinase k digestion and salt precipitation, according to aljanabi and martinez. the first fragment of the mtdna control region was sequenced for 412 utaka specimens (179 copadichromis sp. virginalis kajose, genbank accession ef211832-ef211945 and ef647210-ef647271; 55 c. quadrimaculatus, genbank accession ef647341-ef647438 and ef647578-ef647579; 67 m. eucinostomus, genbank accession ef647356-ef647390, ef647439-ef647460, ef647498-ef647505 and ef647581-ef647582; 70 c. chrysonotus, genbank accession ef647273-ef647340, and ef647571-ef647572; 41 c. borleyi, genbank accession ef647470-ef647497, ef647520-ef647531, and ef647548), using published primers by meyer et al.. we additionally sequenced the second fragment of the control region using the primers by salzburger et al. and lee et al. for 14 individuals, selected on the basis of the results of the phylogenetic reconstruction for the first fragment of the control region. polymerase chain reactions (pcrs) were carried out in 25 l buffered reaction mixtures, containing 5 l template dna, 5 l of each primer (2 m), 200 m of each dntp, 2.5 l of 10x buffer (1 mm mgcl2), and 0.65 units of red taq polymerase (sigma aldrich). pcrs were performed under the following conditions: 94c for 120 s, followed by 35 cycles of 94c for 60 s, 52c for 60 s, 72c for 120 s, followed by 72c for 10 min. pcr products were purified following the tmqiaquick pcr purification kit protocol and sequenced on an abi 3130 automatic sequencer (applied biosystems) using standard protocols. a total of 179 c. sp. virginalis kajose, 230 c. chrysonotus, 252 c. quadrimaculatus, and 344 m. eucinostomus individuals were screened for genetic variation at nine microsatellite markers: pzeb1, pzeb3, pzeb4, pzeb5, unh002, tmom5, tmom11, tmom27, and ume003. pcrs were performed under the following conditions: 94c for 120 s, followed by 5 cycles of 94c for 45 s; 55c for 45 s; 72c for 45 s, followed by 30 cycles of 90c for 30 s; 55c for 30 s; 72c for 30 s, followed by 72c for 10 min. 10 l reaction mixes included 1 l template dna, 0.5 m of each primer, 200 m of each dntp, 0.26 units taq polymerase (sigma aldrich, germany), 1 l 10 reaction buffer (sigma aldrich). pcr amplification products were run on 6% denaturing polyacrylamide gels using an alf express dna sequencer (amersham pharmacia biotech). fragment sizes were scored with alfwin fragment analyser v1.0 (amersham pharmacia biotech), using m13mp8 dna standards as external references, following van oppen et al.. for the reconstruction of the phylogenetic relationships of the utaka, two datasets were analysed. the program collapse v1.2 was used to reduce this dataset to one individual sequence per haplotype for further analyses. gtr+g+i model was the best-fitted model of sequence evolution inferred by modeltest v3.7 according to the akaike information criterion (aic). a maximum likelihood (ml) heuristic search was performed with phyml starting from a neighbour-joining (nj) tree. parameters of the tree and of the substitution model were optimised sequentially until no increase in likelihood was found. based on the results of the short control region phylogenetic reconstruction, we performed a second, more computationally intensive phylogenetic analysis with a smaller dataset containing 47 representatives of the different main lineages in the lake malawi cichlid flock (both new sequences and sequences extracted from genbank) to test the interrelationships between these main lineages. the final dataset, 837 bp long, was first run through modeltest, which selected the trn+i+g model (aic criterion). phylogenetic inferences were carried out using maximum-parsimony (mp, 100 replicates starting from random stepwise addition trees; tbr branch swapping) with different transition-transversion weights (1: 1, 2: 1 and 3: 1) in paup*v4.0. ml reconstructions (100 replicates starting from random stepwise addition trees; tbr branch swapping) were run in paup*. sequential searches were performed by reestimating the substitution model parameters upon the best tree found and then running a new search with these parameters. this was done until no change in the likelihood of the tree or in the estimated parameters was found. support for the internal branches in the ml tree was assessed by analysing 100 bootstrapped replicates in the program phyml. for bayesian inference (bi) analyses, the gtr+g+i model was used since the trn+g+i is not implemented in mrbayes v.3.1. two runs with four chains for each run were sampled every thousandth generation until the average standard deviation of split frequencies between runs reached ~0.003. inspection of plot of likelihood versus generation revealed that the runs had reached stability and so did the analysis of the potential scale reduction factors. using the shimodaira-hasegawa test, as implemented in paup *, we tested the relative fit of two alternative tree topologies: the forced monophyly of all utaka specimens was compared to the best, unconstrained tree. significance of the difference in log-likelihood between the two trees was assessed by means of the resample estimated log-likelihood test (rell). we estimated the number of populations present in our microsatellite dataset using the program structure [36, 37]. we calculated the posterior probability for different numbers of putative populations (k from 1 to 18 populations) using a model-based assignment. burn-in was set at 100,000 steps followed by 300,000 mcmc iterations at each k. simulations were run five times for each k to check for convergence of the mcmc. we performed clustering both under the admixture model without prior population information and with correlated allele frequencies between populations. to determine the most likely number of clusters, the rate of change in the log probability of data and in the statistic k between successive k values was estimated using structureharvester. first, we assessed the phylogenetic relationships among as many specimens as available from the five utaka species that we collected throughout the lake. for this extensive dataset, we sequenced the short (328 bp) but most variable part of the mtdna control region. by this analysis we aimed to detect specific or geographical patterns among the utaka species studied. second, we attempted to resolve the phylogenetic position of the utaka species within the lake malawi cichlid flock. for this purpose we sequenced the complete mitochondrial control region for representative specimens (n=14) of the previous dataset and included published sequences from species representing the main lineages in the malawian cichlid flock. a total of 115 haplotypes were found amongst the 412 utaka short mtdna control region sequences (figure 2). the ml tree presented two divergent clades within the utaka: a large clade containing circa 70% of all sequences, and a smaller group. individuals (125 c. sp. virginalis kajose individuals out of 179 sequenced clustered within this clade), together with two (out of 67) m. eucinostomus and one (out of 55) c. quadrimaculatus individuals. both mtdna clades were present lake-wide in nearly all localities sampled, and within each lineage distinct geographic structuring was absent. the complete mtdna control region phylogenetic reconstructions using mp (with the different weighting schemes), ml, and bi consistently recovered the 6 main clades among the malawian cichlids (figure 3): (i) a lineage containing most non-mbuna and utaka species (non-mbuna clade hereafter); (ii) a clade containing only copadichromis individuals (virginalis clade hereafter); (iii) a mbuna clade containing all mbuna species plus some deep-water lethrinops species and an aulonocara specimen; (iv) a diplotaxodon clade; (v) a rhamphochromis clade; (vi) a clade containing a. calliptera. for these clades, bootstrap values and posterior probabilities displayed high node support values, except for the virginalis clade, which had a bootstrap support of 73 and a posterior probability of 0.87 (figure 3). the phylogenetic relationships between the clades remained, however, unresolved: their branching order was variable, depending on the reconstruction methods used and was even resolved as a polytomy in the bayesian analysis. the shimodaira-hasegawa test indicated a significant difference in likelihood score between the two topologies examined (p=0.03), giving preference to the unconstrained topology (where utaka are paraphyletic) over the best tree obeying to the monophyly of all utaka specimens analysed. the model-based clustering approach implemented in the program structure yielded estimated ln probabilities for 1 k 18 ranging from 37678 to 34160 with the highest posterior probability and k (= 20.605) for k=3. in the most likely scenario, structure assigned m. eucinostomus and c. chrysonotus to two different groups, while c. quadrimaculatus and c. sp. the phylogenetic inferences of the utaka assemblage performed herein showed that it contains two genetically distant and geographically widespread mtdna lineages. the two lineages have been observed before [5, 10, 13, 14] but this is the first study to reveal the paraphyly not only of the genus copadichromis in individuals from throughout lake malawi, but also of three (of the five analysed) utaka species (based on the short mtdna sequences). in a wider taxonomic context involving the other malawian cichlid lineages, the most abundant of the two lineages in the utaka clustered within the non-mbuna mtdna clade, while the other formed a separate clade containing exclusively utaka specimens, mostly c. sp. virginalis kajose the paraphyly of the utaka does not represent an artefact in our analyses, as corroborated by the long and well-supported branches that connect the non-mbuna and the virginalis clades, as well as by the significant result of the shimodaira-hasegawa test. one possible explanation is that the utaka share ancestral polymorphic alleles and/or represent a truly paraphyletic group containing multiple lineages that have undergone convergent evolution. importantly, the occurrence of two divergent mtdna lineages within the utaka is related neither to taxonomic clustering, nor to geographical structuring. if the two haplogroups observed within the utaka indeed correspond to two ancestral lineages that are genetically isolated for such a long time that their mtdna genotypes have become so deeply diverged, we would expect this to be also reflected in the nuclear genome of the species. however, we did not find any subdivision of nuclear gene pools that corresponds to the deep mtdna divergence, neither across the utaka species, nor within c. sp. virginalis kajose which yields the majority of the individuals in the divergent virginalis clade as well as a large number of individuals in the non-mbuna clade. it thus seems unlikely that the presence of a cryptic species is the cause of the mtdna divergence within c. sp. virginalis kajose. recently published phylogenetic reconstructions using aflp loci [10, 14] also showed a discordance between the nuclear and mitochondrial placement of copadichromis virginalis within the malawi cichlid radiation, supporting our finding that the observed paraphyly of the utaka and of c. sp. virginalis kajose is unlikely to be the result of incomplete ancestral lineage sorting or true paraphyly. alternatively, a disparate pattern of divergence between mitochondrial and nuclear dna among conspecific individuals may be the result of a past hybridisation and introgression event, a process which has been documented in malawian cichlids before (e.g., [4042 ]) and for which evidence is accumulating (e.g., [10, 14, 16, 43]). under this hypothesis we advance two possibilities regarding the original position of the utaka within the malawi cichlid phylogeny. a first scenario assumes that all utaka species formerly constituted a separate ancestral clade within the malawi cichlid flock, corresponding with the current virginalis clade. subsequent unidirectional introgression of mtdna from non-mbuna into the utaka could then explain the observed clustering of utaka specimens within the non-mbuna lineage. this scenario would involve that either all, or the ancestors of the current utaka species, would have been extensively hybridised with a non-mbuna species, resulting in the almost complete replacement of the original mtdna of the utaka. a second scenario assumes that all utaka species initially belonged to the non-mbuna lineage and a species from a distant mtdna lineage hybridised with copadichromis species. interestingly and despite our extensive taxonomic sampling, the maternal species involved in the putative hybridisation event remains unidentified as the virginalis clade only contained representatives of the utaka assemblage. hybridised either has thus far not been subjected to molecular studies or may no longer be present in the lake. empirical evidence for or against the above scenarios can be gained by examining mtdna of supplementary utaka species to validate whether the majority of the taxa cluster is within the non-mbuna clade or within the virginalis clade. the more utaka species cluster within the non-mbuna clade, the less probable becomes the first scenario. regardless of which of the two mtdna lineages is the original or the introgressing one, and irrespective of the maternal species involved in the hybridisation event, our results show that the two mtdna lineages have persisted within the gene pool of copadichromis sp. virginalis kajose for a rather long period, as suggested by the diversity displayed by either of these two lineages (figure 2). it thus suggests that either the population size of this species has remained very high since the hybridisation event (such that genetic drift would represent a lesser issue) or that some other mechanism is maintaining the two lineages within the same species (e.g., balancing or frequency-dependent selection). interspecific gene flow is increasingly recognized as an important factor in shaping speciation (e.g., [1719, 44, 45]). progressively more examples for hybridization are known from african cichlid fish: among lake tanganyika's cichlids evidence is found for ancient introgression (e.g., [15, 4648 ]) and a complete replacement of mtdna in multiple tribes of the cichlid assemblage. from lake malawi, evidence for deep introgression leaving a long-term signal in its haplochromine radiation [10, 14, 43], as well as evidence for more recent natural hybridisation [16, 50, 51] among malawi cichlids, has been provided. in the lake victoria cichlid flock recent or ongoing hybridisation [5254] presumably affects large parts of the species ' genomes by homogenization [54, 55], hampering the reconstruction of its young evolutionary history [54, 56, 57], yet potentially seeding the process of speciation but see. in cameroonian crater lakes the hybridisation of two ancient lineages resulted in the formation of a new and ecologically highly distinct species. also for steatocranus cichlids from the congo basin it was recently shown that ancient as well as recent introgression of genes and hybridisation produced a genomic network that potentially promoted divergence and speciation. our results chime well with previous studies reporting hybridisation in the early stages of a cichlid radiation. our findings reconcile with the recently reported evidence for ancient introgression between mbuna and deep-benthic cichlids at the base of the malawi radiation. virginalis kajose and c. quadrimaculatus, two phenotypically distinct taxa, by our microsatellite markers. however, it has already been reported that the performance of a clustering method may become poor for fst's below 0.05 (, j. pritchard, pers. comm.). the estimates of population differentiation were low in both species (= 0.006 in c. sp. virginalis kajose and =0.007 in c. quadrimaculatus, reported in), and slightly higher among the two species (= 0.01). whether this observation might yield a demonstration of the relative ease of hybridisation among phenotypically well-differentiated taxa [14, 43] or be the result of an insufficient resolution of the markers used, deserves further research.

we present a comprehensive phylogenetic analysis of the utaka, an informal taxonomic group of cichlid species from lake malawi. we analyse both nuclear and mtdna data from five utaka species representing two (copadichromis and mchenga) of the three genera within utaka. within three of the five analysed species we find two very divergent mtdna lineages. these lineages are widespread and occur sympatrically in conspecific individuals in different areas throughout the lake. in a broader taxonomic context including representatives of the main groups within the lake malawi cichlid fauna, we find that one of these lineages clusters within the non-mbuna mtdna clade, while the other forms a separate clade stemming from the base of the malawian cichlid radiation. this second mtdna lineage was only found in utaka individuals, mostly within copadichromis sp. virginalis kajose specimens. the nuclear genes analysed, on the other hand, did not show traces of divergence within each species. we suggest that the discrepancy between the mtdna and the nuclear dna signatures is best explained by a past hybridisation event by which the mtdna of another species introgressed into the ancestral copadichromis sp. virginalis kajose gene pool.

PMC3357950

pubmed-942

who has estimated that there were 171 million people worldwide with diabetes mellitus (dm) in 2000 and predicted that 366 million people will have dm by 2030. the united states centers for disease control and prevention have estimated that 13 million persons in the united states have diagnosed dm, and an additional 5,2 million have the disease but it is not yet being diagnosed. diabetes mellitus is a severe metabolic disease associated with a number of complications including retinopathy, nephropathy and neuropathy. mortality related to dm and its complications result in 3.8 million deaths annually, accounting for 6% of the total worldwide mortality [2, 3]. the mortality risk in the diabetic population in the age of 4049 years is about 67 years and for the age category of 6069 it is about 3-4 years higher, compared to mortality risk, at the same age healthy population. the stage of nephropathy, including the severity of proteinuria is a predictable factor for the mortality risk. approximately 2.5% to 15% of annual healthcare budgets is spent on care of patients with diabetes and associated complications. it has been estimated that programs to identify and treat diabetic retinopathy (dr) would have saved the united states health care budget nearly 400 million dollars annually in the early 1990s,] a figure that would probably be substantially higher today. a lot of studies in dm type 2 between which is the ukpds-study, have underlined the beneficial effect of having the hba1c, not<6%. a diverse array of micro- and macrovascular changes, are induced trough bad metabolic memory and increase, as well as decrease of hba1c (fig. 1). increase of glycated hemoglobin in diabetic population at baseline, and in 6 years follow-up period, has the same impact on the cardiovascular complications, as the decrease of hba1c<6.0% accord increase of glycated hemoglobin in diabetic population at baseline, and in 6 years follow-up period, has the same impact on the cardiovascular complications, as the decrease of hba1c<6.0% retinal vascular changes in diabetic population are the most common cause of blindness among eye diseases. impairment of retinal circulation results in blood flow alternations that affect the delivery of oxygen and metabolic substrates to the tissue. release of oxygen derived free radicals, nitric oxide-mediated neurotoxicity, and delayed cell death lead to neuronal damage. poor glycemic control sets the stage for initiation of the microvascular changes. the molecular basis of dr and maculopathy takes into consideration the role of growth factors and cytokines in the development of diabetic vascular alterations, their specific influence on the cellular interaction between retinal endothelial cells and pericytes, and the role of intravascular blood components. the earliest histological features of dr include neuroretinal damage, capillary basement membrane thickening, loss of pericytes and loss of endothelial cells. the hallmark of proliferative diabetic retinopathy (pdr) is neovascularization (nv) which occurs at the latter stages of the disease and can result in blindness; nv is the consequence of abnormal fibrovascular proliferations with subsequent bleeding and retinal detachment. numerous studies have underlined the role of the angiogenic factors in the progression of dr and include vascular endothelial growth factor (vegf), insuline-like growth factor (igf-1), hepatocyte growth factor (hgf), basic fibroblast growth factor (b-fgf), platelet-derived growth factor (pdgf), pro/inflammatory cytokines and angiopoetins. on the other hand, the major antiangiogenic factors are: pigment epithelium derived factor (pedf), transforming growth factor beta (tgf-beta), thrombospondin (tsp) and somatostatin [57]. the balance among angiogenic and antiangiogenic factors has a determining role in the progression of dr. the early diabetic retinopathy study (edrs) demonstrates the ability of panretinal photocoagulation to reduce the rate of severe visual loss by 50% for eyes with high risk characteristics, defined as nv originating from optic disc (> 1/3 disc diameter), any nv originating from the optic disc with hemorrhage, and nv originating from the retina with vitreous hemorrhage. the edrs showed that patients with (type 2) dm who were older than 40 years of age, with severe nonproliferative dr (defined as hemorrhages in four quadrants, or intraretinal microvascular abnormalities in one quadrant), have benefited from early panretinal photocoagulation. the early vitrectomy diabetic study (evds) showed that early vitrectomy (within 6 months of onset of vitreous haemorrhage) was associated with better results in type 1 diabetes mellitus patients [79]. where are we now in relation to our knowledge in prevention and early treatment of dr? did we follow our investigations in a direction that can give us solution of the main problems, or our investigations do not have a real clinical impact? can we expect better predictive strategies for early diagnosis and treatment? to answer these questions we will summarize our results achieved with currently available diagnostic and therapeutic approaches. a number of advances in science have been translated into real and measurable advances in patient care. the emerging investigations are concentrated on different secretory proteins and their role in the development on pdr. for example secreted acidic proteins rich in cystein (sparc) are isolated from the vitreus fluid and are increased in patients with pdr. furthermore, subretinal injections of recombinant sparc adenovirus, induce pdr-like changes in the rat, thereby providing a relevant animal model for the investigation of pdr. robo4, a member of roundabout (rabo) family, acts as a neuronal guidance receptor and plays some role in vasculogenesis and angiogenesis. several studies have shown its effect on the formation of fibrovascular membranes in patients with pdr, and its role in choroids-retina endothelial (rf/6a) and human retinal pigment epithelial cells. silencing the expression of robo4 in rf 6a and rpe cells inhibited their proliferation and reduced the hypoxic condition tolerance. a number of studies have estimated increased levels of interleukin 8 in vitreous samples from patients with pdr and in patients with more severe large-vessel gliotic obliteration. cystein-rich 61 (cyr61) induces are reported to mediate angiogenesis, and they have been shown to cause proliferation, migration, and synthetic matrix tube formation of rf/6a cells. recent reports indicate that cyr61 acts as an angogenic mediator of ocular nv in vitro and in vivo. it may interact in synergy with vegf in the pathogenesis of pdr. on the other hand, rosiglitazone maleate, an oral peroxsisome-proliferating activated receptor gamma agonist and oral insulin sensitising agent with potential antiangiogenic activity, delays onset of pdr. the golden standard of therapy in dm is insulin, and previous studies have emphasized its inhibitory effect on the progression of the dr. however, recent information suggests this to be controversial because insulin can stimulate vegf and hypoxia-inducible factor-1 (hif-1) expression in retinal pigment epithelial cells. vegf is considered as a main factor for the development of the pdr, but recently, a 634c/g polymorphisms in the vegf gene was shown to be associated with pdr. the expression of vegf, vegfr1 and vegfr2 levels is much higher in patients with pdr and dm, than in diabetics with pdr and dm type 2. studies that have analyzed the expression of integrins and their localization within the endothelium (in membranes from proliferative dr isolated during the vitreoretinal surgery) may provide new possibilities for the future treatment of the proliferative dr. the results suggest an essential role of integrins alfa and beta3 in the pathogenesis of pdr. a number of studies have shown the beneficial effect of intravitreal anti-vegf therapy. the results were achieved between 13 weeks in the patients with retinal neovascularization elsewhere and neovascularization of the disc. assessment of the short-term safety and efficacy of inravitreally administrated bevacizumab (an anti-vegf agent) as an adjunctive treatment for pdr, especially in the severe cases with iris nv, has been estimated in a number of studies [1720]. these studies confirmed regression of the nv, but the main disadvantage of the anti/vegf drug administration is the need of repeated injections in order to prevent recurrences of the nv. glucocortcoids (e.g., triamcinolon acetonide) that are among the drugs used for intravitreal application can influence diabetic macular edema (dme). the pathogenesis of pdr is multifactorial involving both angiogenic and inflammatory processes. when administrated together with anti-vegf drugs (bevacizumab, ranibizumab) can induce nv and dme regression. the results achieved from the studies may make this procedure an important adjunctive treatment in the management of selected cases with severe pdr. the long-term results of intravitreal bevacizumab (avastin), alone, in patients with pdr do not reveal any safety concerns [22, 23]. for instance, laser photocoagulation, while superior to intravitreal triamcinolone (ivt) or anti-vegf drugs, reduces but does not eliminate the risk of continued visual loss and is destructive to the retina. focal/grid laser is more effective than ivt with fewer side effects within the initial 2 years. laser or ivt are both more likely to improve the va over three years compared to the expected untreated course. clinical investigations include those which evaluated the efficacy of intravitreal injection of bevacizumab in preventing panretinal photocoagulation, macular thickening and visual dysfunction in eyes with severe pdr. the results showed that the best corrected visual acuity increased after the combined antivegf-laser treatment while the central foveal thickness decreased. intravitreal injection of bevacizumab can also be used in patients with pdr, that are undergoing pars plana plana vitrectomy (ppv). the intravitreal administration of bevacizumab (before ppv for pdr) facilitates surgery and may decrease the rate of postoperative vitreous hemorrhage and improve visual acuity [24, 25]. ranibizumab (lucentis) is an affinity-matured recombinant humanized immunoglobulin monoclonal antibody fragment with a molecular weight of 48 kd that binds to and inhibits the biologic activity of all isoforms of human vegf by preventing interaction with its receptors. the fragment is one third the size of a full-length antibody and readily penetrates all layers of the retina after intravitreal injection, consequently decreasing cell proliferation and vascular permeability. the latest studies have also shown the positive effect of the drug, applied intravitreal in cases of dme. the funding was through national eye institute that sponsored cooperative agreement that was initiated in september 2002. at the moment there are eleven on-going protocols that should estimate the results of different therapeutic approaches in cases of dr. the objective is to develop a collaborative network to facilitate multicenter clinical research on dr, dme and associated conditions (fig. 2). 2searching for the results of different treatment modalities in cases of diabetic macular edema, according to the drcr network protocols searching for the results of different treatment modalities in cases of diabetic macular edema, according to the drcr network protocols the goals of the drcr are: involvement of community based practices as well as academic or university-based centres. collaborate with industry to facilitate investigations and pursue opportunities otherwise not possible and to do so in a manner consistent with the networks dedication to academic integrity and optimal clinical performance. involvement of community based practices as well as academic or university-based centres. collaborate with industry to facilitate investigations and pursue opportunities otherwise not possible and to do so in a manner consistent with the networks dedication to academic integrity and optimal clinical performance. this will include the effects of cataract surgery upon dme and the effects of intravitreal plasmin to cause a posterior vitreous detachment upon dme. with the advent of new sophisticated techniques and diagnostic tools, the latest classification of the dr seems to be insufficient. triggered on the molecular level, the non-responders to the standard therapy should be provided a strategy of personalized patient treatment. although individualized therapy planning seems to be expensive, we should consider the fact that the diabetic complications lead to economic and psycho-social problems. the complicated treatment phase, including the sophisticated and expensive techniques of ppv, using different kind of drug substitutes do not always provide us satisfactory results. the main problem at this point is not only the associated complications of the treatment, but the chronicity of the disease. the long-term results of the surgery are not always stabile over time, and a lot of late complications can not be avoided. thus there is a need for new diagnostic and therapeutic approaches to be instituted much earlier at a time when there is no evidence of retinal damage. hypertension, insulin resistance, dyslipidemia and obesity, are commonly found in a combination with this stage of pdr. new diagnostic images, that can discover early diabetic changes, can provide useful informations for the novel dr treatment. pathology specific biomarkers, dna analysis in conditions of chronic diseases diagnostic and treatment, do not deliver us only information about the general concept of treatment, but also help us in the individual-treatment, individual drug response and benefits according to this strategy of individual healthcare. there is a need of collaborative network, to facilitate multicenter clinical activities, and thereby development of advanced treatment algorithms for dr and dme. the multidisciplinary approach to chronic diseases, such as dr can initiate research activities in different fields that could create the model of personal-based-patient-care. this means, we should gain new strategies to the optimal concept that includes: recognize the population at risk of the diseasefollow-up before the initial changes become manifestedsearch for new approaches that can prevent the development of the diseaseearly treatmentoptimal follow-up recognize the population at risk of the disease follow-up before the initial changes become manifested search for new approaches that can prevent the development of the disease

retinal vasculature changes in diabetic patients are most common cause of blindness among eye diseases. numerous studies have explored the role of the agiogenic factors in the progression of diabetic retinopathy (dr). the balance between angiogenic and antiangiogenic factors has a determining role in the dr progression. current treatment modalities include laser photocoagulation, intravitreal drug application, and pars plana vitrectomy (ppv). these maneuvers are employed with occurrence of advanced retinal changes. new diagnostic approaches can provide better information for the initial retinal changes thereby requiring a new dr classification and treatment guidelines. the results that are expected from diabetic retinopathy clinical research network (drcr) are at the level where prediction and prevention can not be made. innovative molecular-imaging technology, can pave the way for application of novel clinical approaches. identification of pathology-specific biomarkers and their application to diagnosis and treatment, support the individualized treatment algorithms.

PMC3405300

pubmed-943

prostate cancer is a common health problem that in the majority of cases starts to develop at the age of 50 years, reaching its peak at 6070 years of age. a variation in its incidence and prevalence exists between western, asian and arabic populations. the highest incidence is in the usa and canada followed by european countries, then the asian population and the lowest incidence is in arabic population [1, 2]. the incidence of prostate cancer was reported to be about 100127/100.000 men in the usa, while only 3.1, 3.3 and 6.5/100.000 men in saudi arabia, oman and kuwait. the marked difference in the incidence and prevalence of prostate cancer between western and arabic countries is very interesting and can be attributed to different explanations. all arabic countries are actually islamic countries where muslims constitute the majority of the population. they adopt male circumcision in the first few years of life as an obligatory event in relation to the religion of islam. interestingly, many recent studies confirmed the association between circumcision and the lower incidence of penile viral infections. uganda trial reported that applying male circumcision results in a reduced incidence of hsv2 infection by 25%, hpv infection by 35% and hiv infection by 5060% [7, 8]. morris recently reported that male circumcision is a surgical vaccine that guards against genital and urinary bacterial and viral infection and protects against penile and prostate cancer. in a study of 20,243 men in finland, infection with hpv18 was associated with a 2.6fold increase in risk of prostate cancer (p<0.005). for hpv16, some studies reported that uncircumcised men have more than twice the incidence of prostate cancer compared with circumcised men [1214] which explains why prostate cancer is relatively rare amongst jews. of men operated on for prostatic obstruction, 1.8% of obstructions were cancerous in jews (circumcised), compared with 19% of non jews (uncircumcised). a study in the uk in 1996 found an odds ratio for the reduction in risk of prostate cancer in circumcised men to be 0.62. table 1 illustrates some recent publications studying the association between male circumcision, stds and prostate cancer. publications studying the association of circumcision with sexually transmitted diseases and prostate cancer the age standardized incidence rate of prostate cancer is lower in asian countries than in the united states and european countries. however, the incidence rate in asians living in the united states is substantially higher than that in those living in their homelands. migration studies have shown an increase in prostate cancer incidence in asian men after emigration to the united states. this observation suggests that environmental factors and changes in lifestyles, particularly in dietary practices, can affect the aetiology of prostate cancer. a higher intake of vegetables in the arabic population may be also a factor that contributes to the lower incidence of prostate cancer. a recent study from egypt confirmed higher vegetable intake to be of significant value in reduction of the risk of prostate cancer. a recent systematic review confirmed that a diet low in fat and meat and rich in vegetables and fruits is recommended to lower the risk for prostate cancer. studied the difference in serum testosterone between arab and german groups of patients with diseases other than prostate cancer and was able to demonstrate a significantly lower level of testosterone in the arab population in the middle age group (< 30 years), pointing out that the relatively lower level of serum testosterone in that age group may be protective in the long run. in another study, the same group published the difference in levels of serum testosterone between arabs and american caucasians and reported lower levels o in arabic men in all age groups, with a higher significance in the middle age groups (< 30 years). schistosomiasis is a parasitic disease that is endemic in tropical countries with more than half of the cases located in africa and middle east. animal studies on the effect of schistosomal infection on the testes confirmed that schistosomal infection results in a significant decrease in the level of serum testosterone [2427]. another endemic problem that emerged in egypt, secondary to schistosomiasis, is caused by the treatment itself. in the mid20 century, the egyptian ministry of health began to conduct mass treatment in rural areas and for every suspected infection using tartar emetic intravenous injection. at that time, the dangers of exposure to human blood were n't considered and disposable needles and syringes were not available [28, 29]. frank et al. reported a direct relationship between parenteral treatment of schistosomiasis and the country wide prevalence of antibodies to hepatitis c infection. many studies [30, 31] confirmed the association between hepatitis c infection and chronic liver disease with the occurrence of hypogonadotropic hypogonadism manifested by low serum level of gonadotropic hormones and serum testosterone. table 2 illustrates findings in publications correlating serum testosterone in arabic and western populations. publications correlating serum testosterone in arabic and western populations serum psa is a widely used parameter for the early detection and monitoring of prostate cancer. in western countries men with total psa<2.5 ng/ml have a low probability of having prostate cancer, while those with psa>10 ng/ml have>50% chance of having prostate cancer [3234]. a recent paper from kuwait, demonstrated that for cases with psa>10 ng/ml, prostate cancer was still of a low probability, with the majority of cases with elevated psa only suffering from prostatitis or bph. they could detected a psa value of>50 ng/ml to carry 100% specificity for prostate cancer in the arabic population. a recent paper from saudi arabia studying the incidence of prostatic adenocarcinoma in patients admitted to king abdul aziz university hospital showed that prostate cancer was detected in 28.5% of patients with psa>4 ng/ml, which is considered lower than the expected incidence for their patients median age group of 68 years. table 3 demonstrates the difference in incidence between the findings in the saudi arabia study with a canadian and a multi institutional studies. incidence of prostate cancer in different recent studies another arabic study compared age adjusted serum level of psa of the arabic population with that of usa caucasians and the japanese population. they showed that usa whites tend to have the highest total psa, followed by japanese population and finally the lowest psa level, adjusted to age, was present in arabic population. psa density (psad) is another important parameter that was introduced by benston and colleagues as a useful tool to increase specificity of the detection of prostate cancer. different cut off values of psad ranging from 0.1 to 0.15 was proposed by urologists aiming to increase the specificity of prostate cancer detection in the grey zone area of total psa level, and thus avoid unnecessary prostatic biopsies [4144]. contrary to studies on the western population, a recent arabic study, trying to include psad as a mean to avoid unnecessary prostatic biopsy, concluded that in patients with total psa<10 ng/ml, values of psad<0.32 strongly suggest benign disease. the aim of our work was to report the pattern of prostate cancer presentation in alexandria university that as a tertiary referral center, provides care for uro oncology cases. data collection for all patients diagnosed with prostate cancer at alexandria universityin egypt through the year 2012 was done. all possible information including patients age, clinical presentation, dre findings and serum total psa and prostate volume were evaluated. analytical analysis was done using two tailed fisher exact test and two tailed unpaired t test. we found 950 patients diagnosed with prostate cancer in our database through the year 2012. mean serum total psa, prostate volume and psad were 149 ng/ml, 63 grams and 3.1 ng/ml/gm respectively. the remaining group was presenting with luts, including 23 patients who presented initially with back pain. patients characteristics only 7 patients were found to fulfill the criteria of low risk prostate cancer namely, psa<10, t1c and gleason grade<7. symptomatic patients were significantly associated with finding suspicious nodules during dre (p 0.002) and higher psa density (p 0.01). psa density of 0.15 did not yield any significant difference between both symptomatic and asymptomatic groups. the median psa density for the whole group was 0.425. when we used that value as the cutoff value, symptomatic patients had significantly higher psa density values. table 5 shows the analytical analysis for the studied groups. analytical analysis for asymptomatic and symptomatic cases only 54 patients (25%) were diagnosed through surveillance programs for prostate cancer (annual total psa and dre) without having any symptoms. thirty three patients had suspicious findings on dre and 42 patients had serum total psa>10 ng/ml. the striking features in those groups were the high psa density and that most of the cases (46 patients) had a gleason grade>6. irritative urinary symptoms were the main presenting complaints in our patients and the same findings of markedly higher psa density and higher gleason grade were noted. using the suggested western psa density value of 0.15 did not give any significant difference between surveillance and symptomatic groups, as the majority of cases had a psa density>0.15. we decided to use psa density value of 0.425 as the cut off value, being as it was the median value from the all 216 cases. the surveillance group had 65% of cases with a value<0.425, while the symptomatic group had 46% of patients with values<0.425 (p 0.01). that in the arabic population, a high psa density value up to 0.32 may present benign disease. our study also confirms the pivotal role of dre in the diagnosis of patients with prostate cancer as 77% of our cases had a suspicious finding during examination. it was clear that patients diagnosed with prostate cancer in egypt, regardless of their mode of presentation and whether screened or not, tend to have a high serum total psa, psa density, abnormal findings on dre and higher gleason grade, reflecting the aggressive nature of prostate cancer in the egyptian population (using the western definitions). a recent paper from nigeria showed that abnormal dre can predict higher gleason grades. our study shows that prostate cancer may not be a single disease throughout the world and racial, religious and life style conditions may be contributing factors that affect the nature and presentation of the disease. applying western definitions would mean that nearly all of our cases already had a metastatic and advanced disease, while really only 11% of them had metastatic deposits. our reports confirm that egyptian men with prostate cancer had higher psa, psa density and higher gleason grade at initial diagnosis and tailored risk stratification may be needed to allow proper management for our cases. egyptian men with prostate cancer have a markedly high psa density and gleason grade at diagnosis.

introductionprostate cancer is a common health problem that in the majority of cases starts to develop at the age of 50 years, reaching its peak at 6070 years of age. a variation in its incidence and prevalence exists between western, asian and arabic populations.the aim of our work was to report the pattern of prostate cancer presentation in alexandria university that as a tertiary referral center provides care for uro oncology cases. material and methodsdata collection for all patients diagnosed with prostate cancer at alexandria university in egypt through the year 2012 was done. resultsthe mean age of the patients was 67. mean serum total psa, prostate volume and psad were 149 ng/ml, 63 grams and 3.1 ng/ml/gm respectively. 25% of patients were asymptomatic diagnosed accidentally during screening for prostate cancer. the remaining group was presenting with luts, including 23 patients who presented initially with back pain. conclusionsegyptian men with prostate cancer have a markedly high psa density and gleason grade at diagnosis.

PMC4310882

pubmed-944

the supporting cells of the organ of corti are structurally and electrically coupled together by gap junctions (jahnke, 1975; gulley and reese, 1976; iurato et al., 1976; hama and saito, 1977; santos-sacchi and dallos, 1983; kikuchi et al., 1995). such gap junctional coupling among the supporting cells provides for electrical and metabolic uniformity; cochlear homeostasis is believed to rely on intercellular coupling (santos-sacchi, 1985, 1986, 1991; kikuchi et al., 1995). gap junction channels are distinguished from other ionic channels since the integration of two aligned hemichannels from adjacent cells is required for normal function. in early work, hypertonic solutions, which cause cell and tissue shrinkage, were found to uncouple gap junctions in several different preparations (barr et al., 1965, 1968; goodenough and gilula, 1974; loewenstein et al., 1967). more recently, hypotonic treatments, which cause cell swelling, were determined to either increase (kimelberg and kettenmann, 1990) or decrease (ngezahayo and kolb, 1990) gap junctional coupling. these effects could have been due to a variety of factors, including direct mechanical influences, changes in nonjunctional resistance, and modulation of intracellular factors that are known to uncouple cells. in the study of ngezahayo and kolb (1990), where junctional conductance was studied directly, the decrease in coupling was abolished by 5 mm egta in nominally ca-free internal solutions, and was linked to the activity of pkc. in the present report, we used the whole-cell voltage clamp technique to examine the effects of turgor pressure on junctional coupling of isolated pairs or small groups of cochlear supporting cells. both input capacitance (santos-sacchi, 1991; bigiani and roper, 1995) and transjunctional conductance measures were used to gauge intercellular communication. we report that data obtained with both techniques indicate that positive intracellular pressure, which is known to induce membrane tension, uncouples gap junctions of supporting cells in corti's organ. detailed experimental methods can be found in previous reports (santos-sacchi, 1991; sato and santos-sacchi, 1994). in brief, isolated supporting cells or cell aggregates were freshly obtained from the organ of corti of the guinea pig cochlea by shaking for 515 min in nominally ca-free leibovitz medium containing 1 mg/ml trypsin. to reduce the voltage-dependent ionic currents from nonjunctional membrane during double voltage clamp experiments, cells were perfused with an ionic blocking solution containing (mm): 100 nacl, 20 tea, 20 cscl, 1.25 cocl2, 1.48 mgcl2, 10 hepes, ph 7.2, 300 mosm. in initial experiments, a modified leibovitz medium was used for measurement of input capacitance (cin) with a single pipette voltage clamp containing (mm): 136.9 nacl, 5.37 kcl, 1.25 cacl2, 1.48 mgcl2, 10 hepes, ph 7.2, 300 mosm. pipette solutions were composed of (mm): 140 kcl, 10 egta or bapta, 2 mgcl2, and 10 hepes, ph 7.2. for double voltage clamp recording, patch electrodes had initial resistances of 2.54 m, corresponding to 12 m in diameter. series resistance (rs) after whole cell configuration was estimated from the current in response to 10-mv steps (huang and santos-sacchi, 1993). in single hensen cells, where rs could be unequivocally determined after whole cell configuration, the average value was 7.16 0.43 m (mean sem, n=48). cells were typically held at 80 mv, within the hensen cell's linear current voltage range (santos-sacchi, 1991). currents were filtered at 10 khz with a four-pole bessel filter (axon instruments, foster city, ca). intracellular pressure was modified either through the patch pipette with a syringe connected to the teflon tubing attached to the patch pipette holder or by changing osmolarity with y-tube pipette pressure was monitored via a t-connector to a pressure monitor (world precision instruments, inc., all experiments were video tape recorded and performed at room temperature. since the input capacitance can be measured by a single pipette voltage clamp and is correlated with junctional conductance (santos-sacchi, 1991; bigiani and roper, 1995), it can be conveniently used to study gap junctional coupling under conditions of less cellular damage than the double voltage clamp technique. input capacitance, in conjunction with input resistance (rin), was continually measured on line to monitor junctional coupling. cin and rin were determined from the transient charge and steady state current, respectively, induced by small (10 mv) test pulses with duration of 18 the clamp time constant at the holding potential; measures were made at 13 hz (santos-sacchi, 1991). 1\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation*}c_{in}=\frac{q_{in}}{v_{test}}\end{equation*}\end{document}2\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation*}r_{in}=\frac{v_{test}}{{\delta}i_{{\infty}}},\end{equation*}\end{document} where 3\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation*}q_{in}={\int_ {0}^{{\infty}}}i_{c}dt.\end{equation*}\end{document} qin is the charge moved, vtest is the voltage of the test pulse, ic is the capacitive current induced by the test pulse, and i is the current difference between the steady state current induced by the test pulse and the holding current at the holding potential. for the double voltage clamp, each cell in a cell pair was separately voltage clamped using 200a and 200b patch clamps (axon instruments). both cells were clamped at the same holding potentials and a test pulse (10 mv, 10 ms) superimposed only on cell 1. the transjunctional current (ij) is equal to the current difference (i2) in cell 2 caused by the test pulses applied to cell 1. the transjunctional conductance (gj) can be calculated by: 4\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation*}g_{j}=\frac{-{\delta}i_{2}}{v_{test}},\end{equation*}\end{document} where vtest is the test pulse voltage applied to cell 1. data collection and analysis were performed with an in-house developed windows-based whole-cell voltage clamp program, jclamp (http: //www.med.yale.edu/surgery/otolar/santos/jclamp.html), using a digidata 1200 board (axon instruments). in some experiments, gj was measured online at 24 hz and the corresponding video images of recorded cells were digitally captured every 510 s under software (jclamp) control. the captured images were printed at 1,700 and the plane cell areas calculated. to gauge membrane stress, area strain (a/a0) was calculated, where a is the change of cell area after pressure or osmotic treatment and a0 is the original cell area. hensen cells can be easily distinguished from other inner ear supporting cells by their prominent lipid vacuoles. the number of cells comprising isolates of hensen cells can be determined under the light microscope, and corresponds to the isolate's cin since hensen cells are well coupled electrically. although the size of hensen cells is variable, the distributions of cin for one, two, and three hensen cells, whose numbers were visually confirmed, were quite distinct (fig. the peaks of the isolate distributions were clearly separated at 31.03 0.86, 64.75 1.5, and 103.9 3.05 pf, corresponding to one, two, and three cell contributions, respectively. the number of cells within isolates can also be confirmed using uncoupling agents, such as co2, octanol, or, as we now find, positive turgor pressure, to uncouple the cells. when cells fully uncoupled, cin reached single cell capacitance levels (e.g., figs. 2 and 3). the correlation of cin with degree of cell coupling is illustrated by real measures of cin in a coupled two-cell electrical model (fig. cin of the electrical model was a monotonic function of transjunctional resistance or conductance, indicating the validity of cin as an indicator of cell coupling. positive turgor pressure induced either by osmolarity changes or directly via the patch pipette decreased cin of cell pairs or three-cell groups (figs. 2 and 3), but did not reduce single cell capacitance (fig. this indicates that positive turgor pressure uncouples gap junctions between adjacent hensen cells. in fig. 2 a, bath application of hypo-osmotic solution (150 mosm) caused a hensen cell pair to swell (insets) and decreased cin of the pair to single cell levels. the uncoupling induced by increased turgor pressure is reversible since return to normal osmolarity solution often restored initial cin values; subsequent reperfusion with hypo-osmotic solution remained effective as an uncoupling stimulus (fig. 2 b). in single cells, while the same hypo-osmotic treatment caused cell swelling, cin remained stable (fig. 4 b). 3 illustrates the uncoupling effect of cell turgor pressure change induced by patch pipette pressure. as turgor pressure was directly increased to 1.2 kpa via the patch pipette, cin decreased to almost single cell levels (after an initial delay possibly due to pipette plugging), and immediately began to return when the pressure was released (fig. the cells could be permanently uncoupled during the application of prolonged, continuous positive pressure (fig. the uncoupling effect of positive turgor pressure was found in 40 of 42 cell pairs, or three-cell groups. as with osmolarity change, direct application of positive turgor pressure via the patch pipette also did not decrease the measured capacitance in single hensen cells despite cell swelling (fig. 4 a, insets). although cin can be easily measured by single pipette voltage clamp to gauge the degree of cell coupling, transjunctional conductance can not be measured directly since transjunctional voltage and current are unknown. additionally, a quantitative estimate of degree of coupling based on cin is not easily established since cin is a nonlinear function of transjunctional conductance (see fig to further investigate the uncoupling effect of positive turgor pressure on gap junctions in hensen cells, the transjunctional conductance was directly assessed with a double voltage clamp technique, and corresponding changes of the cell plane surface areas (a/a0) (i.e., an indicator of membrane strain) were simultaneously measured. cell areas increased in concert with decreases of transjunctional conductance as positive turgor pressure was delivered to the cells. the changes in cell area were observable before gap junctional uncoupling and occurred faster than gj decay (figs. 5 and 6). however, unlike pressure changes induced by pipette pressure, hypo-osmotic shocks produced changes in gj and cell areas that were quite fast. with extracellular perfusion of a 150-mosm solution, 6 b, and the average value was 5.1 1.86 s (n=6). in fig. 6 b, the rise time constant of membrane strain was 4.43 s. the average rise time constant of membrane strain is estimated to be close to or less than that of the average gj decay since in most cases the swelling fully occurred within the 510-s video capture rate. in most, but not all, cases, it was noted that after membrane tension stabilized, transjunctional conductance likewise stabilized (fig. the correlated and reciprocal changes in gj and membrane strain (a/a0) were reversible and repeatable (fig. 6 a), strongly indicating that gj decreases were relative to increases of membrane strain; i.e., membrane tension. it should be noted that the latency to gj change after a/ a0 change is possibly due to the absence of significant membrane stress during the initial cell inflation, which clearly (based on the magnitude of cell enlargement) was accompanied by membrane unfolding. uncoupling of hensen cell gap junctions by membrane stress was not inhibited by using pipette solutions containing 50 m h-7 (dihydrochloride; calbiochem corp., la jolla, ca), a broad-based serine/threonine kinase inhibitor (boulis and davis, 1990) (figs. these data imply that the uncoupling effect of positive turgor pressure on inner ear gap junctions is independent of protein kinases, and that the effect is different from previous observations that cell volume changes induced uncoupling of gap junctions via the pkc pathway (ngezahayo and kolb, 1990). nevertheless, cell swelling induced by hypo-osmotic shocks has been linked to increases of another uncoupling agent, intracellular ca (hoffmann and simonsen, 1989; suzuki et al., however, uncoupling by ca, which occurs at millimolar intracellular concentrations in hensen cells (sato and santos-sacchi, 1994), can be ruled out since pipette solutions contained 10 mm bapta, a fast highly selective calcium chelating reagent, and extracellular and intracellular solutions were nominally ca free. considering all evidence, the observed uncoupling effect of positive turgor pressure on inner ear gap junctions, which is fast (within seconds), correlated with changes of membrane strain, and independent of protein kinases and ca, is likely to occur via direct mechanical effects on the plasmalemma; i.e., membrane tension. the effect of membrane tension on gap junctional conductance was further studied by increasing turgor pressure in cell 1 and measuring ij in cell 2 at different membrane potentials (fig. gap junctional conductance in hensen cells at a holding potential of 80 mv was 52.9 12.1 ns (n=51). as the turgor pressure in cell 1 was increased, ij decreased (fig. 7 a). the junctional conductance at different membrane potentials reduced in parallel when the turgor pressure was increased. in those cell pairs where turgor pressure alterations were successfully applied without losing the cells, gj at 80 mv holding potential decreased 38.3 9.5% from 50.5 14 ns (n=9) at a turgor pressure of 1.41 0.05 kpa. 7. in this case, as the cells were depolarized, gj decreased (fig. 7 b). other vm dependencies of transjunctional conductance were also found, including vm insensitivity and an increase with depolarization. we provide evidence, based on input capacitance and double voltage clamp measures, that junctional coupling is sensitive to positive turgor pressure-induced membrane tension. turgor pressure has been used to induce membrane tension in a wide variety of cells, including the outer hair cell (ohc), where it has been shown that motility and motility-related gating current characteristics are directly altered (iwasa, 1993; gale and ashmore, 1994; kakehata and santos-sacchi, 1995). membrane tension (possibly acting via cytoskeletal interactions) is also known to gate stretch-activated ionic channels (yang and sachs, 1989), which have been observed in outer hair cells (ding et al., 1991; iwasa et al., it is possible that membrane tension also alters gating characteristics of supporting cell gap junctions. we show, however, that unlike stretch channels, inner ear gap junctional conductance decreases with membrane stress. recently, it has been postulated that gap junction channels possess two distinct gating mechanisms, namely, a voltage gating mechanism and a chemical gating mechanism (bukauskas et al., 1995; bukauskas and peracchia, 1997; chemical uncoupling agents, such as co2, h, and ca, may act on sensor elements from the cytoplasmic side. supporting cell coupling has been shown to be sensitive to a variety of chemical uncoupling agents (santos-sacchi, 1985; 1991), and we now report that supporting cell coupling is voltage dependent as well. the existence of voltage-dependent gap junctional conductance may account in part for previous reports of temperature-induced depolarization on supporting cell coupling ratios (santos-sacchi, 1986). interestingly, junctional voltage dependence is unaffected by concomitant tension-induced junctional conductance change, possibly indicating that an independent tension gating mechanism may exist. gap junctions consist of two aligned transmembrane hemichannels (connexons), one from each cell (revel et al., 1984; goodenough et al., 1988; bennett et al., 1991). each of these hemichannels is formed by six connexin subunits (kumar and gilula, 1996; perkins et al., 1997). our data indicate that membrane stress acts on inner ear gap junctions in a manner independent of ca, ph, and protein kinases. the rapid and reversible nature of the uncoupling also indicates that the mechanism is not due to some sort of mechanical destruction of the channels. while there may be other unknown links between membrane stress and junctional conductance, it is conceivable that tension may gate gap junction channels by a conformational change in connexon structure, possibly causing only the stressed membrane's hemichannel to close. gap junction connexins represent a family of homologous proteins that have differing voltage gating characteristics (harris et al., 1981; spray et al., 1981; bennett et al., 1991 cx26 was found in gap junctions of the rat (kikuchi et al., 1995) and gerbil (forge et al., 1997) more recently, cx26, cx30, cx32, and cx43 have been localized to supporting cell regions of the rat cochlea (lautermann et al., 1997). such diversity of connexins within the organ may provide for a variety of junctional communication characteristics; for example, rectifying junctional conductance. indeed, in addition to our direct observation that voltage-dependent junctional communication exists in the supporting cells, we have preliminary evidence that junctional rectification occurs. directional flow of ions mediated by rectified gap junctions may be crucial for normal cochlea homeostasis (see below). since the mid 1980 's, gap junctional coupling however, input capacitance and resistance reflect the degree of electrical coupling and can be conveniently measured using a single voltage clamp (santos-sacchi, 1991; sato and santos-sacchi, 1994; bigiani and roper, 1995).. 1 b, inset), and assuming that the individual cells have the same input impedance, the following equations are obtained (bigiani and roper, 1995), 5\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation*}c_{in}=\frac{(2r_{m}r_{j}+2r^{2}_{m}+r^{2}_{j})r^{2}_{m}}{(2r_{m}r_{s}+r^{2}_{m}+r_{s}r_{j}+r_{m}r_{j})^{2}}c_{m},\end{equation*}\end{document}6\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation*}r_{in}=\frac{r_{s}r_{j}+2r_{s}r_{m}+r_{j}r_{m}+r^{2}_{m}}{r_{j}+2r_{m}},\end{equation*}\end{document} where rs and rm are electrode series resistance and nonjunctional membrane resistance, respectively, and cm is single cell capacitance (see fig since rm is not readily available from recordings, we can solve eqs. 5 and 6 to remove rm. rj can be finally expressed: 7\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0 in \begin{document} \begin{equation*}r_{j}= \left{\mid} \frac{c_{in}r^{2}_{in}+4c_{m}r_{s}r_{in}-2c_{m}r^{2}_{s}-2c_{m}r^{2}_{in}}{\sqrt{2c^{2}_{m}r_{s}r_{in}+c_{in}c_{m}r^{2}_{in}-c^{2}_{m}r^{2}_{s}-c^{2}_{m}r^{2}_{in} }} \right{\mid} .\end{equation*}\end{document} cin, rin, and rs are readily obtained from recordings. 8 illustrates the measurement of these parameters during an uncoupling event, and the bottom panel shows the estimated gj based on those data. it should be noted that rs changes can also produce changes in cin and rin. for example, to obtain the observed maximum change in cin, an order of magnitude increase of rs would be required in this case. in our experiments, changes solely in rs required to produce a comparable change in cin were not observed. series resistance remained constant, being 7.79 0.49 m (n= 7) for cell pairs that were well coupled and 6.34 1.13 m after those same cells were uncoupled with positive pipette pressure. finally, how might the turgor pressure dependence of junctional coupling in the organ of corti affect cochlear function? in vivo, the organ of corti, comprising hair cells and supporting cells, is bathed in two different media, high k endolymph apically and low k perilymph basally. since the receptor current through hair cells is predominantly carried by k, an accumulation of k within the perilymphatic space along the basolateral region of the hair cells is unavoidable. this would potentially depolarize hair cells with disastrous consequences for both forward and reverse sensory transduction. in the mammal, forward transduction (gating of stereociliar transduction channels) relies on the large driving force present across the hair cell's apical plasma membrane. voltage gradients across the apical membranes of inner and outer hair cells (i.e., endolymphatic potential minus membrane potential) range from 125 to 150 mv, and drive the k-based receptor currents. reduction of this gradient (e.g., by membrane depolarization) will reduce the magnitude of receptor potentials and synaptic output. reverse transduction is a phenomenon that is restricted to the outer hair cell and is believed to provide for the enhanced high frequency selectivity and sensitivity enjoyed by mammals. ohcs, which are additionally mechanically active, possess lateral membrane motors that are driven by voltage (santos-sacchi and dilger, 1988); the cell's mechanical response provides feedback into the basilar membrane, thereby enhancing the stimulus to the primary receptor cells, the inner hair cells (for review see ruggero and santos-sacchi, 1997). not only will depolarization of the ohc alter the driving force for the mechanical response, but the function relating mechanical response to voltage will be shifted along the voltage axis as well, resulting in an altered gain for the cochlear amplifier (santos-sacchi et al., 1998). a nutritive and k sinking role for gap junctions in the avascular organ of corti has been proposed (santos-sacchi, 1985, 1991; santos-sacchi and dallos, 1983). inner ear supporting cells have been shown to share plasmalemmal voltage-dependent conductances due to the high degree of cell coupling (santos-sacchi, 1991). the magnitude and stability of their resting potentials is pronounced (close to 100 mv), and likely depends on cell coupling since isolated cell resting input conductance is only 1 ns. at the normal resting potential of this cellular syncytium, an inward rectifier appears continuously activated and may result in k removal from perilymphatic spaces. it should be noted that the large perilymphatic fluid spaces may provide little support in sinking k or directing its movement, since hair cell regions that are likely to experience k elevations are not directly exposed to those spaces. inner hair cells are closely surrounded by supporting cells, and the region of the ohcs that possesses voltage-dependent conductances (e.g., outward k) is restricted to the basal pole of the cell (santos-sacchi et al., 1997), which is surrounded by a deiters cell cup. (1995) provided morphological evidence detailing epithelial and connective tissue gap junctional systems within the cochlea that may complete the mechanism responsible for recycling k from the perilymphatic space near hair cells to the k-rich endolymph via the stria vascularis. the maintenance of normal fluid space architecture within the inner ear requires fine osmotic control; imbalances can lead to serious auditory and vestibular problems (e.g., meniere's disease). while at present we do not know the normal physiological significance of tension-dependent gap junctional communication, it is likely that fluid balance disorders in the inner ear will affect gap junctional communication, thus compromising sensory function by indirectly modifying hair cell activity. the cell numbers (1, 2, or 3) in the isolates were determined under the light microscope, and the cin was obtained at the holding potential 80 mv. each bar represents the number of isolates within a bin width of 10 pf. the lines plotted over each histogram represent the fitted gaussian distribution for the three isolate groups. (b) cin was measured for a coupled two-cell electrical model as transjunctional resistance (rj) was changed. rs, 4.7 m; rm, 500 m; cm, 33 pf. (a) when an extracellular 150 mosm solution was perfused (indicated by the horizontal bar) the cell pair swelled (see insets). rs, 6.5 m. (insets) captured cell images before and during the perfusion of the hypo-osmotic solution. (b) decreases of cin due to hypo-osmotic challenge were reversible and repeatable. in this case, the cells finally fully uncoupled and cin remained at the single cell capacitance level. rs, 3.8 m. hensen cells uncouple as turgor pressure is directly increased via the patch pipette. (a) in this example, increased pipette pressure caused cin to decrease to single cell levels after an initial delay (possibly due to pipette plugging), but returned to the original level soon after pipette pressure was released. rs, 9.76 m. (b) a hensen cell pair uncoupled after prolonged application of pressure. rs, 8.5 m. changes of turgor pressure caused either directly via the patch pipette (a) or by perfusion of hypo-osmotic solutions (b) do not decrease the measured capacitance in a single hensen cell. rs: (a) 8.4 m, (b) 4.4 m. positive turgor pressure-induced reduction in transjunctional conductance is correlated with increases in membrane strain (a/a0). transjunctional conductance was measured with a double voltage clamp technique, and membrane strains were calculated from captured images (see methods). (a) positive pipette pressure induced an increase in membrane strain and concomitantly reduced gj. (b) perfusion of 150 mosm solution also induced an increase in membrane strain and concomitantly reduced gj. (a) hypo-osmotic solutions induced reversible, concomitant changes in transjunctional conductance and membrane strain. (b) the time course of the uncoupling effect caused by hypo-osmotic shock is compared with the increase of membrane strain. the beginning of treatment is indicated by an arrow and continued during the observed period. turgor pressure does not affect membrane potential (vm or vi-o) dependence of gap junctions in hensen cells. (a) voltage stimulus protocols for each cell and current trace recorded in cell 2 are plotted (only five traces are shown for clarity). each cell in a hensen cell pair was separately voltage clamped at the same holding potential of 80 mv. voltage steps from 140 to 70 mv for 100 ms in 10-mv increments were simultaneously delivered into both cells except for 10 mv, 10-ms test pulses superimposed on cell 1 only. three current traces from cell 2 at different pressures were zeroed and superimposed in the middle. rs at 0 kpa, 12.5 m; at 1.2 kpa, 12.7 m; at 2 kpa, 12.2 m. transjunctional conductance is estimated by cin and rin in single pipette voltage clamp. rs, 8.73 m. (bottom) gj is estimated from cin and rin measurements from the cell pair data based on the coupled two-cell model. cm was set as half of cin, 27.5 pf, at zero applied pressure, and rs was 8.5 m. the change in estimated gj corresponds well to changes in cin. the magnitude of gj is larger than the largest obtained under double voltage clamp (500 ns), and may be due to the fact that cell impedances are not actually identical as required in the model .

the effects of turgor pressure-induced membrane tension on junctional coupling of hensen cell isolates from the inner ear were evaluated by input capacitance or transjunctional conductance measurement techniques. turgor pressure was altered by changing either pipette pressure or the osmolarities of extracellular solutions. both positive pipette pressure and extracellular applications of hypotonic solutions, which caused cell size to concomitantly increase, uncoupled the cells as indicated by reduced input capacitance and transjunctional conductance. these changes were, in many cases, reversible and repeatable. intracellular application of 50 m h-7, a broad-based protein kinase inhibitor, and 10 mm bapta did not block the uncoupling effect of positive turgor pressure on inner ear gap junctions. the transjunctional conductance at a holding potential of 80 mv was 53.6 5.8 ns (mean sem, n=9) and decreased 40% at a turgor pressure of 1.41 0.05 kpa. considering the coincident kinetics of cell deformation and uncoupling, we speculate that mechanical forces work directly on gap junctions of the inner ear. these results suggest that pathologies that induce imbalances in cochlear osmotic pressure regulation may compromise normal cochlear homeostasis.

PMC2229429

pubmed-945

post-operative radiotherapy is an established adjuvant treatment after radical hysterectomy with intermediate- and high-risk early-stage cervical cancer patients [1, 2]. a conventional radiation technique for post-operative cervical cancer patients is whole-pelvic radiation therapy (wprt) requiring four static photon fields. this technique exposes most of the contents of the true pelvis, including the small bowel as well as the target volume. the highly conformal technique of intensity-modulated radiation therapy (imrt) has the potential for delivering the required radiation dose to the target volume, while avoiding surrounding normal tissues, and its effectiveness has been validated in several anatomical sites such as head-and-neck [3, 4] or prostate cancer treatment [5, 6]. however, the advantage of using the complex imrt technique in the field of gynecologic cancer has not yet been determined [79] and demands a prospective clinical trial for its validation. the radiation therapy oncology group (rtog) launched a multi-institutional prospective phase ii trial (rtog 0418) using imrt for post-operative endometrial and cervical patients in order to determine whether imrt could reduce short-term bowel toxicity. recently, a positive preliminary result was presented. in japan, because of the concern about possible severe late bowel complications related to the combination of surgery and radiotherapy, and because of a positive result from adjuvant chemotherapy alone for intermediate- and high-risk post-operative cervical cancer patients, several institutions dare not use adjuvant radiation therapy for early-stage intermediate- or high-risk post-operative cervical cancer patients. since the rtog 0418 trial included not only cervical cancer but also endometrial cancer patients, it was decided that the effectiveness and safety of imrt should be validated including only post-operative cervical cancer patients in a japanese multicenter prospective clinical trial. as a preparation for this clinical trial, it was regarded as important to ascertain the current status of imrt for post-operative cervical cancer in japan. the object of this study was to perform a surveillance study of imrt for post-operative cervical cancer in a working group within the radiation therapy study group (rtsg) of the japan clinical oncology group (jcog). in 2013, the working group conducted a surveillance using a questionnaire asking about the precise methods of conducting imrt. six leading academic institutions in the field of gynecologic oncology, who have already commenced applying imrt for post-operative cervical cancer patients in clinical practice, were selected for the current survey. data collection included: (i) the technical environment for imrt; (ii) patient preparation before taking planning computed tomography (ct); (iii) the technique for determining the target volume and the normal structure; (iv) the prescription dose, the dose constraint for organs at risk (oars), how to define the prescription dose; and (v) how patients were set up in daily treatment. half of the institutions used conventional static imrt, while the others used volumetric-modulated arc therapy (vmat). one institution used a 15-mv photon beam, which could deliver radiation to deep-seated organs. all except one institution used either a vacuum cushion or a shell for patient body fixation, which was important for highly precise radiation therapy as imrt., vosveld 9a, 2110 wijnegem, belgium), which was capable of fixing a patient's hips, legs and feet. according to the rtog 0418 protocol, in contrast to the rtog protocol, most of the institutions did not visualize the vaginal cuff before taking planning ct. 2.a bar graph showing the way of patient preparation before taking planning ct. a bar graph showing the way of patient preparation before taking planning ct. figure 3 shows how physicians define the clinical target volume (ctv). for the elective lymph node region, all institutions used the guideline for pelvic nodal ctv created by the japan clinical oncology group gynecologic cancer study group (jcog gcsg). for contouring the vaginal cuff, the paracolpium and the parametrium, which may contain microscopic cancer cells after radical hysterectomy, four institutions used the rtog guideline for reference, while two institutions used the jcog gcsg guideline for the intact uterus with modification, because the uterus no longer existed after surgery. two institutions used the fusion ct images taken with the bladder full and empty according to the rtog protocol to account for the internal motion of the ctv vaginal cuff, while others contoured the ctv vaginal cuff based on one ct series, adding a 510 mm internal margin. 3.a bar graph showing which guidelines physicians used as a reference when contouring the target volumes. a bar graph showing which guidelines physicians used as a reference when contouring the target volumes. the organs which were commonly selected as oars were the rectum, the bladder, the femoral head, and the small bowel/peritoneal cavity. one institution contoured the bowel loop, four the peritoneal cavity, and one did not contour either the bowel loop or the peritoneal cavity. 4.a bar graph showing which normal structures were selected as organs as risk (oars). a bar graph showing which normal structures the total prescription dose was either 50 gy/25 fr in 2 gy per fraction or 50.4 gy/28 fr in 1.8 gy per fraction. five institutions used the planning target volume (ptv) dmean (mean dose of the ptv) as a prescription point, while one institution used ptv d95 (lowest dose encompassing 95% of the ptv). as for the dose constraint for oars, one institution did not set any specific dose constraint. in this institution the attending physician checked the dose distribution and paid careful attention to hot spots in oar. the other five institutions used the individual dose constraint definition, and this is summarized in table 1. preliminary compliance rates of dose constraints for oars for five to eleven patients treated during the study period are also summarized in table 1. table 1.dose constraint for oarinstitution a (adherence rate, n=10)institution b (adherence rate, n=10)institution c (adherence rate, n=11)institution d (adherence rate, n=5)institution e (adherence rate, n=5)rectumv50 gy<40%, dmax<55 gy (100%)v50 gy<35% (80%)v40 gy<60% (18.2%)v50 gy<35% (100%)v40 gy<80% (40%)bladderv45 gy<50%, dmax<55 gy (80%)v45 gy<70%, v50 gy<35% (80%)v45 gy<35% (36.4%)v50 gy<35% (100%)v45 gy<35% (60%)small bowel/peritoneumv40 gy<40%, v55 gy 30% (45.5%)v40 gy<30% (80%)v40 gy<30% (80%)femoral headv30 gy<20% (80%)v30 gy<15% (20%)v30 gy<15% (36.4%)dmean<30 gy (80%)pelvic bonev20 gy<80% (80%)v10 gy<90%, v40 gy<37% (20%)cauda equinadmax<50 gy (40%)oar=organ at risk. fig. 5.a bar graph showing the prescription dose and the definition of prescription dose. dose constraint for oar a bar graph showing the prescription dose and the definition of prescription dose. with regard to the daily set-up of image-guided radiation therapy (igrt), three institutions took cone-beam ct (cbct) every day, one three times a week and two once a week. three institutions encouraged patients to empty the rectum every day before treatment, whereas no instruction concerning rectum emptying was given to patients in the other three institutions; however, in two out of these three institutions, cbct was taken every day and if large amounts of gas or stools were found, patients were asked to empty the rectum before irradiation. figure 6 shows a typical dose distribution of imrt for post-operative cervical cancer patients from the six institutions participating in this study. 6.a typical dose distribution of imrt for postoperative cervical cancer patients from six institutions participating in this study: institutions a f (a f). a typical dose distribution of imrt for postoperative cervical cancer patients from six institutions participating in this study: institutions a planning and delivery of radiation therapy have changed dramatically over the past several decades, and imrt is one of the most complicated and error-prone techniques, and thus requires thorough quality assurance programs, not only for multicenter clinical trials but for daily practical use. as a preparation for a future clinical trial using adjuvant imrt for post-operative cervical cancer patients, it was considered to be important to know the current status of imrt in terms of post-operative radiotherapy for cervical cancer in japan; therefore, the current surveillance study was performed. half of the institutions used vmat, for which it was shown that the treatment time will be shortened compared with conventional static imrt for pelvic irradiation, but a dosimetric benefit would not be expected. the high-energy photon beam has an advantage of delivering photons to deep-seated organs with less attenuation; however, it brings with it a concern about creating neutrons along with photons. in the latest patterns of care study (pcs) for cervical cancer in japan, the most appropriate machine energy should be discussed for developing the protocol of a future clinical trial. although visualization of the vaginal cuff was recommended in the rtog protocol, most institutions did not visualize them. it was supposed that inserting markers into the vaginal cuff would require manpower, time and patient endurance of pain. one institution inserted a small piece of gauze soaked with a contrast agent into the vaginal cuff, whereas it was not permitted to insert gauze into the vagina in the rtog protocol because it would potentially cause anatomical changes relative to the surrounding structures. it was considered to be feasible if only a small piece of gauze was manipulated; however, the impact from volume changes of the bladder and the rectum is considered to be more significant. therefore, inserting a small piece of gauze with a contrast agent will be included in the protocol of a future clinical trial. it was supposed that when the bladder was filled with urine, the bowel would be pushed away from the small pelvis and it would protect the small bowel from radiation exposure. however, reproducibility of bladder filling is problematic because the pelvic nerve plexus has already been damaged to varying degrees by radical hysterectomy; therefore, monitoring daily bladder filling by some means needs to be considered to ensure the accuracy of the treatment. the reason why the rtog guideline was not used as a reference to contour the ctv lymph node was that the definition of the ctv lymph node according to the jcog first, in the jcog gcsg guideline, the cranial margin was set at the bifurcation of the aorta, not based on the bony structure as in the rtog guideline. it would be difficult to categorize the recurrence as a regional (pelvic recurrence) or a distant (para-aortic nodal) failure when the previous pelvic field was constructed based on the bony anatomy. second, the japanese guideline involved the adipose connective tissue between the iliopsoas muscles and the lateral surface of the vertebral body, which was not included in the rtog guideline. this area was also included in the atlas of taylor et al. [18, 19]. therefore, the current rtog definition may be insufficient in terms of lateral expansion of the ctv for the internal iliac node. as for the ctv vaginal cuff, while four institutions used the rtog guideline, two institutions used the jcog gcsg guideline for the intact uterus. not all institutions used the rtog guideline, because the rtog guideline did not describe the paracolpium and the parametrium in detail. although, theoretically, the jcog gcsg guideline for the intact uterus was not appropriate for contouring the post-operative female pelvis, the jcog therefore, this guideline can be used as a reference in contouring the paracolpium and the parametrium after making some modification. we are now creating a consensus-based guideline for ctv vaginal cuff as well as the paracolpium and the parametrium (which may contain microscopic cancer cells, even after r0 radical hysterectomy), because there exists no such consensus guideline other than the rtog guideline. with regard to the internal organ motion of the ctv vaginal cuff, two institutions used the fusion ct with the bladder full and empty according to the rtog protocol, while others contoured the ctv vaginal cuff based on one ct series, adding 510 mm internal margin to ctv vaginal cuff. the vagina is sandwiched by the rectum and the bladder, whose volume will potentially vary from time to time; therefore, it was considered to be important to make a consensus on the internal margin for the ctv vaginal cuff. whereas all but one institution selected the small bowel or the peritoneal cavity as an oar, with four institutions contouring the peritoneal cavity and one the bowel loop, it was not standardized as to whether to contour only the bowel loop itself or the peritoneal cavity as well. in the current study, four institutions contoured the peritoneal cavity and one the bowel loop. the dose volume relationship will not be reliable if it is not consistent whether the bowel loop or the peritoneal cavity is contoured. volume relationship was found between chronic gastrointestinal (gi) complications and dose to the small bowel loops, whereas no parameter for the peritoneal cavity was significantly associated with gi complications. on the other hand, some modification of the definition will be required if the peritoneal cavity is to be used as an oar, because part of the bowels are embedded in the retroperitoneal space, such as the ascending or the descending colon. therefore, we will also develop a consensus-based definition of what constitutes normal structures for the post-operative cervical cancer patient. most institutions did not employ ptv d95 as the prescription point for concern about possible dose escalation compared with the conventional dose prescription according to icru report 50. consequently, the rtog 0418 protocol, in which ptv d97 was used as a prescription dose, was considered to be a more aggressive prescription dose in our working group. the dose constraint for oars and preliminary compliance rates of dose constraints for oars are summarized in table 1. because every institution used individual dose constraints, and contouring of the small bowel/peritoneum was not uniform, a large variation in the actual compliance rate was found. rtog 0418 reported that 6676% of patients did not meet the dose criteria for the bladder and the rectum, and the dose constraint was loosened in the next rtog 1203 protocol because it was considered to be too strict. it is, therefore, important to set achievable and clinically relevant dose constraints as well as to develop a consensus of contours for oars, especially the small bowel/peritoneum, for a future clinical trial using imrt for post-operative cervical cancer patients. the six institutions that contributed to this study were equipped with modern linear accelerators capable of doing cbct for daily set-up. it is very important to ensure accurate daily patient set-up when using imrt, which can generate a very steep and complicated dose distribution, especially when applied in such a large field as the pelvic region. if imrt is to become a standard therapy for post-operative cervical cancer, which is unfortunately still a relatively common cancer in our country, many institutions will need to update their accelerators with modern machines; this will contribute to improving the quality of radiation therapy in our country. the valuable information reported here was derived from six leading institutions in the field of gynecological oncology. this study was partially supported by the cancer research development fund (23-a-13, 26-a-4 and 26-a-28). funding to pay the open access publication charges for this article was provided by the cancer research development fund (23-a-13, 26-a-4 and 26-a-28).

intensity-modulated radiation therapy (imrt) was recently introduced to the field of gynecologic malignancies; however, its value is not yet validated. a clinical trial is in preparation to investigate the efficacy and feasibility of imrt for postoperative cervical cancer. the object of this study was to perform a surveillance study of imrt for post-operative cervical cancer. a questionnaire regarding the precise methods of conducting imrt was sent to six institutions that had already introduced imrt for post-operative cervical cancer, and the data were analyzed. half of the institutions used static imrt and the others used volumetric-modulated arc therapy (vmat). most institutions used body-immobilizing devices for patient fixation. most institutions instructed patients to fill their bladder before undergoing planning ct or daily treatment. while one institution inserted metallic markers and another one used radio-contrast soaked gauze to visualize the vaginal cuff, the other institutions used nothing for vaginal cuff visualization. most institutions defined the clinical target volumes according to the japan clinical oncology group or the radiation therapy oncology group guidelines. only one institution used a prescribed dose based on 95% of the ptv (d95), while the rest used the mean dose (dmean). this valuable information from six leading institutions will be utilized in a future prospective clinical trial.

PMC4497393

pubmed-946

maternal depression has adverse effects on infant behavioral, emotional, and cognitive development [17]. studies investigating the impact of postpartum depression on child development have largely relied on correlative studies in humans. however, investigation into the mechanisms mediating the transmission of negative affect from depressed mother to child has been impeded by the lack of useful animal models. we previously characterized a mouse model with deficits in maternal care which exhibits depression-like behaviors during the postpartum period (gabrd mice). here we utilize this mouse model to investigate the mechanisms underlying the negative impact of maternal depression-like behaviors on offspring development. deficits in offspring development associated with maternal depression are correlated with elevated levels of stress hormones in both the mother and the fetus. treatment of pregnant women with exogenous glucocorticoids results in deficits in child development similar to those related to postpartum depression, suggesting that the stress response may mediate the adverse effects of maternal depression on offspring development (for review see). the body's physiological response to stress is mediated by the hypothalamic-pituitary-adrenal (hpa) axis and involves the release of crh from the hypothalamus, which triggers the release of adrenocorticotropic hormone (acth) from the pituitary, ultimately resulting in glucocorticoid (corticosterone in mice and cortisol in humans) release from the adrenal gland. although basal levels of corticosterone increase throughout pregnancy and lactation, stress-induced elevations in stress hormones are suppressed (for review see), which is thought to protect the fetus from the negative effects of exposure to high levels of glucocorticoids [13, 14]. accumulating evidence suggests that dysregulation of the hpa axis plays a role in postpartum depression. the hpa axis may be hyperresponsive in postpartum depression, as indicated by elevated levels of cortisol, although these findings are controversial [16, 17]. more convincing evidence exists for increased levels of crh [18, 19] and acth associated with postpartum depression. accordingly, it has been suggested that crh levels are increased in women with postpartum depression and may even be used as a diagnostic criteria for postpartum depression. elevated levels of stress hormones in the depressed mother result in elevated levels of stress hormones in the infant and higher levels of stress hormones are associated with decreased maternal care and offspring anxiety. therefore, it is reasonable to hypothesize that dysregulation of the hpa axis may play a role not only in postpartum depression but also in the negative impact of maternal depression on offspring development. consistent with this hypothesis administration of exogenous corticosterone to the dams during the postpartum period induced behavioral abnormalities in the offspring. the activity of the hpa axis is governed by crh neurons in the paraventricular nucleus (pvn) of the hypothalamus (for review see). the activity of these neurons, and thus activity of the hpa axis, is tightly regulated by robust gabaergic inhibition (for review see [2426]), including tonic gabaergic inhibition mediated by subunit-containing gabaars. interestingly, we demonstrated that a mouse model deficient in the gabaar subunit (gabrd mice) exhibit depression-like behaviors exclusively during the postpartum period and deficits in maternal behaviors. a recent study confirmed these findings demonstrating that gabrd dams provide fragmented maternal care and their offspring exhibit phenotypes similar to those subjected to early life stress. in this study, we utilized this mouse model (gabrd mice), which exhibit abnormal/fragmented maternal care and depression-like behavior during the postpartum period [8, 28], to investigate the impact of maternal depression-like behavior on offspring development and the involvement of stress-related steroid hormones. it is well known that maternal depression negatively impacts child development in humans [17]. here we reproduce analogous deficits in offspring development associated with maternal depression-like behavior in gabrd dams which can be mimicked with unpredictable stress or exogenous corticosterone administration in wild type mice. further, blocking crh signaling with antalarmin during pregnancy in gabrd mice prevents the adverse behavioral effects on the juvenile offspring. this study demonstrates the utility of gabrd mice in investigating the pathophysiological mechanisms of postpartum depression and implicates hyperexcitability of the hpa axis in postpartum depression-like behavior and the negative impact on offspring development. adult (3 months old) c57/bl6 and gabrd mice were housed at the university of california, los angeles (ucla), division of laboratory animal medicine. the animals (4/cage) were housed in clear plastic cages in a temperature- and humidity-controlled environment with a 12 h light/dark cycle (light on at 6 a.m.) and were maintained on an ad libitum diet of lab chow and water. animals were handled according to protocols approved by the ucla, chancellor's animal research committee (arc). for the pregnancy experiments, c57/bl6 and gabrd adult female mice the female was checked for a vaginal plug and placed into a separated home cage. the pregnant female was individually housed with the single litter until the pups were weaned. for the cross-fostering experiments, the mothers were removed from the native litter and swapped with a surrogate (either wild type or gabrd) immediately following delivery. the juvenile offspring remained in the home cage with the mother (or surrogate) and littermates until all behavioral testing was complete. this study utilized a stress paradigm devised by another research team, which they termed chronic ultramild stress [29, 30] and has been shown to elevate corticosterone levels during pregnancy [29, 30]. in the current study, this stress paradigm is referred to as unpredictable stress (us). wild type and gabrd mice were subjected to unpredictable stress from d14 to d21 of pregnancy as previously described [29, 30]. wild type and gabrd mice at d14 of pregnancy were randomly assigned to two groups: group 1 (stressed) were subjected to an unpredictable stressor (cage tilt, confinement in a small cage, overnight light exposure for a single night, soiled cage for a single 24-hour period, and difficult access to food) during the dark period for 7 consecutive days until d21 of pregnancy unless parturition occurred before at which time subjection to the stressors was immediately ceased. the stressors were alternated to prevent habituation to a single stressor. the periods of stress were separated by stress-free intervals of at least 12 hours. group 2 (controls) were maintained in their home cage without subjection to the unpredictable stressors. behavioral tests were performed on wild type and gabrd dams at 48 hrs postpartum, a time point which has previously been demonstrated to be associated with abnormal postpartum and maternal behaviors in gabrd mice. behavioral tests in juvenile mice began at p21 with the open field test followed by the more stressful forced swim test 24 hrs later. p21 was chosen as the time point to test the impact of maternal behavior on the behavior of the offspring since this time point is prior to weaning and at this time the offspring still share a home cage with the mother. depression-like behavior was assessed in wild type and gabrd dams subjected to unpredictable stress (us) at 48 hrs postpartum and in cross-fostered, cort, and antalarmin-treated offspring at approximately p21 using the forced swim test as previously described [8, 31]. briefly, each mouse was placed individually in a glass cylinder (21 cm 12 cm), containing 9 cm of room temperature water (2225c), in which there is no escape. the latency to immobility and the total duration of immobility throughout the 6 min forced swim test were measured. the mouse was considered to be immobile when it ceased swimming and remained floating motionless, except for infrequent movements of a single hindlimb to maintain being afloat. anxiety-like behavior was assessed in cross-fostered, cort, and antalarmin-treated juvenile offspring at p21 using the open field test. animals were tested in the same testing area, under bright light, with no visual cues. the apparatus was cleaned with ethanol and water in between animals to prevent olfactory cues. the mice were placed into the center of the open field, which consists of a plexiglass container (40 30 40 cm) with a grid of squares (10 7) on the bottom. the amount of time spent in the center (6 3) squares was measured over a single 10 min testing period. the total number of lines crossed (beam breaks) during the 10 min test was also counted. the acute stress paradigm was utilized to measure stress-induced elevations in corticosterone in virgin and postpartum wild type and gabrd mice. an adapted protocol of the co2 exposure paradigm in adult female wild type and gabrd mice was used as an acute stressor. exposure to 35% co2 for 2 min virgin and postpartum (48 hrs) wild type and gabrd mice were randomly assigned to two groups: group 1 (stressed) which were subjected to a single episode of co2 stress (35% for 2 min) and group 2 (controls) which were handled in a similar way as group 1 except they received air instead of air enriched with co2. all animals were handled similarly in which their home cage was inserted into a larger ventilation box where co2 (or air in the case of controls) was administered. blood was collected for corticosterone measurements from wild type and gabrd mice 30 min following acute co2 stress and compared to controls. mice were anesthetized with isoflurane before whole blood was collected from experimental groups by retro-orbital bleeding between 12 and 14 hrs. corticosterone levels were measured by enzyme immunoassay according to manufacturer's specifications (enzo life sciences) as described previously [27, 3436]. briefly, triplicate 5 l plasma samples were assayed and compared to a standard curve using a spectrophotometer (at 450 nm). intra-assay variability of the corticosterone assay was 7.8 ng/ml between paired samples and the interassay variability was 4.4 ng/ml for the same samples between assays. wild type mice at day 14 (d14) of pregnancy were briefly anaesthetized with halothane until unresponsive to a foot pinch and were either sham implanted or implanted with a 21-day release 10 mg corticosterone pellet (innovative research of america, sarasota, fl). the hair from the incision site on the back of the neck was clipped and swabbed with ethanol and iodine prior to making the incision. a small 1 cm incision was made on the back of the neck and a small slow-release pellet (or nothing for sham) was placed underneath the skin using forceps without touching the external area. pup survival was determined at postnatal day 7 (p7) and offspring behavior was assessed in the juveniles at p21. antalarmin was administered to gabrd mice from d14 to d21 of pregnancy in the drinking water to minimize the handling of the animals. at day 14 of pregnancy, the normal drinking water was replaced with the antalarmin solution (10 mg antalarmin/10 l ethanol/100 ml drinking water). the animals were maintained on either vehicle (10 l ethanol/100 ml drinking water) or antalarmin until d21 of pregnancy or immediately after parturition (if before d21) at which time the animals were returned to normal drinking water. this treatment strategy was previously shown to block elevations in corticosterone levels [35, 36]. pup survival was assessed at p7 and offspring behavior was assessed at p21. a one-way anova with tukey's post hoc multiple comparisons test was used to determine statistical significance for comparing more than two experimental groups. student's t-test was used to determine statistical significance between two experimental groups. here we utilized gabrd mice to investigate the impact of maternal depression-like behaviors on offspring behavior. to determine if the maternal behavior, per se, directly mediates the deficits in offspring behavior, we performed cross-fostering experiments. immediately following delivery, the natural mothers of wild type or gabrd litters were replaced with a surrogate wild type or gabrd mother and the behavior of the cross-fostered animals was then assessed in the juvenile offspring at age p21 (figure 1(a)). our data demonstrate that juvenile wild type or gabrd mice reared by mice exhibiting depression-like behavior during the postpartum period (gabrd mice) exhibit increased anxiety-like behavior in the open field test compared to juvenile wild type or gabrd mice reared by surrogate wild type mothers (table 1; figure 1(b)). juvenile mice reared by gabrd mice spent less time in the center squares of the open field test compared to juvenile mice reared by wild type mothers (table 1; figure 1(b)) (n=1217 mice per experimental group; denotes p<0.05 using a one-way anova with tukey's multiple comparisons test; f(3,54)=7.778). however, there is no significant difference in the locomotor behavior, indicated by the number of lines crossed, between offspring reared by wild type and gabrd mothers (table 1; figure 1(c)) (n=1217 mice per experimental group; denotes p<0.05 using a one-way anova with tukey's multiple comparisons test; f(3,54)=2.819). these results suggest that maternal depression-like behaviors negatively impact offspring development, resulting in increased anxiety-like behavior. in addition, both wild type and gabrd juvenile mice, reared by gabrd mothers, exhibit an increase in depression-like behavior compared to mice reared by surrogate wild type mothers. juvenile mice reared by gabrd mice exhibit a decreased latency to immobility and an increase in the total time spent immobile in the forced swim test compared to juvenile mice reared by wild type mothers (table 1; figures 1(d) and 1(e)) (n=1012 mice per experimental group; denotes p<0.05 using a one-way anova with tukey's multiple comparisons test; latency: f(3,39)=4.386; total time: f(3,39)=12.61). these data demonstrate that maternal depression-like behaviors in gabrd mice during the postpartum period negatively impact offspring development and validate the use of this model for investigating the mechanisms mediating the negative impact of maternal depression on offspring development. we proposed that the abnormal postpartum behaviors in gabrd mice may be associated with altered stress reactivity during the postpartum period. postpartum gabrd mice exhibit an increase in corticosterone levels following acute co2 stress (384.6 27.6 ng/ml) compared to postpartum wild type mice (81.0 10.4 ng/ml), virgin wild type mice (241.0 45.3 ng/ml), or virgin gabrd mice (225.6 32.7 ng/ml) (figure 2). however, there is no significant difference in circulating corticosterone levels between unstressed postpartum gabrd mice (20.5 4.5 ng/ml), postpartum wild type mice (16.8 4.1 ng/ml), virgin gabrd mice (59.9 10.6 ng/ml), or virgin wild type mice (30.7 5.3 ng/ml) (figure 2) (n=717 mice per experimental group; denotes p<0.05 using a one-way anova with tukey's multiple comparisons test; f(8,93)=20.94). if altered stress reactivity plays a role in maternal depression-like behaviors in gabrd mice, then we hypothesized that chronic stress would be sufficient to induce the same behavioral disturbances in wild type mice during the postpartum period. wild type mice subjected to unpredictable stress (us) from d14 to d21 of pregnancy (figure 3(a)) exhibit a decrease in the survival rate of their pups compared to unstressed controls (table 1; figure 3(b)) (n: wild type=12 mothers, 100 pups; wild type us=9 mothers, 73 pups; denotes p<0.05 using student's t-test). decreased pup survival is exacerbated in gabrd mice subjected to us compared to unstressed gabrd mice (table 1; figure 3(b)) (n: gabrd=12 mothers, 84 pups; gabrd us: 5 mothers, 36 pups; denotes p<0.05 using student's t-test). dams subjected to us also fail to build a nest and keep the pups at an increased distance from the mother (data not shown), similar to gabrd mice. these abnormal maternal behaviors in wild type mice subjected to the us paradigm are associated with depression-like behaviors in the dams (figures 3(c)-3(d)). wild type mice subjected to unpredictable stress exhibit a decreased latency to immobility and an increased total time spent immobile in the forced swim test at 48 hours postpartum compared to unstressed postpartum wild type mice (table 1; figures 3(c)-3(d)) (n=59 for each experimental group; denotes p<0.05 using a one-way anova with tukey's multiple comparisons test; latency: f(3,22)=2.729; total time: f(3,22)=7.874). these data are consistent with the hypothesis that altered stress reactivity plays a role in mediating abnormal postpartum behaviors. to investigate whether altered stress reactivity in gabrd mice plays a role in mediating the negative impact of maternal depression-like behaviors on offspring development, we either sham-implanted wild type mice or implanted them with a slow-release 10 mg corticosterone pellet on day 14 of pregnancy and assessed offspring behavior at p21 (figure 4(a)). we determined that corticosterone treatment does not interfere with pup delivery or litter size (7.3 0.9 pups) compared to sham implanted mice (6.0 0.8 pups) (n: sham=9 mothers, 54 pups; cort: 10 mothers, 73 pups; denotes p<0.05 using student's t-test). note: litter sizes were determined at the time of delivery since there is a decreased survival rate of the pups born to corticosterone implanted mice (figure 4(b)); however, all pups were alive at the time of delivery. corticosterone levels are significantly elevated in the corticosterone implanted dams at 48 hours postpartum (192.8 50.3 ng/ml) compared to sham implanted mice (29.8 3.2 ng/ml), postpartum wild type controls (16.8 4.1 ng/ml), or stressed postpartum wild type mice (81.0 10.4 ng/ml) (n=1015 mice per experimental group; denotes p<0.05 using student's t-test). corticosterone treatment in the mothers at d14 was sufficient to induce abnormal postpartum behaviors in wild type mice, such as inability to build a proper nest (data not shown) and an increase in pup mortality rate due to cannibalism or neglect (table 1; figure 4(b)), similar to that seen in gabrd mice (n: sham=9 mothers, 54 pups; cort: 10 mothers, 73 pups; denotes p<0.05 using student's t-test). corticosterone treatment in the mother was also sufficient to induce behavioral deficits in their juvenile offspring. juvenile mice (p21) reared by mothers treated with corticosterone spent less time in the center squares of the open field test compared to juvenile mice reared by sham implanted wild type mothers (table 1; figure 4(c)) (n=8 mice per experimental group, 2 mice per litter in 4 different litters; denotes p<0.05 using student's t-test), indicative of anxiety-like behavior. in addition, corticosterone treatment alters locomotor behavior in the offspring of corticosterone-treated mothers, evident from the increased number of lines crossed in the open field test compared to sham implanted mothers (table 1; figure 4(c)) (n=8 mice per experimental group, 2 mice per litter in 4 different litters; denotes p<0.05 using student's t-test). corticosterone treatment in wild type mothers also induced depression-like behavior in the offspring. offspring reared by corticosterone implanted wild type mothers spend an increased total time immobile during the forced swim test compared to juvenile mice reared by sham implanted wild type mothers (table 1; figure 4(d)) (n=8 mice per experimental group, 2 mice per litter in 4 different litters; denotes p<0.05 using student's t-test). these data support the hypothesis that stress hormones, specifically corticosterone, mediate the negative impact of maternal depression-like behaviors on offspring behavior in the mouse. if altered stress reactivity in gabrd mothers plays a role in the impact of maternal depression-like behaviors on offspring development, we hypothesized that inhibiting the stress response in the mother with the corticotropin-releasing hormone (crh) antagonist, antalarmin, would decrease the anxiety- and depression-like behaviors in the juvenile offspring (figure 5(a)). we did not observe any changes in litter size associated with antalarmin treatment (7.6 0.9 pups) compared to vehicle treatment (6.5 0.8 pups). this dose of antalarmin was sufficient to decrease the stress-induced circulating corticosterone levels in postpartum gabrd mice (101.5 12.0 ng/ml) to levels similar to postpartum wild type mice (81.0 10.4 ng/ml) which is significantly lower than the stress-induced levels in postpartum gabrd mice (384.6 27.6 ng/ml). gabrd dams treated with antalarmin exhibit an increase in pup survival compared to controls (table 1; figure 5(b)) (n: gabrd vehicle=8 mothers, 52 pups; gabrd+antalarmin: 9 mothers, 68 pups; significance was determined as p<0.05 using student's t-test). further, antalarmin treatment in the mother was also sufficient to ameliorate the mood disorders in juvenile mice reared by gabrd mothers. juvenile mice (p21) reared by gabrd mothers treated with antalarmin spent more time in the center squares of the open field test, which is indicative of decreased anxiety levels, compared to juvenile mice reared by vehicle-treated gabrd mothers (table 1; figure 5(c)) (n=810 offspring per experimental group, 2 mice per litter in 4-5 different litters; significance was determined as p<0.05 using student's t-test). there was no significant difference in the number of lines crossed in the open field test between the offspring of antalarmin-treated and vehicle-treated gabrd mothers (table 1; figure 5(c)). similarly, offspring reared by antalarmin-treated gabrd mothers exhibit decreased depression-like behavior, evident by an increased latency to immobility and decreased total time spent immobile compared to offspring reared by vehicle-treated gabrd mothers (table 1; figure 5(d)) (n=810 mice per experimental group, 2 mice per litter in 4-5 different litters; significance was determined as p<0.05 using student's t-test). these data demonstrate that inhibiting the stress response, such as with the crh antagonist, antalarmin, is therapeutic in ameliorating the abnormal postpartum behaviors in gabrd mothers as well as preventing the negative impact of maternal depression-like behaviors on offspring development. this study highlights the utility of a unique mouse model to investigate the underlying mechanisms mediating the pathophysiology of postpartum depression and the mechanism(s) through which postpartum depression negatively impacts offspring development. it is generally accepted that both genetic and environmental factors play a role in the pathophysiology of postpartum depression. however, either the current animal models exhibit a genetic predisposition for depression-like behavior or the behavior is environmentally induced. here we describe a genetic mouse model exhibiting depression-like behavior that is restricted to the postpartum period, which is aggravated by environmental stress similar to the human condition. this is the first genetic mouse model which exhibits a predisposition to postpartum depression-like behavior in which there is also an environmental component. therefore, we feel that this is a useful model for studying the mechanisms mediating postpartum depression-like behavior and the accompanying deficits in offspring development. clearly, hormone changes throughout pregnancy and the postpartum period trigger the onset of postpartum depression. however, gonadal hormone levels do not appear to be significantly altered in women with postpartum depression [3941], suggesting that women must be predisposed to the disorder. during pregnancy, levels of estrogen and progesterone steadily increase due to placental production of these hormones, which decrease abruptly with the removal of the placenta. however, no change in estrogen or progesterone levels has consistently been shown to be associated with postpartum depression. there are numerous other hormonal changes that occur during pregnancy, including changes in oxytocin, prolactin, and cortisol levels (for review see). however, no alterations out of the physiological range were found for prolactin [41, 43], oxytocin, or vasopressin associated with postpartum depression. hypercortisolism has been suggested to play a role in the pathophysiology of postpartum depression, since major depression is also associated with hypercortisolism. normally, the stress-induced activation of the hpa axis is suppressed during pregnancy (, for review see), consistent with our observations in postpartum wild type mice (figure 2). altered levels of cortisol [11, 18, 48], acth, and crh have been associated with postpartum depression. researchers have gone so far as to say that elevated crh levels may be used as a diagnostic criterion for postpartum depression. however, other studies have failed to reproduce these results ([ 41, 43], for review see). here we demonstrate hyperresponsivity of the hpa axis associated with depression-like behaviors during the postpartum period in a mouse model, similar to what has been observed in women with postpartum depression. consistent with a role for hpa axis hyperresponsiveness in postpartum depression-like behaviors, this study supports a role for elevated corticosterone in the pathophysiology of postpartum mood disorders, since physiological stress is sufficient to induce depression-like behavior during the postpartum period in mice (figure 3). previous studies have demonstrated that corticosterone alters maternal care and induces postpartum depression-like behaviors in the dams. here we demonstrate that exogenous corticosterone treatment in wild type mice during pregnancy results in a robust decrease in pup survival (figure 4). this has previously been observed, albeit not to the same extent as in the current study. the discrepancy may be due to a prolonged exposure in our study to levels of corticosterone normally found in stress. however, we can not rule out potential abnormalities in the pups due to corticosterone exposure which may impact pup mortality. interestingly, our study demonstrates that physiological stress in the dams is sufficient to increase pup mortality in both wild type and gabrd mice (figure 3(b)). however, unpredictable stress does not alter depression-like behaviors in postpartum gabrd although it increases depression-like behaviors in wild type mice (figures 3(c) and 3(d)). we interpret these data to indicate that physiological stress is incapable of altering depression-like behaviors in postpartum gabrd mice in which corticosterone levels are already elevated, demonstrating a potential ceiling effect. similarly, our results demonstrate that the crh antagonist, antalarmin, increases pup survival in gabrd mice (figure 5). these data demonstrate a direct role of stress hormones in postpartum mood disorders and may contribute to the negative impact of maternal depression on offspring development. children exposed to mothers with postpartum depression exhibit deficits in cognitive development, motor, and emotional development (for review see). many mechanisms have been proposed to mediate the negative association between maternal depression and offspring development, including environmental and genetic components. it is also possible that there is a direct, biochemical component of maternal depression which impacts offspring development. deficits in child development associated with maternal depression are correlated with elevated cortisol levels in the mother [53, 54], suggesting that the stress hormones may play a role in the negative impact of maternal depression on child development. consistent with this theory, we demonstrate behavioral deficits in mice reared by gabrd mothers, which exhibit hyperresponsiveness of the hpa axis, and offspring reared by wild type mice subjected to us or treated with exogenous corticosterone. further, corticosterone treatment in dams has previously been shown to result in adverse behavioral effects in the offspring, which could be a direct effect of corticosterone levels in the offspring which may alter subsequent hpa axis activity. these data support a direct role of stress hormones in both the pathophysiology of postpartum depression and the negative impact of maternal depression on offspring development. in the current study, the mouse models which exhibit abnormal postpartum behaviors and a negative impact offspring development, including gabrd mice and wild type dams subjected to unpredictable stress or treated with exogenous corticosterone, exhibit a high degree of pup mortality due to cannibalism and/or neglect. this is in contrast to the human condition of postpartum depression which is not typically associated with infant mortality or infanticide. neonaticide and infanticide are more commonly associated with postpartum psychosis. however, this is difficult to assess in mice and requires further study. due to the high level of pup mortality in the mouse models exhibiting abnormal postpartum behaviors and a negative impact offspring development, including gabrd mice and wild type dams subjected to unpredictable stress or treated with exogenous corticosterone, we can not rule out both the impact of changing litter size on maternal behaviors and the impact on offspring development. relevant to the current study, smaller litter size is associated with more directed maternal care [57, 58]. further, mice reared in smaller litters also exhibit decreased anxiety-like behaviors during adulthood. these findings are in contrast to the current study where we observe a decreased litter size in gabrd litters associated with abnormal maternal care and increased anxiety- and depression-like behaviors in the offspring reared by gabrd mice (figure 1). thus, it does not appear that litter size impacts the findings in the current study. this study directly demonstrates the negative impact of postpartum depression-like behavior and deficits in maternal care on offspring behavior. further, our data suggest a role for hpa axis dysfunction in mediating the negative impact of maternal mood on offspring behavior.

maternal depression has been shown to negatively impact offspring development. investigation into the impact of maternal depression and offspring behavior has relied on correlative studies in humans. further investigation into the underlying mechanisms has been hindered by the lack of useful animal models. we previously characterized a mouse model which exhibits depression-like behaviors restricted to the postpartum period and abnormal/fragmented maternal care (gabrd/ mice). here we utilized this unique mouse model to investigate the mechanism(s) through which maternal depression-like behaviors adversely impact offspring development. cross-fostering experiments reveal increased anxiety-like and depression-like behaviors in mice reared by gabrd/ mothers. wild type and gabrd/ mice subjected to unpredictable stress during late pregnancy exhibit decreased pup survival and depression-like behavior in the postpartum period. exogenous corticosterone treatment in wild type mice during late pregnancy is sufficient to decrease pup survival and induce anxiety-like and depression-like behaviors in the offspring. further, the abnormal behaviors in juvenile mice reared by gabrd/ mice are alleviated by treatment of the mothers with the corticotropin-releasing hormone (crh) antagonist, antalarmin. these studies suggest that hyperresponsiveness of the hpa axis is associated with postpartum depression and may mediate the adverse effects of maternal depression on offspring behavior.

PMC4706939

pubmed-947

increasingly, gerontological researchers, practitioners, policy makers, and planners are concerning themselves with the growing importance of aging in place. aging in place does not have one single definition but broadly is considered to be the ability to continue to live in the environment of one's choice, even when declining competence reduces or threatens independence, while allowing for consumer choice in the types of services delivered. lawler suggests that aging in place strategies can minimize inappropriate care and work best as a comprehensive and holistic approach to the needs of aging individuals and communities. lau and colleagues have conceptualized a framework for aging in place safely and acknowledge the importance of multiple factors, including the biological and psychological characteristics of the individual, the network of social support, formal services, the need for medical services, and the structure of the home and neighborhood. this and other frameworks clearly recognize that aging in place strategies must consider not only the personal (micro) environment, including housing, but also community and structural components as well [4, 5]. before embarking on a discussion of elder-friendly communities, it is important to discuss a number of theoretical frameworks and conceptualizations from gerontology that help inform our understanding of aging in place. there are numerous frameworks that are relevant to aging in place including ecological theory, person in environment, and social inclusion/exclusion. in addition, the area of environmental gerontology has specific relevance to this discussion. ecological theory suggests that there is a mutual relationship and mutual reciprocity between individuals and their environment and that this interaction occurs at multiple levels, including the micro-, exo-, mezzo-, macro-, and chronosystems levels. ecological theory is important for the concept of aging in place as it suggests that individuals interact with multiple levels of environment in their day-to-day lives. older people must not only interact with microenvironments such as their home and immediate family, but also with broader systems that can equally influence their ability to age in place. this perspective, like ecological theory, acknowledges that the environment interacts with individuals at multiple levels and suggests that the environment is not a static backdrop but rather continually changes. from the person-in-environment perspective, the older person must continually take from the environment what he or she needs, control what can be modified, and adapt to conditions that can not be changed. also of relevance to, the theory of social inclusion/exclusion examines the role of older people and highlights the social costs when individuals, families, or communities are excluded from or become disengaged from larger society due to characteristics such as poverty, gender, ethnicity, or neighborhood. scharf and colleagues conceptualize the inclusion and exclusion of older people as associated with three key themes: participation and integration (beyond the labor market), spatial segregation, and institutional disengagement. of particular interest in our exploration of aging in place is the thematic area of participation and integration. posit that participation and integration not only include older people's involvement in community life, but also are associated with their social capital, including civic participation, and the nature of social networks and mutuality/reciprocity. in addition to several theoretical frameworks, the field of environmental gerontology has specific relevance to the topic of aging in place. wahl and weisman suggest that environmental gerontology's (eg) theories and findings can and should influence the development of age-friendly communities. for example, eg is concerned with the role of neighborhoods and the influence those neighborhoods have on opportunities and constraints of residents. at a more macrolevel, eg recognizes the community as a locus of aging with a sociophysical and policy perspective. with regard to elder-friendly communities, we can draw upon the work of lawton who posited that the environment has three major functions of maintenance, stimulation, and support. maintenance is concerned with the consistency and predictability of one's environment, while stimulation is concerned with the effect of stimuli on behavior. finally, support is concerned with the environment's potential to compensate for diminished or lost competencies. in recent years, the concept of elder-friendly communities has become central to the notion of aging in place. described in various ways, an elder-friendly community is a place where people can live their entire lives, if they so desire, rather than having to relocate and lose their social capital [14, page 6]. an elder-friendly community examines the environment in more macro-level terms as places where older people are actively involved, valued, and supported by an infrastructure that accommodates their needs. in what was perhaps the first on-line conference focusing on elder-friendly communities, the sierra health foundation suggested that elder-friendly communities are those communities in which age is not considered a barrier to improving lifelong interests and activities, where support and accommodations exist to meet the basic health and social needs of those with age-related disabilities, and where opportunities exist for older adults to develop new sources of fulfillment and engagement. while the literature on elder-friendly communities is to a degree embryonic, several models have been developed in recent years. among these models created in the united states, canada, and europe, for example, feldman and oberlink's work on the advantage initiative demonstrated that elder-friendly communities must address basic needs, optimize well-being, maximize independence, and promote civic engagement. the city of calgary elder-friendly community project noted that feeling safe, being valued and respected, staying active, and building community were important elements of an elder-friendly community. the world health organization (who) has established international guidelines for age-friendly communities that include the encouragement of active aging by optimizing opportunities for health, participation, and security in order to enhance people's quality of life as they age. according to the who, an age-friendly city adapts its structures and services to be accessible to, and inclusive of, older people with varying needs and capacities. while various models have emerged identifying aspects key to the concept of elder-friendliness, a consistent theme found in the literature is associated with social interaction or social connectedness. an elder-friendly community will assist older adults to maintain social connectedness while deepening existing relationships. such a community will recognize the social capital of these relationships that in turn result in contribution. the concept of contribution recognizes the wisdom and experience of older citizens and sees them as more than clients, but rather as active contributors to community well-being. similarly, the calgary project identified as important the active participation of older people in their communities. this premise is consistent with the work of rubinstein and colleagues who found that the ability to actively manage one's environment was a source of well-being for older adults. similarly, the model of age-friendly communities developed by the who clearly recognizes that social participation and social support are strongly associated with overall well-being, allowing elders to exercise their competence and enjoy the respect and esteem of their community. alley and colleagues remind us that a community's respect for older adults, which includes available opportunities, contributes significantly to their quality of life. while social participation and connectedness are important in an elder-friendly community for example, the advantage initiative promotes the importance of civic engagement, including meaningful connections, volunteer and paid opportunities, and the prioritization of aging issues. the who acknowledges that an age-friendly community provides the option for older adults to continue to contribute to their community through civic engagement with both paid and volunteer opportunities and to have the ability to be active in the political process. the benefits of such reciprocity are many, such as an increased sense of purpose and satisfaction for older adults as they engage with the community, while younger community members may benefit from the knowledge and experience older adults bring to the community. as an example, intergenerational programs recognize the knowledge and skills possessed by older adults that can be shared with youth, while providing opportunities for civic engagement for the older person. much of the research on elder-friendly communities has highlighted the multidimensional nature of community life and has not focused primary attention on social connectedness despite the importance of interdependence and engagement as primary qualities of aging in community. for example, the advantage initiative identifies social and civic engagement but used quantitative measures to evaluate communities in three preordained realms. if an age-friendly community is a positive place to grow old, then the views of younger citizens (baby boomers, for example) need to be taken into account. alley and colleagues suggest that in an age-prepared community, processes of planning and advocacy are utilized to foster aging in place, which may be a prospective view of what is needed in planning for future community needs. this process must take into account the views and needs of the citizens who are not yet defined as older adults, but who will bring their own needs and views to the community. the purpose of this paper is to further elucidate the importance of social relationships and social connectedness in developing an elder-friendly community. the process used in the project described here was inclusive of younger adults (age 4065) as well as older adults (65 +) in order to help understand how they envision a community that could support their own aging. community [15, page 8] as one which has assessed its current services and is planning for the needs of future populations. second, the qualitative methodology used in this study allowed for a more naturalistic and personal narrative. padgett acknowledges the importance of meaning making in the narrative process that includes storytelling, conversation, and discourse of naturally occurring speech. this study, therefore, was informed by the perspective of narrative analysis and the use of the spoken and written word in narrating the meaning of social connectedness as we age. in april of 2002, surveys related to assessing the elder friendliness of communities were completed by 5.100 individuals, 65 and over, throughout 10 cities across the united states. in one participating community in western washington, findings suggested that older adults in that community were satisfied with their neighborhoods and participated in religious or cultural activities, and the majority of respondents were engaged in health screening. the vast majority of these respondents had participated in some type of social activity in the past week and slightly fewer than one in three people volunteered. the survey results were promising and positive, yet are now dated and do not reflect the opinions of members of the aging baby boom generation. second, the original survey did not focus specifically on the issue of social connectedness but limited the focus to volunteering and participation in cultural and religious activities. recognizing the need to better refine and focus attention on the importance of social connectedness as part of elder-friendly communities, a city committee responsible for the continuation of the elder-friendly community agenda sponsored a community forum in october of 2009. a community forum using the world caf format was conducted in order to engage community members, 40 years and older, in conversation about the importance of social connectedness in elder-friendly communities. previous research in this area has approached the topic of social connectedness through an a priori definition of social engagement, primarily utilizing quantitative methods for measurement and evaluation. this forum, however, sought to understand social connectedness from those approaching retirement using a more naturalistic method. a second purpose of this forum was to obtain data on what would keep aging boomers in their community as they age. the results of the forum and its applicability to elder-friendly communities and aging in place research are being presented here. the world caf is a concept that was born out of appreciative inquiry, which is a form of research that emphasizes the positive aspects of an experience, particularly how that experience can foster creativity among people. the world caf format involves exchanging ideas and sharing different points of view in a safe, intimate setting with the purpose of coalescing wisdom and experience into learning. a foundational component of the world caf concept is conversations, purposeful conversations that have a reason for taking place, conversations that matter [25, page 4]. they may be initiated to solve a community problem or to envision a preferred future, in this case an elder-friendly community, with a focus on social connectedness. the world caf format places an emphasis on moving from simply talking to taking action. this movement takes place as participants are able to understand the connection between talking and acting, or conversation as action. it was in this context of sharing collective discoveries [25, page 138] that the community forum took place. this study provided an opportunity to test the value of the world caf format as a method for future research. this study was determined to be an exempt study by the university of washington human subjects division. the method employed for this study involved a melding of the world caf format as the structure of the study with a focus group format as the process that informed data collection in the study. first, groups formed, discussed, and reformed with different participants for each of the three main questions that were posed at the forum. second, instead of the more customary audio or video taping of the groups, each table was covered with paper on which participants wrote and/or drew as they discussed the topic at hand. these notes and doodles became the transcript along with notes taken by each table leader. this is consistent with narrative analysis in which both spoken and written words are used in meaning making. finally, groups were given great latitude as to how they addressed the discussion topic for their table. the discussion leaders at each table helped to keep the group on topic and were careful not to inject their opinions into the group discussion. the setting for the study was a community forum for those over 40 years of age living within the school district boundaries of a suburban community in western washington with a population of approximately 37.000, whose residents are predominately caucasian (87%). the forum included refreshments, and people were invited to sit at one of several round tables covered with paper for writing thoughts as they occurred to the participants. the conversation at each table began with the posing of one of three questions, with ample time allowed for each table group to discuss, strategize, and imagine a preferred future in an elder-friendly community. the three questions were as follows (1) what does it mean to you to be socially connected? (2) how can our city help with life transitions that would keep you in this community? these three questions were developed through consensus by the city level committee charged with examining issues and processes that enhance an age-friendly community. the questions were designed to determine how people define and make meaning of being socially connected, to identify aspects of community life that would reinforce continuity with the community versus relocation to another community after retirement, and to ask participants to think about their own value to the community, thereby initiating thought around the idea of social reciprocity. conversation was not limited to only the question at hand, and participants were invited to speak, draw, and write about the broader topic throughout the session. at set times, participants were asked to move to a different table, to be with a different group of people, and to consider a different question, until all three main questions were answered by most of the participants. one member from each table stayed behind during the rotation in order to serve as an ambassador for the previous members, thus assisting in continuity of conversation. a goal was to allow participants to engage creatively as they tackled the questions together. so, rather than gather individual feedback, table leaders encouraged participants to converse with each other and to spend time thinking together about potential solutions to dilemmas as they were raised by group members. once the group session was completed, participants were invited to gather into a large group to debrief and discuss the most important topics from the perspectives of the participants. a purposive sample of people over age 40 was recruited through newspaper ads and invitations from the city parks and recreation department and through the aging in place committee (aip) membership. membership lists from the senior activity center and local faith communities also served as sources for potential participants. the invitation requested community members to participate in a community forum to discuss how to create, promote, and maintain a more elder-friendly community. ultimately, 23 individuals participated in the community forum and ranged in age from midforties to late eighties. participants therefore represented both those who might be identified as baby boomers as well as those who are currently retired and may be defined more traditionally as older adults. we did not collect specific data on age, but some participants offered their age as part of the conversations. since this was originally conceived by the aip committee as a community forum and not a research project, no additional sociodemographic data were collected on socioeconomic status, education, or other typical variables associated with creating a demographic profile. following the world caf community forum, researchers were asked to analyze the data from the event in order for the aip committee to present findings and make recommendations to city government officials. no identifying information about participants was included with the data provided for analysis. using an approach consistent with grounded theory first, they met together and carefully reviewed the data from each of the questions. they used an open coding process for notes of verbal exchanges, drawings and notes from participants, and memos from group leaders. the few illegible writings and unrecognizable doodles were dismissed from the analysis process. as categories began to emerge, the researchers engaged in conversation about meanings and interpretations, until they were satisfied they had a clear understanding of the data. in order to confirm that trustworthiness of the data was maintained, once the themes were identified, the aip committee reviewed the findings and then invited all of the original forum participants to attend a focus group to discuss the findings. the focus group was held in the same location as the community forum approximately two months after the forum was convened and was made up of five individuals (approximately 20% of forum members). like the forum participants, most focus group participants were female and caucasian, with one or two individuals representing communities of color. the focus group participants reviewed, clarified, and added data to the transcripts and confirmed that the themes identified by the researchers were reflective of the community meeting. the review by the focus groups provided credibility and trustworthiness (validity) to the qualitative findings, reinforcing a fit between the respondents ' views and the researchers ' interpretation as well as being confirmatory, for example, demonstrating that the study's findings were not imagined. this process, known as member checking, not only serves to validate findings but is empowering to the participants and reinforces the close relationship between the researchers and the informants in qualitative research. the researchers identified three major themes that emanated directly from the data and were confirmed by the focus group. all three themes emerged from the open coding and were ultimately labeled as follows: social reciprocity, meaningful interactions, and structural needs/barriers. this theme was directly related to the overarching focus on social connectedness but illustrated the importance of added value in these relationships. within the theme of social reciprocity, giving and receiving to/from one's community were both seen to be of equal importance. some participants were currently volunteering or communicated an interest in doing so (giving). while exact ages were not available, it appeared that older adults (65 +) were more likely to be active volunteers than their younger counterparts. baby boomers expressed interest in volunteerism, while older adults may have already engaged in that process if they were interested. many participants expressed an interest in receiving through such things as enhanced educational opportunities (e.g., more age-friendly options from the local community college and public university). the idea of educational opportunities at no or low cost was initially mentioned by younger participants. participants also indicated that venues for creating social connectedness could come from both formal and informal entities. formal entities are those which would require some infrastructure involving an organization or business, such as theater, outdoor concerts, or free movie nights. an example might be the initiation of social activities through city government, the local chamber of commerce, or even a local business. informal entities would include activities that require limited resources, such as the creation of book clubs or neighborhood gatherings. participants also suggested that such activities aimed at increasing social connectedness could be sponsored or influenced by community resources. for example, through the senior activity center, the city might sponsor a new boomer or senior walking group. the city government, for example, could attempt to influence the policy of a not- for- profit community organization regarding how cumbersome and degrading the process is for older adults with limited income to obtain reduced membership fees. for example, a nongovernmental organization such as a church could recruit older volunteers from their congregation to volunteer in local schools. the theme of social reciprocity can and should conceptually occur at multiple levels, such as between governmental and nongovernmental organizations, as well as between individuals and their community. in all aspects of the data, reciprocity (the mutual exchange of commercial or other privileges) was exemplified as the willingness to give and receive in order to foster social connectedness. inherent in the discussion of social reciprocity was the notion that the relationship between the individual older persons may occur at multiple levels of community and environment. relationships and mutual exchange might occur at the level of neighborhood, a community organization, or at the level of city government or policy advocacy. for example, some forum participants suggested helping others by providing space for a communal garden (neighborhood), while some suggested that developing a volunteer position to work as a senior ombudsman related to negotiating city services would be beneficial to the whole (city government level). this exchange improves the well-being of those being helped while fostering a sense of accomplishment and service. while participants discussed the desire to give and receive in order to maintain social connectedness, they were clear, however, that these experiences should be meaningful both to themselves and others. while a high number of forum participants expressed a desire to volunteer in their community, they clearly stated that the activity should be meaningful to them and important to the community. this sentiment communicates the view that these individuals see themselves as having social capital (whether or not it is recognized by others). as one participant put it, we should all volunteer, even if it is in the home the participants shared a collective view that the purpose of volunteering was not to kill time. rather, participants were interested in sharing their passions, time, sense of history, and even sharing personal space to accomplish this end. one participant suggested that people share their gardens with others or help others to do crafts in their homes. participants also viewed volunteering as a way they could advocate for others and for their community. finally, if participants were to be involved in meaningful interactions through volunteering, they wanted to feel appreciated for the work they did. they voiced the concern that organizations often diminished or ignored the value of their time as volunteers and took volunteers for granted. it is important to note here that forum participants did not suggest they wanted to volunteer for the sake of recognition, but rather they felt the need to be valued not taken for granted. the message that was communicated by forum participants was that they desired both the organization/community in which they served as well as themselves to view their contributions as meaningful. while speculative due to a lack of specific data on age, the older participants appeared more settled in their roles as volunteers, as many of them had held these roles for some time. younger adults (boomers) appeared to have more concerns about the meaning they derived from volunteer opportunities and how that may be accomplished. while the majority of participants provided feedback on what or how they could contribute to their community to enhance social connectedness, a similar number of people voiced substantial frustration with the lack of either organized opportunities or communication with potential organizations with which to volunteer. these issues were impediments to social reciprocity as well as to meaningful interaction, and as such were labeled as structural needs or barriers. structural (infrastructural) needs or barriers were those things participants viewed as currently lacking in the community but, if present, would facilitate social reciprocity both in terms of physical and social venues. for example, many forum participants expressed the need for improved methods by which potential volunteers could be connected to opportunities (community entities). the examples that were given included organizations that needed volunteers should return phone calls more promptly to potential volunteers, as well as the need for more personal connections between those requesting volunteers and the people who might be willing to give of their time. they were not interested in having to make numerous inquiries to potential organizations in order to volunteer. the feeling expressed was that there was a lack of reciprocity from the very beginning on the part of agencies or organizations with which these individuals might wish to volunteer. transportation was described as an additional structural barrier and was mentioned frequently in all table conversations. transportation was viewed as an essential element of social connectedness. in areas of both volunteerism as well as overall social connectedness, transportation issues associated with public transit and walkable communities issues concerning transportation included that a lack of reliable, frequent, and accessible transportation created barriers for participants within the community. the view communicated by these participants was that improved transportation can foster and enhance social connectedness by decreasing barriers of distance and reducing the need for use of one's personal vehicle. while younger participants voiced interest in improved transportation as a means toward improved social connectedness and as an environmentally friendly alternative to automobiles, older participants expressed a more urgent need for improved transportation, as well as having a more specific personal need. for example, one couple who was likely in their 70s expressed the need for improved transportation services for their parents (in their 90s) as they identified gaps in transportation services as personally problematic. the purpose of this research project was to analyze data gathered from aging individuals (including baby boomers) on the importance of social connectedness in the creation of elder-friendly communities through a naturalistic method of inquiry. by engaging in a more naturalistic conversation utilizing the world caf format, the participants in the study were able to utilize conversation in meaning making without the confines of any a priori assumptions about social connectedness. the findings from this community forum and the subsequent focus group reinforce earlier data from the original advantage initiative as well as other literature on elder-friendly communities and point to the utility of several important theories. first, these findings echo the original framework from the advantage initiative, which emphasizes the importance of social and civic engagement. the individuals from this community forum, as well as the elder counterparts in the original study, underscored the importance of meaningful connections to family, friends, and neighbors as part of civic engagement. an elder-friendly community needs to find new ways to promote active and continual engagement in community life. the findings from this study parallel the view of scharlach who suggests that as we get older and ever closer to the end of our lives, maintaining social connectedness and deepening existing relationships becomes a priority [14, page 9]. these findings also reinforce the importance of participation and integration, which is a critical element of social exclusion theory. forum participants identified multiple activities associated with social inclusion/exclusion including production (economic or socially valued) activity, political activity to improve or protect the social environment, and social activity that involved engagement with family, friends, and community. scharf and colleagues define participation and integration as older people's embeddedness in social networks and the extent to which older people contribute to or draw upon social capital that exists in their neighborhoods [8, page 316]. thus, our findings related to social reciprocity appear consistent with the major theme from social inclusion/exclusion theory. a community needs to be dynamic in order to support changes in the older citizenry. while the concept of support is typically relevant to adjustment to altered or lost competencies, the concept of support can be extended to include the need for continued and changing modes of social and civic engagement. this study also reinforces both the importance of volunteer opportunities and that those opportunities be purposeful and meaningful. as suggested by scharlach, in an elder-friendly community, older adults are not just seen as clients or passive recipients of services, but active contributors to the well-being of the community [14, page 9]. in the original advantage survey, residents from this community volunteered at a rate substantially lower than the national average for the 10 advantage communities. what we learned from this study was that aging community members held interest and motivation to volunteer or otherwise be engaged in their community. we believe they see themselves as having social capital, but as putnam points out, others may not always share their view. the environmental function of stimulation is relevant here as participants seemed to look to their community for stimuli for enhanced social well-being and to elicit new and relevant social and leisure behaviors. older participants appeared more likely to have volunteer and community activities in place, while younger adults (boomers) were perhaps seeking out methods for accomplishing that goal. both younger and older participants also noted structural barriers to social connectedness and social integration, supporting alley et al. who suggest that while communities may be able to support aging in place, they may also contain barriers that make community living difficult for older residents. a recent study of 253 older adults reinforces the importance of organizational structure in volunteerism. tang et al. found organizational support (defined as choice of volunteer activity, training, and ongoing support) to be associated with socioemotional benefits, including perceived contribution and personal benefits. these researchers concluded that the psychological well-being of older adults can be improved through engagement in meaningful volunteer activities and contribution to others [30, page 603], again reinforcing what rubinstein and colleagues noted concerning the connection between well-being and active environmental management. in order for these benefits to occur, however, an elder-friendly community must work to eliminate structural and organizational barriers to volunteerism and social connectedness. as scharf and colleagues assert, participation and integration are enhanced by good public service such as access to reliable transportation. to not provide such services serves to reinforce the social exclusion of older people. the identification of structural barriers also reinforces the person-in-environment perspective that the needs of older people change over time and must be successfully navigated in order to maintain social integration. if a community is to be elder-friendly, the infrastructure needs to be consistent and predictable at the very least, while at the same time dynamic in its ability to provide stimulation and support. community-based research is particularly useful when it is able to identify problems and move toward a resolution of that issue. researchers can partner with communities to study areas of interest, interpret results, and assist in the empowerment of community members to make changes. the findings from this study have already resulted in community level change efforts related to volunteerism. an annual volunteer fair was initiated in 2010 with the goal of creating a venue to match older volunteers with community level volunteer opportunities. this newly formed activity grew directly out of the identification of structural barriers in this research and was created through a partnership of senior advocates, the community's aip committee, and local organizations, including the area hospital. in the first year of operation, more than 80% of older adults were successfully matched with local organizations, thus improving social connectedness, integration, and reciprocity in a direct and clear way. this event has now been established as an annual event sponsored by seniors, city government, and other community entities. its goal is to improve civic engagement among older residents, thus fostering the connectedness between older residents and organizations that serve the community. in addition to the importance of civic engagement, the philosophy of aging in place supports the continued importance of maximizing independence for not only the frail and disabled, but for aging adults of all abilities. in particular, these findings point to the need for accessible and available transportation, an issue that city officials and community advocates should attempt to improve through partnerships. as feldman and oberlink noted in their original findings, transportation and safety are fundamental factors that enable older adults to stay connected to the community [17, page 5]. rosenbloom suggests that transportation in elder-friendly communities will need to be planned to provide more customized services, linking residential concentrations with important destinations, including volunteer opportunity destinations. the project findings noted that while all participants voiced the need for improved transportation services, the kinds of services desired may change with age. the lack of this kind of transportation was clearly identified as a major structural barrier reinforcing social exclusion and needs to be considered as future planning takes place. with impending cuts to public transportation, the aging in place committee is examining potential alternatives to improve transportation through private and voluntary means. the results of this study provide important information on social connectedness in elder-friendly communities. first, as a qualitative and naturalistic study, the findings are the specific views of those individuals involved and can not be generalized to any larger population of aging adults. second, a further limitation is that those who responded to the invitation to participate in the community forum may have had a greater interest in the topic, or a vested interest in having their voices heard as compared to those who did not or could not attend. however, the study results provide a new dimension to the subject area and support previous studies and theories on aging in place, thus adding to the picture of what needs to be done to support the creation of elder-friendly communities. because of the homogeneous makeup of forum participants, the voices of other communities such as communities of color were not clearly heard. it must be acknowledged that the opinions and concerns of this group do not likely represent all older adults in this community. finally, because sociodemographic data was not collected on individuals, distinctions between older (65 +) and younger participants are based upon educated guesses about participant's age. the results of this study reinforce the importance of social connectedness, participation, and integration in creating and maintaining elder-friendly communities and suggest that the findings are areas of concern not just for the old-old, but for recent and soon-to-be retired individuals wishing to maintain life satisfaction. the study suggests the possibility of using more nontraditional research techniques for gathering community level data such as the kinds of findings generated from the world caf process. while creating and fostering elder-friendly communities can be a long and ongoing process, small incremental change can occur from such studies as is illustrated by the case of the annual volunteer fair now established in this community. if a national agenda of enabling our aging population to age in place is to be accomplished, creating elder-friendly communities has a logical and important role. scharf et al. suggest an important association between social connectedness and quality of life. they found that older people who rated their quality of life as good were less likely to experience social exclusion. for aging in place to happen successfully, with older adults being continually valued and integrated into community life, city officials, policy makers, and gerontological researchers will need to collaborate in order to move these ideas from research to reality.

the purpose of this paper is to further elucidate the importance of social relationships and social connectedness with aging in place and in developing elder-friendly communities. the process used in this study was inclusive of younger adults (age 4065) as well as older adults (65 +) in order to further understand how they envision a community that could support their own aging in place. a community forum, using the world caf format, was conducted in order to engage community members, 40 years and older, in conversation about the importance of social connectedness in elder-friendly communities. a second purpose of this forum was to obtain data on what would keep aging boomers in their community as they age. three major themes emerged from qualitative analysis of the forum: social reciprocity, meaningful interactions, and structural needs/barriers. the results of this study reinforce the importance of social connectedness in creating and maintaining elder-friendly communities for older adults, as well as soon-to-be retired individuals, wishing to maintain life connectedness to their community. the study suggests the possibility of using more nontraditional research techniques (such as the world caf process) for gathering community level data.

PMC3227400

pubmed-948

lotus (nelumbo nucifera) belongs to the family of nelumbonaceae, all parts of which are edible in various forms. it is generally consumed as vegetable; chiefly the stem part is processed in different forms such as roasted, pickled, dried, and fried. the plant exhibits multiple nutritional and medicinal properties, hence considered as a popular health food. the alkaloid (liensinine) extracted from the stem is effective in treating arrhythmia, sunstroke, fever, dysentery, diarrhea, dizziness, and stomach problems. its seeds find applications in folk remedies as a diuretic, cooling agent, antiemetic, and an antidote in the treatment of tissue inflammation and cancer. biochemically, the rhizomes are composed of proteins, fats, carbohydrates, and minerals and are a good source of energy. starch is considered to contribute to the textural properties of various foods and has several industrial applications as a thickener, stabilizer, adhesive, gelling, and water retention agent. lotus is loaded with starch, which is commercially available in china and japan having numerous industrial applications as thickening agent in food products. scanning electron microscopy of lotus stem starches revealed small rounded and typical oval shaped granules with a smooth surface. swelling, solubility, and water absorption were improved with increasing temperature from 50 to 90c. consumer preferences lead to the consumption of lss in preparations such as breakfast meals, fast foods, and traditional confectioneries and also the utilization of lss as potential food additives. processes such as extrusion, baking, and pressure-cooking which employ heat-moisture treatment with pressure and shear have foremost applications in production of snack foods, ready-to-eat (rte) foods, breakfast cereals, and porridge. high proportion of starch is employed in formulations involving gums, salts, and sugars as potential ingredients to carry out reaction with starch in presence of water subsequently modifying its physicochemical properties. hence, this interaction between additives and starch is of immense concern to food scientists. hydrocolloids (gums) are utilized in food industries since they improve stability, modify textural profile, and reduce the retrogradation rate of the starch. mandala and bayas investigated the effect of xanthan gum on swelling power, solubility index, and granules status of wheat starch dispersion (2% w/w). according to swelling power values and granules dimensions at 75c, xanthan addition enhanced swelling. foods containing starches hold usual nature of changing the physicochemical properties frequently by addition of salt and sugar during food processing and storage. rice starch with high content of amylopectin exhibits better swelling properties, while salt addition reduces it considerably. sugar affects color, flavor, dimension, hardness, and surface finish of the food product. sugar tends to disperse the protein and starch molecules making the product fragile, thereby preventing the formation of a continuous mass. jin et al. reported decreased water solubility index of corn meal on sugar addition due to inhibited degradation of the starch molecules in presence of sucrose. so far, there is no information on the effect of such additives on the physical properties of lotus stem starch. hence, the aim of this study was to determine the effect of additives, that is, acacia gum, nacl, and sucrose on the physical properties such as water absorption (wa), water absorption index (wai), and water solubility index (wsi) of lotus stem starch (lss) employing response surface methodology (rsm). sucrose was procured from sisco research laboratories private limited, mumbai, acacia gum was procured from merck specialties private limited, and nacl was procured from loba chemie private limited, mumbai. starch granules in native form were extracted using the method of wei et al. with slight modification. lotus stems were washed and then peeled to scrape off the outer skin and the peeled stems were then cut into small pieces. the small pieces were then homogenized with ice-cold water in a home blender followed by squeezing of the homogenate through four layers of cheese-cloth by hand. the fibrous residue was blended and squeezed twice with ice-cold water to facilitate the release of starch granules from the fibers. the starch was then extracted by washing four times with distilled water and centrifuged at 3200 rpm for 10 min. the yellow gel-like layer formed on top of the packed white starch granule 2.5 g of sample (ws) was taken in 25 ml of distilled water in a preweighed test tube. contents were stirred using vortex shaker occasionally with a holding period of 30 minutes and finally centrifuged at 3000 rpm for 15 minutes. the water absorption (wa) was calculated by the ratio of hydrated starch (waw) and weight of samples (ws): (1)wa=wawws100 (g/g). for samples containing acacia gum, nacl, and sucrose, lss powder was mixed with these three additives and the procedure was continued as it was described for pure starch suspensions. wai and wsi were determined using a modification of the method of leach et al.. starch dispersion of 2.5% was put in centrifugal tubes and heated in a water bath at temperature of 90c for 15 minutes. in order to prevent granules ' sedimentation during heating, stirring was applied periodically using glass stirrers. after heating, samples were centrifuged (3000 rpm, 10 min). both phases were dried at 105c for 24 h and the dry solids in precipitated paste (wdp) and supernatant (wds) were calculated. wai is the ratio of the weight of swollen starch granules after centrifugation (g) to their dry mass (g): (2)wai=wpwdp (g/g). wsi is the ratio of dry mass of solubles in supernatant to the dry mass of whole starch sample (ws): (3)wsi=wdsws100 (g/g). similarly, as executed for calculating wa, lss powder was mixed with three additives and the procedure was continued as it was described for pure starch suspensions. water solubility index (wsi) was calculated based on the assumption also mentioned in a study by mandala and bayas that the total amount of additives remained in the supernatant. wsi was calculated after subtracting dry weight of acacia gum, nacl, and sucrose from dry mass of soluble substances in supernatant. the experiment was designed according to a rotatable central composite surface response model with three variables and three levels. the three independent variables were acacia gum (0.51.5%), salt (0.51.5%), and sucrose (1030%). the three levels were coded as 1, 0, and 1, for the statistical analyses (table 1). the numbers of design points were obtained using the statistical software package, that is, design expert 8.0 (stat-ease, inc.) based on the number of independent variables. the range of additives levels was selected on the basis of formulations normally used in starch-based breakfast cereals and processed foods [17, 21]. as lss involved three independent variables, the total number of combinations made was twenty in which the number of design points was 14 with six center point replications. the center points for these designs were selected with additives at levels expected to yield satisfactory results. for each response, analysis of variance (anova) was conducted to determine significant differences among various additive combinations. wa and wai were maximized and wsi was minimized to obtain the most desirable optimized formulation of additives. effect of three additives on lss was studied and optimized combination was obtained. the experimental design with three independent variables and the respective responses for the lss are given in table 2. since wa, wai, and wsi are important properties to assess the behavior of starch in food system containing water as one of the most essential ingredients, these parameters were taken as responses. the regression analysis of the responses was conducted by fitting 2fi model as suitable for the respective response. analysis of variance was carried out to assess the significance of hypothesis for selected model and responses (table 3). 2fi model was highly suitable for wa (p<0.0001), wai (p=0.0001), and wsi (p=0.0003) response in lss. the p values given in the parenthesis for each response are for the model significance. the p value indicates the p>f-value which should be less than 0.05 for model to be significant; otherwise the model can not be used for further routing or prediction. multiple regression equations as obtained for all the three responses of lss with actual factors are represented as follows. further, the optimization of variable levels was achieved by desirable maximization or minimization of the necessary response along the fitted 2fi models by numerical optimization procedure of design expert software. the effect of change in the levels of selected additives on the response parameters is represented in figure 1. as can be seen from table 2, maximum response for wa (72.50%) minimum response for wa (41.00%) was obtained with acacia gum0.16%, sucrose20%, and nacl1%. effect of acacia gum on wa of lss was highly significant (p<0.05) leading to increase in the latter value with increasing content of the former (see (4)). interaction effect of acacia gum with nacl and also with sucrose was also found significant (p<0.05) for wa. the coefficient of determination (r) was obtained to be 0.9486 for wa (table 3). according to henika, the effects of three independent variables on wa of lss are depicted in response surface plots (figures 1(a)-1(b)). maximum response for wai (15.20%) was obtained with acacia gum1.50%, sucrose10%, and nacl0.50%. minimum response for wai (8.30%) acacia gum and nacl individually and their interaction were found to affect wai of lss significantly (p<0.05, see (5)). interaction of nacl and acacia gum reduced the wai value of lss. a regression equation which had a good fitting capacity for wai of lss the effects of independent variables on wai of lss are depicted in response surface plots (figure 1(c)). in case of wsi, maximum response for wsi (19.00%) minimum response for water solubility index (0.20%) was obtained with acacia gum1%, sucrose36.82%, and nacl1%. sucrose was found as significant factor (p<0.05) affecting the value (see (6)). high coefficient of determination (r=0.8192) for wsi of lss was obtained. finally in lss, the variables were optimized based on the maximization of the wa and wai and minimization of wsi values. the solutions were required to maximize the desirability function for the given criteria by being at random starting points. lss thus treated with optimized level of additives were verified for the predicted values and the actual values for the responses. actual and predicted values were compared using student's t-test and the p value (< 0.05) suggested no significant difference between the two. hence, the fitted models are best suitable for predicting the responses of the study (table 4). for obtaining optimized solution of formulation for the development of appropriate final product, responses were suitably maximized and minimized while performing rsm. wa and wai of any starch of edible purpose should be high enough for proper gelatinization during heating in the presence of moisture for making it digestible. if this happens then the yield of starch could reduce and also desired property of starch may change. hence, wsi of starch should be very less. with this view, wa and wai were kept maximized in rsm and wsi was kept minimized. among the combinations studied in the present study, the combination which yielded maximum desirability was selected. water absorption (wa) is the ratio of the wet weight of the sediment starch gel to its dry weight. water absorption (wa) of starch is an imperative parameter for expressing the interaction of native starch with water in processing of food products to improve yield and consistency and impart desired body and textural characteristics to the food. when unheated native lss was used to estimate water absorbed by granules, significant increase was observed with increasing acacia gum content, which can be explained by the fact that hydrocolloids promotes swelling. wa of lss was restricted by the interaction effect of gum with salt and sugars. the reason may be attributed to the limited water availability to starch granule in the presence of sugar and salt. sucrose addition to starch dispersion significantly decreased water absorption attributed to the fact that sucrose has a higher number of oh groups which makes it more hydrophilic, thus limiting water availability to lss. teixeira et al. reported decrease in water uptake (~60%) by cassava starch with sugar (2%) addition. likewise, chen et al. also reported significant decrease in swelling of potato starch and flaxseed polysaccharide potato starch (fg-ps) complexes due to the addition of sucrose (24%) owing to reduced water activity and formation of more bonding water which was unable to take part in absorption process. no significant effect of nacl was observed on wa which can be supported by the study of zhu et al., which concluded that if no heat is applied, no significant influence of nacl on the particle size distribution of wheat starch granules was observed. hence, it can be interpreted that salts can not be classified as swelling inhibitor or promoter as such, since it is the function of temperature at which the swelling is observed. swelling power is simple analytical test to measure the water uptake during the gelatinization of starch. amylopectin is considered as sole contributor to water absorption and subsequent swelling and pasting of starch granules, whereas amylose tends to retard this phenomenon. on heating at 90c for 30 min, significant changes in water absorption index (wai) or swelling power of lss were administered by addition of acacia gum and nacl and by interaction thereof. effect of additives was investigated on gelatinized starch existing in two forms, that is, continuous phase (amylose/amylopectin matrix) and the dispersed phase (starch granules). heating of starch dispersion causes swelling of granules, which influences the properties of both continuous and dispersed phases. increase in wai with acacia gum addition can be supported by the study of mandala and bayas which reported that xanthan addition (0.09%) to wheat starch enhanced swelling of starch dispersion (2%) during heating at 90c for 30 min. the following assumption about the role of gums in starch systems supports the finding of mandala and bayas. according to abdulmola et al., starch molecules can interact and a network can be created at concentrations well below this leads to force enhancement applied to them, facilitating swelling and amylose solubilisation and its exudation. further increase in temperature causes leaked amylose and the xanthan in the continuous phase to create a film around the granules which further inhibit swelling. effect of hydrocolloids addition on swelling of starch granules during heating was also reported by chaisawang and suphantharika suggesting that addition of guar or xanthan gum (0.073%) slightly enhanced swelling of native tapioca starch (1.25%) granules at temperatures that ranged from 60 to 90c. decrease in wai of lss with increasing nacl content occurred due to competition between salts and starch for available water molecules at high temperatures. also, electrostatic interaction between starch and ions from nacl has ability to limit the swelling of starch granules. similarly, zhu et al. reported limited promoting effect of nacl on wheat starch at higher temperatures concluding that swelling inhibiting effect of nacl is largely temperature dependent. significant decrease in swelling power of rice starch subjected to heating at 75c on addition of 0.1 m nacl solution further sustains the results. similar influence of hydrocolloid and salt on swelling power of rice starch has been reported by samutsri and suphantharika. addition of salts (0.1 m) significantly decreased water uptake of rice starch complexed with xanthan and guar gum in 19: 1 (w/w) ratio of starch and gum. hence, conclusion can be drawn that effect of nacl on reduction of swelling power of rice starch follows the order of the hofmeister series which is a classification of ions in order of their ability to salt-out or salt-in proteins. spies and hoseney proposed that stabilization of amorphous region of the starch granules occurs when sugar molecules possibly restrict the mobility and flexibility of starch chains by forming bridges with more starch chains. this kind of sugar-starch chain interaction could be the reason for observed reduced amylose leaching. richardson et al. conducted a study to investigate the effect of sucrose (12 or 24%, w/w) on solubility index of wheat starch dispersion (8%) which revealed delayed amylose leakage and granule fragmentation above 50c. in the presence of acacia gum, nacl, and sucrose, the physical properties of lotus stem starch might be governed by hydrocolloid-salt, hydrocolloid-sugar interaction and individual effect of these additives. desired or optimum level of these additives was obtained by using rsm, which returned 1.5% of acacia gum, 0.5% of nacl, and 30% of sucrose with a desirability of 0.844. wa, wai, and wsi are 65.42%, 14.95, and 2.74%, respectively. in presence of acacia gum, swelling power of starch increased. nacl can not be classified as swelling inhibitor or promoter as such, since it is the function of temperature at which the swelling is observed. sucrose was found to inhibit water absorption by limiting the water availability to starch in the presence of acacia gum, thus reducing the swelling promoting effect of the latter. sucrose also retarded amylose leaching (wsi) by mechanism of building bridges with starch chains and reducing their flexibility. hence, results of present investigation suggest important practical inferences in applications of assayed additives in starch-based food products containing lotus stem starch as one of the major ingredients which is highly popular as functional food in east asian part of the world.

consumer preferences in east asian part of the world pave the way for consumption of lotus stem starch (lss) in preparations such as breakfast meals, fast foods, and traditional confectioneries. the present study envisaged the investigation and optimization of additives, that is, acacia gum, sodium chloride (nacl), and sucrose, on water absorption (wa), water absorption index (wai), and water solubility index (wsi) of lss employing response surface methodology (rsm). acacia gum resulted in increased water uptake and swelling of starch; however, nacl reduced the swelling power of starch by making water unavailable to starch and also due to starch-ion electrostatic interaction. sucrose restricted the water absorption by binding free water and decreased amylose leaching by building bridges with starch chains and thus forming rigid structure.

PMC4745554

pubmed-949

this is a non-controlled, longitudinal study, carried out in a sample of 150 children 12-15 months of age in three health units of the municipality of rio de janeiro who received the combined mmr vaccine according to the basic immunisation schedule and nip routine procedures. healthy male and female children, living in the city of rio de janeiro, with no significant past medical history, were selected for this study. the criteria for not including into the study were: (i) subjects with a history of measles, rubella and/or mumps, (ii) subjects who had already received mmr vaccination documented in the vaccination card, (iii) subjects with a history of receiving blood transfusion or blood products, including immunoglobulin in the past one year previous to the study, (iv) subjects who presented skin lesions at sites of venipuncture and (v) subjects who had used corticosteroids (except topical or aerosol) during the last six months or reported use of immunosuppressive drugs. two blood samples were collected: before and 42 days after vaccination (minimum acceptable 30 days and maximum 60 days). volunteers who did not seroconvert from seronegative to seropositive or who had inconclusive results for any of the antigens were vaccinated again and a third blood sample was collected within the same time range recommended for the second blood collection. medical records were prepared using the teleform workgroup 2008 program, v.10.2, which allowed capture of the scanned data, without manual typing and creation of a database. vaccine used on the study-the mmr received in bulk from gsk, formulated and distributed by bio-manguinhos, oswaldo cruz foundation (fiocruz), single lot (072vva007z), in 10 dose presentation, february/2007 production date and valid for two years, was used on the study. each 0.5 ml dose of reconstituted vaccine contained at least 1,000 50% cell culture infectious dose (ccid50) of attenuated measles virus, schwarz strain, at least 1,000 ccid50 of attenuated rubella virus wistar ra27/3 strain and at least 5,000 ccid50 of attenuated mumps virus, rit 4385 strain, derived from jeryl lynn strain. for each component of the vaccine batch used in the study, the potencies at temperatures varying from 2c to 8c were, in log10, 4.26 (measles), 5.28 (mumps) and 4.02 (rubella) and, after storage at 37c, 3.96, 4.99 and 3.85, respectively. the vaccine administered into each volunteer was diluted at the time of study enrollment, according to the nip procedures and each volunteer received only the first dose from each vial. laboratory methods-blood samples were placed into an insulated box kept between 4-10c from time of collection until arrival at the laboratory. the maximum time between blood collection and arrival at the laboratory was 6 h. igg antibodies against measles, mumps and rubella were determined at the reference laboratory for measles and rubella, oswaldo cruz institute/fiocruz using an enzyme immunoassay (eia) with a commercial kit from siemens (enzygnost igg). the results for optical density were converted to international units or units per millilitre of serum using a table provided by the manufacturer and categorised as negative for rubella, mumps and measles if<4.0 iu/ml,<231 u/ml and<150 miu/ml, respectively. these cut-offs were used in all studies referred to in the bibliographical references. additionally, results were categorised as seronegative, inconclusive and seropositive, according to the kit instructions. at the end of 2010, samples were sent for retesting for the mumps component at the gsk biologicals laboratory (rixensart, belgium) using the same methodology (eia) and diagnostic kit used in the reference laboratory at fiocruz. igm antibodies against mumps were measured after revaccination of children seronegative after the first dose in order to detect primary immune failures. data analysis-a database for the study was created using the statistical package for social sciences program v.17. the distribution of absolute and relative frequencies of subjects were tabulated by sex, age group, health units, interval between date of vaccination and date of blood sample collection. immunogenicity of the mmr vaccine was assessed primarily in terms of the percentage of baseline seronegative children who seroconverted for antibodies against measles, mumps and rubella viruses (that is, developed antibody levels cut-off for seropositivity after vaccination). criteria for protocol adherence were: children seronegative before vaccination with available serologic test results before and after vaccination and blood collected from 30-60 days after vaccination. we constructed 95% confidence intervals (ci) for proportions using winpepi (abramson 2011). immunogenicity was also evaluated by geometric mean titre (gmts) after vaccination and the magnitude of the immune response could be assessed against the minimum antibody levels for seropositivity. ethical aspects-the mmr vaccine administered to volunteers was the same used in the nip routine, having already gone through immunogenicity and reactogenicity clinical studies before registration and use on a large scale. the study protocol was approved by the research ethical committee of the municipal health secretariat of rio de janeiro (protocol 48/08). from may-august 2008, 165 children were enrolled, of which 150 were eligible and 146 (96.7%) had blood samples obtained before and after vaccination. there was a slight male predominance (55%) and most children (92.7%) were from 12-15 months of age. the intervals between vaccination and blood sampling after vaccination ranged from 34-73 days and of 146 volunteers who had a second blood sample collected, 97.9% had intervals from 30-60 days. before vaccination, there was one child seropositive for measles, one for mumps and one for rubella and 143 children were susceptible to measles, mumps and rubella. according to the kit instructions and in children with adherence to protocol, 86 (60.1%) were seropositive for mumps, 42 (29.4%) inconclusive and 15 (10.5%) seronegative. for all children, with exception of one child seropositive to mumps before vaccination, 88 (60.7%) were seropositive and the number with negative or inconclusive results after vaccination was 57 (39.3%). considering 231 u/ml as the cut-off, in children with adherence to protocol, seroconversion for mumps after first vaccination was 89.5% (95% ci: 83.3; 94.0) and 130/145, 89.7% (95% ci: 83.5; 94.1), for all available children who were seronegative before vaccination (table i). table iresults of mumps serology (enzyme immunoassay) after the first dose of combined vaccine against measles, mumps and rubellaigg serologycut-off 231 u/ml n (%) according to kit n (%) negative15 (10.5)15 (10.5)inconclusive-42 (29.4)positive128 (89.5)86 (60.1) total143 (100)143 (100) post-vaccination gmts were 2,234 (95% ci 2039.4; 2447.7) mui/ml for measles, 596.6 (95% ci 517.2; 688.3) u/ml for mumps and 50.1 (45.1; 55.8) iu/ml for rubella. when contrasted with cut-off values for seropositivity, the gmts indicated considerably larger magnitude of the immune response for rubella (cut-off: 4.0 iu/ml) neither health unit nor time of blood collection after vaccination was relevant regarding immunogenicity (data not shown). of the 58 children seronegative or with inconclusive serology for mumps after vaccination, 57 were eligible for revaccination (1 child was excluded due to a 2nd dose of mmr vaccine received during a mmr campaign, registered on the vaccination card). blood samples were collected after the second vaccine dose in 54 children (94.7%) and were not collected in three children due to parent/tutor refusal. the interval between vaccination and revaccination ranged from 203-249 days, with a mean of 221 days [standard deviation (sd): 11.6] and a median of 220 days. the interval between revaccination and third blood collection ranged from 31-64 days, with a mean of 39 days (sd: 6.2) and a median of 37 days. after revaccination, all children had high igg titres for mumps, above 1,200 u/ml, with exception of one child, who had a titre of 457 u/ml. measles and rubella antibody titres showed a modest rise (table ii). table iiigg (elisa) geometric mean titres (gmt) after vaccination and revaccination and revaccination/vaccination ratios, for measles, mumps and rubellaigg titresnafter vaccination gmt (95% ci)after revaccination gmt (95% ci)gmt ratio revaccination/vaccination (95% ci)measles (miu/ml) 542,155.5 (1,828.0; 2,541.6)5,692.1 (4,815.5; 6,728.4)2.6 (2.3; 3.1)mumps (u/ml) 54247.6 (214.3; 286.0)3,157.0 (2,684.9; 3,712.0)12.8 (10.3; 15.8)rubella (ui/ml) 5441.3 (33.8; 50.4)151.0 (134.2; 169.9)3.7 (3.0; 4.5) moreover, after revaccination, igm for mumps was negative in 52/54 (96.3%) children and in two children results were inconclusive; for measles, 50/54 (92.6%) were igm negative and four were inconclusive; for rubella, 53/54 (98.1%) were igm negative and one was inconclusive. due to the high percentage of negative and inconclusive results for mumps after the first mmr vaccination, according to the kit instructions, the pre and post-vaccination samples were sent for blind retesting for mumps at the gsk in rixensart, belgium. mumps immunogenicity has been highly variable across mmr studies with features similar to the current study, that is, with the same jeryl-lynn based vaccine from gsk, after the first dose (mmr used alone or with simultaneous administration of varicella vaccines), with similar age at vaccination, using the same enzygnostkit and 231 u/ml cut-off for seropositivity. of note, all assumed 231 u/ml for mumps indicated seropositive results (usonis et al. studies from 1999-2002 presented the highest immunogenicity. a study in 2011 in germany showed a gmt for mumps of 523.7 u/ml, with 71.3% seroconversion (rmke et al. efficacy trials represent the best scenarios of vaccine performance under controlled conditions and are commonly required before a new vaccine is licensed. they are measured usually as immune responses and when there are correlates of protection (that is, a cut-off of antibodies above which there will be protection against the disease), it is possible to estimate vaccine effectiveness from immunogenicity data. this is the case for measles and rubella. in the case of mumps, there are no correlates of protection, so it is not possible to estimate effectiveness-that is, the magnitude of reduction of disease rates attributable to vaccination under real life conditions. many studies evaluated effectiveness of the mumps component in populations vaccinated with jeryl-lynn based vaccines. the recent mumps outbreak in new york and new jersey reported by the centers for disease control and prevention (cdc 2010) estimated a variation on mumps vaccine effectiveness from 73-91% after one dose and from 79-95% after two doses. even so, overall vaccine effectiveness is not questioned, as the annual number of mumps cases in the united states of america decreased from 186,000 in 1967, when the vaccine was introduced, to less than 500, in the early 2000s. waning immunity, that is, decrease on seroprotection since time of last vaccination, seems to be an important factor for vaccination failure, both for one and two-dose vaccinees. waning may also partly explain why vaccination effectiveness has been in general lower than efficacy, which is usually assessed after shorter follow-up. there is a trade-off between mumps vaccine immunogenicity and reactogenicity, mainly regarding aseptic meningitis. clearly, the jeryl lynn based vaccines are the safest, although probably not the most immunogenic. this evaluation should be done by each country, according to epidemiological considerations and degree of tolerance for adverse events. it should be noted that brazil has had a negative experience regarding adverse events with mmr campaigns using mumps strains other than jeryl-lynn (dourado et al. the strong response to mumps revaccination on the current study, with a very high after revaccination/after vaccination gmt ratio, suggests insufficient power of this vaccine to induce strong mumps immune response after one dose, but the booster response is reassuring concerning seroconversion after two doses. moreover, after revaccination, 96.3% of children were igm negative for mumps, again suggesting a secondary immune response, with the caveat that the blood collection was taken from one-two months after revaccination, when igm levels are expected to be on the decrease. however, high igm levels after disease in unvaccinated subjects are maintained for several weeks or months (cdc 2012). assessment of igm levels after mmr first dose was found in only one bibliographical reference, using the hoshino mumps strain, the elisa ibl kit, with blood collected four-seven weeks after vaccination and igm seropositivity was found in 71% of children (tabatabaei 2013). limitations in the accuracy of the mumps laboratory test, particularly its sensitivity (81%), may have contributed to the suboptimal mumps immunogenicity results (backhouse et al. these considerations assumed no relevant virus circulation, which seemed reasonable, even though reporting of mumps cases is not mandatory in brazil, except outbreaks, which are usually perceived in health care units and are reported on the national notification system, which was not the case. the results of this study are in agreement with two previous studies done by our group, the first already referred (silva et al. 2011), which included 1,769 children, and a second not yet published, but with final report approved, which included 183 children 12-18 months of age, using the same methodology and cut-offs. in the absence of accepted correlates of protection for mumps, no definite statements regarding protection against disease can be derived from the results of this study. however, the data here provided strengthen the need of a second mmr dose to ensure maximum protection against mumps. serological and epidemiological studies after two doses should be implemented to know if further doses and at which intervals are needed. data from the current study confirm the high immunogenicity of the mmr measles and rubella components with one dose and suggest lower immunogenicity of the mumps component. after a second dose, the main and immediate implication of these findings is the reassurance that two mmr doses are highly immunogenic for all antigens included on the vaccine. as there is no serological correlate of protection for mumps, the implications of immunogenicity data should be considered cautiously, but they may be useful regarding immunisation practices and guidance, taking into account other variables, such as epidemiological data.

a non-controlled longitudinal study was conducted to evaluate the combined vaccine against measles, mumps and rubella (mmr) immunogenicity in 150 children vaccinated in the routine of three health units in the city of rio de janeiro, brazil, 2008-2009, without other vaccines administered during the period from 30 days before to 30 days after vaccination. a previous study conducted in brazil in 2007, in 1,769 children ranging from 12-15 months of age vaccinated against yellow fever and mmr simultaneously or at intervals of 30 days or more between doses, had shown low seroconversion for mumps regardless of the interval between administration of the two vaccines. the current study showed 89.5% (95% confidence interval: 83.3; 94.0) seroconversion rate for mumps. all children seroconverted for measles and rubella. after revaccination, high antibody titres and seroconversion rates were achieved against mumps. the results of this study and others suggest that two mmr doses confer optimal immunoresponses for all three antigens and the possible need for additional doses should be studied taking into account not only serological, but also epidemiological data, as there is no serological correlate of protection for mumps.

PMC4131786

pubmed-950

cardiac resynchronization therapy (crt) is an important device-based, non-pharmacological approach that has shown to improve the outcome in selected patients with chronic heart failure (hf). large randomized trials have demonstrated that crt improves left ventricular (lv) function and reduces both morbidity and mortality rates. until recently, crt was indicated in patients with advanced hf [new york heart association (nyha) functional class iii iv], reduced lv systolic function [ejection fraction (ef) 35%], evidence of electrical dyssynchrony (qrs duration 120 ms), receiving optimal medical therapy, and who were in sinus rhythm (sr). in the last esc recommendations, for the first time, atrial fibrillation (af) patients, who constitute an important subgroup of hf patients treated with crt, have been considered as eligible to receive crt on the condition that the effects of underlying rhythm be neutralized by atrio-ventricular junction (avj) ablation. the aha/acc/hrs guidelines also favourably considered crt in hf patients with af, without however emphasizing the possible need for aggressive rate control. also, these guidelines remain imprecise in defining differentiated approaches according to the forms of af other than permanent. the present review explains, in the first instance, in which way af interferes with adequate crt. secondly, a brief overview on the effects of crt in af patients is presented, followed by some recommendations, based on current evidence, on the most adequate approach according to patient characteristics, emphasizing the extent of atrial arrhythmic [atrial tachycardia (at)/af] burden. it is important to point out that these recommendations remain unsupported by evidence derived from randomized controlled trials (rcts), which are much needed. atrial fibrillation (whether permanent, persistent, or paroxysmal) poses a number of challenges for adequate crt delivery. an intrinsic, intermediate-to-high, irregular spontaneous af rhythm reduces the percentage of effectively biventricular paced captured beats (bvp%). even in a patient who has normal rate af, phases of effective biventricular capture alternate with phases of competing af rhythm which causes spontaneous, fusion, or pseudo-fusion beats (figure 1) this suggests that the global effective crt-dose may be markedly reduced compared with atrial-synchronous rhythm with a short av interval (as is achieved during sr) since the number of effective biventricular captured beats are reduced. moreover, in af patients, during exertion, spontaneous ventricular rate tends to override bvp rates, determining a further reduction of paced beats precisely when patients are most in need of having biventricular capture, thus greatly limiting functional capacity. another problem is the possible negative impact on prognosis of using combinations of negative chronotropic therapy to achieve adequate rate control. in fact, some studies have indirectly suggested that the use of either digoxin or amiodarone in hf may increase morbidity and mortality. a patient with af and hf treated with crt, spontaneous irregular intrinsic beats alternate with fusion and pseudo-fusion beats, thus markedly reducing effective crt. adequate management of af and other atrial arrhythmias is primarily based on defining the at/af burden and how it impacts negatively on crt delivery. defining atrial arrhythmic burden is derived from integrating clinical, device-derived data, as well as instrumental findings (such as echocardiographic measures). any hf patient with a history of atrial arrhythmias requires particular attention, especially in the first months of crt, in order to ensure that resynchronization be adequately delivered. from a clinical standpoint, it is important to identify symptoms such as palpitations, and more importantly, worsening effort dyspnoea which may suggest that the resynchronization effect is reduced because of the interference of the underlying atrial rhythm. these clinical aspects should be substantiated by instrumental echocardiographic data, which may show unchanged or further progression of lv dysfunction expressed through increased ventricular volumes and further ef reduction. retrieving relevant information (bvp%, duration, and numbers of mode switch episodes, etc.) through device monitoring (figure 2) may complement clinical and echocardiographic data and, thus, provide a more complete picture on the extent of af/at burden in each patient. these different aspects, all obtainable during a routine outpatient visit, allow provision of an approximation of the effective af/at burden influencing crt delivery. recently, kamath pointed out the importance of an accurate evaluation of crt-dose using sophisticated 12-lead holter monitoring, which seemed to be more accurate than conventional device-based information. different aspects of crt patients with af before and after avj ablation, which may be appreciated through device features. in a 59-year-old female with permanent af treated with a crt-d device, avj ablation yielded the following improvements: better functional status as shown by the number of hours of activity per day; maximization of bvp%; and improvement of heart rate variability profile. rate control strategy encompasses treatment options which effectively reduce and regularize heart rate in patients who usually have permanent af or a persistent af which can not be readily cardioverted to sr. first, lowering heart rate to intermediate-to-low rate allows better diastolic filling and increases stroke volume in hearts with conserved frank the recourse to rate control drugs and/or activation of device-based algorithms is reasonable as first-line approach when af/at burden is low/intermediate. rate control drugs considered effective in hf patients with depressed lv function include digoxin, amiodarone, and beta-blockers. however, more recent findings derived from randomized trials have suggested caution in the use of digoxin and amiodarone in patients with hf. some device-derived features may be helpful to improve rate control and thus improve crt delivery these features include ventricular rate regularization (vrr) which consists in performing bvp, which overrides intrinsic rhythm, through faster ventricular-paced depolarization allowing to reduce short cycles through retrograde concealed penetration of the av node. the benefits of rate control achieved by activating vrr function is well established in patients with chronic af and no or only mild hf treated with a single chamber right ventricular (rv) pacing. in these patients, vrr has been demonstrated to confer acute haemodynamic benefits, to restore autonomic balance, and to provide a more regular rhythm during exercise, thus potentially improving functional status. ventricular sense response (also called trigger function) which triggers lv pacing after a premature rv sense event is detected: this option may be activated in all crt devices of the latest generation. in the context of crt, the effectiveness of such rate control and rate regularization algorithms combined with the use of rate control drugs has not been investigated in an rct. findings derived from different large observational cohort studies on the effects of crt in patients with permanent af have yielded contrasting results. one of these studies observed that treatment combining negative chronotropic drugs and activation of device features (vrr and trigger mode), even if permitting 85% of biventricular stimulation, did not yield significant long-term improvements in functional status, lvef, or lv end-systolic volume (lvesv) reduction. the ineffectiveness of this approach further found confirmation through another more extensive multicentre european study, which reported relatively high death rate, particularly occurring for worsening progressive hf in af patients treated with negative chronotropic drugs. quite differently, other smaller studies have advocated that to achieve good results after crt in terms of survival, aggressive rate control strategy is not necessary. it is worth emphasizing, however, that when the survival curves of the hf patients with af treated with a combined device-based/drug regimen are compared, yearly death rate for any cause is considered to be remarkably high, amounting to over 14%/year in both separate cohorts of non-ablated patients (figure 3). comparison of kaplan meier analysis for freedom from death for any cause between the gasparini et al. and the khadjooi et al. sr, sinus rhythm; af, atrial fibrillation; af-drugs: atrial fibrillation with preserved av node conduction; af-abl, atrial fibrillation group with ablated av node. it therefore follows that in hf patients treated with crt who present a high or intermediate at/af burden, the pursuit of an aggressive treatment strategy, such as avj ablation, is warranted. atrio-ventricular junction ablation is commonly performed in patients with symptomatic, drug-refractory, fast, permanent af as part of the conventional ablate and pace strategy, and has been shown to confer symptomatic relief. atrio-ventricular junction ablation in individuals with af treated with crt has mainly been confined to selected patients in whom high-rate af or at jeopardizes satisfactory biventricular stimulation, and in crt-implantable cardioverter defibrillator (icd) recipients determines inappropriate icd interventions. the problem of inappropriate icd therapies during af, constituting 30% of all icd interventions, has an important negative impact on the quality of life of patients and may be completely resolved after avj ablation. however, in the context of crt in hf patients with concomitant af, a growing amount of evidence has demonstrated that avj ablation may be useful to optimize crt delivery by eliminating the deleterious haemodynamic effects of a competing, irregular, spontaneous intrinsic rhythm. the mustic af randomized trial, besides being the first randomized trial demonstrating possible benefits of crt in hf patients with permanent af and conventional indication for crt, also showed that in these patients, the preferred mode of ventricular stimulation was biventricular as opposed to rv. the study enrolled af patients with either slow-rate af or those who underwent ablation of the av node; the effects between pacing modes were compared using a crossover design with two 3-month periods. although no result was found in the intention-to-treat analysis between the two modes (because of high numbers of dropouts), hf patients who completed the study improved in terms of functional status with bvp. two other prospective studies investigated the effects of pacing mode in the management of af with rapid ventricular rates following avj ablation. rate control achieved following avj ablation significantly improved symptoms and functional status with no difference between the pacing modalities, whether lv or rv, but in a population with much better lv function. the pave trial further confirmed the benefits of the ablate and pace approach using different pacing modes. the latter study observed a greater benefit of the bvp mode in patients with depressed lvef (45%) and/or in nyha functional class iii. further observational studies have investigated the acute and short-term effects of avj ablation in hf patients with af treated with crt and have demonstrated an increase in global lv function, a reduction of mitral regurgitation, and an increase in exercise capacity; others have confirmed the chronic effects of crt in this patient subgroup, reporting improvements in nyha class, exercise capacity, and global lv function. it is important to stress that these benefits appear to be confined to af patients with previous avj ablation or spontaneous low-rate af. one large observational prospective investigation specifically evaluated the effects of avj ablation on crt delivery using a pre-defined protocol. this study showed that only those af patients who underwent avj ablation (and thus approaching 100% effective bvp) showed significant improvements in lvef, lvesv, and exercise capacity. furthermore, a significantly higher proportion of responders (response defined as a 10% reduction in lvesv) were observed in the avj ablation group (68%) compared with the non-ablated group (18%) at 12 months. as later observed by the same groups in a more extensive observational multicentre study, crt combined with avj ablation conferred a significant reduction of deaths for any cause compared with crt alone, particularly by reducing deaths for progressive hf. taken together, based on current observational data on af populations treated with crt, the benefits of avj ablation in allowing appropriate crt delivery seem to outweigh the risks associated with creating pacemaker dependency. the peculiarity of crt devices (using an rv and an lv pacing leads) should, theoretically, at least reduce the risks of pacemaker dependency related to lead fractures or malfunction. nonetheless, the fear of pacemaker dependency remains a limiting aspect for the wider diffusion of avj ablation. further studies are of course needed to investigate the extensive benefits of adding avj ablation to crt in hf patients with af following a prospective, randomized, multicentre design. the avert-af (atrio-ventricular junction ablation followed by resynchronization therapy in patients with chf and af) and the an-art study (av node ablation in crt) are both concerned with establishing whether avj ablation coupled with bvp may significantly improve functional capacity compared with pharmacological therapy in hf patients with permanent af and depressed ef. it should be stated that such studies aiming to assess the effects of crt according to soft/subjective endpoints may add little to the current recommendations. there is a great need to design rcts with strong endpoints, even though such designs may be difficult to implement for ethical and financial reasons. a reasonable and up-dated approach to investigate the effects of crt in this group involves using device-based surrogates of major clinical events. device-based remote continuous patient monitoring may provide information on a daily basis of the patients ' functional and clinical status, before the onset of an overt clinical event becomes manifest. but before these parameters may be validated as surrogates for strong endpoints of major cardiovascular events, better definition of their accuracy is needed. atrial fibrillation and other atrial rhythm disturbances in patients with hf may have an important negative impact on the clinical benefit conveyed by crt, if these are not appropriately managed. careful overall evaluation is mandatory to define precisely the at/af burden in order to articulate tailored diagnostic and therapeutic strategies. on the basis of recent observational data, in patients presenting intermediate or elevated at/af burden, avj ablation may represent a fundamental tool to achieve full crt delivery and, thus, confer marked improvements in global cardiac function, and, further, in survival. more studies are necessary to further support the recourse to avj ablation in this situation. efforts should also be dedicated towards establishing tailored treatment approaches to adequately manage different atrial rhythm issues in hf patients treated with crt. funding to pay the open access publication charges for this article was provided by electrophysiology and pacing unit, irccs istituto clinico humanitas, rozzano, milano.

cardiac resynchronization therapy (crt) is an important device-based, non-pharmacological approach that has shown, in large randomized trials, to improve left ventricular (lv) function and reduce both morbidity and mortality rates in selected patients affected by advanced heart failure (hf): new york heart association (nyha) functional class iii iv, reduced lv systolic function with an ejection fraction (ef) 35%, qrs duration 120 ms, on optimal medical therapy, and who were in sinus rhythm. for the first time, the latest esc and aha/acc/hrs guidelines have considered atrial fibrillation (af) patients, who constitute an important subgroup of hf patients, as eligible to receive crt. nevertheless, these guidelines did not include a strategy for defining differentiated approaches according to af duration or burden. in this review, the authors explain in which way af may interfere with adequate crt delivery, how to manage different af burden, and finally present a brief overview on the effects of crt in af patients.

PMC2768583

pubmed-951

carcinomas producing granulocyte colony-stimulating factor (g-csf) are extremely malignant and have a poor prognosis [1, 2]. thus, a liposarcoma producing g-csf is particularly rare and has only been described in a few case reports [1, 3]. here, the tumor also showed spontaneous rupture, which has not been previously reported for retroperitoneal liposarcoma. positron emission tomography using f-fluorodeoxyglucose (f-fdg-pet) is used to detect tumors based on elevated glucose metabolism in carcinoma cells. it has also been shown that f-fdg uptake by bone marrow is correlated with the peripheral blood neutrophil count, since uptake reflects increased marrow metabolism. we performed f-fdg-pet with computed tomography (pet/ct) for imaging of the g-csf-producing tumor and found high f-fdg uptake by the tumor itself and diffuse uptake throughout the bone marrow. these are the first pet/ct images of a g-csf-producing tumor in a urological disease. the patient was a 63-year-old man with a history of hypertension and no specific family history. he was taken to the emergency department of our hospital due to significant aggravation of the pain in late july 2008. an abdominal ct imaging (fig. 1) revealed a non-fatty, non-fibrotic, and non-calcified right retroperitoneal tumor of 15 cm in size and retroperitoneal hemorrhage. hemorrhagic shock occurred due to spontaneous rupture of the retroperitoneal tumor during the examination, and an emergency transcatheter arterial embolization was conducted. the nutrient vessels were the superior and inferior suprarenal arteries, inferior phrenic artery, renal capsular artery, and the first lumbar artery. three days after admission, the patient was transferred to our department for a more detailed examination of the retroperitoneal tumor. a routine peripheral hematological examination indicated a high white blood cell (wbc) count of 37,820/l and a strong left-shift of the differential leukocyte count, with a neutrophil count of 34,794/l. however, no infection appeared to be present based on physiological and biochemical findings, and endocrinological tests showed no abnormal findings. since the wbc count remained high after the transfer to our department, further tests were carried out at the department of hematological internal medicine, based on the suspicion of a g-csf-producing tumor or a comorbid hematological disease. in parallel, elevated uptake of<f-fdg correlated with the tumor site (maximum standardized uptake value, suvmax, was 18.5) and the case was diagnosed as malignant. pet/ct imaging also indicated diffuse uptake throughout the bone marrow, and particularly elevated uptake in the vertebral bone (suvmax: 10.5) (fig. 2). however, bone scintigraphy showed no bone metastasis, and bone marrow biopsy revealed little fat, significant hyperplasia, and increased neutrophils, but no findings of metastasis or bone marrow involvement. based on all the above findings and the high serum g-csf level of 2,670 pg/ml (normal<8 pg/ml), the cause of the increased wbc count was determined to be a g-csf-producing tumor. a malignant tumor was suspected and right nephrectomy and retroperitoneal tumorectomy were conducted in mid-september. however, since the tumor had infiltrated into the inferior vena cava, diaphragm and abdominal wall already, only partial resection of the tumor was performed. histopathological tests using hematoxylin-eosin staining showed findings for malignant fibrous histiocytoma mixed with those for well-differentiated liposarcoma (fig. the tumor was diagnosed as dedifferentiated liposarcoma based on the diagnostic criteria of the world health classification (who) [5, 6]. the postoperative peripheral wbc count was 21,000/l and the serum g-csf level was 1,170 pg/ml, both of which showed a transient decrease. with informed consent from the patient, only symptomatic therapy, but no chemotherapy or radiotherapy, was carried out while observing the prognosis. liposarcomas are morphologically subdivided into five main subgroups: well-differentiated, myxoid, round cell, pleomorphic, and dedifferentiated. dedifferentiated liposarcoma was first recognized by evans in 1979 and was defined in 2002 as a tumor with a clear-cut and well-differentiated component clearly separated from or occupying a large area beside a poorly differentiated component [5, 6]. among 78 cases of retroperitoneal liposarcoma, fabre-guillevin et al. described 52 cases of dedifferentiated liposarcoma with a differentiated component with features of malignant fibrous histiocytoma (36 undifferentiated pleomorphic sarcoma; 14 fibrosarcoma; 1 myxofibrosarcoma; 1 osteosarcoma; and/or 4 leiomyosarcoma), 21 of well-differentiated liposarcoma, 2 of myxoid liposarcoma, 2 of round cell liposarcoma, and 1 of pleomorphic liposarcoma. the cases of dedifferentiated liposarcoma had local recurrence and metastasis at rates of about 40 and 20%, respectively, and a poorer clinical outcome compared to well-differentiated liposarcoma [6, 7]. the production of g-csf has been reported in cases of hepatocellular carcinoma, pancreatic cancer, and gastric cancer [8, 9, 10]. g-csf-producing carcinomas show aggressive growth and a very poor prognosis [8, 9, 10]. there have only been a few previous case reports of a g-csf-producing liposarcoma [1, 3]. six cases of liposarcoma accompanied by leukocytosis have been described in the english literature. however, the g-csf level has only been evaluated in 3 cases: in an upper arm dedifferentiated liposarcoma described by sakamoto et al., in a mesenteric pleomorphic liposarcoma reported by nakamura et al., and in the current case. in all 3 cases, the g-csf level and peripheral wbc counts were correlated with the clinical course. the g-csf level and wbc count showed a transient postoperative decrease in our patient, but this was followed by an aggressive clinical course and the wbc count was 145,800/l at the time the patient died. no hematological diseases such as leukemia or a leukemoid reaction were observed and bone marrow biopsy only showed hyperplasia. these findings led to the diagnosis of a g-csf-producing retroperitoneal dedifferentiated liposarcoma. this is an extremely rare clinical presentation, with the description of spontaneous rupture of a renal liposarcoma by mazuch et al. the rapid aggressive growth of the g-csf-producing tumor may have caused the rupture, and palliative surgery was performed because of the rupture. pet/ct is useful for the evaluation of malignant lesions. in cases of carcinoma, diffuse f-fdg uptake in bone marrow is often indicative of bone metastasis or bone marrow involvement. in our case, pet/ct showed diffuse f-fdg uptake throughout the bone marrow, which indicated multiple bone metastases. described 2 cases of g-csf-producing lung cancer that showed diffuse f-fdg uptake throughout the bone marrow. commented that diffuse elevated f-fdg uptake in bone marrow may be helpful in the diagnosis of a g-csf-producing tumor. bone metastasis is a differential diagnosis, and in our case bone scintigraphy and bone marrow biopsy were useful for ruling out bone metastasis. murata et al. showed that f-fdg uptake by bone marrow is strongly correlated with the wbc count (especially neutrophil count) and suggested that f-fdg uptake by bone marrow reflects marrow metabolism, which is mainly regulated by granulocyte progenitors and stimulated by endogenous hematopoietic growth factors. found increased f-fdg uptake in patients treated with g-csf and showed that the increased uptake returned to the pretreatment value approximately 1 month after discontinuation of g-csf. the serum g-csf level is likely to be higher in patients with a g-csf-producing tumor than in those treated with g-csf thus, for a g-csf-producing tumor, at least 1 month is needed before a pet/ct evaluation after removal of the primary lesion. other conditions associated with diffuse f-fdg uptake in the bone marrow include marrow hyperplasia resulting from hemolytic/iron-deficiency/blood-loss anemia. in addition, van de weile et al. examined the relationship between bone marrow f-fdg uptake and levels of serum cytokines. in patients with non-small cell lung carcinomas, which are known to produce cytokines, f-fdg uptake by bone marrow thus, bone marrow f-fdg uptake in patients with a g-csf-producing tumor may also be related to cytokines such as tgf-. in conclusion, we encountered a rare case of a g-csf-producing retroperitoneal dedifferentiated liposarcoma that underwent spontaneous rupture due to rapid aggressive growth. this case is also the first report of pet/ct imaging of a g-csf-producing tumor in a urological disease. in such cases, one needs to be careful to avoid a misdiagnosis of bone metastasis, but imaging may be useful for differential diagnosis of a g-csf-producing tumor in patients with abnormally high wbc counts in the absence of infection.

the patient was a 63-year-old man who was referred to our hospital as an emergency case with the chief complaint of abdominal pain. an abdominal ct revealed a right retroperitoneal tumor of 15 cm and retroperitoneal bleeding. after a transcatheter arterial embolization was performed, the patient was transferred to our department. there was no infective focus. the white blood cell (wbc) count (37,820/l, normal range<8 pg/ml) and the serum granulocyte colony-stimulating factor (g-csf) level (2,670 pg/ml, normal range<8 pg/ml) were high. bone marrow biopsy revealed little fat, significant hyperplasia, and predominantly increased neutrophils, but no findings of bone metastasis or bone marrow involvement. a g-csf-producing tumor was diagnosed and right nephrectomy and retroperitoneal tumorectomy were performed. however, the tumor had infiltrated into the inferior vena cava, diaphragm and abdominal wall, and only part of the tumor could be removed. in histopathological tests, hematoxylin-eosin staining showed malignant fibrous histiocytoma-like findings mixed with those for well-differentiated liposarcoma, and the case was diagnosed as dedifferentiated liposarcoma. preoperative 18f-fdg-pet computed tomography showed diffuse 18f-fdg uptake throughout the bone marrow and elevated uptake at the tumor site. however, since bone biopsy and bone scintigraphy indicated no bone metastasis or bone marrow involvement, we concluded that pet/ct imaging gave false-positive results in the bone marrow. this is the first report of pet/ct imaging of a g-csf-producing tumor in a urological disease. the imaging results may be useful for differential diagnosis for this tumor in patients with high wbc counts without infection.

PMC3085038

pubmed-952

supracondylar fractures of the humerus represent 50-70% of all elbow fracture in children in the first decade of life.1 current method of treatment of this fracture is based on gartland classification. flynn et al., reported the incidence of cubitus varus deformity after treatment was 5%, whereas arino et al., reported that it was almost 21%, ulnar nerve deficit was found in 15% of patients who were treated with medial and lateral pin as per the report of chai.2345 many different methods are described such as close reduction and long arm cast or slab, dunlop skin traction, olecranon traction, but all of these methods had large complication rate.126789101112 the current preferred method of treatment for displaced supracondylar fracture has been close reduction and percutaneous pin fixation. this method has given excellent results reported by various authors.101112131415 thus, i conducted this retrospective study to compare whether lateral pin construct, if placed properly, can provide the same stability like medial and lateral pin fixation, at the same time avoiding the possibility of iatrogenic ulnar nerve palsy.161718 this retrospective study was carried out at orthopaedics department of m. m. medical college from july 2005 to july 2010. a written informed consent was obtained from all the patients (by their parents).1920 in this study, 170 children with grade iii close supracondylar fractures of humerus were included. the patients were aged between 1.5 years and 13 years with the mean age of 7.76 years. the time of operation ranges from the 1 day of injury to the 8 day of injury with the mean time of operation being 4.6 days. the patients were evaluated as described by flynn and the results compared with the contra lateral normal elbow.2 under general anaesthesia, using c-arm fluoroscopy closed reductions were done.21 when satisfactory reduction had been achieved, then fixations were done by k-wires of 1.5 or 2.0 mm size and well-padded above-elbow posterior back-slabs were applied. [figures 1a-c] [figures 2a-c]. the patients were carefully observed for 12-72 hours (average 58 hours) and then discharged. the above-elbow plaster of paris (pop) back slabs were kept for two to three weeks and the pins and slab were removed in the outpatient (opd) clinic. elbow range of motion (roms) was started after removing the pop back slab. the follow-ups were arranged as follows: the first follow-up on the 7 day to inspect the wound; the second follow-up on the second week for wound inspection or suture removal and to see the pin configuration., x-rays were taken to see the callus formation; if callus is formed, then we remove the pop and pins and to start physiotherapy; the third follow-up on the 4 week and the fourth follow-up on the 8 week post-operatively to see the rom and carrying angle of the elbow, and the final follow-up on the 6 months post-operatively to see the final result of the study. (a) pre-operative a-p and lateral radiographs showing supracondylar fracture of humerus of 4-year-old child, (b) post-operative anteroposterior radiographs of supracondylar fracture of humerus showing with crossed k-wire fixation, (c) post-operative lateral radiographs of supracondylar fracture of humerus showing with crossed k-wire fixation (a) pre-operative a-p radiograph showing supracondylar fracture of humerus of 6-year-old child, (b) pre-operative lateral radiograph showing supracondylar fracture of humerus of 6-year-old child, (c) post-operative a-p and lateral radiographs of supracondylar fracture of humerus showing with 2 lateral k-wire fixation the protocol was approved by institutional ethics committee and thus meets the standards of the declaration of helsinki in its revised version of 1975 and amendments made to it in 1983, 1989 and 1996 (jama 1997;277:925-6). the protocol was approved by institutional ethics committee and thus meets the standards of the declaration of helsinki in its revised version of 1975 and amendments made to it in 1983, 1989 and 1996 (jama 1997;277:925-6). there were 170 children in this study, 97 children were male and 73 children were females. there were 103 left-sided and 67 right-sided fractures. among 170 children, 102 children had injury during playing, 44 children had met with a road traffic accident and 24 had a fall from a height. the extension types were 158 (92.94%) and flexion types 12 (7.05%). 85 (50%) cases were treated by two lateral parallel k-wires and 85 (50%) by cross k-wires. preoperatively, six cases had nerve injuries (median nerve three, ulnar nerve two and radial nerve one) and there were no cases of vascular injuries. post-operatively, eight patients (4.70%) got ulnar nerve injury in the crossed k-wire group (n=85). six (3.52%) patients got pin tract infection, four in the crossed k-wire group (n=85), and two in the lateral k-wire group (n=85), which were superficial and healed after removing pins and oral antibiotic administration. there were no ulnar nerve injuries in the patient treated by inserting only lateral two k-wires. callus formations were seen in all patients at the 2-3 weeks post-operatively before removing the k-wires. results were analysed using flynn's criteria.2 all patients were followed at 8 week, 16 week and the 24 week, postoperatively. however, comparison between two groups showed that all categories such as 8 weeks, 16 weeks and 24 weeks with respective excellent, good, fair and poor were not found statistically significant. in cases of ulnar nerve injuries and pin tract infections all patients achieved complete radiographic healing at a mean of 4 weeks (range: 3-6 weeks). in a subjective measure of outcome at follow-up, in the crossed k-wire group (n=85), the results were excellent in 88.23% and good in 5.88% patients, and in the lateral k-wire group, the results were excellent in 91.75% and good in 7.05% patients. no patients or parents reported their out-come as not satisfied. at follow-up, all patients went on to osseous union and regained a full range of movement after rehabilitation. during this study, complications like vascular injury, compartment syndrome, myositis ossifications, significant mal-union and non-union were not red. distal pin migration was seen in five (2.94%) patients, loss of reduction was seen in six (3.52%), which was not significant and did not require re-reduction and re-pinning. comparison between two groups such as cross k-wire group (85) and lateral k-wire group (n=85) by using the chi square test showed that in case of 8 weeks with (p-values=0.89), in 16 weeks (p=0.91) and 24 weeks (p=0.85) with respective excellent, good, fair and poor categories were not found statistically significant. the mean baumann angle loss in the medial-lateral pin fixation group and the 2-lateral pin fixation group was 5.96 and 5.30, respectively. analyses of the baumann angle loss showed no significant difference between medial lateral pin fixation and 2-lateral pin fixation management of displaced extension type iii supracondylar fracture of humerus treated by close reduction and percutaneous pin fixation has consistently given satisfactory result compared to other method of treatment. however, controversy persists regarding the adequate pin fixation technique comparing medio-lateral and lateral pin fixation. in this study, not much difference between both fixation methods in terms of stability was found but there is an evidence of iatrogenic ulnar nerve injury (4.70%) in medio-lateral pin fixation group. iatrogenic ulnar nerve injuries in this study were most likely neuropraxia (sunderland type 1) since all of them recovered without exploration or repair within 3.5 months post-operatively. complete transaction of the nerve or neurotmesis was not seen in this study. in cases of ulnar nerve injuries and pin tract infections also not found statistically significant.22 the medio-lateral pin fixation method supposed to have the advantage of better fracture stability, although iatrogenic ulnar injury can occur with this technique. pin fixation from lateral side has the advantage of avoiding ulnar nerve injury but this construct has been thought to be biomechanically less stable. lee ss et al., and ziouts et al., reported that medial and lateral entry provides greater torsional rigidity than lateral entry pin fixation does.1920 the total strength of this construct is not only related to pin entry but mainly to divergence of the pins in different column and number of pins. the greater strength seen with the divergence of the pins was related to the location of the interaction of the two pins and the fact that the greater amount of divergence between the two pins allow for some purchase in the medial and lateral column.1920 there are some authors who advocated the use of the third wire to prevent the displacement of the distal fragment.2324 the use of a third pin requires the medial pin to enter the joint and thus increases the risk of joint penetration and infection. skaggs et al.,13 found no ulnar nerve palsy and no reduction was lost in 124 children managed with only lateral-entry pins. in an other study of skaggs et al.,14 of 204 children who had a gartland type-3 fracture, 51 were treated with lateral pins only and 153 were treated with crossed pins. the configuration of the pins did not affect the baumann's angle in gartland type 3 fractures. the most common complication in the treatment of closed reduction and percutaneous pinning of displaced supracondylar fractures of the humerus is iatrogenic ulnar nerve palsy with the use of medial pin.1826272829 the rate of ulnar nerve injuries varies in different studies. lyons et al.,29 have reported this number as 6%, royce et al.,27 as 3%, agus et al.,28 as 58%. it is found that postoperative nerve palsies after percutaneous pinning was with direct injury to the nerve, not after manipulation of closed reduction.11262730 skaggs et al.,14 noted the incidence of ulnar nerve injury as 4% in patients whom the pins were applied without hyper flexion of the elbow and as 15% in whom the medial pin was applied with the elbow hyperflexed. it is also showed that lateral-pins decrease the rate of ulnar nerve injury when compared with medial-pins. in the present study, there was no incidence of ulnar nerve injury where pinning was done from the lateral side; and i did not find any difference in bone healing and stability between lateral-pin insertion and cross-pin insertion as the same treatment protocol was followed for both the groups. skaggs found that the use of lateral-entry pins alone was effective for even the most unstable supracondylar humeral fractures and they saw no iatrogenic ulnar nerve injuries, and no reduction was lost.1314 in the present study, iatrogenic nerve injury was seen in eight patients (4.70%) where pinning was done from cross pin insertion. although most of the ulnar nerve injuries recover spontaneously between 4 months and 6 months, permanent damage has been reported in the literature.2731 lyons et al.,18 observed spontaneous functional recovery after the removal of medial pin. pathological electromyographic measurements can be detected in most of ulnar nerve injuries during the early postoperative period. in this study, the results of both lateral and cross pin insertion groups at 8 post-operative week showed excellent results in around 70% of patients. at the final follow-up, these excellent results were seen in around 90% of the cases. in post-operative period those patients who had good or fair results were having severe soft tissue injuries or repeated closed reduction. khan obtained 88% excellent, 4% good and 4% poor results in his study.32 tiwari observed 88% satisfactory results, among which 42% were excellent, in his series of late-presenting supracondylar fractures of humerus in children.33 these two studies are comparable to our study. cubitus varus deformity is the most common problem seen after the treatment of supracondylar fractures. the cause of the deformity is coronal rotation, or tilting of the distal fragment.34 some investigators believed that varus deformity is due to epiphyseal growth disturbance or rotation of the distal fragment.35 smith suggested that residual medial tilt after reduction is the most important factor in varus angulations, with isolated rotational deformities being corrected by compensatory rotation at the shoulder.36 this concept has become popular in understanding the sequel of alteration in carrying angle.37 in this series, six patients (3.52%) had nerve injury preoperatively, out of which three had median, two ulnar and one radial. eight patients got ulnar nerve injuries post-operatively, which is 4.70% of the total number. the incidence of postoperative has been estimated to range from 5% to 19%.38 culp recommends that initial observation and supportive therapy for neural injury associated with a closed, displaced, supracondylar fracture of the humerus; and that if there is no clinical or electromyography evidence of return of neural function at five months after injury, exploration and neurolysis should be performed. if the nerve is in continuity, the prognosis after neurolysis is excellent.39 in my study, six (3.52%) patients developed pin-tract infections, which were superficial and healed after removing pins and administration of oral antibiotics. pirone found superficial pin-tract infection in 2% of cases with no deep infection and septic arthritis.40 in the present series, the distal pin migration was seen in five (2.94%) patients and loss of reduction in six (3.52%), which were not significant and so required no re-reduction and re-pinning. gordon observed pin-tract migration in 6% of cases and lee noticed the loss of reduction in 7% of cases.1219 lee et al.,41 stated that the lateral pinning technique was found to be more beneficial than the medial and lateral crossed pinning technique for supracondylar fractures of the humerus in children, on the basis of current evidences. avoiding the worst clinical scenario (permanent ulnar nerve palsy) might be more important and affordable than obtaining favourable clinical results (stable fixation) at the potential cost of disastrous complications. dua et al.,42 proposed that closed reduction and crossed pinning of displaced supracondylar fractures of humerus in children is a safe and effective method even with delayed presentation. erpelding et al.,43 stated that open treatment of distal humeral fractures with an extensor mechanism-on approach results in excellent healing, a mean elbow flexion-extension arc exceeding 100, and maintenance of 90% of elbow extension strength compared with that of the contra lateral, normal elbow. woratanara et al.,44 stated that lateral pinning is preferable to cross pinning for fixation of pediatric supracondylar humerus fractures as a result of decreased risk of ulnar nerve injury. the main goal of the treatment of displaced paediatric supracondylar humerus fractures is to achieve an anatomic reduction. this reduction should be supported by a fixation with a good stability and less morbidity. when all these are taken into consideration, we believe that closed reduction and percutaneous lateral pinning is an efficient, reliable and safe method.

background: supracondylar fractures are the commonest elbow injury in children. most displaced supracondylar fractures are manipulated and held with a medial/lateral entry or two lateral kirschner wires. it was the purpose of this study to investigate the treatment of this injury in this unique patient population. materials and methods: this study was conducted in the department of orthopaedic surgery in m. m. medical college from july 2005 to july 2010. one hundred seventy patients were recruited from emergency and outpatient department having closed displaced supracondylar fractures of humerus in children. they were treated either with medial-lateral pin fixation (n=85) or with 2-lateral pin fixation (n=85). all patients were operated under general anaesthesia. all patients were followed for 6 months. results were analysed using flynn's criteria.statistical analysis used: chi square test. chi square calculator was used as a software. results:all children achieved union in a mean time of 4 weeks (range: 3-6 weeks). post-operatively, eight patients (4.70%) got ulnar nerve injury and six (3.52%) patients got pin tract infection. comparison between two groups such as cross k-wire group (85) and lateral k-wire group (n=85) by using the chi square test showed that in case of 8 weeks with (p-values=0.89), in 16 weeks (p=0.91) and 24 weeks (p=0.85) with respective excellent, good, fair and poor categories were not found statistically significant. conclusion:the lateral percutaneous pinning technique of displaced supracondylar fractures of the humerus offers a viable alternative to the crossed pinning group as it offers the same stability without the incipient risk of iatrogenic ulnar nerve injury.

PMC3883234

pubmed-953

cervix cancer is one of the leading causes of cancer morbidity among women worldwide and it is the second most common cancer among women in developing countries. except for early stage, cancer cervix is usually treated with radiotherapy which consists of external beam radiotherapy followed by intracavitary brachytherapy. bladder and rectum are the main organs at risk in the treatment of cervical intracavitary brachytherapy whose doses are ensured not to exceed the limits of tolerance. several authors have shown that the dose distribution is affected by the geometry of the intracavitary applicators, age of patient, disease stage, length and angulation of the tandem, size of the ovoids and tandem [2, 3], volume of bladder and rectum. similarly dwell position of source, vaginal packing, balloon inflation technique [6, 7] and proximity of the applicator to the bladder and rectum do affect the dose to organs at risk. it is a well known fact that the abdominal and pelvic organs move during respiration and can therefore lead to changes in the dose to organs at risk. although there are many studies related to critical organ dose variation with respect to the factors as stated above, there is no study which evaluates the possible dose alteration induced by respiration in intracavitary brachytherapy. in this study, an effort has been made to evaluate the effect of respiration on the doses to bladder and rectum for patients undergoing cervical intracavitary brachytherapy. fifteen cervix cancer patients of figo stage iib-iiib were included in this study. external beam radiotherapy was delivered to pelvis to a total dose of 50 gy in two phases, 40 gy in 22 fractions by four field box technique in first phase and 10 gy in 5 fractions with midline shield in the second phase. subsequently all of them were treated with high dose rate intracavitary brachytherapy to a total dose of 21 gy delivered in three equal fractions at an interval of 1 week. during the procedure, a foley catheter was inserted into the bladder and its balloon was filled with 7 cc of radio-opaque fluid (omnipaque). the insertion of intracavitary applicators was performed under general anaesthesia and in each application an intrauterine tandem was place inside the uterine cavity and the ovoids in the vagina at the level of fornices. five patients were kept on continuous bladder drainage while the rest had their foley s tube clamped to allow formation of full bladder. all the patients were scanned with philips brilliance big bore 16 slice ct scanner following intracavitary applicator insertion. the movement of the patient s chest was monitored using bellows during the scanning procedure. the respiratory bellows (philips medical systems, cleveland, oh) was tied around the chest at thoraco-abdominal junction. as the belt gets stretched with every respiratory effort, the pressure transducer produces disturbance in electrical voltage which is displayed in the monitor as waveforms corresponding to different phases of respiration. four sets of ct image datasets each of 3 mm slice thicknesses with applicator in place were acquired at different phases of a respiratory cycle. the brachytherapy applicators were reconstructed from the ct image datasets and treatment plans were generated for all the four ct datasets on the planning system. in each treatment session, a dose of 7 gy was prescribed to point a. doses to icru 38 bladder (ibrp) and rectal (irrp) reference points were evaluated in all the four ct datasets. besides, another point was taken at the center of the foley s balloon chosen to represent the mean dose to the bladder (bmean). repeated mea-sures analysis was used to compare the differences in ibrp, irrp and mean bladder dose in all the four respiratory phases. figure 1a shows the topogram of a patient with intracavitary applicator and 1b displays the respiratory waveform displayed on the philips 4d ct console. table 1 shows the mean, range and median of the maximum dose to ibrp in all the four different respiratory states for full and empty bladder. similarly, table 2 shows the mean, range and median doses of the mean doses to the bladder for full and empty bladder. the maximum dose to the icru 38 rectal reference point in the four different respiratory phases is displayed in table 3. the maximum observed variation in doses to ibrp for full and empty bladder among the four respiratory phases was 15% and 3% respectively. similarly the maximum observed variation in doses to irrp for full and empty bladder among the four respiratory phases was 12% and 8% respectively. the mean variation between the doses to full bladder at different phases of respiration is 4% whereas it is less than 1% with empty bladder. the dose to icru rectal reference point is within 3% for both full and empty bladder. no statistical significance was observed between different respiratory phases with ibrp, irrp and bmean for full and empty bladder condition. dose to icru bladder reference point (ibrp) mean dose to bladder (bmean) dose to icru rectal reference point (irrp) a) topogram of a patient with applicator, b) respiratory waveform displayed on philips 4d ct console the doses to bladder and rectum are the major determining factor for late complications in intracavitary brachytherapy. there are several studies illustrating the different ways of reducing the dose to these organs at risk. jain et al have demonstrated that proper vaginal packing and patient anatomy can minimize the dose to the bladder and rectum. have shown that the bladder and rectal doses can be reduced by adjusting the dwell positions and relative dwell times of source positions. studies have also shown that inflating the balloon of a foley catheter significantly reduces the dose to rectum and bladder [6, 7]. quantified the displacement of points identified on the anterior uterine body, posterior cervix and upper vagina. they found that rectal filling may affect cervical position, while bladder filling has more impact on uterine body position, highlighting the need for specific instructions on bladder and rectal filling for treatment. chan et al. studied the internal movement of tumour, cervix, and uterus using serial cinematic magnetic resonance imaging scans and point-of-interest analysis. both inter- and intra-scan motion was greatest at the fundus of the uterus, less along the canal, and least at the cervical os. the isotropic internal target margins required to encompass 90% of the interscan motion were 4 cm at the fundus and 1.5 cm at the os. in contrast, smaller margins of 1 cm and 0.45 cm, respectively, were adequate to encompass the intrascan motion alone. currently 4d ct is being used extensively in imaging organs with evident internal movement. in many institutions, mri guided 3d optimization significantly improved the target coverage while retaining the constraints to organs at risk [1114]. in this study, we have used the 4d ct datasets to analyze the bladder and rectal dose at different respiratory phases. we noted that there is a difference between the doses to rectum and bladder at different phases of respiration. to the best of our knowledge, this is a unique study addressing the issue of organ motion with respiration in brachytherapy of cervix cancer. in our practice, the intracavitary treatment plan based on point a prescription icru points are preferably chosen for treatment evaluation than the 3d volume dvh parameters to avoid the possible dosimetric errors introduced by organ contours in the latter method. in our study, the variation of dose distribution in bladder is more with full bladder than when it is empty. filling status of bladder also affects the rectal dose variation. an important observation from our study is that the dose to ibrp for those patients with full bladder was less as compared to those with empty bladder. this may be due to the fact that when the bladder is full, the foley s balloon falls farther from the applicator. the individual ibrp dose variation was of the order of 15% as compared to 3% for full and empty bladder respectively. this indicates that the foley s balloon get displaced with respiration when the bladder is distended but remain restricted in position in an empty bladder. the individual dose to irrp for different respiratory phases with full bladder is 12% as compared to 8% with empty bladder condition. hence, the radiation oncologist could be advised to consider a possible variation in the doses to the bladder and rectum while planning intracavitary brachytherapy. one of the limitations of this study is that the study has been performed with ct based brachytherapy whereas gec-estro recommends mri based brachytherapy that enables accurate delineation of bladder and rectal volumes. our study shows that respiration affects the bladder and rectal doses in cervix cancer brachytherapy. the dose to irrp was found to be affected with both full and empty bladder whereas the dose to ibrp was found to be affected more with full bladder than with empty bladder. even though the mean variation in dose to bladder and rectum was within 4%, large individual dose difference was observed in some patients. hence it is advisable to reduce the critical organ dose to account for the dose variation introduced by respiratory motion.

purposein cervical intracavitary brachytherapy, it is mandatory to evaluate if the doses to bladder and rectum are within tolerance limits. in this study, an effort has been made to evaluate the effect of respiration on the doses to bladder and rectum in patients undergoing brachytherapy. material and methodsfifteen patients with cervix cancer treated with concurrent chemoradiation followed by intracavitary brachytherapy were included in this study. at the time of brachytherapy, all patients underwent 4d computed tomography (ct) imaging. five out of fifteen patients were scanned with empty bladder while the rest had full bladder during sectional imaging. four sets of pelvic ct image datasets with applicators in place were acquired at equal interval in a complete respiratory cycle. treatment plans were generated for all the ct datasets on a platotm sunrise planning system. a dose of 7 gy was prescribed to point a. doses to icru (report no.38) bladder (ibrp) and rectal (irrp) reference points were calculated in all the ct datasets. resultsthe mean of maximum dose to ibrp at four different respiratory phases for full and empty bladder were 53.38 19.20%, 55.75 16.71%, 56.13 17.70%, 57.50 17.48% and 60.93 15.18%, 60.29 16.28%, 60.86 15.90%, 60.82 15.42% of the prescribed dose respectively. similarly, maximum dose to irrp for full and empty bladder were 55.50 18.66%, 57.38 14.81%, 58.00 14.97%, 58.38 17.28% and 71.96 6.90%, 71.58 7.52%, 68.92 6.21%, 71.45 7.16% respectively. conclusionsour study shows that respiration affects the dose distribution to the bladder and rectum in intracavitary brachytherapy of cervix cancer. it is advisable to reduce the critical organ dose to account for the dose variation introduced by respiratory motion.

PMC5183644

pubmed-954

despite the significant improvement in adhesive systems, they are not capable of preventing the formation of microgaps at the dentinal margins of composite restorations. even when immediate complete marginal sealing was established, degradation of resin-dentin interface can occur rapidly over time. also, the plaque accumulation containing microorganisms on the composite surface is more than that of the enamel surface and other restorative materials. additionally, some active microorganisms may be left in the cavity due to lack of definitive and reliable assessment criteria for detection of carious dentin and complete elimination of microorganisms in the cavity. particularly, this problem is more serious by increasing predilection to a minimally invasive tissue-saving dentistry. subsequently, the adjunctive treatment with antibacterial agents during dentin bonding would be beneficial for preventing the detrimental effects caused by residual bacteria or by microleakage. the self-etching primer systems have become popular in adhesive dentistry due to their advantages over etch-and-rinse adhesives including less technique sensitivity; reduced postoperative sensitivity; and elimination of etching, washing, and drying as a separate step. however, demineralized smear layer possibly containing microorganisms is incorporated into the hybrid layer, so the effect of antibacterial activity is of major importance in these adhesives. the antibacterial activity is provided by two types of materials, disinfecting solution as agent releasing type and adhesive containing antibacterial monomer as a nonagent releasing type. unpolymerized mdpb exhibits strong bactericidal activity similar to a disinfecting solution, such as chlorhexidine digluconate (ch). this name was given due to the fact that the agent is immobilized at the adhesive-dentin interface after copolymerization with other monomers. this unique antibacterial effect is long lasting and it may have no adverse effect on mechanical properties and bonding efficiency of the adhesive. commercially available product containing mdpb is clearfil protect bond (pb) which is derived by addition of this monomer to the self-etching primer adhesive, clearfil se bond (se) as its parent. the latter contains no antibacterial agent. apart from antibacterial effect of ch, it functions as a matrix metalloproteinases (mmps) inhibitor. this additional effect of ch can prevent collagen degradation at the bonding interface over time. mmps are a class of metal-dependent endopeptidases that were expressed by human pulp fibroblasts in dental extracellular matrix. these enzymes can be activated during dentin demineralization process following carious lesions formation or application of the acidic adhesives. the beneficial preservation effect of ch on the bond stability was reported in restorations bonded by simplified etch-and-rinse and self-etch adhesives. the naked collagen fibrils at the base of the hybrid layer following incomplete resin penetration are susceptible to degradation by mmps. if ch has no adverse effect on dentin bond strength of two self-etch adhesives with or without antibacterial monomer, it may preserve bonding interface of these adhesives. furthermore, little information is available on the effect of ch on the long-term bond strength of these adhesives, particularly the antibacterial self-etching adhesive. the simultaneous application of two types of antibacterial materials may improve the durability of the self-etch adhesive. therefore, the aim of the present study was to test the null hypothesis that adjunctive use of ch with a self-etch adhesive (se) and a self-etch adhesive containing mdpb (pb) has no effect on immediate (24 h) bond strength to dentin, or after water storage for 6 months plus thermocycling. the teeth were stored in 1% chloramine t solution for 2 weeks, then in distilled water at 4c before use. after removing the roots, the midcoronal dentin surfaces were exposed by removing the occlusal enamel with a diamond saw under a water spray. the dentin surfaces were examined under a stereomicroscope (carl zeiss inc, oberkochen, germany) to ensure that no enamel or pulp tissue remained. the flat dentin surfaces were polished with 600-grit silicone carbide abrasive paper (snam abrasives pvt. after ultrasonic cleaning, rinsing, and drying; the adhesive tape was applied to the prepared surfaces to limit the bonding area. the prepared teeth were randomly divided into four main groups of 20 teeth each according to the adhesive system used: se and pb. each adhesive was assigned to two control and experimental groups. in the latter groups, 2% chlorhexidine digluconate solution (ch, consepsis, ultradent, usa) was applied on the dentin surface prior to application of acidic primer of the adhesives. all of the materials were used according to manufacturer's instructions [table 1]. materials used in the current study the resin composite (filtek z250, 3 m, usa) was placed on the cured adhesives using a cylindrical split mold with a height of 2.5 mm and surface diameter of 2 mm in two increments of 1 and 1.5 mm. each increment was light-cured for 40 s at 600 mw/cm with a light-curing unit (vip junior, bisco, schaumburg, il, usa). half of the specimens (n=10) in each group were stored in tap water at 37c for 24 h and the other half (n=10) were stored in tap water containing 0.4% sodium azide with a stable ph at 37c for 6 months and additionally thermocycled 1,000 times (between 5 and 55c with 20 s dwell times) during 6 months prior to bond strength testing. the shear test was performed using universal testing machine (instron z020, zwick, roell, germany). a knife-edge shearing rod at a crosshead speed 1 mm/min was used to load the specimens until fracture. shear bond strength in mpa was calculated from the peak load at failure divided by the specimen's surface area. one specimen from the control and experimental groups pretreated with ch and bonded with the self-etch adhesive (pb) were prepared for sem examination. approximately, 2-mm thick disks were prepared from each specimen with accutom-50 cut-off machine (struers, denmark) prior to examination with scanning electron microscope (sem, xl30, philips, netherland) operating at 17 kv. all data were analyzed with three-way analysis of variance (anova) for the effect of adhesive system, ch, and storage time. for each adhesive, a two-way anova was done to evaluate the interaction effect of storage time and ch, and t-test was done to compare the effect of two factors. all tests were done at a 0.05 level of significance and all analyses were performed using spss version 11.5 software (chicago, il, usa). after testing, the fracture modes were evaluated using a stereomicroscope (carl zeiss inc, oberkochen, germany) at 10 and classified according to the predominant mode of fracture including: 1) adhesive, 2) cohesive in dentin, 3) cohesive in composite, and 4) mixed, a combination of adhesive and cohesive. the mean strength and standard deviations in the eight experimental groups are summarized in figure 1. three-way anova showed that bond strength was significantly influenced by the adhesive type (p<0.001) and time (p=0.004). the effect of ch, the interactions between adhesive type and time, between adhesive type and ch, and the interaction among all the three factors was not significant (p>0.05). however, the interaction between ch and time was statistically significant (p<0.001). this interaction was statistically significant for each adhesive, se (p=0.02) and pb (p=0.006). the bond strength of pb was significantly lower than that of se in two time periods (p=0.001). the mean shear bond strength for eight tested groups the bond strength of two adhesives was significantly reduced after aging (p=0.004). for two adhesives (se, pb), ch had a significantly negative effect on the initial bond strength (p=0.04 and 0.004, respectively), but bond strength was not altered over the 6 months of storage (p=0.84 and 0.70, respectively). after a 6-month period, bond strength of two adhesives associated to ch did not vary with their control groups (p=0.25 and 0.48). representative sem images of the dentin/restoration interface for the control and experimental groups are shown in figures 2 and 3. the frequency of fracture modes of eight groups a representative scanning electron microscope (sem) image of a bonded interface in control group (self-etch adhesive), where gap-free interface can be observed (250). c=composite, d=dentin a representative sem image of a bonded interface in experimental group (self-etch adhesive+chlorhexidine). in spite of a potential benefit for antibacterial activity, it is noteworthy that achieving effective durable bonding to dentin is still one of the most critical factors affecting long-term clinical success of a restoration. hence, in the current study, the effect of two types of antibacterial agents on the bond strength of the two self-etch adhesives to dentin was tested in two time periods. the present results revealed that although the ch application prior to the two tested adhesives significantly decreased the immediate bond strength, this bond was stable after aging in water; there was no significant difference between the control and experimental groups treated with ch after 6 months of storage. the adverse effect of ch solution on initial bonding performance of se was previously reported by ercan et al. also, meiers and shook reported that ch application prior to acidic primer of syntac significantly decreased its bond strength to dentin. however, in other studies, no adverse effect was found on sealing ability of se and bond strength of se to dentin. in a study by siso et al., although there was no significant difference between the control and ch-treated groups, the latter group revealed a higher value of microleakage than that of the control group bonded by se. it was reported that the application of ch prior to self-etch adhesives (clearfil tri s bond and se) did not affect immediate bond strength resin composite to dentin. the self-etch adhesives used in this study contain a functional acidic monomer, 10-mdp. it was demonstrated that dissociated ch cations could bind to phosphate groups and calcium of the hydroxyapatite. the remaining cations might form bonds with phosphate anions of 10-mdp molecules of the self-etch adhesives after application of the adhesive on the surface treated with ch. this inhibitory effect on 10-mdp may impair the bonding ability of this functional acidic monomer (10-mdp) to dentinal calcium, reducing the bond strength to dentin. according to our results, a significant decrease in bond strength of the two adhesives although self-etch adhesives are capable of simultaneous etching and priming, a discrepancy in the depth of demineralization and resin infiltration may occur in these adhesives. the disintegration of these collagens could contribute to a decrease in the dentin bond strength of two self-etch adhesives observed in the current study after aging. moreover, it has been shown that mild versions of self-etch adhesives can activate mmps, resulting in degradation of suboptimal infiltrated collagen and loss of bond strength. the results of the present study revealed that although ch application compromised the immediate bond strength, it diminished loss of bond strength after aging. ch was applied on the smear layer-covered dentin, and then covered with acidic primer and adhesive resin may protect the unprotected collagen created during bonding procedures. the beneficial effect of 2% ch on stability of hybrid layer formed by an etch-and-rinse and a self-etch adhesives was reported in a recent study. few studies have so far reported the effect of ch application prior to two-step self-etch adhesives on long-term bonding effectiveness to dentin. in a study by campos et al., the preservative effect of ch 2% on bond strength of an all-in-one self-etch adhesive, clearfil tri s bond, to dentin was reported during a 6-month aging period. however, mobarak has recently demonstrated that pretreatment with 2 or 5% ch is not able to diminish the loss of bond strength of se to normal dentin over 2-year aging in artificial saliva under simulated intrapulpal pressure. these divergent results may be due to the difference in the ch concentration, aging condition, and aging duration time, bonding substrate, and testing method. the results of the current study indicated that pb showed significantly lower bond strength to dentin than that of se. the nonuniform distribution of naf fillers may contribute to a relative poor mechanical property in some areas of bonding interface, resulting in lower bond strength of pb compared to se. contrary to our results, some studies have suggested that incorporation of antibacterial monomer into primer did not adversely affect the dentinal bond strength of the adhesive. when evaluating the fracture modes of the eight tested groups in this study, the higher number of the adhesive fracture was detected in two groups of pb+ch and control group of pb (24 h). it seems that the higher number of the adhesive fracture was associated to the lower bond strength of these groups. further studies should investigate the interaction of ch with other two-step self-etch adhesives and the long-term effect of ch on bond strength of these adhesives to dentin. within the limitations of the current study, it may be concluded that although ch was capable of decreasing the loss of bond strength of the two-step self-etch adhesives to dentin over time, it compromised the initial bonding of the self-etch adhesives. thus, further in vitro and in vivo studies should be conducted before the combined application of ch, and two-step self-etch adhesives can be recommended in clinical practice.

background: considering the possibility of remaining bacteria in the cavity or invading via microgaps, the use of antibacterial agents in adhesive restoration may be beneficial. this study evaluated the effect of chlorhexidine on immediate and long-term shear bond strength of adhesives with and without antibacterial agent to dentin. materials and methods: in this in vitro study, the occlusal surfaces of 80 intact human premolars were removed to expose the flat midcoronal dentin. the teeth were assigned to four groups. two adhesive systems, clearfil se bond (se) and clearfil protect bond (pb) were used according to manufacturer's instructions as the control groups. in the experimental groups, 2% chlorhexidine was applied prior to acidic primer of two adhesives. then, resin composite was applied. half of the specimens in each group were submitted to shear bond test after 24 h without thermocycling, and the other half were submitted to water storage for 6 months and thermocycling before testing. the data was analyzed using three-way analysis of variance (anova) and t-test (= 0.05). results: chlorhexidine application significantly decreased the initial bond strength (bs) of the two self-etch adhesives to dentin (p<0.05). there was a significant reduction in bs of se and pb after aging compared to initial bonding (p<0.05). however, there was no significant difference between bs of the control and chlorhexidine-treated groups for the tested adhesives after aging. pb showed a lower bs than se in two time periods (p<0.05). conclusion: chlorhexidine was capable of diminishing the loss of bs of these adhesives over time. however, considering the negative effect of chlorhexidine on the initial bs, the benefits of chlorhexidine associated with these adhesives can not possibly be used.

PMC3872633

pubmed-955

striated muscle cells contain a complex, highly ordered cytoskeleton, mainly composed of interdigitating thick myosin and thin actin filaments. muscle contraction and relaxation occurs by the sliding of myosin filaments past actin filaments under the strict regulation of their accessory proteins, which are responsible for their assembly and maintenance as well as the regulation of contractile activity. myosin-binding protein-c (mybp-c) comprises a family of accessory proteins of the thick myosin filaments that encompasses ~2% of the total myofibrillar protein. to date, two major roles have been attributed to mybp-c; it contributes to the regular organization and stabilization of thick filaments and modulates the formation of cross-bridges between myosin and actin, via direct interactions with both filamentous systems (as reviewed in [3, 4]). within the sarcomere, mybp-c localizes to the c-zone, the cross-bridges containing region of the a-band, in 79 transverse stripes that are ~43 nm apart [57]. three mybp-c isoforms have been identified; cardiac, slow skeletal, and fast skeletal (cmybp-c, smybp-c, and fmybp-c), encoded by different genes localizing to human chromosomes 11, 12, and 19, respectively [8, 9]. the core structure of mybp-c is composed of seven immunoglobulin (ig) domains and three fibronectin type iii (fn-iii) repeats, numbered from the nh2-terminus as c1c10 (figure 1;). a proline/alanine- (pro/ala-) rich motif and a conserved linker region, termed mybp-c or m-motif, flank the first ig domain, c1. cmybp-c possesses three unique features, including an additional ig domain at the extreme nh2-terminus (termed c0), a 9-residue long insertion within the m-motif containing consensus phosphorylation sites and a 28-amino acid long loop in the middle of domain c5 [8, 11]. conversely, smybp-c comprises a subfamily of four alternatively spliced variants (v), v1v4, differing from one another due to the retention or exclusion of select exons encoding three novel insertions. these are located at the very nh2-terminus within the pro/ala- rich motif, the middle of domain c7, and the very cooh-terminus of the molecule, following the c10 domain (figure 1;). the presence of these insertions may result in different topographies and possibly functions of the smybp-c variants that contain them [12, 13]. consistent with this, v1, which carries the cooh-terminal insertion preferentially localizes to the periphery of the m-band, rather than the c-zone, where it codistributes with its binding partners obscurin and titin (our unpublished observations) and plays key roles in the structural integrity of m- and a-bands. the last ig domain (c10) of smybp-c v1 interacts with the second ig domain (ig2) of obscurin. although the presence of the novel cooh-terminal insertion further strengthens this interaction, it is not required for binding. however, this novel motif is both necessary and sufficient to support binding of smybp-c v1 to the last ig domain of titin, m10, which is a hotspot for mutations associated with tibial muscular dystrophy [14, 15]. moreover, our laboratory has recently identified novel phosphorylation sites within the pro/ala- rich motif of the nh2-terminus of smybp-c, specifically ser-59, located in the novel nh2-terminal insertion and ser-62 are targets of pka, while ser-83 and thr-84 are targets of pkc; ser-204 is a substrate for both pka and pkc. similar to its cardiac counterpart, phosphorylation of these sites may be essential in regulating the activities of smybp-c. nevertheless, despite the differences in the core structure of the cardiac, slow, and fast mybp-c proteins, they share a ~50% homology and a ~65% identity. although mybp-c was identified over forty years ago as a contaminant of purified myosin, a better understanding of its physiology has emerged in the last two decades, paralleling the discovery of mutations in cmybp-c that result in the development of familial hypertrophic cardiomyopathy (fhc; [18, 19]). consequently, most of our knowledge originates from studies focusing on the cardiac form of the protein. this review will focus on the role of mybp-c in regulating the formation of actomyosin cross-bridges via its direct interaction with both myosin and actin filaments. myosin is a hexameric protein consisting of two heavy chains (mhc) and two light chains (regulatory and essential, rlc and elc, resp.). a dimer of heavy chains forms a coiled-coil helix that constitutes the rod or light meromyosin (lmm) segment of myosin. toward its nh2-terminus, the helix unwinds and each mhc gives rise to a catalytic head, referred to as subfragment 1 (s1) that has atpase activity and participates in the formation of cross-bridges with filamentous actin [21, 22]. the lever arm, referred to as subfragment 2 (s2), separates the s1 segment from the rod portion, and transduces the chemical energy from the hydrolysis of atp into mechanical movement along the thin filament. a pair of rlc and elc binds in tandem to each head of the s1 segment modulating the speed and force of contraction [24, 25]. in addition to myosin light chains and actin, myosin is intimately associated with mybp-c (table 1 and figure 2;). the cooh-terminal c10 domain of all three mybp-c isoforms harbors binding sites for the lmm portion of myosin. charged residues r1064, e1079, n1083, r1100, and r1101 present on the surface of the c10 domain of mybp-c support an electrostatic interaction with residues 15541581 of the rod domain of myosin that has a modest affinity of ~3.5 m [28, 29]. moreover, through domains c8c10, mybp-c interacts with titin, a giant sarcomeric protein spanning half a sarcomere, which is also tightly bound to the thick myosin filament. specifically, mybp-c binds to the first ig domain of an 11-domain superrepeat present in the a-band portion of titin. thus, it has been speculated that the repetitive binding of mybp-c to the a-band portion of titin likely contributes to its periodicity within the c-zone. consistent with these findings, a number of studies have further shown that the cooh-terminal c8c10 domains are necessary and sufficient to target mybp-c to the a-band [44, 45]. while binding of the cooh-terminus of mybp-c to the rod domain of myosin may contribute to the maintenance and stability of the thick filament, (reviewed in [3, 4]), binding of the nh2-terminus of mybp-c to myosin may mediate contractile regulation a number of biochemical and structural studies have implicated the nh2-terminal portion of cmybp-c, containing domains c0c2, in binding to the s2 region of myosin. specifically, the m-motif located between the c1 and c2 ig domains has been shown to bind directly to the nh2-terminal 126 residues of the s2 fragment with an affinity of ~4.3 m; this interaction is abolished by phosphorylation of specific ser residues within the m-motif of cmybp-c via camp-dependent protein kinase [4649]. similarly, the c2 domain has been also shown to interact with the same 126 residues of the nh2-terminus of the s2 fragment, albeit with considerably lower affinity (~1.1 mm), compared to the m-motif. interestingly, molecular modeling has suggested that the interaction between the s2 portion of myosin and the c2 domain of cmybp-c is mediated by polar residues. on the other hand, the c1 domain of cmybp-c has been reported to interact with the hinge region between the s1 heads and the s2 fragment, in close proximity to the myosin light chains, while a recent study reported that the first ig domain of cmybp-c, c0, binds to the rlcs with an affinity of ~3.2 m. in support of this, overexpressed c0 targets to the a-band independently of the rest of the molecule, likely through its interaction with the rlcs. taken together, these findings strongly suggest that cmybp-c may regulate the position and thus proximity of the myosin s1 heads relative to the actin filament, through its interaction with the s2 region and the rlcs, therefore affecting the formation of actomyosin cross-bridges. in addition to the mounting experimental support indicating the intimate association of the nh2-terminus of cmybp-c with the s2 region of myosin, there is also significant evidence supporting its direct interaction with actin thin filaments. early studies have shown that purified full-length cmybp-c binds to filamentous actin (f-actin) [36, 50]. however, reconstituted thin filaments (i.e. preincubated with troponin and tropomyosin) fail to bind purified full length cmybp-c in the presence of edta, a ca chelating agent, but binding is restored upon addition of ca [36, 50]. further studies have recently begun to identify the actin-binding domain(s) of cmybp-c through biochemical and structural approaches. neutron scattering experiments have indicated that recombinant c0c2 binds to f-actin in a repetitive manner, stabilizing it, while biochemical studies have shown that recombinant c0 is capable of directly interacting with f-actin. moreover, bacterially expressed c1-c2 associates with naked f-actin as well as reconstituted thin filaments, likely through the m-motif, exhibiting a binding affinity of ~10 m to naked f-actin [40, 41]. this finding has been also substantiated by a recent study that used negative stain electron microscopy and three-dimensional reconstruction to show that bacterially expressed c0c3 is well ordered on actin filaments near subdomain 1. noticeably, some of the above studies report linear, nonsaturating binding, suggesting the presence of weak electrostatic interactions, as exemplified by the calculated micromolar affinity. consistent with this, these binding reactions depend largely on ph and ionic strength and are regulated by phosphorylation of cmybp-c. thus, an increase in ph or ionic strength significantly decreases the capacity of the aforementioned cmybp-c peptides to bind to f-actin [4042]. similarly, pretreatment of these recombinant peptides with pka results in a dramatic reduction of their ability to associate with f-actin. importantly, a recent study by rybakova and colleagues investigated the actin-binding capabilities of cmybp-c across its entire length. in support of previously published studies discussed above, the authors found that the nh2-terminal domains of cmybp-c bind to f-actin in a linear, nonsaturating manner, likely mediated by weak electrostatic interactions. however, recombinant full-length cmybp-c, expressed in the baculovirus system, supported a direct and saturating interaction with f-actin with a calculated kd of ~4.3 m. moreover, constructs lacking domains c0c5 exhibited similar binding properties as full-length cmybp-c; this observation prompted the authors to conclude that the weak actin binding mediated by the nh2-terminus of the molecule does not contribute significantly to the overall affinity of cmybp-c for f-actin. moreover, the authors further showed that the actin binding supported by the cooh-terminal c6c10 domains, unlike the nh2-terminal c0c2 domains, is independent of the regulatory elements, troponin and tropomyosin, of thin filaments, the levels of ca, and the phosphorylation status of the m-motif. although the cooh-terminus of cmybp-c exhibits a higher affinity for f-actin, the minimal binding region and its physiological relevance are still elusive. these open questions are especially important because the cooh-terminal domains c6c10 also harbor binding sites for myosin and titin filaments (discussed above). indeed, space constraints would favor dynamic rather than simultaneous interactions of the cooh-terminus of cmybp-c with the actin, myosin, and titin filaments. however, three-dimensional reconstruction of thick filaments demonstrated that domains c7c10 of cmybp-c run along the length of the myosin rod, suggesting that its interaction with the lmm portion of myosin is not of dynamic nature. collectively these studies suggest the presence of various but weak binding sites for f-actin in the nh2-terminus of cmybp-c as well as the presence of a high affinity, saturating binding site in the cooh-terminus of the molecule. obviously, more work is required before we can obtain a clear understanding of the physiological relevance of these interactions during muscle contraction and relaxation. early work has shown that the skeletal isoforms of mybp-c also interact directly with both thick and thin filaments; however, their binding is much less characterized (figure 3 and table 2). similar to cmybp-c, the skeletal isoforms bind to the rod portion of sarcomeric myosin through their c10 domain and to the s2 region through their nh2-terminus. further characterization of the nh2-terminal binding has shown that the first two ig domains of smybp-c (c1-c2) bind to the s2 region with an affinity of ~2.2 m [26, 34]. in addition, native skeletal mybp-c (presumably containing a mixture of the slow and fast isoforms) coaggregates with f-actin in a ca sensitive manner. it becomes apparent from the above studies that our knowledge on the interaction of slow or fast mybp-c with myosin and actin filaments is limited. the identification of multiple mutations in the cardiac isoform that have been causally linked to the development of fhc is a major contributing factor that has shifted the interest of researchers towards cmybp-c; however, this has recently changed, as mutations in smybp-c were identified in patients suffering from distal arthrogryposis type 1 (da1), a disorder characterized by congenital contractures of the hands and feet (; please see below). more importantly, the molecular diversity of the skeletal isoforms further complicates the relevant studies as there are at least five different skeletal forms of mybp-c (one fast and four slow variants) that share homologous or common sequences, contain novel insertions in the nh2- and cooh-termini, and may coexist in the same muscle, fiber, or even sarcomere. further detailed investigation is, therefore, necessary to evaluate the ability of each isoform to bind to myosin and actin filaments as well as the physiological significance of these interactions. during the last decade, an overwhelming number of mutations (~200) have been identified in the mybpc3 gene, which encodes the human cardiac mybp-c protein. these include missense, nonsense, deletion/insertion, and frame-shift mutations and have been causally linked to the development of modest and late onset familial hypertrophic cardiomyopathy (fch) [5658]. the majority of the nonsense and frame-shift mutations result in truncated forms of the protein, that lack the cooh-terminus harboring binding sites for lmm, actin and titin, and thus negate the ability of cmybp-c to associate with these filaments and regulate contractile activity. interestingly, these truncated peptides are often undetectable in patient biopsies, possibly due to transcriptional misregulation or rapid degradation. contrary to non-sense and frame shift mutations, missense mutations are generally associated with a less severe cardiomyopathic phenotype and do not affect the structure or stability of the protein although some of them have been suggested to weaken myosin binding. moreover, recent genome wide linkage analysis revealed that mutations in the mybpc1 gene that encodes the human skeletal mybp-c slow protein lead to the development of distal arthrogryposis type 1, an autosomal dominant disorder characterized by congenital contractures of the hands and feet. two missense mutations have been identified to date, w236r and y856h, which are present in the m-motif and c8 domain, respectively. evaluation of skeletal muscle biopsies obtained from affected individuals revealed that slow twitch fibers were significantly smaller than fast twitch fibers. importantly, the locations of these two mutations indicate possible alterations in the ability of smybp-c to interact with myosin or actin via its nh2-terminus and to associate with the thick and titin filaments through its cooh-terminus. the causal involvement of the mybp-c family of proteins in the development of cardiac and skeletal myopathies, as exemplified by the aforementioned studies, clearly indicates that the members of this multifaceted and complex family are essential components and key regulators of muscle structure and function. through its dynamic interactions with myosin and actin filaments, mybp-c affects the formation and cycling of cross-bridges in three distinct ways: (i) by maintaining the normal structure of myosin and actin filaments, (ii) by regulating the rate at which myosin and actin interact, and (iii) by modulating the atpase activity of myosin. consistent with this, the addition of skeletal mybp-c reduces the critical concentration necessary for myosin polymerization in vitro and results in the formation of longer and more uniform thick filaments [61, 62]. in addition, the nh2-terminal region c0c2 of cmybp-c supports actin bundling in vitro, as evidenced by the increased turbidity of f-actin in the solution and the formation of significantly thicker actin filaments, as visualized by electron microscopy. notably, the effect of cmybp-c on actin bundling is dependent on ph and regulated by phosphorylation events mediated by pka. moreover, in vitro motility assays, examining the sliding of actin filaments over myosin heads in the presence of full-length or truncated forms of cmybp-c revealed a significant reduction in their sliding velocity [40, 63, 64]. notably, the degree of reduction was also dependent on the presence of the actin regulatory proteins, troponin, and tropomyosin; in their presence, there was a lesser effect on the decrease of actin motility over full-length myosin [63, 65], hmm or s1 heads. mybp-c also modulates the atpase activity of myosin, though in an indirect way, via interactions with both myosin and actin filaments supported by its nh2-terminus. in agreement with this, purified smybp-c inhibited the atpase activity of actin-activated myosin; however, native cmybp-c moderately enhanced the actin-activated atpase activity of myosin [66, 67]. surprisingly though, recombinant cmybp-c containing the c0c2 region inhibited the atpase activity of myosin in the presence of actin. the majority of the aforementioned studies have been performed with native proteins obtained from different species or types of muscles, presumably due to ease of purification. however, alyonycheva et al. have convincingly shown that the strength of the interaction between myosin and mybp-c is considerably increased when both proteins are purified from the same source. as such, cmybp-c and skeletal mybp-c bound with higher affinity to cardiac and skeletal myosin, respectively. as our knowledge about the molecular and functional properties of mybp-c broadens, it becomes apparent that through interactions supported by its cooh and nh2 termini, mybp-c contributes to the maintenance of the normal structure of thick filaments, and the regulation of cross-bridges formation and cycling. it has, therefore, been suggested that mybp-c may serve as a linker, whereby its cooh-terminus is necessary for anchoring it to lmm in the a-band, leaving the nh2-terminus available to modulate contractility through dynamic interactions with the head region of myosin and actin. consequently, calaghan and colleagues proposed the tether model to explain how mybp-c modulates the formation of actomyosin cross-bridges. this model suggests that both myosin binding sites on mybp-c act together by limiting the movement of the s1 heads relative to lmm and actin, thereby restricting the formation of actomyosin cross-bridges. phosphorylation of select ser residues within the m-motif of cmybp-c, mediated mainly by pka, regulates the interaction between cmybp-c and myosin by acting as an on-off switch. consequently, when in the on or dephosphorylated state, the nh2-terminus of cmybp-c binds to myosin and limits its interaction with actin, when in the off or phosphorylated state, myosin is free to interact with actin and form cross-bridges. the tether model is centered on the dynamic and highly regulated interaction of mybp-c with myosin and actin filaments. however, it is necessary to understand the orientation of mybp-c within the sarcomere, as well. two proposed models for the topology of mybp-c have been suggested: a collar model in which a trimer of mybp-c molecules wraps around the rod domain of myosin, and an axial model, where the cooh-terminus of mybp-c runs along the length of the rod domain. a stipulation of the collar model is that mybp-c is required to dimerize. in support of this, it has been shown that mybp-c dimerizes through interactions mediated by domains c5 and c7 with c8 and c10, respectively [43, 72]. on the other hand, x-ray diffraction in combination with molecular and computational modeling studies support the axial model, suggesting that the last three domains of mybp-c lay along the length of the myosin rod [71, 73]. recently, a study using electron microscopy and three-dimensional reconstructions of cardiac thick filaments further demonstrated that the cooh-terminus of cmybp-c runs along the rod domain surface of thick filaments. although mybp-c dimerization is not essential for the arrangement of mybp-c along the length of the myosin rod, the axial model does not negate its dimerization. nevertheless, in both models the nh2-terminus of mybp-c extends into the interfilament space where it is accessible to bind to the s2 portion of myosin and actin. physiological dynamic interactions of mybp-c with myosin and actin are possible as the distance between thick and thin filaments is ~916 nm and the length of extended mybp-c is ~40 nm. therefore, mybp-c is capable to span the interfilament space, enabling interactions with both actin and myosin filaments. it is not without precedence that regulation of contractility occurs through dynamic interactions with both actin and myosin filaments, as the essential and regulatory light chains bind tightly to myosin, but also interact with actin [25, 76, 77]. additionally, twitchin, a protein similar to titin, characterized in c. elegans as a thick filament regulatory protein, interacts with both myosin and actin in a phosphorylation-dependent manner to modulate cross-bridges formation [78, 79]. however, current models (as described above) to explain the mechanism of contractile regulation through dynamic binding to myosin and actin are lacking. undoubtedly, more work is necessary in order to understand the precise mechanisms by which mybp-c modulates contractility.

myosin-binding protein-c (mybp-c) is a family of accessory proteins of striated muscles that contributes to the assembly and stabilization of thick filaments, and regulates the formation of actomyosin cross-bridges, via direct interactions with both thick myosin and thin actin filaments. three distinct mybp-c isoforms have been characterized; cardiac, slow skeletal, and fast skeletal. numerous mutations in the gene for cardiac mybp-c (cmybp-c) have been associated with familial hypertrophic cardiomyopathy (fhc) and have led to increased interest in the regulation and roles of the cardiac isoform. this review will summarize our current knowledge on mybp-c and its role in modulating contractility, focusing on its interactions with both myosin and actin filaments in cardiac and skeletal muscles.

PMC3196898

pubmed-956

the converge consortium has obtained a large number of samples (including 5,303 cases of mdd and 5,337 controls, all chinese subjects) for inclusion in genome-wide association studies (gwas). snp rs35936514 in the lhpp gene has been reported to be significantly associated with mdd at a genome-wide level. lhpp encodes an enzyme known as phospholysine phosphohistidine inorganic pyrophosphate phosphatase (lhpp), which was originally purified from swine brain tissue and shows high levels of expression in the brain [3, 4]. this region of the genome has been implicated in the etiology of mdd using a combination of linkage and association analysis. lhpp or a product of a collinear brain-specific transcript, therefore, may interact with htr1a in the pathogenesis of major depression. a recent study has extended the findings of previous literature by supporting the role of lhpp in risk for mdd. a single study has shown the expression of lhppase to be associated with thyroid function, which could be interesting given that thyroid disorder is thought to mediate the functional regulation of mdd patients. a link between thyroid function and depression any impairment of thyroid function supply to the developing cns causes severe changes to the overall architecture and function of human brain, leading to various neurological dysfunctions [912]. recently, genetic imaging has been used to investigate the association of genetic variation with neuroimaging endophenotypes. genetic imaging is an emerging field that is rapidly identifying genes that influence the brain, cognition, or risk for diseases. along with clinical diagnosis, it has fostered new enthusiasm in depression research, because this approach allows for assessing the neural impact of candidate genes in vivo and thus provides for a new level of evidence [17, 18]. resting-state functional magnetic resonance imaging (rs-fmri) is often used to examine mental disorders. the amplitude of low-frequency fluctuations (alff) is thought to detect the intensity of spontaneous neural activity at rest. it has been considered a reliable and sensitive measure to study both healthy and clinical populations [2022]. the effects of the neural system and their association with lhpp variation in the context of mdd are not yet understood. identifying previously unknown physiological pathways can open new avenues for the development of novel depression drugs. in the present study, the association of lhpp rs35936514 with the resting-state brain activity was measured using alff. it is here hypothesized that variations in rs35936514 may influence spontaneous brain activity at rest in mdd patients. 45 mdd patients (mean age 27.13 sd 12.65 years, range 1359 years, 71% female) were recruited from the outpatients at the department of psychiatry, the first hospital of china medical university, and the mental health center of shenyang. the diagnosis of mdd was confirmed by 2 trained psychiatrists using the structured clinical interview for dsm-iv disorders (scid). scores on the 17-item hamilton rating scale for depression (hamd) were obtained from each participant except for one, who did not complete these evaluations. here, 115 healthy control (hc) subjects (mean age 33.96 sd 14.25 years, range 758 years, 57% female) without any personal history of any axis i disorder or first-degree family member with a major mood or psychotic disorders were recruited through surrounding communities. exclusion criteria for both groups included history of neurological disease, loss of consciousness 5 min, any medical condition that might affect neurovascular function including hypertension, current substance abuse, or dependence, and having contraindications for mri. all participants provided written informed consent after receiving a complete description of the study as approved by the institutional review board of the china medical university. subjects were further divided into two groups: a cc group homozygous for the c allele (23 mdd, 57 hc; mean age=34.06 14.45 years, 60% female) and risk t-carrier group (ct/tt genotypes;=22 mdd, 58 hc; mean age=30.02 13.57 years, 61% female). genotype frequencies were consistent with hardy-weinberg equilibrium expectation (mdd: =2.894, p=0.08; hc: =2.52, p=0.11). the mri data were acquired using a ge signa hdx 3.0 t mri scanner (general electric, us) in the department of radiology of the first hospital of china medical university. soft pads and earplugs were used when scanning to restrict head motion and reduce scanner noise. participants were asked to relax with their eyes closed but remain awake throughout the resting-state scan. three-dimensional t1-weighted images were obtained using a fast spoiled gradient-echo (fspgr) sequence: repetition time (tr)=7.1 ms, echo time (te)=3.2 ms, field of view (fov)=24 cm 24 cm, flip angle=15, matrix=240 240, slice thickness=1 mm, and no gap. functional images were acquired using a gradient echo planar imaging (epi): tr=2000 ms, te=30 ms, fov=24 cm 24 cm, flip angle=90, matrix=64 64, slice thickness=3 mm, no gap, and slices=35. the first 10 volumes of each subject were discarded to allow participants to adapt to the scanning environment. the remaining data were preprocessed including slice timing, head motion correction, and spatial normalization to the standard montreal neurological institute (mni) space (resampling to 3 3 3 mm). subsequently, the images were spatially smoothed with a 6 mm full-width at half-maximum (fwhm) gaussian kernel. participants with head motion less than 3.0 mm in any dimension or 3 rotation at any point during the course of the scan were included for further analysis. subsequent data preprocessing included removal of linear trends and temporal filtering (band pass, 0.010.08 hz) to reduce the effects of low-frequency drift and high-frequency noise. the square root was calculated at each frequency of the power spectrum and averaged square root was between 0.01 and 0.08 hz. the alff of each voxel was divided by the global mean alff value within a brain mask. resting-state fmri data analysis toolkit (rest) (http://www.restfmri.net/) was performed for further data processing and alff analysis. demographic data (sex, age, education, and hamd) were analyzed using chi-square tests and two-way analysis of variance (anova) with diagnostic group (hc, mdd) and genotype group (cc, ct/tt) as between subject factors. a two-sample t-test was used to compare the durations of illness across genotypes within the mdd group. a voxelwise anova (2 2 anova: diagnosis genotypes) was used to determine the effects of diagnosis and genotype on alff values, and age and sex were considered as covariates. post hoc t-test was used to explore the details of the main effects and interactions. correction for multiple comparisons was based on monte carlo simulation [alphasim, analysis of functional neuroimages (afni), cluster size>3321 mm]. pearson correlation analyses and spearman correlation analyses were performed to determine the correlation of alff values between regions showing significant differences with the hamd scores, education, and illness duration in the two diagnostic groups. there was no significant effect of diagnosis, genotype, or interaction between diagnosis and genotype for age, sex, or education. the effect of diagnosis in hamd was significant, with significantly higher hamd scores in the mdd group than in the hc group. two-sample t-tests showed no difference in the duration of illness between the mdd subgroups (table 1). the influence of diagnosis and genotypes on alff in the cc and ct/tt genetic subgroups in the mdd and control subjects is listed in table 2. after performing a two-way anova on the alff maps, a significant main diagnosis group effect was observed (f=56.39, p<0.001, corrected). mdd patients showed significantly increased alff in the left middle temporal gyrus and left occipital cortex compared with individuals in hc group (figure 1). there was also a significant main effect of genotype (f=31.80, p<0.001, corrected). the t-carrier group showed increased alff in the left superior temporal gyrus (figure 2). significant diagnosis by genotype interaction was noted (f=22.24, p<0.001, corrected). among patients with mdd, the t-carrier group had significantly lower alff values in the bilateral lingual gyri than the cc group (f=2.92, p=0.006). within-genotype comparisons showed that t-carriers with mdd had significantly lower alff in the bilateral lingual gyri than healthy controls who were t-carriers (f=4.82, p=0.02). in addition, alff were highest in the brain regions of the bilateral dorsal lateral frontal cortex (dlpfc) and left medial prefrontal gyrus (mpfc) in mdd rs35936514 t-carriers compared to mdd rs35936514 cc homozygotes and both control genotype groups (p<0.001) (figure 3). correlation analyses did not show any significant associations between alff and hamd scores, illness duration, or education in mdd or hc groups. based on the large number of samples studied on lhpp rs35936514 associated with mdd in chinese subjects, it was here noted that lhpp was associated with thyroid disorders, which may affect depression. multiple studies have reported that both hypo- and hyperthyroidism may potentially increase the risk of cognitive impairment and neurodegeneration. the association between hypothyroidism and depression might be explained by higher rates of hypercortisolism in depression, which might lead to changes in the hypothalamic-pituitary-thyroid/adrenal (hpt/hpa) axes. dysfunctional hpt/hpa axes regulation might be a trait in mdd patients, producing changes in the monoaminergic pathways that modulate hormonal responses. autoimmune thyroiditis which is marked by the presence of thyroid antibodies with normal or abnormal thyroid hormone level is also a major cause for mdd. a bidirectional association between depression and the immune system has been also reported [33, 34]. depression has been linked with increased inflammatory markers and depression risk alleles have been found to be associated with regulating genes of the immune response. however, the current work did not show apparent relationship between lhpp, thyroid disorders, and mdd. our present study may provide a new imaging genetics approach to explore their relationships. in order to assess the effects on the neural system associated with lhpp variation and mdd, the current work demonstrated that lhpp rs35936514 snp polymorphism may influence the spontaneous brain activity in individual subjects with mdd and also healthy controls. the spontaneous low-frequency fluctuations (lff) of blood oxygenation level-dependent (bold) signals at rest have been identified as a biological measure of baseline spontaneous activity in the brain [21, 22, 36]. abnormal alff can reflect pathophysiological states in the regional brain area and may help to locate specific impaired brain regions during the resting state. as an additional advantage, alff can be used to assess neuronal activity within the entire brain. in particular, by rs-fmri, the cortico-limbic-striatal circuits (including the pfc, hippocampus, amygdala, and striatum) have been implicated in the dysfunctional regulation in mdd. the current study showed significant interactions between diagnosis and genotype in the bilateral lingual gyri, bilateral dlpfc, and left mpfc. the alterations of alff in the mdd t-carrier group suggest that the risk t allele is associated with abnormal spontaneous activity in mdd. more research is needed to identify the mechanisms by which these regional activities in individuals with mdd may be more vulnerable to the effects of variations in rs35936514, which render them more vulnerable to genetic liability. however, it remains unknown when and how this interaction could change alff, although many factors could influence the status of mdd [3840], which are well established as a risk for mdd. furthermore, expression of multiple genes involved in mdd, and their interaction with the lhpp genotype could make a change in the neural circuits of mdd patients. the parts of the brain in the mpfc are believed to be the neural site of self-referential processing, like the anterior node of the default mode network (dmn). alterations to the mpfc functional networks are involved in the development of mdd [43, 44]. altered resting state in the dlpfc of mdd could adversely affect activity during emotional or cognitive regulation. the lingual gyrus has been reported to participate in the visual recognition network and to play a role in the perception of emotions [45, 46]. many studies have reported decreased activity in the lingual gyrus [45, 47, 48] in patients with mdd. this may be attributable to different roles and levels of expression of the lhpp gene in different regions of the brain, different structural or functional bases (activities) of different brain regions, the combined effects of the t-carrier genotype, and other unknown factors. in this study, independent of genotype, increased alff was observed in the left middle temporal gyrus and left occipital cortex among mdd participants compared to that among hcs. the increased spontaneous activity in the temporal and occipital lobe within mdd patients was consistent with the findings of rs-fmri studies examining depression [4951]. genotype was found to have an effect on alff in the left superior temporal gyrus. it was higher among t-carriers of the lhpp rs35936514 polymorphism in both the hc and mdd groups than in cc individuals within the respective diagnostic groups. it is here suggested that the mdd rs35936514 t-carriers may function as one of several factors that can be used to diagnose mdd. it is possible that variation in lhpp may contribute to the different alff values in temporal lobe in individuals with or at risk for mdd. in this way, the subgroup with mdd that carries the t allele may be a risk factor to abnormal brain activity. however, exploratory analyses did not reveal significant correlations between alff values in regions of significant group differences and clinical factors within each group. we speculate that the relative small sample size or other clinical factors may limit our ability to detect the relation. one lhpp snp, rs11245316, was found to confer risk of mdd by qtl-specific association analysis. however, these data do not provide preliminary indications of how genetic variation in high-risk regions influences susceptibility to neural circuits abnormalities in patients with mdd. first, many snps and genes implicated in mdd were not included in the study. gene-gene interaction studies and even pathway studies are needed to search for valid mdd-associated genetic and neuroimaging biomarkers. second, owing to the small sample size, the study placed individuals with ct and tt genotype in a single group. as a result, no analysis of the alff values among 3 genotype groups could be performed, and the effect of the risk t allele could not be established as dominant or codominant. a larger-sample study should be performed to further investigate the effect of genes. in conclusion, the present study demonstrated for the first time that lhpp rs35936514 ct/tt genotype may affect regional brain activity in mdd patients. this suggests that the influence of lhpp variation may be one mechanism that contributes to the neural circuits of mdd.

a single-nucleotide polymorphism at the lhpp gene (rs35936514) has been reported in genome-wide association studies to be associated with major depressive disorder (mdd). however, the neural system effects of rs35936514 that mediate the association are unknown. the present work explores whether the lhpp rs35936514 polymorphism moderates brain regional activity in mdd. a total of 160 subjects were studied: a cc group homozygous for the c allele (23 individuals with mdd and 57 controls) and a t-carrier group carrying the high risk t allele (ct/tt genotypes; 22 mdd and 58 controls). all participants underwent resting-state functional magnetic resonance imaging (rs-fmri) scanning. brain activity was assessed using the amplitudes of low-frequency fluctuations (alff). mdd patients showed a significant increased alff in the left middle temporal gyrus and occipital cortex. the t-carrier group showed increased alff in the left superior temporal gyrus. significant diagnosis genotype interaction was noted in the bilateral lingual gyri, bilateral dorsal lateral prefrontal cortex (dlpfc), and left medial prefrontal cortex (mpfc) (p<0.05, corrected). results demonstrated that mdd patients with lhpp rs35936514 ct/tt genotype may influence the regional brain activity. these findings implicate the effects of the rs35936514 variation on the neural system in mdd.

PMC5097818

pubmed-957

the dietary magnesium intake tends to be lower than that recommended worldwide even in western countries. several factors have been identified to be trigger for lowering magnesium intake including the waterborne magnesium factor, the loss of magnesium during food refining, and the magnesium content of vegetarian diets, as well as various metabolic situations such as hypertension, pregnancy, osteoporosis, drug therapy, alcoholism, stress, and even cardiac trauma. magnesium is the most abundant intracellular divalent cation that is important for physiological processes including neuromuscular function and maintenance of cardiovascular tone. the importance of magnesium intake in relation to cardiovascular diseases has been increasingly described, so that its intake can be inversely related to the risk of hypertension and type 2 diabetes mellitus and may improve serum lipid profiles. recent surveys in sweden [3, 4] have indicated that vegetarian diets provide a more adequate intake of magnesium, as illustrated in table 4. note that the magnesium intake of men and women on vegetarian diets was considerably greater than the rda requirement; this was attributed to the high intake of vegetables and whole-grain cereals by these individuals. recent studies have shown that some components of the mediterranean diet such as vegetables, legumes, and nuts are rich sources of magnesium; therefore, it seems that the high adherence to the mediterranean diet is associated with high consumption of magnesium. although the main dietary regimen of iranian population is mediterranean diet components especially the major sources of magnesium, the rate of coronary artery disease (cad) risk factors among this population has been considerably high. therefore, in the present study, we tried to determine effect of mediterranean dietary approach on laboratory parameters related to cad risk factors with the focus on serum magnesium concentration among iranian patients with cad. baseline characteristics and clinical data of 102 consecutive patients with the diagnosis of cad and candidates for isolated coronary artery bypass surgery were entered into the study. studied data were collected by the review of clinical recorded files in the day of admission. in this study, cad was considered significant if there was a 75% or greater stenosis in the cross-sectional diameter and 50% or greater stenosis in the luminal view. blood samples were drawn from the corresponding peripheral vein into vacutainer tubes after 1214 h of overnight fasting and before operation. plasma glucose concentrations were assessed by means of a glucose hexokinase method (pars azmoon kits accredited by bioactiva diagnostica, germany). serum total cholesterol, hdl cholesterol, and triglycerides were measured via enzymatic techniques (pars azmoon kits accredited by bioactivadiagnostica, germany). the friedewald formula was used to calculate low density lipoprotein (ldl) cholesterol, except when the triglyceride level was>blood pressure was calculated as the mean of two measurements, performed in the sitting position after 5 minutes of rest, using a random-zero sphygmomanometer (hawksley-gelman, lancing, sussex, uk). c-reactive protein (crp) level was measured by immunoturbidimetry (pars azmun, iran), and lipoprotein(a) was measured using tint eliza (biopool, usa). creatinine was measured with jaffe reaction (parsazmon kit, tehran, iran) using an autoanalyzer (hitachi, tokyo, japan). studied patients were also interviewed on admission to surgical ward and before operation to report the consumption of food items listed as the number of times per day, per month, or per year during the previous year. we assessed nutritional status by a validated food frequency questionnaire (ffq), previously validated in iran and a 24-hour dietary recall questionnaire to record the types, amounts, and frequencies of foods consumed. sum of the consumption of each of several food items was used to determine the overall consumption of the food group to which each item belonged [9, 10]. the diet score was calculated on the basis of mediterranean diet quality index (med-dqi) that the construction of the score for this index (table 1). the index assigns a score of 0, 1, or 2 according to the daily intake of each of the seven components and then final score was reported as a summation of all nutrient scores ranged between 0 and 14. we divided studied patients into two groups according to the final score: the groups with final score lower than 5 (n=35) and those who had final score between 5 and 10 (n=67). we compared categorized variables between the two above groups using chi-square test or fisher's exact test if required. multivariate linear regression analysis was used to investigate the potential confounding effects of patients ' characteristics and clinical data on the association between serum magnesium concentration and mediterranean regimen score. all the statistical analyses were performed using spss version 13.0 (spss inc., chicago, il, usa). studied patients had the mean age of 58.8 year (ranged between 38 and 78 years) and 40.2% of them were male. hyperlipidemia and hypertension were observed in 75.5% and 56.9% of patients, respectively, and 51.0% of them were diabetics. half of the studied patients had moderate functional class, but three coronary arteries were involved in the majority of them (table 2). comparison of the concentrations of serum laboratory parameters between the two groups with lower mediterranean dietary score and group with higher dietary score showed no significant differences between the concentration of albumin, last fasting blood sugar, last creatinine, and lipid profiles (table 3); however, serum magnesium concentration in the group with dietary score between 5 and 10 was lower than that in group with higher score (p=0.026). linear multivariate regression analysis also showed that the lower mediterranean dietary score was a predictor for serum magnesium concentration after adjusting for confounders (table 4). recent studies could confirm the cardioprotective role of intravenous magnesium for prevention of cardiac arrhythmia, pump dysfunction, and death in patients with cad, especially in the immediate postinfarction period [12, 13]. magnesium can inhibit the influx of calcium in vascular smooth muscle cells and therefore inhibit arrhythmic recurrence and the production of il-6 and mmp-1 after reperfusion and prevent the increase of myocardial lesions caused by calcium overload on myocytes. however, effect of dietary intake of this mineral on control and inhibition of cad risk factors has been questioned. some researchers have been hypnotized that the low intake of magnesium may be related to the least degree of cardiovascular disease [15, 16]. some others could show that the dietary magnesium intake can be inversely associated with fasting serum insulin, plasma high density lipoprotein-cholesterol, and systolic and diastolic blood pressure. in the present study, we showed the potential effect of mediterranean dietary regimen on increasing of serum magnesium concentration that can mainly lead to the favorite control and prevention of cad risk factors. we have shown that not only intravenous magnesium administration can be related to the lower risk of poor outcome in patients with cad, but also these effects can be obtained by dietary intake of this mineral via mediterranean dietary regimen. in a similar study by schrder, it was found that the high consumption of vegetables, fruits, legumes, nuts, fish, cereals, and olive oil as main components of mediterranean regimen led to high absorption of magnesiumin obesity and type 2 diabetes. also, singh showed that the increased intake of dietary magnesium in association with the general effects of a nutritious diet can offer protection against cardiovascular deaths among high-risk individuals predisposed to cad. furthermore, in another study, it was observed that the intake of dietary magnesium was associated with a reduced risk of cad; however, associations between dietary magnesium and coronary events occurring after fifteen years of follow-up were modest. it seems that the mediterranean dietary regimen and its components as rich pools of magnesiummight optimize micronutrient status in main body organs especially cardiovascular system. in the present study, it was also found that the patients ' baseline characteristics and even severity of cad had no relationship with serum magnesium concentration in multivariate analysis. similarly, in a study by mataix et al., the risk of hypomagnesemia was not associated with any of the other factors that were investigated such as gender, educational level, obesity, smoking habits, alcohol consumption, and physical exercise. however, some studies showed that the magnesium intake could decrease in advanced age and in men for each of the different race or ethnic groups. it seems that the serum magnesium changes are mainly dependant on nutritional habit, metabolicprocesses, inflammatory biomarkers, and other related-potential mechanisms that should be investigated in future studies. in conclusion, taking mediterranean dietary regimen can be associated with increased level of serum magnesium concentration, and thus this regimen can be cardioprotective because of its effects on serum magnesium.

background. recent studies confirmed cardioprotective role of intravenous magnesium for the prevention of cardiac events, but effect of dietary intake of this mineral via recommended dietary regimens on control and inhibition of coronary artery disease (cad) risk factors has been questioned. the aim of the present study was to determine effect of mediterranean dietary approach on serum magnesium concentration among iranian patients with cad. method. baseline characteristics and clinical data of 102 consecutive patients with the diagnosis of cad and candidates for isolated coronary artery bypass surgery were entered into the study. laboratory parameters especially serum magnesium concentration were measured after 1214 h of overnight fasting and before operation. nutritional status was assessed by food frequency questionnaire and the diet score was calculated on the basis of mediterranean diet quality index (med-dqi). results. no significant differences were found in the concentrations of albumin, last fasting blood sugar, last creatinine, and lipid profiles between the groups with mediterranean dietary score<5 and the group with higher dietary score; however, serum magnesium concentration in the first group was higher than that in the group with higher dietary score. linear multivariate regression analysis showed that the lower mediterranean dietary score was a predictor for serum magnesium concentration after adjusting for confounders. conclusion. taking mediterranean dietary regimen can be associated with increased level of serum magnesium concentration, and thus this regimen can be cardioprotective because of its effects on serum magnesium.

PMC3838832

pubmed-958

human papillomavirus (hpv) is the most common sexually transmitted infection (sti) among young adult women, and it plays a critical role in the development of cervical cancer [1, 2]. biologic susceptibility to hpv acquisition and immune competence for clearance of an hpv infection could be affected by common, treatable vaginal infections that disrupt the intricately balanced vaginal ecosystem and its innate protective mechanisms against infection and disease. bacterial vaginosis and trichomoniasis are associated with high levels of anaerobic microorganisms and their byproducts, that can damage vaginal epithelium, degrade cervical mucus, and cleave immunoglobulin a [36]. despite the similar effects of bacterial vaginosis and trichomoniasis on the vaginal environment, there is increasing evidence only for an association between hpv and bacterial vaginosis, albeit of unknown directionality [79]. bacterial vaginosis and trichomoniasis are categorized, along with vulvovaginal candidiasis, under the somewhat misnomer of vaginitis. unlike bacterial vaginosis and trichomoniasis, studies on vulvovaginal candidiasis are complicated by its lack of a clear laboratory definition, since the etiologic agent may exist as part of the commensal microbiota in healthy women. candida albicans is the most prevalent species in most cases of asymptomatic colonization and vulvovaginal candidiasis; however certain species of candida are more pathogenic than others and induce hyphae formation and have enhanced proteolytic activity and antigen modulation, enabling candida to penetrate the mucosal surface and induce mucosal swelling, erythema, and exfoliation of cells [1012]. the role of vaginal infections in hpv pathogenesis has been evaluated in primarily cross-sectional studies and has yielded conflicting results [8, 9, 1319]. the purpose of the current study was to evaluate associations for vaginal infections with prevalent hpv, incident hpv, and clearance of hpv in a large, prospective cohort of hiv-infected and high-risk, hiv-uninfected women. the hiv epidemiology research study (hers) was a longitudinal multi-site investigation of the biological, psychological, and social effects of hiv infection among us women. briefly, from april 1993 through january 1995, 871 hiv-infected women and 439 high-risk hiv-uninfected women were enrolled at four study sites: bronx, ny; baltimore, md; detroit, mi; and providence, ri. institutional review boards at each participating medical center and at the centers for disease control and prevention approved the study protocol. women aged 1655 years were eligible for participation if they had a history of injection drug use or high-risk sexual behavior, were fluent in either english or spanish, and if hiv-seropositive, reported no history of clinical aids-defining conditions. at each study site, hiv-seronegative women were frequency matched to hiv-seropositive women on hiv status (1: 2 ratio) and hiv risk behavior (1: 1, sexual behavior to injection drug use). women completed up to 15 study visits scheduled 6 months apart that consisted of an interview, physical and pelvic examination, and blood, urine, oral, and cervicovaginal specimen collection. hpv testing used polymerase chain reaction (pcr) to amplify virus obtained by cervicovaginal lavage. the amplified product was identified by hybridization with a generic hpv probe and probes specific to 26 hpv types: 6, 11, 16, 18, 26, 31, 33, 34, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 66, 68, 73, 82, 83, and 84. samples with missing (n=17) or negative (n=72) test results for hybridization with the control, b-globin probe were considered of unsatisfactory quality, and the hpv results were set to missing. for the purposes of the current analysis, visit-specific prevalent hpv infection was defined by detection of any hpv dna by general probe at a given visit. incident infection was defined as detection of a new type of hpv not present at any prior visit. thus, hpv type-specific negative women at enrollment were at risk for infection with the respective hpv type. women who were hpv-negative by all type-specific probes and generic probe prior to testing positive by generic probe only (i.e., untypable infection) were classified as having incident hpv at that visit. for the evaluation of time to hpv clearance, women contributed event times for each incident hpv type. time to clearance was defined as the interval from the type-specific incident visit to the first of two consecutive type-specific negative visits. the flagging negative visit was required to avoid misclassification from fluctuating detection of hpv. hpv infections that were followed by a single hpv-negative, final study visit were censored at that negative visit. hpv infections that were positive at the last study visit had 6 months added to ensure comparability of assumptions about clearance of hpv infection for censored and noncensored observations. hpv infections spanning a single missing visit were assumed to persist over the unobserved period. presence of bacterial vaginosis was determined by nugent scoring of a gram-stained slide. a nugent score of 7 to 10 defined bacterial vaginosis, whereas a score of 0 to 6 defined normal-intermediate vaginal flora. for visits without a nugent score (< 1%), presence of vaginal candida colonization was based on a positive culture for candida species or detection of yeast or pseudohyphae on gram stain. presence of trichomoniasis was determined by detection of trichomonas on wet mount (all visits) or on culture (visits 4 and above). women were eligible for the current analysis if they had baseline data on hpv dna and bacterial vaginosis, vulvovaginal candidiasis, or trichomoniasis test results (n=1256). of these women, 108 who did not have a cervix at baseline and one woman who had cervical treatment and a hysterectomy within 6 months before and after enrollment, respectively, were excluded. for simplicity, an additional 11 women who seroconverted to hiv-seropositive during follow-up were excluded, leaving 1136 subjects for this analysis. person-time was censored at the date of cervical treatment (e.g., excisional biopsy, cryotherapy) or hysterectomy for 111 subjects. potential confounders of the relationship between vaginal infections and prevalent hpv, incident hpv, and clearance of hpv were selected based on the literature and a proposed causal directed acyclic diagram (not shown) used for identification of sufficient sets of confounders. the a priori model for prevalent hpv and incident hpv included sexual risk factors (new or multiple male sex partners and frequency of condom use), illicit drug use, cigarette smoking, use of hormonal contraceptives, immunologic predisposing factors (hiv status, cd4 count, and viral load), and demographic indicators for hpv prevalence in the usa (age, race, and socioeconomic status) [1, 25]. a priori models for time to hpv clearance also included a covariate indicating coinfection with another hpv type within the type-specific duration and its cancer risk classification. all statistical models included the study design variables: study site and risk behavior cohort (sexual or injection drug use). in addition, models included a covariate for visit number, year of visit, or year of hpv onset to account for period effects. in addition to the variables selected a priori, other factors related to cervicovaginal health were evaluated in bivariate and multivariable analysis using sas versus 9.2 (sas institute inc., these factors included potential hormonal influences (e.g., pregnancy, postmenopausal state, and estrogen replacement therapy), sexual behavior (e.g., sexual frequency), genital tract infections (e.g., herpes simplex virus-2 serostatus at baseline culture and pcr testing for chlamydia and gonorrhea ceased after the third study visit due to very low frequency in this population), and other risk behaviors or conditions (e.g., alcohol drinking and hepatitis c serostatus at baseline). the importance of a covariate was evaluated in terms of the causal directed acyclic graph, collinearity with other covariates, and its coefficient magnitude, statistical significance, and impact on the effect estimates for vaginal infections upon addition to and removal from the statistical model. a generalized linear mixed model with a random intercept and a slope based on a toeplitz matrix was used to model associations between current vaginal infections and prevalent hpv while accounting for between-subject variation and repeated measures correlation. to evaluate associations between vaginal infections at the preceding visit and incident hpv, as well as to explore the temporal relationship between hpv and bacterial vaginosis, the case-crossover analytic approach was used. the case-crossover approach is useful for identifying associations between recent, time-varying exposures and an incident outcome while adjusting for all time-independent factors by comparing each woman's case (e.g., incident hpv) visits to her own noncase visits. to evaluate temporality, separate case-crossover models were run for incident hpv and incident bacterial vaginosis that were adjusted for a few shared time-dependent predictors: a combined indicator for hiv status and cd4 category (with 4 categories: hiv-negative and hiv-positive with cd4>500, cd4 200500, and cd4<200) and self-reported crack or cocaine use, new or multiple sex partners, and consistency of condom use since the prior study visit. analyses of time to clearance of a type-specific hpv infection used the wei, lin, and weissfeld extended cox model. hazard ratios less than 1.0 indicate lower clearance rates (i.e., longer duration of infection). vaginal infections, and most of the covariates, were modeled as time-dependent variables allowing the instantaneous effects to vary with changes in the time-dependent variables. upon finalizing the main effects models, modification of the effect estimates for vaginal infections across levels of biologically relevant covariates (e.g., hiv status, concurrent vaginal infections) was evaluated for magnitude and significance (p<0.15) using the wald and likelihood ratio tests. due to effect measure modification of the association between certain vaginal infections and hpv outcomes by hiv status, tabular results are presented separately for hiv-infected and hiv-uninfected women. at baseline, hiv-infected women (n=756) were similar to hiv-uninfected women (n=380) in age, number of live births, cigarette smoking status, and prevalence of bacterial vaginosis, trichomonas vaginalis, chlamydia trachomatis, and neisseria gonorrhoeae infections (table 1). the hiv-uninfected women were more likely to have recently used crack cocaine or injection drugs and had multiple sex partners in the prior 6 months. the hiv-infected women were more likely to be of african american race, of lower educational level, and seropositive for hepatitis c and herpes simplex 2. hiv-infected women were also more likely to be vaginally colonized with candida species and have detectable hpv. the prevalence of hpv among hiv-infected women (64%) was over twice that of hiv-uninfected women (29%), and hiv-infected women were more likely to be coinfected with multiple hpv strains (table 1). however, among women with hpv, the proportion with untypable hpv infections was greater among hiv-uninfected women (30.3%) than among hiv-infected women (20.3%). at enrollment, most of the hiv-infected women (83%) had cd4 counts above 200 cells/l. enrollment was completed before the onset of the highly active antiretroviral therapy (haart) era, so none of the women were on haart regimens at baseline. however, one-third of the hiv-infected women were taking antiretrovirals. the prevalence of hpv infection at visits 115 ranged from 38% to 56% (4968% for hiv-infected women and 1232% for hiv-uninfected women). longitudinal, multivariable analysis for prevalent hpv demonstrated a strong association with hiv status and immunodeficiency. hiv-infected women with cd4 cell counts below 200 cells/l had greater odds for prevalent hpv compared to hiv-infected women with cd4 counts above 500 cells/l (adjusted odds ratio (aor)=1.53, 95% ci: 1.25, 1.86). there was no modification of the odds ratio for bacterial vaginosis on prevalent hpv by hiv status (table 2). in a model adjusted for a combined indicator of hiv status and cd4 group, bacterial vaginosis was significantly associated with increased odds for prevalent hpv among all hers women (aor=1.14, 95% ci: 1.04, 1.26). hiv status significantly modified the associations among trichomoniasis, vaginal candida colonization, and hpv prevalence. in a model for hiv-uninfected women, there was no significant association for trichomoniasis or vaginal candida colonization with prevalent hpv (table 2). however, among hiv-infected women, there was a significant interaction between trichomoniasis and vaginal candida colonization: trichomoniasis was significantly associated with decreased odds for prevalent hpv among women without vaginal candida colonization, and vaginal candida colonization was significantly associated with increased odds for prevalent hpv among women with trichomoniasis (table 2). when vaginal candida colonization was removed from the model, trichomoniasis remained significantly associated with decreased odds for prevalent hpv among the hiv-infected women (aor=0.85, 95% ci: 0.73, 0.98). in contrast, when trichomoniasis was removed from the model, vaginal candida colonization was not associated with prevalent hpv (aor=1.02, 95% ci: 0.92, 1.12). incident hpv infections occurred at 14% of follow-up visits (17.4% among hiv-infected women and 7.5% among high-risk, hiv-uninfected women). there was no modification of the odds ratio for prior bacterial vaginosis on incident hpv by hiv status (table 3). in a model adjusted for a combined indicator of hiv status and cd4 group, bacterial vaginosis at the preceding visit was significantly associated with 24% increased odds for incident hpv among all hers women (aor=1.24, 95% ci: 1.04, 1.47) (table 3). bacterial vaginosis at the current visit was similarly associated with incident hpv (aor=1.25, 95% ci: 1.05, 1.48). in contrast, a case-crossover analysis for the outcome of incident bacterial vaginosis found that hpv at the current visit (aor=1.46, 95% ci: 1.12, 1.90), but not at the preceding visit (aor=1.05, 95% ci: 0.80, 1.37), was predictive of incident bacterial vaginosis among all women. trichomoniasis and vaginal candida colonization was not significantly associated with incident hpv, regardless of timing of vaginal infection relevant to hpv infection (table 3). the majority of incident hpv infections cleared over subsequent study visits (63.1% clearance among hiv-infected women and 74.8% among hiv-uninfected women). among hiv-infected women, those with cd4 counts below 200 cells/l were half as likely to clear an hpv infection as those with cd4 counts above 500 cells/l (adjusted hazards ratio (ahr)=0.51, 95% ci: 0.40, 0.64). hiv status and cd4 category did not significantly modify the hazard ratios for vaginal infections with hpv clearance. however, there was a statistical interaction between bacterial vaginosis and vaginal candida colonization (table 4). among all hers women with vaginal candida colonization, bacterial vaginosis was associated with a lower rate of hpv clearance (ahr=0.65, 95% ci: 0.51, 0.83), and vice versa, among women with bacterial vaginosis, vaginal candida colonization was associated with delayed hpv clearance (ahr=0.73, 95% ci: 0.58, 0.93). when vaginal candida colonization was removed from the model, bacterial vaginosis remained significantly associated with delayed clearance of hpv among all hers women (ahr=0.84, 95% ci: 0.72, 0.97). in contrast, when bacterial vaginosis was removed from the model, vaginal candida colonization was not associated with hpv clearance (ahr=0.93, 95% ci: 0.81, 1.08). we did not detect a significant association between trichomoniasis and time to clearance of hpv infection, regardless of hiv status (table 4). consistent with a recent meta-analysis of primarily cross-sectional studies, bacterial vaginosis was associated with increased odds for prevalent hpv among women in hers. furthermore, in this large, prospective study, bacterial vaginosis was associated with increased odds for incident hpv and delayed clearance of hpv. the former finding is consistent with a reported increase in hpv incidence among women with bacterial vaginosis in the women's interagency hiv study (wihs). prior attempts to decipher the temporal relationship between bacterial vaginosis and hpv yielded conflicting findings [8, 9]. in this study, bacterial vaginosis, whether considered at the preceding or current visit, was associated with incident hpv, whereas, in an alternate multivariable model for the outcome of incident bacterial vaginosis, only hpv at the current visit was significant. these findings are in accord with biologic plausibility since, unlike most cervical hpv infections, bacterial vaginosis causes major changes in the local environment leading to degradation of innate defenses. while trichomoniasis infection was not significantly associated with hpv outcomes among hiv-uninfected women in hers, hiv-infected women with trichomoniasis had decreased odds for prevalent hpv. a similar finding was reported from a wihs analysis which combined 1736 hiv-infected and 493 hiv-uninfected women into one analytic group. in that study, detection of trichomoniasis on wet mount was associated with lower prevalence, increased incidence, and shorter duration of hpv infection. in the current study, we did not detect a significant effect of trichomoniasis on incident hpv or clearance of infection; however, this may have been due to lower statistical power. smaller studies among only hiv-uninfected women are more consistent with our findings among the hiv-uninfected women in suggesting absence of an association between trichomoniasis and prevalent, incident, or persistent hpv [14, 15, 18]. the apparent difference by hiv status in associations for trichomoniasis with hpv prevalence and duration may be attributable to the strong effect of hiv on duration of hpv infection relative to the minimal, if any, effect of hiv on duration of trichomoniasis. explicitly, as the duration of hpv infection increases, the proportion of hpv-positive visits that have trichomoniasis present decrease, giving the appearance of a protective association. the presence of vaginal candida colonization alone was not significantly associated with hpv outcomes; however when concurrent with bacterial vaginosis or trichomoniasis, it negatively impacted their effects on certain hpv outcomes. the decreased odds for prevalent hpv among women with trichomoniasis was not observed among women with vaginal candida colonization, and the association between bacterial vaginosis and delayed clearance of an hpv infection was strongest among women with vaginal candida colonization. standard antibiotic treatment for bacterial vaginosis or trichomoniasis may precipitate a vaginal yeast infection, and a high incidence of asymptomatic and symptomatic vulvovaginal yeast infection has been reported in patients with recurrent bacterial vaginosis. therefore, concurrent vaginal candida colonization may indicate more severe or difficult-to-treat bacterial vaginosis or trichomoniasis. the hers cohort, two-thirds of which are hiv-infected, represents a population at increased risk for vaginal infections and hpv. as such, there are two primary concerns regarding these study's findings: (1) the potential dependency of the results on hiv disease and (2) the generalizability of the results to the general population of women. several methods were used to reduce potential confounding of the relationships between vaginal infections and hpv by hiv-associated factors: (1) by design, hiv-infected women were frequency matched at enrollment to hiv-uninfected women on sexual and injection drug use behavior, (2) self-reported risk behavior between study visits was included in multivariable analyses, (3) models among all hers women were evaluated for statistical interaction with a composite variable of hiv status and cd4 count, and (4) separate models were built for hiv-infected and hiv-uninfected women, with the former adjusted for cd4 category and logarithm of viral load. despite these efforts, disentangling the interrelationships between hiv disease and concurrent vaginal infections was limited by decreased statistical power to detect significant associations among hiv-uninfected women separately. while the hers cohort is a population at high-risk for genital tract infections, the primary findings involve two infections for which the general female population is also at high risk: bacterial vaginosis and hpv. moreover, there is mounting evidence for an association between bacterial vaginosis and hpv across diverse study populations. this is the second large, prospective study among hiv-infected and hiv-uninfected women to report consistent findings for bacterial vaginosis with increased prevalent and incident hpv. in the current study, bacterial vaginosis while there is mounting evidence for an association between bacterial vaginosis and hpv, a temporal association has remained unclear [79]. in the current study, we found that bacterial vaginosis, whether considered at the preceding or current study visit, was associated with incident hpv, whereas hpv at the current, but not preceding, visit was associated with incident bacterial vaginosis. in summary, the current study suggests bacterial vaginosis may predispose a woman to hpv infection and delayed hpv clearance, underscoring the importance of prevention and successful treatment of bacterial vaginosis. bacterial vaginosis is associated with increased odds for prevalent and incident hpv as well as delayed clearance among women in the hiv epidemiology research study.

objective. to evaluate associations between common vaginal infections and human papillomavirus (hpv). study design. data from up to 15 visits on 756 hiv-infected women and 380 high-risk hiv-uninfected women enrolled in the hiv epidemiology research study (hers) were evaluated for associations of bacterial vaginosis, trichomoniasis, and vaginal candida colonization with prevalent hpv, incident hpv, and clearance of hpv in multivariate analysis. results. bacterial vaginosis (bv) was associated with increased odds for prevalent (aor=1.14, 95% ci: 1.04, 1.26) and incident (aor=1.24, 95% ci: 1.04, 1.47) hpv and with delayed clearance of infection (ahr=0.84, 95% ci: 0.72, 0.97). whereas bv at the preceding or current visit was associated with incident hpv, in an alternate model for the outcome of incident bv, hpv at the current, but not preceding, visit was associated with incident bv. conclusion. these findings underscore the importance of prevention and successful treatment of bacterial vaginosis.

PMC3159014

pubmed-959

psychobehavioral approaches attempt to identify the underlying cognitive dissonance creating the intense emotional discomfort that can trigger seizures. a recent review of psychobehavioral therapy for epilepsy recommends case reports as a research design to explore specific psychological mediators of seizure self-control. this case report illustrates how a psychobehavioral intervention addresses cognitive dissonance in order to decrease internal stressors of seizures and was prepared according to the consensus-based care guidelines for standardized clinical case reporting. his magnetic resonance imaging (mri) shows extensive bilateral gray matter band heterotopia (fig. 1). electroclinical day- and nighttime seizures captured during video-eeg studies displayed seizures with loss of consciousness consisting of irregular shaking of his body correlating with epileptiform activity originating from bilateral parasaggital regions synchronously and tonic posturing of arms originating from the left hemisphere. before losing consciousness, he feels a loss of sensation in some of his body parts, usually starting from his right arm. these findings suggest localization-related epilepsy originating from the left hemisphere and bilateral parasagittal regions with simple and complex partial as well as secondarily generalized seizures. he experiences predominantly nocturnal seizures that occur in clusters of up to four complex partial seizures once or twice per week and daytime seizures once or twice per month. a. 's parents had installed an acoustic monitoring device in his bedroom and reported that this baseline seizure frequency had remained unchanged for> 3 years prior to the beginning of the intervention. a. is currently taking valproate, topiramate, and lamotrigine and complies with the modified atkins diet protocol. past medication failures have included carbamazepine, levetiracetam, clobazam, and oxcarbazepine. during previous psychological assessments, he has been diagnosed with a verbal and auditory learning disability as well as expressive language impairment. because of parental worries that challenges would increase a. 's seizure frequency, a. received parental support for doing his schoolwork, and he was never expected to contribute to household chores. as a result, a. was deprived of opportunities to build his confidence in his own achievements and pictures himself as broken otherwise, a. is in good health; he has never used alcohol or tobacco. a. and his parents consented to the use of these data for this case report. to resolve the resultant conflict between insecurity with his performance, fear of failure, and high expectations of himself, a. actively avoided responsibility and demanded help rather than engaging in self-reliant problem-solving behaviors. this defense mechanism that incorporated learned helplessness and secondary gain issues was exacerbated by an anticipated challenge at school or social anxiety. after an initial assessment period comprising two sessions of 3 h each, a treatment plan was formulated including the workbook taking control of your epilepsy, daily journaling, and daily relaxation exercises. the participant received training in abdominal breathing and visualization techniques (imaginary journey and an attention-focusing exercise) to address early seizure warning symptoms. the initial assessment was followed by weekly counseling sessions of 1030 min that were conducted over the phone for 12 months, and monthly sessions were conducted for a 6-month follow-up period. a. engaged in a daily guided relaxation practice after returning from school and listened to a nonguided relaxation tape before going to bed. he reports that he is able to interrupt warning signs of the early onset of a seizure by utilizing deep diaphragmatic breathing, visualization techniques, and self-affirmation statements of personal choice (e.g., peace and release). a. 's parents report that he took on responsibility for the completion of his schoolwork and studies in a self-motivated and timely fashion, all of which helped him to prevent the buildup of anxiety. after a nighttime seizure, he can reliably identify the behavioral strategy which he could have employed in order to avoid the buildup of distress during the day prior to the seizure. a. admitted that it was, at times, easier to have a seizure rather than to address the situational accumulation of seizure triggers by taking responsibility and implementing the appropriate proactive behaviors. hence, motivational exercises were employed to allow a. to deliberately weigh the motivational factors and obstacles regarding prior set goals (e.g., an early bed rest) to facilitate informed changes of his present decision-making habits. while he considered himself broken at the beginning of the intervention, he has developed a perspective on life in which he feels that he can shape his future according to his preferences if he follows through with taking responsibility for the acquisition of self-organizational skills (see patient perspective in table 1). by the end of the follow-up period his nighttime seizures dropped to 12 clusters per month after 6 months of participating in the intervention. since the internalization of the therapeutic principles is an active process that requires continuous learning, motivation, and compliance of the individual, effects are usually expected 36 months after the beginning of the intervention, especially since it takes an individual at least 12 weeks to finish the 12 sessions of the workbook. this seizure frequency has remained stable during the remaining 6 months of the intervention and the 6-month follow-up period (fig. (3 months after the follow-up period), the seizure frequency has still remained the same despite increasing stressors that arise within the framework of a. 's successful admission to college. the implementation of the therapeutic strategies correlated with a cessation of daytime seizures and a clinically significant drop of nighttime seizures. literature suggests that seizure frequency may be positively influenced by the restoration of balance within the autonomic nervous system by the regular practice of relaxation exercises and proactive avoidance of emotional distress during wakefulness, thereby preventing the buildup of seizure activity. furthermore, a. would regularly wake up during the night when preictal or early ictal phenomena arose; he would then react with the application of previously practiced countermeasures. literature indicates that evidence for the enhancement of psychological well-being by psychobehavioral therapy is more established, while reliable evidence on its role on seizure control is scarce. the a/r intervention may represent the most comprehensively developed cognitive behavioral approach to epilepsy that has repeatedly been correlated with a reduction of seizure frequency in individuals with complex partial seizure disorders in uncontrolled prospective and retrospective studies, ,. the possibility of false negative self-reports of seizure occurrence can not be ruled out but seems unlikely because the patient and his parents have been very conscientious in keeping a seizure diary for a long time. because of the uncontrolled nature of a case report, nonspecific effects of attention and natural fluctuations of seizure frequency that could have contributed to improve seizure frequency can not be factored into the interpretation of the presented data. however, the long and stable seizure frequency prior to the intervention makes natural fluctuations unlikely. furthermore, the decreased seizure frequency has remained stable during follow-up and post follow-up with decreased and no contact with the a/r counselor which makes unspecific effects of attention implausible. in this case of a 16-year-old male, the acquisition of self-organizational skills and the development of seizure interruption techniques correlated with a clinically significant drop of seizures and an increased sense of being in control of his seizures. the case exemplifies that motivational strategies may be applied to facilitate the regulation of lifestyle-related seizure precipitants. future prospective studies are needed to further investigate the psychological mediators of an individual's increasing sense of seizure self-control and its actual relationship with seizure frequency.

a recent review of psychobehavioral therapy for epilepsy recommends case reports as a research design to explore specific psychological mediators of psychobehavioral interventions for epilepsy that address the bidirectional relationship between psychological states and seizures. the report was prepared according to the consensus-based care guidelines for standardized clinical case reporting.this is a case of a 16-year-old male individual with a diagnosed seizure disorder and learning disability who continued to have daytime and nighttime seizures on a regular basis despite exhausting of available conventional treatment options. a psychological assessment led to the working hypothesis that cognitive dissonance between fear of failure and high expectations of self had led to a broken self-image and active avoidance of responsibility that resulted in intense emotional distress which correlated with the occurrence of seizures. this working hypothesis resulted in a treatment plan that employed the acquisition of self-organizational skills and relaxation techniques as the main therapeutic strategy. motivational strategies were employed to facilitate the regulation of lifestyle-related seizure precipitants. in this case, the acquisition of self-organizational skills and the development of seizure interruption techniques correlated with a clinically significant decrease of seizures. methodological limitations of the interpretation of the presented data are discussed.

PMC4307961

pubmed-960

nowadays, new treatments for schizophrenia are more ambitious, aiming to improve not only psychotic symptoms, but also quality of life and social reinsertion. we briefly but critically outline advances in the treatment of schizophrenia from the mid-1970s up to the present. for many years, it was widely accepted that any effective drugs for schizophrenia would also induce extrapyramidal side effects (eps), and the term neuroleptic was originally used to describe such neurologic side effects. however, adverse effects, such as movement disorders and sedation, are problematic and can result in noncompliance with medication. positive symptoms, such as delusions, hallucinations, and thought disorders, are more often experienced in the acute phases of the illness than are negative symptoms, such as poverty of speech, lack of motivation, apathy, and inability to express emotions.1 however, negative symptoms are probably the more disabling, and may not respond as well to typical antipsychotic drugs. in addition to efficacy issues, safety of medication also influences the choice of antipsychotic agent. atypical antipsychotic drugs, by definition, differ from typical antipsychotic agents in producing significantly fewer eps and carrying a lower risk of tardive dyskinesia in vulnerable clinical populations at doses that result in comparable control of psychosis. effects on neuronal survival and plasticity, together with decreased neurotoxicity, might also contribute to their clinical advantages over typical neuroleptic drugs.2 the atypical drugs differ from the typicals in their mechanism of action, but not all share the same mechanism (tables 1&2). clozapine, the prototype of these agents, has been found to improve delusions and hallucinations in patients who fail to respond to other antipsychotic drugs, and to reduce the risk of suicide. these agents have been found to increase cortical dopamine and acetylcholine release, as well as to have a variety of effects on the glutamatergic system not shared by the typical agents.2 the term atypical was then accepted as including the characteristics common to those antipsychotic drugs developed more recently, including absence of hyperprolactinemia, greater efficacy in treating positive and negative symptoms as well as symptoms of disorganization, and absence of tardive dyskinesia or dystonia after being administered chronically.3,4 at least in clinical circles, most would agree that clozapine, risperidone, olanzapine, quetiapine, sertindole, ziprasidone, aripiprazole, and amisulpride are atypical, even though many of those agreeing to the above list may disagree on the criteria of definition. when compared with older antipsychotic drugs, atypical antipsychotics show fewer eps and require less concomitant anticholinergic use, even when controlling for the high doses of haloperidol that have been conventionally used in such studies.5 the second most commonly shared feature is that most of the newer atypical antipsychotics show either no, or only transient, prolactin elevation. the two notable exceptions in this regard are risperidone and amisulpride, and it is now understood that this exception may largely be attributed to these drugs having a higher peripheral/central distribution ratio, thereby leading to excessive dopamine blockade in the pituitary that lies outside of the blood brain barrier.6 conventional antipsychotics continue to be the first choice when just cost of treatment is considered, which remains important in poor regions. it is likely that the next generation of treatments will have to move beyond reliance on a single drug as the sole treatment for the multidimensional characteristics of schizophrenia. most new antipsychotics introduced onto the market in the past two decades (eg, risperidone and olanzapine) have been multireceptor-acting agents, especially having concomitant 5-ht2 receptor antagonism.7 a notable exception to this has been amisulpride, a benzamide derivative that has high and similar affinities for the dopamine d2 and d3 receptor subtypes and is devoid of any significant affinity to other receptor systems.8 yet, amisulpride shows most of the attributes of atypicals, ie, a lower risk of eps, a somewhat greater improvement in positive and negative symptoms, and better overall outcome in longer-term follow-up studies compared with more conventional serotonin-dopamine or multireceptor atypical antipsychotics.9,10 amisulpride is a highly selective dopamine d2-like receptor antagonist (ki=2.8 nmol/l for d2 receptors and ki=3.2 nmol/l for d3 receptors), with several orders of magnitude higher affinity for d2/d3 receptors than any other receptor population.8 a positron emission tomography study of amisulpride-treated patients found no significant binding to 5-ht2 a receptors.10 in clinical trials, amisulpride has shown therapeutic benefit, with a profile of side effects similar to that of placebo.9 this and its highly specific receptor profile make it ideally suited to test whether antipsychotic efficacy and a low incidence of eps may be achieved purely by selective action at limbic cortical dopamine d2/d3 receptors in vivo.11 some meta-analyses have identified clozapine, amisulpride, risperidone, and olanzapine as being significantly more effective than first generation (typical) antipsychotics and other second-generation (atypical) antipsychotics.1012 clinically, amisulpride is characterized by a side effect profile most resembling that of an atypical antipsychotic due to its low eps burden.9 however, like risperidone and first-generation antipsychotic drugs, amisulpride causes large elevations in serum prolactin levels, most likely due to its potent d2/d3 antagonist properties.13 thus, despite having a pharmacologic profile reminiscent of a typical antipsychotic in that it exhibits high d2 affinity and low 5-ht2a affinity, amisulpride therapeutically resembles atypical antipsychotics. the identification of the 5-ht7a receptor as a target blocked by amisulpride suggests a plausible explanation for its antidepressant efficacy. changes in 5-ht7 receptor function have been shown to result from chronic antidepressant treatment.14,15 the 5-ht7a receptor antagonism, and not d2/d3 receptor antagonism, likely underlies the antidepressant actions of amisulpride.16 moreover, 5-ht7 receptor antagonists and presently approved antidepressants also appear to have similar effects on hippocampal neurogenesis.17 patients with schizophrenia have been found to have increased somatic morbidity and mortality risks relative to the general population.18,19 weight gain might contribute to their risk of morbidity and mortality by leading to an increase in lipid dysregulation, hypertension, type 2 diabetes mellitus, cardiovascular disease, and other related diseases.20,21 in addition, being overweight usually leads to lower self-image and self-esteem, decreased quality of life, and social disadvantages, and is associated with medication noncompliance.2224 recently, metabolic syndrome in patients with schizophrenia has drawn enormous attention from researchers. previous studies showed that approximately 28.7%60.0% of patients with schizophrenia-related disorders have metabolic syndrome.25,26 most studies show that the prevalence of metabolic syndrome in patients with schizophrenia or schizophrenia-related disorders is higher than that in the normal population.26,27 choosing the right antipsychotic is one of the most challenging issues when treating schizophrenia. next to efficacy issues, safety of medication, including subjectively distressing side effects (sedation, hypersalivation or dry mouth, akathisia, sexual dysfunction) with negative medical consequences (weight gain, orthostatic hypotension, diabetes, hyperprolactinemia, corrected qt prolongation) and life-threatening adverse events (agranulocytosis, neuroleptic malignant syndrome), also influence the choice of medication.28,29 atypical is a term widely used to describe some antipsychotics with specific characteristics, such as minimal risk of acute and chronic movement disorders and less sedation.30 the atypical antipsychotic drugs are also thought to be more effective than conventional drugs in the treatment of negative symptoms in schizophrenia, although this has not yet been adequately established.31 at present, new antipsychotics are routinely investigated for their possible effect on negative symptoms. in spite of their better tolerability profile, catie (the clinical antipsychotic trials of intervention effectiveness) showed a high dropout rate with atypical antipsychotics because of either inefficacy or intolerable side effects.32 the atypical antipsychotic drugs are a class of agents that have become the most widely used to treat a variety of psychoses because of their superiority with regard to eps. the major concern regarding the safety of the atypical antipsychotics is related to their propensity to induce weight gain and alter glucose and lipid metabolism. their main clinical advantage beyond low eps is their ability to improve cognition (to some extent), which is one of the key deficits in schizophrenia. further study is needed to define their mechanism of action, particularly with regard to long-term effects on neuronal plasticity and survival. several studies have found that amisulpride and risperidone are better tolerated than haloperidol with regard to eps.33,34 weight gain was also shown to be significantly greater with risperidone than with amisulpride (1.4 kg versus 0.4 kg, p=0.026).35 in a six-month treatment period, significantly fewer amisulpride-treated patients presented a weight increase of 7% or higher than that of baseline compared with those receiving risperidone (18% versus 34%).36 additional evidence for decreased levels of weight gain in amisulpride-treated patients relative to olanzapine-treated patients comes from both an eight-week study (weight gain in the olanzapine versus the amisulpride group 2.7+3.9 kg versus 0.9+3.2 kg, respectively) and a six-month study (weight gain in the olanzapine versus the amisulpride group 3.9 kg+5.3 versus 1.6+4.9 kg, respectively).37,38 recently, a meta-analysis of all randomized and double-blind studies demonstrated that amisulpride treatment was significantly associated with relatively low weight gain.39 collectively, these findings suggest that amisulpride is an atypical antipsychotic drug with a lower risk of weight gain. both amisulpride and ziprasidone were preferred to olanzapine in patients who had recently experienced weight gain.40,41 this makes sense because second-generation antipsychotics do not appear to differ regarding efficacy, but both amisulpride and ziprasidone have been shown to cause less weight gain than other compounds.38,42,43 during the treatment course, the amisulpride-treated patients showed significantly decreased fasting triglyceride, total cholesterol, glucose, and insulin resistance levels, decreased diastolic blood pressure and pulse rate, and a significant increase in high-density lipoprotein cholesterol levels after switching to amisulpride (all with a p<0.05). the prevalence of metabolic syndrome in amisulpride-treated patients also decreased significantly from 65.2% to 30.4% (mcnemar test, p<0.0005). these findings suggest that switching to amisulpride could be an effective treatment of overweight or obese psychiatric patients treated previously with other second-generation antipsychotics.44 in addition to weight reduction, this study showed that the lipid profiles in these overweight or obese patients also improved significantly. a growing body of evidence indicates that use of some atypical antipsychotics, including clozapine and olanzapine, may be linked to impairment in some health-related lipid indices. for instance, in a prospective study, schizophrenia patients treated with olanzapine and clozapine for four weeks showed significantly elevated triglyceride and total cholesterol levels and decreased high-density lipoprotein cholesterol levels.45 amisulpride is a unique atypical antipsychotic that selectively blocks d2 and d3 receptors presynaptically in the frontal cortex, possibly enhancing dopaminergic transmission, and postsynaptically in the limbic areas, possibly reducing it. thus, dopaminergic overactivity in the frontal cortex and underactivity in the limbic areas, can be treated simultaneously, alleviating both positive and negative symptoms of schizophrenia, respectively.46 additionally, the finding that amisulpride is a highly effective antidepressant via antagonism at 5-ht7 receptors would make its mechanism of action a unique one relative to other approved antidepressant drugs, and supports the development and/or testing of more selective 5-ht7 receptor antagonists to treat depression in humans,16 including their effects on negative symptoms, cognitive dysfunction, mood, morbidity and mortality, work and social function, quality of life, and family and societal burden. other studies have demonstrated superiority over other antipsychotic agents, such as the atypical risperidone, in terms of social interactions and performance, as well as in terms of relevance of the therapeutic effect observed. in addition to low levels of eps, as with all atypicals, amisulpride also shows a low incidence of side effects, such as weight gain, that may contribute to improved compliance and enhanced long-term efficacy.46 schizophrenia is a chronic disorder that results in significant social, psychologic, and occupational dysfunction. not surprisingly, schizophrenic patients score very poorly on subjective and objective measures of quality of life, even when compared with patients with other chronic psychiatric conditions, such as depression or anxiety disorders, and long-term functional impairment is very frequent.47 quality of life measures are especially important when treating patients with chronic conditions, such as schizophrenia, which significantly impair their way of life. every aspect of everyday life is affected, including where they live and work, what activities they can perform, and how they interact with other people. ideally, schizophrenia treatment should protect against relapse, which then becomes the foundation for improvements in quality of life and level of functioning.48 the atypical antipsychotics lack many of the problems associated with traditional treatments, and evidence suggests that they may be associated with a higher subjective quality of life (table 3).49 however, the atypical antipsychotics may also differ from each other on a whole range of factors. amisulpride has a good safety and tolerability profile, with fewer eps than the conventional antipsychotics and a low incidence of anticholinergic side effects. as for other antipsychotics, corrected qt warnings are stated on the labeling for some countries, even though postmarketing surveillance for amisulpride shows no cause for concern.50 improvement in quality of life and social functioning, with consequent reintegration into society, is clearly a major goal of treatment for schizophrenia. the improved safety and tolerability profile of the atypical antipsychotics, combined with their benefits on negative symptoms and cognitive impairment, should help achieve this aim.51 the atypical antipsychotic, amisulpride, has an improved safety and tolerability profile, and has been shown to be significantly more effective than placebo and haloperidol on a number of quality of life and social functioning scales, including the global assessment of functioning, the quality of life scale, the functional status questionnaire, and the psychosocial aptitude rating scale.50 in conclusion, amisulpride, in addition to its proven clinical efficacy, may help reintegration of the schizophrenic patient back into social inclusion. the negative symptoms of schizophrenia are characterized by poverty of speech, blunted affect, lack of initiative, poor motivation, and a general slowness and underactivity, all of which result in social withdrawal. this may reflect the fact that new-generation antipsychotics have more distinct differences in their safety and tolerability profiles than in their efficacy characteristics. although this knowledge helps to guide clinicians in drug choice, the translation of clinical trial findings into individual patient needs remains a daunting challenge. recent data indicate that, in the case of the atypical antipsychotic amisulpride, these properties can be translated into a better quality of life, and enhanced social functioning and reintegration into society.

the introduction of the atypical antipsychotic drugs represents an important advance in the treatment of schizophrenia, because the therapeutic efficacy, tolerability, and safety profiles of these agents seem to be superior to that of the classical neuroleptics. as would be predicted from the pharmacologic profile of a pure d2/d3 receptor blocker, amisulpride is an atypical antipsychotic agent, effective for positive and negative symptoms, which can bring about additional improvement in the social functioning and quality of life of patients with schizophrenia. amisulpride is effective in acute schizophrenia as determined by clinical global impression scores. the major concern regarding the safety of the atypical antipsychotics is related to their propensity to induce weight gain and alter glucose and lipid metabolism. amisulpride has one of the lowest potentials for weight gain of all the antipsychotic agents, and is associated with clearly lower use of antiparkinsonian medication and with fewer dropouts due to adverse events than conventional antipsychotics. amisulpride is well tolerated with regard to anxiety and insomnia, and not notably different from placebo. amisulpride has a pronounced prolactin-elevating effect which appears to be independent of dosage and duration of administration. hyperprolactinemia rapidly reverses following amisulpride discontinuation. amisulpride benefits patients with negative symptoms, and is the only antipsychotic to demonstrate efficacy in patients with predominantly negative symptoms. amisulpride maintains its efficacy when used for medium/long-term treatment, as demonstrated in studies of up to 12 months. in terms of relevance of the effects, superiority is observed for quality of life, social adaptation, and functioning, as measured by the quality of life and functional status questionnaire scales. in conclusion, amisulpride is an antipsychotic agent with proven efficacy and good tolerability. moreover, this drug can help people with schizophrenia to attain social reinsertion.

PMC3108712

pubmed-961

in 2007, the world health organization (who) launched its initiative for the global elimination of congenital syphilis, with the goal that by 2015 at least 90% of pregnant women are tested for syphilis and at least 90% of seropositive pregnant women receive adequate treatment. subsequently in september 2010, paho member states approved a strategy and plan of action for the elimination of mother-to-child transmission (mtct) of hiv and congenital syphilis by resolution 50/12 at the 50th directing council meeting [2, 3]. one of the three targets set was reduce the incidence of congenital syphilis to 0.5 cases or less per 1,000 live births by 2015. the caribbean community (caricom) health ministers endorsed this initiative. in adults syphilis is a complex sexually transmitted disease that has a highly variable clinical course. clinically the primary lesion (chancre) presents mainly as an anogenital ulcer that appears 990 days after exposure, although extra-anogenital sites such as the lip, tongue, and tonsils from oral sex and kissing, nipple from kissing or wet nursing of infected babies, and finger with minor abrasion from touching infectious lesions can occur. secondary syphilis presents with generalised rash affecting the palms and soles, generalised lymphadenopathy, and orogenital mucosal lesions, including snail tract ulcers and condylomata lata. gummatous syphilis (sometimes known as benign tertiary syphilis) can involve organs or supporting structures and can result in infiltrative or destructive lesions leading to granulomatous lesions or ulcers (e.g., skin) or perforation/collapse of structure (e.g., palate and nasal septum) or organomegaly. late neurosyphilis can cause meningovascular syphilis leading to stroke syndromes and parenchymal involvement leading to general paresis and tabes dorsalis. cardiovascular syphilis involves the aortic arch, which can lead to angina from coronary ostitis, aortic incompetence, and aortic aneurysm. there is sparse data reflecting the true occurrence of congenital syphilis (cs) in trinidad. in 1990 ali reported that during the period from january 1985 to december 1988, 28 cases of cs were diagnosed shortly after birth at one major hospital in trinidad with an average annual incidence of 1.5 per 1000 live births, seen at that particular institution. subsequently paho reported 45 cases of congenital syphilis in 2009 for the entire country, a rate of 2.3 per 1000 live births; both pieces of evidence suggest that cs remains a public health challenge. diagnosis and prevention of mother-to-child transmission of syphilis (mtct) are feasible, inexpensive, and cost-effective in nearly every situation evaluated. in fact congenital syphilis is preventable if a single dose of 2.4 million units of benzathine penicillin g is given to a pregnant woman with the infection during the first two trimesters of pregnancy [911]. the challenge is to find such women. in order to monitor progress towards the goals of the elimination initiative in the region, paho requested that member countries gather information on several key programmatic elements. in trinidad there are two tier systems of health care both operating independently of each other, a public health care system (phcs), and a private health care system (prhcs). the phcs is funded by the ministry of health (moh) and therefore all services are free as opposed to the prhcs which is based on a fee for service model. the largest provider for the diagnosis and treatment of sexually transmitted infections is the moh through a dedicated vertical program delivered at the queen's park counselling centre (qpcc). the laboratory services delivered by this program extend to prhcs and therefore offer a reliable source of data from which syphilis occurrence in trinidad and can be estimated. the aim of this study therefore is to describe the current status of acquired syphilis in trinidad, trends over the first decade of the 21st century, and a spatial analysis of syphilis cases, to inform strategies for achieving the elimination of syphilis. the qpcc is a repository for all clinical and laboratory data for syphilis for the entire population of 1.3 million. thus all clinical cases and laboratory investigations associated with a diagnosis of syphilis or cs were eligible for entry into the study. we defined syphilis and cs using the cdc surveillance case definitions, which include clinical features as well as a laboratory diagnosis. in particular for syphilis clinical features included one or more of the following: chancres consistent with primary syphilis, localized or diffuse mucocutaneous lesions often associated with generalized lymphadenopathy and, a reactive serologic test (nontreponemal: venereal disease research laboratory (vdrl) or treponemal (fluorescent treponemal antibody absorbed)). similarly for cs the clinical features occur in an infant or child such as hepatosplenomegaly, rash, condyloma lata, snuffles, jaundice (nonviral hepatitis), pseudoparalysis, anaemia, or oedema (nephrotic syndrome and/or malnutrition) and stigmata (e.g., interstitial keratitis, nerve deafness, anterior bowing of shins, frontal bossing, mulberry molars, hutchinson teeth, saddle nose, rhagades, or clutton joints) in older children. this was followed by laboratory confirmation using the treponema pallidum particle agglutination assay (tppa). the results of all serum samples tested by the laboratory for vdrl between 2009 and 2012 were reviewed. vdrl positive samples using standard methods and quantitative vdrl testing were eligible for entry into the study. a positive vdrl supported by a positive confirmatory test was used to tract the clinical records to ensure that a case of syphilis met the case definitions. from the clinical records, data on age, gender, ethnicity, and address (without street number) we also used secondary data published by the moh for the period 19942009 to demonstrate trends. this data reports only the recorded number of cases for each calendar year and published by the moh in the annual reports of the moh. the street addresses of established syphilis cases diagnosed, between 01/01/2010 and 31/12/2012 only, were collected and mapped using arcmap 10.0 gis software (esri, redlands, ca, usa). the geocoded addresses were transformed into a density map, using the spatial analyst kernel density tool areas with the highest density of syphilis (defined as areas with greater than ten cases per square mile over the three-year period) and were designated as hot spots. in addition we calculated a standardized incidence ratio (sir) with 95% confidence intervals (ci) using the approach by vandenbroucke. all data was stored, retrieved, and analysed under strict confidential cover using spss versus 18. data published by the ministry of health (moh) for the period 20042009 revealed a decline in aggregate cases of syphilis during this period (figure 1). this decline was observed in both males and females. since there were no published data by the moh for 20102012 we reviewed the laboratory records at the qpcc to determine the number of laboratory confirmed cases for the period (20102012). in addition in order to validate the most recent data published by the moh, that is, 2009, we also reviewed the clinical and laboratory records at the qpcc for 2009. we found the number of cases of syphilis for 2009 was 507 and not 130 as reported by the moh. overall the number of cases of syphilis for the period 200912 averaged 428, with cumulative incidence rates varying between 39 and 29 per 100 000 population for this period. there were 64 confirmed cases, 37 (57.8%) males and 27 (42.2%) females. more than half (14, 52%) of the females who tested positive came from samples received from prenatal clinics. for the first time data were collected on the sexual orientation of the positive cases. there were more cases of syphilis among heterosexuals (47, 73.4%) than homosexuals (11, 17.2%) or bisexuals (6, 9.4%). these numbers should be interpreted cautiously since it is reasonable to assume (although unknown) that the heterosexual population is much larger than bisexuals or homosexuals. in addition risk taking behaviour, attitudes, and practices among these groups are unknown. using only data reported by moh in fact it appears that in 2009 trinidad had almost reached the target of 0.5 cases per 1000 live births. three hot spots were identified (beetham gardens, san juan/laventille, and arima) using the kernel method (figure 3). arima has the second highest population density of 2890 km on the island; both beetham gardens and san juan/laventille also have high population densities of>1500 km. hence all the areas identified as hot spots were urban and densely populated. the major finding of the study was the high levels of occurrence of syphilis still occurring in parts of trinidad. in fact between 2009 and 2012 the average number of cases per annum was 428 with an incidence rate as high as 39 per 100 000 population. the highest rates of syphilis were predominantly found in urban men and women in their sexually most active years, that is, between the ages of 15 and 35. on average, women become infected at a younger age than men. consequently, the prevalence of active syphilis is higher among young women than their male peers, a finding that has also been observed for hiv, herpes simplex virus type 2 (hsv-2), and other stds [1517]. however in developed countries such as the usa, for instance, there were 46 042 cases of syphilis in 2011 and the rate of primary and secondary syphilis increased by 3.8% among men (from 7.9 to 8.2 cases per 100,000 men) during 2010-2011 [18, 19]. on the other hand, the rate decreased to 9.1% among women (from 1.1 to 1.0 cases per 100,000 women), while in western europe rates have fell below 5 per 100 000 in the majority of countries [2123]. the implication of this finding is clearly that young women are at high risk, and this is of special concern as a high proportion of women become pregnant or commence childbearing before reaching 20 years of age, thus sustaining prenatal transmission and congenital syphilis. these findings support the implementation of educational programs among adolescents to encourage safer sexual behavior and improved treatment seeking behaviour. based on this evidence we advocate for adolescent sexual health education programs to be implemented in the school curriculum. another important finding of the study is the large discrepancy in data reported by the moh for 2009, that is, 130 reported cases versus 507 cases actually collected from a review of laboratory data. national health data are required for planning and evaluation of service delivery [2426]. this planning and evaluation is critical in developing countries where the majority of health services are provided through national programs and the limited funds must be used efficiently and effectively [24, 25, 27]. in these settings, high data quality is important to ensure that decisions reflect program needs and direct health professional education priorities [2528]. poor data quality not only contributes to poor decisions and loss of confidence in the systems but also threatens the validity of impact evaluation studies. while the study did not focus on the reasons for this discrepancy, several studies have reported inconsistencies in data reporting as well as poor support mechanisms to ensure data quality as it traverses several levels of governmental bureaucracy [3032]. for example a study done in nepal found that data obtained from the facility registers were lower than the data reported at the district level, showing a tendency of overreporting to the higher levels. other studies showed that errors in reporting were due to lack of supervision and feedback from the superior levels as well as inadequate incentives to health workers [3032]. the quality of data, and consequently of the information system, must be seen in a broader perspective focusing not only on technicalities (data collection tools and the reporting system) but also on support mechanisms. another important finding was the widening gap in the occurrence of syphilis among men and women. historically more men than women contracted the disease but by 1997-98 women began exceeding men and a decade latter this gap was the largest. this finding has two implications; first it is more likely for the disease to be sustained and propagated if women continue to be harbouring the infectious agent and are agents for transmission and secondly the risk of cs remains. while significant gains have been made with congenital syphilis, as of 2009 we are still above the targeted incidence of congenital syphilis, that is, 0.5 cases/1,000 live births. three hot spots were identified using spatial analysis techniques, as well as a sir of 1.6. the sir is generally calculated to provide an estimate of the likelihood that an excess of cases exists in the population of concern (hot spots) compared to the general or reference population. the measure is typically interpreted as an excess number of cases in this instance as much as 60%. there were several limitations to the study mainly the unavailability and difficulty to retrieve all data, which may hinder the exact burden of the disease. in addition because case definitions for congenital syphilis vary widely by country, measuring the attainment of the specific targets of the global initiative may be difficult. in a young child, the possibility of sexual abuse should be considered as a cause of acquired rather than congenital syphilis, depending on the clinical picture; this data could not be determined. although congenital syphilis includes cases of syphilitic stillbirths we were unaware of the existence of such cases and hence it is not included in the study. in conclusion the occurrence of syphilis in trinidad continues to be high; this is against a background in which syphilis screening and treatment can cost less than us $1 per syphilis test and us $0.50 per penicillin dose, and health economists estimate that this is among the most cost-effective public health interventions in existence. in addition elimination of congenital syphilis as a public health problem by 2015 is an important and attainable component of global efforts to achieve the millennium development goals (mdgs) 4 (reduce child mortality), 5 (improve maternal health), and 6 (combat hiv/aids, malaria, and other diseases).

objective. to describe the current epidemiological features of syphilis and congenital syphilis in trinidad, 20092012. methods. all laboratory confirmed syphilis cases diagnosed through a vertical program in the ministry of health, between 1/1/2009 and 31/12/2012, were identified. all relevant data were collected including address which was geocoded and mapped using arcgis 10.0 (esri). both spatial techniques and standardized incidence ratios were used to determine hot spots. results. the annual cumulative incidence rate for syphilis remains high varying from 39 per 100 000 population in 2009 to 29 per 100 000 in 2012. we identified 3 hot spots, in urban areas of trinidad. young men and particularly young women in childbearing age 1535 living in urban high density populations were commonly infected groups. conclusion. the incidence of syphilis continues to be very high in trinidad. new initiatives will have to be formulated in order to attain the global initiative to eradicate syphilis by 2015.

PMC4519538

pubmed-962

posterior chamber phakic intraocular lens (piol) implantation is generally accepted as an effective and reversible treatment method for high myopia with preservation of lens-regulating capability. in the ophthalmology clinic, the implantable collamer lens (icl, staar surgical, nidau, switzerland) is the main posterior chamber piols; it is designed to be placed between the iris and the anterior surface of the lens and is fixed to the ciliary sulcus by four haptics, thus preventing its contact with the lens. although piol implantation has a good clinical efficacy in the correction of high myopia, some short-term and long-term complications have been reported. this complication may be caused by direct contact of the icl with the lens due to a low vault or insufficient circulation of the aqueous humor. the incidence of cataract after icl implantation ranges between 0.61% and 2.7%, depending on different follow-up periods in different studies [24]. high intraocular pressure is a major concern after icl implantation, particularly for early icl models [58]. to avoid postoperative elevation of intraocular pressure, peripheral iridectomy however, iridectomy may lead to pain and intraoperative bleeding and increase difficulty performing the operation. to avoid peripheral iridectomy, the v4c-icl model with a 0.36 mm central hole has been designed based on the v4-icl model. because the central hole of the v4c-icl facilitates the outflow of the aqueous humor, peripheral iridectomy is not required. in addition, compared with the traditional v4-icl model, the v4c-icl model exhibits similar low-, middle-, and high-frequency contrast sensitivity and higher-order aberrations under the conditions of various pupil sizes, and the subjective symptoms such as glare or halo were also essentially equivalent, thus suggesting that the v4c-icl model has good safety and efficacy [9, 10]. although studies have investigated the effect of v4c-icl implantation on patients ' visual function status, to our knowledge, the effect of v4c-icl implantation on visual activity in the complex environment of the patients ' daily lives has not been studied. it remains to be determined whether this subjective symptom can affect the patient's daily visual activity. therefore, in this study, we conducted a questionnaire to investigate the effects of v4c-icl implantation on myopic patients ' vision-related daily activities. this retrospective study collected data from 42 consecutive patients (82 eyes) with complete clinical data who underwent v4c-icl implantation at the affiliated hospital of zunyi medical college (zunyi, china) between november 2014 and november 2015. all patients were followed up for more than 3 months (range, 3 to 6 months; mean, 4.62 1.23 months). the patients ' average age was 24.04 4.75 years (range, 1835 years). the average preoperative sphere power was 10.21 3.02 diopter (d) (range, 4.0 d to 15.0 d), and the average cylinder power was 2.48 0.91 d (range, 1.25 d to 4.5 d). the average spherical equivalent (se) was 11.55 3.52 d (range, 5.75 d to 16.25 d). the preoperative and postoperative uncorrected visual acuity (ucva) and best corrected visual acuity (bcva) of the patients were recorded using the decimal method and converted into the logmar (logarithm of the minimal angle of resolution) equivalence. ubm was used to detect the central and peripheral vault at 3 months after operation. three months after operation, all patients were asked by the same doctor to evaluate the visual function. the questionnaire was designed based on the visual function evaluation questionnaire used by the corneal diseases and excimer laser research unit, university of dundee, scotland, with slight modifications. since some patients complained of halos after v4c-icl implantation, we also investigated the effect on the visual functions of the presence of a halo in patients during the follow-up. this study was performed according to the declaration of helsinki, and all patients gave their informed consent after a comprehensive explanation of the possible risk of v4c-icl implantation. inclusion criteria were bcva of 0.5 or above and refractive stability for more than 2 years. exclusion criteria were age<18 years; anterior chamber depth<2.8 mm, ecd<2000/mm; corneal diseases; and a history of eye diseases affecting visual function such as glaucoma, cataract, retinal diseases, uveitis, and ocular trauma. the size of the v4c-icl was determined by the horizontal white-to-white corneal diameter and anterior chamber depth of the patients. the power of v4c-icl was calculated using the software provided by the manufacturer (staar surgical). fifteen minutes before operation, compound tropicamide eye drops were applied to dilate the pupils, followed by topical anesthesia with 0.4% oxybuprocaine hydrochloride. for patients implanted with v4c-icl for astigmatism, the main incision site was created at the position of 135 and an auxiliary incision site was made at the position of 45. after introduction of viscoelastic materials to maintain the anterior chamber, the v4c-icl was slowly pushed into the anterior chamber using an injector cartridge. then, the haptics of the icl was enclaved into the anterior chamber via the main and auxiliary incision sites using the manipulator. numerical data are presented as mean sd. analyses were performed using spss v17.0 software. repeated analysis of variance was used to analyze the differences in intraocular pressure as well as ecd at different timepoints before and after the operation. independent student's t-test was used to analyze the differences in the central vault and peripheral vault and the score of visual analog scale between different groups. the average preoperative spherical equivalent was 11.55 3.52 d (range, 5.75 d to 16.25 d). the average spherical equivalent was 0.12 0.33 d (range, 1.00 to 0.50 d) and the residual astigmatism was 0.18 0.32 d (range, 0.25 to 1.25 d) at 3 months after patients operations, excluding those patients with preexisting astigmatism who did not receive the implanted astigmatic v4c-icl model and required preservation of astigmatic power. the average preoperative ucva was 1.47 0.23 logmar (range, 1.15 to 2.0). the average ucva was 0.03 0.08 logmar (range, 0.18 to 0.30) at 3 months after operation. the uncorrected visual acuity had increased significantly at 3 months after operation compared with before operation (p<0.001). at 3 months after operation, ucva in 80 eyes (98%) was equal to or better than preoperative bcva. the average bcva at 3 months after operation was 0.03 0.07 logmar (range, 0.18 to 0.22), which was significantly better than preoperative bcva (average, 0.08 0.10 logmar; range, 0.08 to 0.40) (p=0.029). the average preoperative intraocular pressure (iop) was 13.35 2.34 mmhg (range, 9.7 to 18.0 mmhg). one day after operation, the iop was slightly increased (average, 14.26 3.20 mmhg; range, 10.0 to 25.0 mmhg). only one eye had an iop>21 mmhg, and the iop returned to the normal level (14 mmhg) without any special treatment at the second day after surgery. at 1 month after operation, the iop was stable (average, 13.46 1.74 mmhg; range, 10.0 to 17.2 mmhg). at 3 months after operation, the average iop was 13.42 2.19 mmhg (range, 10.0 to 17.0 mmhg), which was not significantly different from that before (and at 1 month) and after operation (figure 1). ecd was 2857.76 295.60 before operation, 2745.59 384.11 at 1 month after operation, and 2719.30 363.02 at 3 months after operation. compared with the preoperative value, ecd at 1 month and 3 months after operation decreased by 3.92% (p=0.144) and 4.83% (p=0.065), respectively (figure 2). we investigated the peripheral vault (the perpendicular line between the end of suspensory ligament and the icl) and the central vault (the perpendicular line between the surface of the anterior lens capsule and the icl surface) at 3 months after icl implantation (figure 3). the peripheral vault was 0.25 0.17 mm (range, 0.03 to 0.92 mm) at 2 o'clock, 0.29 0.21 mm (range, 0.05 to 0.92 mm) at 4 o'clock, 0.23 0.12 mm (range, 0.06 to 0.64 mm) at 8 o'clock, and 0.22 0.13 mm (range, 0.01 to 0.80 mm) at 10 o'clock. the central vault was 0.42 0.22 mm (range, 0.13 to 0.90 mm), which was significantly higher than the peripheral vaults (p=0.000, 0.003, 0.000, and 0.000, resp.). in one eye, the peripheral vault at 2 o'clock and 4 o'clock was as high as 0.92 mm, and the peripheral vault at 8 o'clock and 10 o'clock was 0.46 mm and 0.44 mm, respectively. the high vault value may be due to icl tilt after implantation. during the 6-month follow-up, the patients had visual acuity of 0.08, normal iop (1116 mmhg), no endothelial damage, and normal daily visual activities. the patients were required to fill out a questionnaire about visual functions at 3 months after operation. complete questionnaires were returned by 42 patients and all of these patients answered the questionnaire satisfactorily. in table 1, items 1-2 evaluated near vision, items 35 evaluated far vision, item 6 was for night vision, and items 711 were for middle-distance vision. we evaluated the daily activities associated with near vision, far vision, and middle-distance vision. apart from one patient who had a difficulty reading computer screens, all patients had satisfactory or very satisfactory results. we also investigated whether the patients had a halo after operation. during the early postoperative follow-up period, halos occurred in 23 patients (54.8%). with time, halos gradually disappeared at 3 months after operation without any treatments. the patients were categorized into two groups: patients with halos and patients without halos. the average age in patients with halos was 25.5 4.3 years, which was not significantly different from those without halos (23.2 5.0 years) (p=0.702). there were no significant differences in the scores of visual functions between the two groups (p>0.05, table 2). posterior chamber piol implantation is an effective refractive surgery that has been widely accepted. however, elevated intraocular pressure is a common postoperative complication after piol implantation, even in patients with preoperative or intraoperative iridectomy. to simplify the icl implantation procedure and improve postoperative aqueous circulation, clinical studies have shown that v4c-icl implantation is effective in the treatment of moderate and high myopia [9, 12, 13]. in the present study, we found that (after v4c-icl implantation) ucva was equal to or better than preoperative bcva in 80 eyes (98%) with high myopia. almost all eyes had bcva equal to or better than preoperative bcva by one line or more. the postoperative ucva and bcva were 0.03 0.08 logmar and 0.03 0.07 logmar, respectively. the efficacy index and safety index were 1.27 and 1.28, respectively, which were consistent with previous studies [9, 1214]. these findings suggest that v4c-icl is a safe and effective treatment for high myopia, and it significantly simplifies the icl implantation procedure. during postoperative follow-up periods, intraocular pressure was stable and only slightly increased on postoperative day 1. due to the presence of the central hole, v4c-icl implantation avoids preoperative or intraoperative peripheral iridectomy, thus reducing iop elevation caused by surgical stimulation, depigmentation, and pupillary block. in the present study, we found that the ecd at 1 month and 3 months after operation were reduced by 3.92% and 4.83%, respectively. compared with the preoperative value, postoperative ecd was not significantly decreased, which was consistent with the results reported by shimizu et al.. although we found that v4c-icl implantation did not produce a short-term effect on ecd, it remains to be determined whether aqueous outflow through the central hole of the v4c-icl has a long-term effect on ecd. further studies with long-term follow-ups and large sample sizes should be performed. vault is one of the most important indices for evaluating the postoperative effect of the icl implantation. in the present study, we performed ubm to observe the relative position between the v4c-icl and the lens and found that no v4c-icl was in direct contact with the lens. the peripheral vault at each measured site was significantly lower than the central vault. this may occur because the center of the v4c-icl is designed to be thinner than the periphery. evaluation of visual quality is a comprehensive method to evaluate the effect of refractive surgery and has been widely used. it has been reported that there is no significant difference in visual outcome between v4c-icl and v4-icl implantation [9, 10]. however, these studies only investigated the effect of v4c-icl and c4-icl implantation on contrast sensitivity and higher-order aberration. there were no reports about whether it affects the daily visual functions after implantation of v4c-icl. in the present study, we investigated the effect of v4c-icl implantation on vision-related daily activities during follow-up. we found that apart from one patient (2.4%) who had a difficulty in reading computer screen, all patients had satisfactory or very satisfactory results. in addition, we found that there were tilt icl in one eye of another patient. during the 6-month follow-up, the patients with tilt icl had visual acuity of 0.08, normal iop (1116 mmhg), no endothelial damage, and normal daily visual activities. in our short-term follow-up, even the tilt of the v4c-icl could not bring a serious impact on the patient. of course it was necessary to observe the patient for a long term. during the early postoperative follow-up period, halos occurred in more than 50% (54.8%, 23 patients) of patients. with time there were no significant differences in the scores of visual functions between patients with or without halos. these findings suggest that halos do not affect postoperative vision-related daily activities. in conclusion, we investigated the vision-related daily activities at 3 months after v4c-icl implantation. we found that the patients were satisfied with their vision-related daily life activities, and the presence of central hole of v4c-icl did not affect vision-associated daily activities. postoperative intraocular pressure was stable, and no vision-associated complications were found during the follow-up period. however, because v4c-icl implantation alters aqueous circulation, it remains to be determined whether v4c-icl implantation produces long-term effect on intraocular pressure and ecd.

the new type implantable collamer lens with a central hole (v4c-icl) is widely used to treat myopia. however, halos occur in some patients after surgery. the aim is to evaluate the effect of v4c-icl implantation on vision-related daily activities. this retrospective study included 42 patients. uncorrected visual acuity (ucva), best corrected visual acuity (bcva), intraocular pressure (iop), endothelial cell density (ecd), and vault were recorded and vision-related daily activities were evaluated at 3 months after operation. the average spherical equivalent was 0.12 0.33 d at 3 months after operation. ucva equal to or better than preoperative bcva occurred in 98% of eyes. the average bcva at 3 months after operation was 0.03 0.07 logmar, which was significantly better than preoperative bcva (0.08 0.10 logmar) (p=0.029). apart from one patient (2.4%) who had difficulty reading computer screens, all patients had satisfactory or very satisfactory results. during the early postoperation, halos occurred in 23 patients (54.8%). however there were no significant differences in the scores of visual functions between patients with and without halos (p>0.05). patients were very satisfied with their vision-related daily activities at 3 months after operation. the central hole of v4c-icl does not affect patients ' vision-related daily activities.

PMC5124673

pubmed-963

since the middle of the 1990s, numerous mergers and consolidations have taken place in the health insurance industry. in fact, the annual number of mergers among managed care companies averaged 43 during the 19942005 period and ranged from a low of 27 in 1995 to a high of 66 in 1996 (kaiser family foundation). economic theory suggests that mergers among companies may confer net benefits upon society because of cost efficiencies that result from scale and scope economies. however, economic theory also indicates that horizontal mergers may allow the newly combined company to enjoy the advantage of increased market power. the increased market power may translate into higher prices received for products and/or lower prices paid for inputs. theory suggests that the unbridled exercise of market power in output or input markets can generate net social losses. in a series of annual reports, the american medical association (ama 2003, 2004, 2005, 2006) has collected and reported information on the concentration of health insurers in various metropolitan areas of the united states. based on this data, the ama finds a health insurance industry that is characterized by a few dominant health insurers and believes that these dominant firms may engage in monopoly and monopsony behavior. hence, this organization warns that health insurers may raise insurance premiums and thereby reduce the number of insured individuals. in addition, the ama stresses that health insurers may lower reimbursement rates paid to health care providers and, as a result, cause contrived shortages of medical care. governor rendall of pennsylvania claims that a merger between the state s two largest health insurers, highmark inc. and the governor notes that employers in pennsylvania would be less likely to secure low premiums for health insurance if the merger is allowed. moreover, nurses at landmark medical center in rhode island recently protested outside the office of blue cross/blue shield of rhode island because of the health insurer s failure to pay provider rates that sufficiently compensates for the quality of medical care delivered at the health care facility (malinowsky 2006). but are these concerns about the market power of health insurers truly justified on efficiency grounds or do they represent a situation where health care providers are attempting to protect their monopoly rents? specifically, it is unclear if lower health care provider reimbursement rates by health insurers reflect the exercise of monopsony or monopoly-busting power. if the former, economic theory suggests that the lower price results in efficiency losses. but if the latter, efficiency gains occur from lower prices according to theory. pauly (1998) and, subsequently, feldman and wholey (2001) explain that the only way to determine if health insurers exercise monopsony or monopoly-busting power is by empirically examining the impact of health insurer buying power on the utilization rate of medical services, an input in the production of health insurance. if it can be shown empirically that greater buying power of health insurers is related to lower (increased) utilization of medical services, then the evidence provides support for monopsony (monopoly-busting) behavior. feldman and wholey (2001) provide the only empirical test, to date, for the monopsony versus monopoly-busting theories of health insurer buying power. specifically, they investigate if health maintenance organizations (hmos) possess monopsony power in the markets for ambulatory and inpatient hospital services. using a data set containing all hmos in the u.s. over the period from 1985 to 1997 and multiple regression analysis, they investigate the importance of an individual hmo, as a single buyer, on the price paid and utilization of inpatient and ambulatory care. buying power for hospital services is measured by the percentage of inpatient days in the market area purchased by each hmo. ambulatory buying power is measured by the number of ambulatory visits purchased by each hmo per 1,000 active physicians in the market area. feldman and wholey control for a host of supply and demand factors in the regression equation and find thathmobuying power lowers hospital price and increases hospital output. thus, their evidence suggests that hmos use their buying clout to bust the monopoly power of hospitals. in addition, they determine that hmo buying power has no statistical impact on the price or output of physician care, perhaps because the physician services market is much more fragmented than the hospital services industry. overall, feldman and wholey conclude that hmos do not exercise monopsony power and that they may have improved efficiency in the hospital services industry through their monopoly-busting power. while the study by feldman and wholey offers valuable insights, it is already dated and fails to consider the role of preferred provider organizations (ppos). feldman and wholey s study covers the years from 1985 to 1997 but many consolidations among health insurers have taken place since that time. the increased consolidations may have conferred greater buying clout such that a single health insurer can more easily exploit its monopsony position. also, feldman and wholey do not consider if ppos possess monopsony power. as a result, this study uses more recent data to explore the monopsony power of hmos and ppos in the hospital services market. the analysis is conducted at the metropolitan level over the years from 2001 to 2004. six different measures of hospital output are employed and the buying power of the two types of mcos is measured by the herfindahl/hirschman index (hhi) of market concentration based on the health insurers enrollment shares in the various metropolitan areas. an instrumental variables approach is employed to control for the possibility that health insurer concentration is endogenous at the industry level. the empirical results consistently and strongly suggest that hmos and ppos do not exercise monopsony power in the typical hospital services market. indeed, some evidence is found to support the monopoly-busting theory of health insurer concentration. the sample used to investigate the impact of mco buying power on various measures of output in the hospital services industry has its roots in the series of reports issued by the ama. the ama began collecting and disseminating information on the concentration of health insurers at the state and metropolitan levels in 2001. since that time the ama has issued three additional reports. each report contains data for the hhi separately for the hmo and ppo segments of the health insurance industry in various metropolitan statistical areas (msas) of the u.s.1 the number of msas covered in the ama reports has increased over time, growing from 40 in 2001 to 294 in 2004. several reasons have been offered for treating hmo and ppo plans as distinctly different health insurance products (us v. aetna 1999). first, the two plans differ in terms of benefit design, costs, and other factors. for example, hmo plans are more restrictive with features such as gatekeepers and tighter provider networks. in addition, hmos are known to provide greater preventive care benefits and place limits on treatment options. second, employers and employees often perceive ppo and hmo plans as different products that meet different needs and appeal to different types of enrollees. in fact, enrollees who drop an hmo plan are disproportionately more likely to choose another hmo plan. finally, the price elasticity of demand for hmo plans has been found to be relatively low suggesting that very few consumers may switch to ppo plans given an increase in price. as a result, hmo and ppo plans are considered as two different insurance products in the empirical analysis. the relevant geographical market (rgm) for health insurance is assumed to be the msa in the ama reports because kopit (2004), and others, have argued that the health insurance marketplace is local in nature since employers and consumers/patients want access to nearby health care provider networks. for example, an employer in philadelphia would be unlikely to switch to a health insurer with an established network of providers in boston. in their research on the impact of competitive behavior on the profitability of hmos, pauly et al. (2002) assume that the metropolitan area represents the relevant geographical market for health insurance. in the forthcoming empirical analysis, the msa is also treated as the rgm for hospital services. while this definition of the rgm is not without its weaknesses (dravove and white 1994), urban hospital markets are commonly defined in this manner for empirical work (joskow 1980; manheim et al. but because msas like chicago, los angeles, or new york city may be too large for an individual hospital services market, the empirical test is also performed using a subset of msas that contain fewer than 2 million people to check the sensitivity of the results. (2006) argue that treating the metropolitan hospital services industry rather than the individual hospital as the unit of analysis offers a benefit to the econometrician. they note that quality of care and patient case-mix both differ considerably across hospitals within a given geographical area as a result of individual hospitals specializing in the delivery of certain services. 485) write; hospital a admits more fevers of unknown origin and performs more combined liver kidney transplants, hip replacements, and coronary by-pass grafts and hospital b has more tooth extractions, vaginal deliveries without complications, and circumcisions, it is an unfair comparison. by focusing on the msa as the unit of analysis, these supply-side differences across individual hospitals within a given geographical area are averaged out to some degree, thereby allowing a cleaner isolation of the relationship between market factors and hospital services at the msa level (keeler and ying 1996). as the msa level, the distribution of treatments and quality of care is likely to be more comparable because of closer similarities in the underlying types and severities of illnesses across areas. because a panel data set helps to control for unobservable heterogeneity, the sample is defined as those msas with at least 2 years of health insurer concentration data over the period from 2001 to 2004. that restriction results in a total maximum sample size of 344 msa observations over the 4 year period with a maximum cross-sectional sample of 86 msas. while the total number of observations seems relatively small, the 86 msas account for over 50% of the population in the u.s. thus, the panel data set potentially conveys a considerable amount of useful information on the behavior of health insurers and health care providers. health forum (various years) provides data for six indicators of hospital output. four direct measures of hospital services are employed: total admissions, total inpatient days, total surgeries, and total outpatient visits. revenue data necessary for determining price are unavailable so only the impact of health insurer buyer power on output can be empirically observed. because the monopsony and monopoly busting theories both predict that insurers buying power should reduce providers prices, it seems perfectly acceptable to examine the impact of insurer concentration on quantity without examining its impact on price. to check the consistency of the findings, the total number of hospital personnel and labor costs are specified as indirect indicators of hospital services. the expectation is that total labor costs and number of personnel change in the same direction as the direct measures of hospital output given the derived nature of medical inputs such as labor in production. to isolate the pair-wise correlation between the various measures of health insurer concentration and each indicator of hospital services, it is important to control for as many supply and demand factors as possible. however, the lack of consistent time-series/cross-sectional data forced us to be parsimonious in our selection of independent variables. control variables in the basic model are population, per capita income, and the number of hospitals in the msa along with a set of time and metropolitan dummy variables. data for population and income come from the bureau of economic analysis and health forum (various years) provides the data for the number of hospitals. no expectations are made regarding the sign of the estimated coefficients on population and income per capita because they simply control for a host of factors influencing hospital services that relate to scale or physical environment and socioeconomic conditions. however, the sign of the estimated coefficient on the number of community hospitals is expected to be positive for two reasons. market demand is not perfectly inelastic, the greater supply will be associated with an increased quantity of hospital services. second, a single hospital is likely to hold less market power when more hospitals exist in the market area so the quantity of services is expected to be higher. in an alternative specification, but with fewer observations, the following are specified as additional control variables: the percentage of the population under 15, the percentage of the population over 65, the percentage of the population with a bachelors degree, the percentage of the population that is white, the percentage of the population that is poor, the unemployment rate, and median value of housing in each msa during each year. these variables should help control for the impact of age distribution, education, wealth, and health insurance coverage, among other effects, on the demand for hospital services. based upon a review of the literature, scanlon et al. (2006) conclude that the omission of market characteristics such as these affect the conclusions drawn from empirical studies concerning the effects of health insurance competition. as a final test, the alternative model is estimated with a sample of msas with fewer than 2 million people because the true rgm for hospital services may be less broadly defined in practice than some of the very large msas. that is, both the level of hospital services, variously measured, and the degree of health insurer concentration may be jointly influenced by some unobservable factors. for example, the expansion decisions of both hospitals and health insurers may be jointly influenced by the health status of people in their market areas. therefore the concentration of the hmo and ppo submarkets is modeled in the first stage of the two stage multiple regression analysis. because employer-sponsored health insurance remains the dominant form of private health insurance in the u.s., the number of firms in the msa serves as one instrument to capture the size of the employer market. experimentation shows that this variable fits the first stage model best when specified in quadratic form (in logs). indicators of firm size distribution have previously been used as instrumental variables in the prediction of the number or market penetration of health insurers (dranove et al. the size distribution of firms in the msa is captured in the estimation equation by the average number of employees per firm. data for the number of firms and employees are obtained from the msa business patterns website at the u.s. census bureau (various years). finally, the penetration rate of hmos at the state level in the previous year is specified as an additional instrument. the prior year state hmo penetration may indicate the extent to which state regulations such as guarantee issue, community-rated premiums, and state insurance mandates hinder the entry and scale of health insurers. the expectation is that a greater hmo penetration at the state level is associated with a lower level of insurer concentration (more health insurers) at the msa level. all of these variables are likely to be correlated with health insurer concentration but not the level of hospital output a necessary property for a good instrumental variable. all variables are expressed in log-form in both the first and second stage regression equations. notice that the average hhi for the hmo and ppo product markets exceeds the threshold for a highly concentrated market of 1,800 as set by the department of justice and federal trade commission. also notice that the hhi varies considerably across msas, with the index ranging from just over 1,000 to well over 9,000. interestingly, the mean hmo-hhi increased from 3,651 in 2001 to 4,323 in 2003 but declined to 4,244 in 2004 (data not shown). also, the mean ppo-hhi increased from 4,115 in 2001 to 4,599 in 2001 but declined to 4,179 in 2004. given that the ama increased the number of msas in their sample from 1 year to the next, it is not possible to draw any meaningful implications about the overall trend in industry concentration from their data. table 1descriptive statisticsvariablemeanstandard deviationminimum valuemaximum valuenumber of observationsadmissions187,613228,6645,0701,341,277344inpatient days1,047,5091,399,99739,4709,884,548344surgeries139,169156,6784,698981,518344outpatient visits2,694,4643,487,177110,06823,545,341344personnel21,58227,618855181,500344labor expenses2,482,7203,367,97257,03425,139,710344population1,869,6732,739,524108,68018,754,585344per capita income30,5046,10314,38549,276344number of community hospitals16.517.91102344hhi for hmos40432065112710000262hhi for ppos4322157913709363265number of firms (employers)43,64462,8732,797533,528338employees per firm16.102.4710.6123.04338hmo state penetration rate in previous year30.0113.826.5053.50344percent young0.0710.0120.0440.112233percent old0.1220.0360.0720.274233percent white0.7320.1110.4910.936233percent with bachelor degrees0.1080.0320.03670.226233percent unemployment7.4671.831414233percent poverty13.5365.527644233median value of owner-occupied housing178,007125,97556,087689,276233 descriptive statistics the descriptive statistics in table 1 also indicate that that a sizeable amount of variation exists in the degree of seller concentration in the various metropolitan hospital services industries. according to the data, although an hhi would be preferred to measure seller concentration for the sake of consistency, the data source provides only the number of community hospitals in each metropolitan area. (2005) show that inferences regarding the broad effect of competition are not sensitive to whether the number of hospitals or hhi is used to measure the intensity of seller competition in the hospital services industry. table 2 provides the regression results for the first stage predicting the hmo and ppo hhis. according to the results, hmo concentration declines with metropolitan size, as measured by population, whereas ppo concentration increases with metropolitan size. interestingly, hmo and ppo concentration both first decline and then increase at some point with respect to the number of firms in the msa. also, greater hmo market penetration at the state level in the previous year tends to be associated with lower hhi and ppo concentration, as expected. finally, the average number of employees per firm evidently influences the number and size distribution of ppos but not hmos. more importantly, the set of instruments in each first stage equation has a statistically significant impact on the hhi as determined by a wald test. in addition, the f-statistics exceeds the threshold value of 10, as set by staiger and stock (1997), for detecting weak instruments. table 2first stage results for hmo and ppo concentrationlog of hmo-hhilog of ppo-hhiconstant39.11(3.93)2.954 (0.16)log of population0.265 (2.39)1.679 (4.45)log of per capita income0.901 (1.16)0.988 (0.87)log of number of hospitals0.066 (0.90)0.047 (0.31)log of number of firms3.026 (3.92)2.320 (1.95)log of number of firms squared0.130 (3.76)0.104 (1.94)log of employees per firm0.151 (0.46)2.826 (5.25)log of state hmo penetration rate in previous year0.336 (2.92)0.546 (1.59)number of observations256259adjusted r0.8980.261f-test for instrumental variables10.96 (prob.=0.0000)17.91 (prob.=0.0000)notes: coefficient estimates with t-statistics reported in parenthesescross-section seemingly unrelated standard errors and covarianceall specifications include msa and time fixed effects indicates statistical significance at the 5% level indicates statistical significance at the 10% level first stage results for hmo and ppo concentration notes: coefficient estimates with t-statistics reported in parentheses cross-section seemingly unrelated standard errors and covariance all specifications include msa and time fixed effects indicates statistical significance at the 5% level indicates statistical significance at the 10% level selected findings for the second stage results are reported in tables 3 and 4. recall that monopsony theory predicts a negative coefficient estimate on the hmo-hhi, and ppo-hhi. in contrast, positive coefficient estimates on these concentration variables provide empirical support for the monopoly-busting theory. looking first at the results for the basic equation reported at the top of table 3, we can see that the estimated coefficient on the hmo-hhi has a negative sign in only two of the six equations. in addition, the hmo-hhi possesses a positive and statistically significant coefficient estimate in the inpatient days equations. while the coefficient estimates on the ppo-hhi are negative in 4 of the 6 equations, they are not different from zero at conventional levels of statistical significance. in addition, the ppo-hhi has a positive and statistically significant coefficient estimate in the outpatient visits equation. taken alone these regression results indicate that health insurers do not possess monopsony power on the buyer side of the hospital services market. table 3abbreviated two stage least square resultsmeasure of outputestimated coefficient (t-statistic) on hmo-hhiestimated coefficient (t-statistic) on ppo-hhiadjusted rnumber of observationsbasic modeladmissions0.059(0.82)0.021(0.41)0.998252inpatient days0.150(2.81)0.015(0.22)0.997252surgeries0.003(0.02)0.030(0.38)0.995252outpatient visits0.091(0.52)0.238(2.25)0.987252hospital personnel0.110(1.17)0.093(1.60)0.990252labor expenses0.020(0.18)0.056(0.81)0.997252alternative modeladmissions0.081(0.92)0.010(0.18)0.997172inpatient days0.181(2.71)0.003(0.04)0.996172surgeries0.008(0.08)0.033(0.67)0.995172outpatient visits0.220(0.82)0.199(2.20)0.989172hospital personnel0.015(0.34)0.024(0.46)0.996172labor expenses0.069(0.65)0.005(0.07)0.997172 all continuous variables expressed as logs. control variables include population, per capita income, number of hospitals, and a set of metropolitan dummy variables same as above plus percent young, percent old, percent white, percent with bachelor degrees, percent unemployed, percent poverty, and median value of owner-occupied housing (in logs) cross-section seemingly unrelated standard errors and covariance indicates statistical significance at the 5% level indicates statistical significance at the 10% leveltable 4abbreviated two stage least square results for msas with fewer than 2 million peoplemeasure of outputestimated coefficient t-statistic) on hmo-hhiestimated coefficient (t-statistic) on ppo-hhiadjusted rnumber of observationsalternative modeladmissions0.041(0.59)0.052(0.67)0.992118inpatient days0.186(2.92)0.042(0.47)0.989118surgeries0.147(1.13)0.114(0.75)0.984118outpatient visits0.097(0.67)0.015(0.19)0.985118hospital personnel0.056(0.59)0.017(0.16)0.989118labor expenses0.006(0.07)0.002(0.02)0.992118 control variables include population, per capita income, number of community hospitals, percent young, percent old, percent white, percent with bachelor degrees, percent unemployed, percent poverty, median value of owner-occupied housing (all in logs), and msa and time fixed effectscross-section seemingly unrelated standard errors and covariance indicates statistical significance at the 5% level indicates statistical significance at the 10% level abbreviated two stage least square results all continuous variables expressed as logs. control variables include population, per capita income, number of hospitals, and a set of metropolitan dummy variables same as above plus percent young, percent old, percent white, percent with bachelor degrees, percent unemployed, percent poverty, and median value of owner-occupied housing (in logs) cross-section seemingly unrelated standard errors and covariance indicates statistical significance at the 5% level indicates statistical significance at the 10% level abbreviated two stage least square results for msas with fewer than 2 million people control variables include population, per capita income, number of community hospitals, percent young, percent old, percent white, percent with bachelor degrees, percent unemployed, percent poverty, median value of owner-occupied housing (all in logs), and msa and time fixed effects cross-section seemingly unrelated standard errors and covariance indicates statistical significance at the 5% level indicates statistical significance at the 10% level once additional control variables are added to the basic model, not much change occurs in the magnitude and statistical significance of the estimated coefficients on the hhis, as shown in the bottom panel of table 3. moreover, no statistical evidence is found to support a monopsony view of health insurer concentration. the number of inpatient days is directly related to greater hmo concentration and a direct relationship exists between ppo concentration and the number of outpatient visits. table 4 shows the multiple regression findings when the sample is limited to msas with fewer than 2 million people. the concern is that the rgm for hospital services market may be too broadly defined in the larger msas. these results closely mirror the results for the larger sample of msas. like before, hmo concentration is directly related to the number of inpatient days. the only difference is that ppo concentration is no longer associated with an increased number of outpatient visits.2 all in all, the empirical results strongly suggest that health insurers do not possess monopsony power. greater hmo concentration is shown to be associated with an increased amount of inpatient services and some evidence indicates that higher ppo concentration and outpatient visits are directly related. more specifically, the estimated elasticities suggest that a 10% increase in hmo concentration is associated with about 1.51.9% more inpatient days. using sample averages, that percentage difference means 3 more patients per day are treated at the typical hospital on an inpatient basis because of 10% greater hmo concentration. it also may mean that hospitals are less likely to release patients quicker and sicker when health insurers are more influential in their market areas. the results also imply a 10% increase in ppo concentrationmay be associated with 2% more outpatient visits. that percentage translates into nearly 9 more outpatient visits per day at the typical hospital. the implication is that hospitals typically reduce some services and thereby raise price in the absence of health insurer buying power.3 following the suggestion of pauly (1998) and lead of feldman and wholey (2001), this paper revisits the question of whether health insurers possess monopsony power. the test is conducted by observing the impact of health insurer concentration on the quantity of hospital services. monopsony (monopoly-busting) theory predicts an inverse (direct) relation between health insurer concentration and hospital services. this paper extends the literature on this topic by investigating the impact of hmo and ppo concentration on six different measures of services offered by metropolitan hospitals during the period 20012004. taking all of the empirical results together the relationships between buyer concentration and the six different measures of hospital services are either direct or statistically insignificant. consequently, it appears that much of the attention being paid to consolidations among health insurers may largely reflect that health care providers are trying to protect their monopoly rents. in fact, the empirical findings suggest that health insurers, when they possess more buyer clout, can negotiate additional inpatient days and outpatient visits without necessarily raising the number of personnel and labor expenses (2006) who find empirically that metropolitan hospitals are more efficient when health insurers dominate the health insurance industry at the state level. one intriguing aspect of the results concerns the finding that hospital services may be influenced differently depending on whether greater buyer concentration shows up in the hmo or ppo segments of the hospital services industry. it appears that increased buyer concentration from hmos puts pressure on hospitals to increase inpatient services whereas ppos may impact the amount of outpatient services. that discrepancy likely reflects that ppo enrollees use outpatient hospital facilities more often than hmo enrollees because the latter typically must obtain prior authorization from their gate-keeper primary care givers. because price data are unavailable for the test, we can not be sure if the ineffectiveness of hmos and ppos, with regard to influencing the provision of some types of hospital services like admissions or surgeries, holds because these buyers could not secure price discounts or because they were unable to affect health care provider behavior.4 or it may be the case that inpatient days and outpatient visits are more discretionary at the margin than the other types of services.

this paper uses metropolitan data to test empirically if health insurers possess monopsony or monopoly-busting power on the buyer-side of the hospital services market. according to theory, monopsony power is indicated by a fall in output, whereas, monopoly-busting power is shown by an increase in output when buyer concentration rises. the empirical results provide evidence that greater health insurer buyer concentration is not associated with monopsony power. instead, some evidence is found to suggest that higher health insurer concentration translates into increased monopoly-busting power. that is, metropolitan hospitals offer increased services when the buyer-side of the hospitals services market is more highly concentrated.

PMC2780653

pubmed-964

systemic lupus erythematosus (sle) is a systemic autoimmune disease with manifestations in multiorgans that are induced by the deposition of circulating autoantibody-autoantigen complexes (immune complexes, ic) and amplified by subsequent infiltration of different types of leukocytes promoting the inflammation. lupus nephritis (ln), a major cause of morbidity and mortality in up to 60% sle patients, is characterized by inflammation of the kidney. ic and subsequent complement activation both induce the activation and damage of renal cells that further release inflammatory factors leading to the infiltration of leukocytes into glomerular, tubulointerstitial, and perivascular regions of the inflamed kidney to amplify the renal inflammation and damage. therefore, leukocyte recruitment to the inflamed kidney is a critical step in the development of ln. chemokines are a group of cytokines with small molecular weight whose main action is the recruitment of leukocyte subsets under homeostatic and pathological conditions. through interacting with chemokine receptors that are expressed on the cell surface as 7-transmembrane proteins coupled with g-protein for signaling transduction, chemokines can induce firm adhesion of targeted cells to the endothelium and direct the movement of targeted cells to their destination according to the concentration gradient of a given chemokine. through this mechanism chemokines chemokines are classified into four subfamilies according to the first two cysteines and the amino acid residues in between at n-terminal end of the polypeptide. based on whether the first two cysteines are adjacent, separated by one residue, or separated by three residues, a chemokine is classified into ccl, cxcl, or cx3cl family, respectively. chemokine receptors are named corresponding to the subfamilies of chemokines as ccr, cxcr, cx3cr, or xcr, respectively. individual chemokines and chemokine receptors discovered to date have been reviewed elsewhere with summarized tables [58]. homeostatic chemokines and chemokine receptors are those important for the homing of progenitor cells and mature immune cells into the primary/secondary immune tissues for the development of the immune system and into peripheral nonimmune tissues for the tissue-specific functions and immune surveillance [5, 9]. examples are ccr7 that is expressed on nave lymphocytes and dendritic cells for recruitment into lymph nodes by ccl19 and ccl21 as part of the normal immune system development; cxcl12 that is important for the retention of cxcr4 hematopoietic stem cells (hscs) in hsc niches in the bone marrow; ccl2 that is critical for ccr2 monocytes emigration from the bone marrow; and cx3cl1 that is essential for cx3cr1 monocytes patrolling along the blood vessels. on the other hand, when there is an infection or injury-induced inflammation, activated immune cells will upregulate some chemokine receptors and migrate into inflamed immune and nonimmune tissues by recognizing correspondingly increased inflammatory chemokines. for example, upon the stimulation of pathogen-associated molecular pattern molecules (pamp) or danger-associated molecular pattern molecules (damp), resident mast cells, macrophages, and dendritic cells will release cytokine signals to induce the upregulation of several inflammatory chemokines expressed by activated endothelial and epithelial cells, such as ccl2, ccl3, ccl4, ccl5, cxcl1, cxcl2, cxcl3, cxcl5, and cxcl8. consequently, circulating immune cells such as neutrophils, monocytes, and effector t cells will migrate into inflamed tissues using related chemokine receptors such as ccr2, ccr1, and cxcr2. chemokines, unlike adhesion molecules broadly shared by different types of immune cells, are selectively used by specific cell populations and have been found to be involved in the migration of leukocytes to nephritic kidney of both sle patients and lupus-prone mice. studies have shown that several immune cell populations are accumulated in the kidney in ln, including various t cell subsets, b cells, plasma cells, nk cells, monocytes/macrophages, dendritic cells (dc), and neutrophils [10, 11]. in this review, we summarize recent studies on the chemokines that are increased in the kidney as ln progresses and the corresponding chemokine receptors used by renal-infiltrating leukocytes in response to the chemokines. we focus on those highlighted by multiple studies, including the chemokines cxcl13, cxcl12, cxcl9, cxcl10, cxcl11, ccl2, ccl3, ccl4, ccl5, and cx3cl1 and chemokine receptors cxcr5, cxcr4, cxcr3, ccr1, ccr2, ccr5, and cx3cr1. inflammatory factors inducing the expression of chemokines, as well as chemokines that may be used as biomarkers for the diagnosis of ln, are also discussed. some chemokines are more commonly related to ln than others based on both studies of lupus-prone mouse models and sle patients, which have been summarized in tables 1 and 2, respectively. to interact with specific chemokine receptors expressed on particular cell populations, these chemokines have diverse biological effects by influencing the migration of different cell populations in both healthy and disease situations. in ln disease, animal model studies suggest that these chemokines contribute to systemic autoimmune responses in immune tissues thus indirectly promoting ln and are involved in local renal inflammation with direct effects. evidence from human studies, on the other hand, suggests the clinical involvement of these chemokines in the development of ln. in the following sections we will discuss each of these ln-related chemokines regarding their biological effects, roles in mouse models of ln, and clinical evidence from studies of lupus nephritic patients. cxcl13, also known as b cell-attracting chemokine 1 (bca1) or b lymphocyte chemoattractant (blc), is the chemokine recognized by cxcr5. cxcl13 is important in directing the trafficking of cxcr5 cells, including b cells, follicular helper cd4 t cells (tfh), cxcr5cd8 t cells, and cxcr5 dc, all involved in humoral immune responses [1214]. both b cells and tfh critical for the formation of germinal centers (gc) depend on cxcr5 to migrate into the b cell follicles in secondary immune tissues [12, 1517]. circulating cxcr5 central or effector memory-like tfh have also been discovered that could migrate to and function in immune and nonimmune tissues [1821]. besides tfh, cxcr5cd8 t cells and cxcr5 dc are also found to facilitate b cell responses [13, 14]. therefore, by attracting different types of cxcr5 immune cells, cxcl13 contributes to b cell responses, especially the generation of high affinity antibody-producing cells in gc. in two commonly used lupus-prone mouse models, nzb/w f1 and mrl/lpr, transcript levels of renal cxcl13 and cxcr5 are consistently increased in aged lupus nephritic mice compared to nonlupus control mice or young mice prior to disease onset [2225], suggesting their involvement in the development of ln. renal macrophages and dc in lupus-prone mice may be the source of cxcl13 in the nephritic kidney [24, 2629], leading to increased migration of cxcr5 b cells and tfh-like cells into the inflamed kidney towards cxcl13 [24, 26, 30]. further studies with nzb/w f1 mice have shown that, among b cell populations, b1 cells compared to b2 cells express much higher cxcr5 and migrate towards cxcl13 more efficiently in vitro [22, 30], and preferentially migrate into the kidney and lung of diseased mice instead of lymphoid tissues. functionally, b1 cells isolated from nzb/w f1 mice, but not b2 cells, can activate t cells in allogeneic mixed lymphocyte reaction. cxcr5 cd4 t cells, on the other hand, have been shown to promote igg production from b1 cells in vitro, suggesting potential interaction of b1 and t cells in situ in the nephritic kidney. however, another study found most renal-infiltrating b cells to be non-class-switched b2 cells in nzb/w f1 mice, leaving the role of renal cxcr5 b in ln an open question. while further studies are required, these results suggest important functions of cxcr5 and its ligand in the development of ln. the critical roles of cxcl13 and cxcr5 cells in the pathogenesis of ln are also evidenced by studies of anti-cxcl13 neutralizing antibodies in mrl/lpr lupus-prone mice and cxcr5-deficiency in b6/lpr lupus-prone mice [31, 32]. renal pathology, including proteinuria and serum creatinine levels, glomerular and perivascular scores, deposition of ic and complement c3, and renal il-1, il-6, il-33, and il-17 protein levels, was significantly lower in the neutralizing antibody-treated mice than controls. systemic autoimmune responses such as the level of circulating anti-double stranded dna (anti-dsdna) antibodies and the ratio of splenic th17/treg were reduced as well, suggesting that the pathogenic role of cxcl13/cxcr5 may not be kidney-specific. as the administration of anti-cxcl13 neutralizing antibodies is not tissue-specific, it is difficult to say if reduced renal pathology is due to the secondary effect of decreased systemic autoimmune responses or the direct effect of blocking cxcl13/cxcr5 signal in the kidney. similar to the cxcl13 blockade study, cxcr5-knockout in b6/lpr mice also downregulated systemic autoimmune reactions, including reduced lymphadenopathy and splenomegaly with reduced gc, b cells, plasma cells, and double negative (dn) t cells in secondary lymphoid organs, as well as reduced circulating igg. importantly, this study also showed reduced infiltration of adoptively transferred dn t cells from cxcr5-deficient b6/lpr mice compared to wild type b6/lpr mice into the kidney of rag1 recipient mice, indicating direct contribution of cxcl13/cxcr5 signal to local renal inflammation in ln. therefore, cxcl13/cxcr5 contributes to the development of ln both systemically in immune tissues and locally in the kidney of lupus-prone mice. evidence from the studies of sle patients with ln further suggests the clinical involvement of cxcl13/cxcr5 in the development of ln. in sle patients with ln, but not in healthy controls (hc), cxcl13 and cxcr5 are highly expressed in the cortex of the kidney. in addition, b cells and tfh-like cells that express cxcr5 have been indicated to infiltrate the nephritic kidney of sle patients and are colocalized with cxcl13-expressing regions. besides chemoattractant functions, cxcl13 may also contribute to ln by activating cxcr5 renal nonimmune cells such as human podocytes to produce proinflammatory molecules. cxcl12, also known as stromal cell-derived factor 1 (sdf-1), is the ligand of chemokine receptor cxcr4. it is involved in the homing, retaining, and survival of cxcr4 hematopoietic stem cells, b cell precursors, and plasma cells in the bone marrow. in addition, cxcl12 maintains the homeostasis of neutrophils, t cells, and b cells in immune and nonimmune tissues [3638]. studies have shown possible involvement of cxcl12 and cxcr4 in ln development. in lupus-prone mouse models, cxcr4 is consistently increased in various immune cell populations including b cells, plasma cells, t cells, neutrophils, and monocytes in the circulation and spleen of diseased mice, suggesting enhanced chemotaxis of these cells towards cxcl12 [3941]. importantly, both cxcl12 and cxcr4 expressions are increased in the kidney of diseased lupus-prone mice, indicating migration of cxcr4 cells into the kidney as ln progresses [23, 24, 40, 42]. in particular, studies have shown the increased accumulation of cxcr4 cells in the kidney of diseased mice, including plasma cells, foxp3 cd4 regulatory t (treg) cells, foxp3 cd4 conventional t cells, and inflammatory monocytes and neutrophils [40, 41, 43]. besides promoting leukocyte infiltration, cxcl12/cxcr4 may also deteriorate ln by directly affecting renal tissue cells. studies have shown that activated parietal epithelial cells (pecs) as glomerular progenitor cells are involved in proliferative glomerulonephritis. normally, pecs possess regenerative potential for the repair of injured kidney [45, 46]. however, in glomerulonephritis, cxcr4 is overexpressed on pecs upon inflammatory stimulation, whereas autoantibodies and inflammatory mediators stimulate cxcl12 production on injured podocytes [42, 44, 47]. consequently, through the interaction between cxcl12 and cxcr4, pecs migrate into the glomerular tuft during the development of ln, where they predominately form hyperplastic lesions in proliferative glomerulonephritis and lead to glomerulosclerosis by secreting extracellular matrix [3, 44]. blocking the interaction of cxcl12/cxcr4 in lupus-prone mice reveals their contributions to both systemic autoimmune responses in secondary lymphoid organs and local renal inflammation. administration of anti-cxcl12 neutralizing antibodies in nzb/w f1 mice led to increased survival rate and reduced renal inflammation including decreased proteinuria and igg deposition. this may be at least partially due to decreased systemic autoimmune reactions, since circulating anti-dsdna igg and b1a subset in the peritoneal cavity and the spleen were reduced, as well as reduced activated cd4 t cells in the spleen and lymph nodes. the direct role of cxcl12/cxcr4 network to recruit immune cells in the lupus nephritic kidney is demonstrated in another study with administration of a cxcr4 antagonist in b6.sleyaa lupus-prone mice. similar to anti-cxcl12 neutralizing antibodies, cxcr4 antagonist ameliorated ln with decreased renal pathological scores and proteinuria and prolonged lifespan. early administration before severe proteinuria also led to reduced splenomegaly and circulating ana igg, suggesting a systemic effect. splenic monocytes, activated t cells, and b cells in marginal zone and follicular and germinal center were similarly reduced. however, late administration after the onset of severe proteinuria did not influence systemic autoimmune responses but led to reduced infiltration of monocytes, neutrophils, and cd4 t cells into the kidney, suggesting a direct effect of cxcl12/cxcr4 in the kidney. in patients with ln, it has been consistently demonstrated that cxcl12 expression is significantly increased in tubules and glomeruli of the kidney, while most circulating cd4 t cells and b cells express cxcr4 in sle patients [6265]. although it is debatable whether its level on b cells is reduced or increased [62, 65], cxcr4-expressing b cells are found to be accumulated in the renal biopsy samples of patients with ln, suggesting involvement of cxcl12/cxcr4 in the kidney of patients with ln. cxcr3 is a chemokine receptor interacting with three interferon-inducible chemokines, cxcl9 (monokine induced by gamma-interferon, mig), cxcl10 (interferon-induced protein of 10 kda, ip-10), and cxcl11 (interferon-inducible t cell alpha chemoattractant, i-tac). several immune cell populations have been reported to express cxcr3, including nk cells, plasmacytoid dc (pdc), conventional dc (cdc), b cells, and activated t cells. among activated t cells, t helper 1 (th1) cells and effector cd8 t cells t helper 17 (th17) cells have also been reported to express cxcr3, although ccr6 is the predominant chemokine receptor on their surface. since the expression of cxcr3 is induced upon activation of immune cells, especially in effector t cell populations, activated immune cells can migrate into inflamed peripheral tissues where cxcr3 ligands are induced. the three cxcr3 ligands under different circumstances have shown redundancy, dominance, collaboration, or antagonism to one another. therefore, cxcr3 and its ligands are mainly associated with the effector stage of immune response and are regulated in a more complex fashion than single paired chemokines/chemokine receptors. cxcr3 and its ligands are involved in the pathogenesis of sle. in lupus-prone mice, most commonly nzb/w f1 mice and mrl/lpr mice, cxcr3-expressing t cells and plasma cells as activated effector populations in the secondary lymphoid organs are increased during the development of ln [41, 48, 66, 74]. importantly, studies have shown that cxcr3 and its ligands are increased in the nephritic kidney of lupus-prone mice, suggesting migration of cxcr3-expressing effector cells from the secondary lymphoid organs into the inflamed kidney [23, 24, 49, 66, 7476]. detailed studies with mrl/lpr and nzb/w f1 mice reveal that cxcr3 is expressed on different renal-infiltrating cells with varied proportions, including cd4 t cells (1533%), cd8 t cells (1033%), b220 cells (including both b cells and pdc, 25%), plasma cells (40%), and macrophages (5%) [23, 48, 49]. all renal cxcr3 t cells are confirmed as cd44 activated t cells, while cd44 nave t cells are cxcr3-negative. in addition, renal-infiltrating cxcr3 plasma cells can secrete igg instead of igm, indicating their pathogenic role in promoting ln. while both cxcr3 and its ligands are increased in the kidney of lupus-prone mice with ln, their deficiencies in lupus-prone mice have shown inconsistent or even contradictory results. however, cxcr3- or cxcl9-deficiency in the nephrotoxic serum nephritis (nsn) model showed reduced nephritic disease with decreased igg deposits and activated t cells and macrophages in the kidney. this suggests that cxcl9 rather than cxcl10 may be critical for cxcr3-dependent cellular infiltration of the kidney in ln. consistent with this, another study showed that cxcl9 in the kidney of diseased mrl/lpr mice was the most abundant chemokine for t cell trafficking. however, circulating antigen-specific igg was also reduced in cxcr3- or cxcl9-deficient nsn model, suggesting that cxcr3/cxcl9 interaction may influence systemic immune responses and indirectly affect kidney pathology. further studies with cxcr3-knockout mrl/lpr and nzb/w f1 mice have shown different effects on the development of ln. with cxcr3-deficiency in mrl/lpr mice, glomerular pathology score was reduced with decreased t cells and macrophages infiltration around glomeruli, ameliorated renal lesion, and decreased proteinuria. ifn-producing t cells and il-17-producing t cells were also reduced in the kidney but not in the spleen or lymph nodes of cxcr3-knockout mrl/lpr mice. importantly, renal igg and c3 deposits and circulating total igg and anti-dsdna igg were not different between cxcr3-knockout and wild type mrl/lpr mice, suggesting a direct effect of cxcr3 and its ligands on the kidney by recruiting activated effector t cells and macrophages. however, in nzb/w f1 mice, cxcr3 deficiency did not change either the infiltration of plasma cells and t cells to the kidney or the course of ln. therefore, further studies are required to determine whether cxcr3 is important for ln development and which ligand(s) are critical for the infiltration of cxcr3 cells to the kidney of lupus-prone mice. despite controversial results from studies of lupus-prone mice, evidence from sle patients still suggests the possible involvement of cxcr3 and its chemokine ligands in the development of ln. patients with active sle compared to hc or patients with inactive disease have reduced cxcr3 cd4 t cells in the circulation, suggesting infiltration of the cells into peripheral tissues. in addition, several studies have shown that, in sle patients with ln, compared to hc or patients without nephritic involvement, cxcr3 cells (mostly t cells) are increased in the kidney and urine, which is correlated with increased expression of renal cxcr3 ligands [26, 66, 67]. moreover, it has been found that cxcr3 cells are accumulated in tubulointerstitial regions and around glomeruli in the kidney of lupus nephritic patients, account for 60% of total infiltrating cells, and are positively correlated with proteinuria. among the three cxcr3 ligands, cxcl10 is most increased in sle patients and localized in the same region as cxcr3 cells in the nephritic kidney. besides cxcr3-expressing t cells, a group of pathogenic cd19 b cells also express cxcr3 at a high level in sle patients and migrate towards cxcl9 in vitro, suggesting their potential to migrate into inflamed peripheral tissues such as the kidney. ccr1 and ccr5 share the same ligands, ccl3 (macrophage inflammatory protein 1-alpha, mip-1) and ccl5 (regulated upon activation, normally t-expressed, and presumably secreted, rantes). ccr1 is expressed on cd34 bone marrow progenitor cells, monocytes, nk cells, t cells, and preferentially on cd45ro activated/memory t cells [50, 77]. ccr5 is expressed on monocytes/macrophages and t cells (both cd4 and cd8 subsets), especially on th1 cells [50, 79]. interestingly, monocytes express a high level of ccr1 but low ccr5, while the expression pattern is the opposite in activated/memory t cells, suggesting selective expression of ccr1 and ccr5 in monocytes and activated t cells, respectively. ccr1 or ccr5 knockout mice have been developed to study their functions in different disease models. even though ccr1 and ccr5 share the same chemokine ligands, studies with unilateral ureteral obstruction and renal ischemia-reperfusion injury models have shown that ccr1 but not ccr5 is essential for t cells, macrophages, and neutrophils infiltration in the tubulointerstitial region of the kidney [8183]. moreover, ccr1-deficient mice have enhanced macrophage and t cell infiltration to the glomerular region of the kidney in a nephrotoxic nephritis model, suggesting that such infiltration is ccr1-independent. ccr1-deficient mice also exhibit increased circulating antigen-specific, th1-biased, pathogenic igg2a response, indicating that ccr1 is also involved in th1-dependent systemic humoral immune response. however, in a host versus graft disease (hvgd) model, ccr1-deficiency shows a protective effect by inhibiting chronic cardiac allograft rejection, which makes the role of ccr1 complicated in different diseases possibly depending on whether humoral immune responses are involved and/or which tissues and immune cell populations are involved. regarding ccr5 deficiency, macrophages from ccr5-knockout mice have reduced ability to produce inflammatory cytokines including il-6, il-1, and tnf, rendering defective bacteria clearance in a listeria monocytogenes infection model. ccr5-deficient t cells, on the other hand, have elevated production of ifn, granulocyte macrophage colony-stimulating factor (gm-csf), and il-4 with enhanced delayed-type hypersensitivity reaction and humoral immune responses following antigen challenge in ccr5-deficient mice. ccr5 also contributes to the recruitment of treg in lymphoid and nonlymphoid tissues, which is important in suppressing effector responses in graft versus host disease- (gvhd-) targeted organs. therefore, ccr5 deficiency in different diseases leads to different outcomes depending on which cell types are critical and whether the initial immune response (in lymphoid organs) or the effector phase (in nonimmune tissues) is involved. in lupus-prone mice, ccr1, ccr5, and their ligands are increased in the kidney during ln development [24, 25, 28, 4952, 54, 75, 76]. studies have shown that, in nephritic nzb/w f1 mice, both renal t cells and monocytes/macrophages have elevated ccr1 expression on the surface. in mrl/lpr mice, the administration of a ccr1 antagonist at late stage improved ln with reduced interstitial lesions including decreased infiltration of t cells and monocytes/macrophages, reduced inflammation-induced proliferating and apoptotic cells, and reduction of tubular atrophy and interstitial fibrosis. however, glomerular igg deposits and different isotypes of circulating anti-dsdna igg reflecting systemic humoral autoimmune response did not change, suggesting a direct effect of ccr1 antagonism on preventing renal infiltration of t cells and macrophages. this was confirmed by reduced renal infiltration of adoptively transferred t cells and macrophages pretreated with the ccr1 antagonist. the role of ccr1 in ln was limited in interstitial region, as glomerular injury and proteinuria were not improved by ccr1-antagonist administration in mrl/lpr mice. in nzb/w f1 mice, the effect of ccr1 antagonist administration at late stage has also been studied. besides the similar effects of ccr1 blocking t cells and macrophage infiltration, the study with nzb/w f1 mice also showed prolonged lifespan and improved glomerular injury including reduced proteinuria. in mrl/lpr mice, the extent of ccr5 expression is debated, as over 50% of renal t cells express ccr5 in one study, whereas only 1% of t cells are shown to express ccr5 in another study [49, 52]. renal-infiltrating macrophages, on the other hand, are ccr5-positive in mrl/lpr mice. contrary to the effects of ccr1 blocking, ccr5 knockout in mrl/lpr mice deteriorated ln with increased proteinuria and tubulointerstitial infiltration of total cd3 t cells and f4/80 macrophages in the kidney. foxp3 tregs were also increased in the kidney of ccr5-knockout mrl/lpr mice, but ln progression was not reversed by the increase of treg cells. systemic humoral immune responses were not affected by ccr5 deficiency, as the circulating anti-dsdna igg and renal igg/c3 deposits were not different between ccr5-knockout and wild type mrl/lpr mice. however, ccr5-knockout mrl/lpr mice exhibited increased splenomegaly and elevated circulating/renal ccl3, suggesting that renal-infiltrating immune cells may use alternative chemokine receptors responding to ccl3 such as ccr1 to migrate into the kidney and promote ln. this study reveals possible roles of ccr5 in negatively regulating ln progression by modulating ccl3 production and controlling lymphoproliferation in the spleen. therefore, it appears that ccr1 and ccr5 may, respectively, promote or attenuate the development of ln in lupus-prone mice. in sle patients, ccr1, ccr5, and their ligands are also increased in the kidney during the development of ln [69, 70, 86, 87]. evidence from sle patients further shows that most ccr1 cells infiltrating in the kidney are cd68 macrophages [63, 69], while ccr5, on the other hand, is expressed on both circulating and renal-infiltrating t cells in sle patients, particularly interstitial infiltrating t cells. ccr2 is expressed on a fraction of monocytes, dendritic cells, nk cells, and t cells, and one of its ligand is ccl2 (monocyte chemoattractant protein-1, mcp-1) [50, 88]. ccr2 expression on monocytes is important for both their egression from bone marrow and extravasation into inflamed tissues. besides chemoattractant function for monocytes, ccr2 and ccl2 t cells from ccr2-deficient mice produce less ifn upon stimulation, while ccl2 is associated with th2 cell polarization and enhances il-4 production by t cells. in lupus-prone mice, ccr2 and ccl2 are increased in the kidney during the development of ln, suggesting the recruitment of ccr2 leukocytes into the inflamed kidney by ccl2 [24, 25, 5456, 89]. using mrl/lpr mice, studies have shown that most renal-infiltrating ccr2 cells are macrophages and not t cells [52, 54]. in addition, ccl2 is mainly expressed in the tubulointerstitial regions of the kidney in lupus-prone mice [55, 89]. by blocking the interaction between ccr2 and ccl2 in mrl/lpr lupus-prone mice, studies have shown that ccl2/ccr2 network contributes to ln development through both systemic and local mechanisms. in both ccl2/ccr2 antagonist experiments and ccl2/ccr2-knockout models, animal lifespan was consistently prolonged with reduced ln including less glomerular and tubulointerstitial infiltration of t cells and macrophages, although severe proteinuria in old mice was not improved [5559]. in addition, the pathology and inflammation in the lung and skin of ccl2/ccr2-deficient mrl/lpr mice were reduced, suggesting the systemic involvement of ccl2/ccr2 in multiperipheral tissues. by further comparing the differences between antagonist and knockout models, it was evident that ccl2/ccr2 antagonists did not improve splenomegaly, lymphadenopathy, and circulating total/autoantibodies, suggesting the local involvement of ccl2/ccr2 in autoimmune target tissues, such as the kidney [56, 58, 59]. in contrast, ccl2/ccr2-knockout mrl/lpr mice exhibited reduced circulating anti-dsdna igg, diminished lymphadenopathy, and decreased percentage of circulating cd8 t cells, suggesting ccl2/ccr2 network also contributes to systemic autoimmune reactions in the immune tissues, through which ln progression was indirectly promoted [55, 57]. interestingly, anti-ccl2 spiegelmer, a ccl2 antagonist, blocked the emigration of monocytes from the bone marrow of mrl/lpr mice, which suggested an additional mechanism of how ccl2/ccr2 may promote ln by facilitating monocytes migration from the bone marrow into the kidney. together, these results suggest the importance of ccr2 and ccl2 in promoting ln. in sle patients, ccr2 and ccl2 expression same as shown in lupus-prone mice, ccl2 is mainly expressed in the tubulointerstitial region of the kidney in sle patients. renal endothelial cells, epithelial cells, and infiltrating leukocytes could be the source of ccl2. in patients with active sle, a small proportion of t cells (both cd4 and cd8) express ccr2 and are reduced in the blood circulation, suggesting their migration from the blood to inflamed peripheral tissues such as the kidney. cx3cl1, also known as fractalkine, is the only chemokine with cx3c-motif discovered to date that interacts with its unique chemokine receptor, cx3cr1. cx3cr1 is expressed on a fraction of monocytes/macrophages, dendritic cells, nk cells, t cells, and particularly cd8 cytotoxic t cells [9093]. different from other chemokines, cx3cl1 possesses a soluble form and a transmembrane form, which function to induce chemotaxis and adhesion of cx3cr1 leukocytes, respectively. cx3cr1/cx3cl1 interaction also provides antiapoptotic signals to sustain the survival of cx3cr1 leukocytes [50, 94, 95]. studies have shown possible involvement of cx3cl1 and cx3cr1 leukocytes in the development of ln. in mrl/lpr mice, cd16 cells in glomeruli are increased with lupus development, with increased protein level of cx3cl1 detectable in glomeruli, interstitial microvasculature, and arterial regions. unlike mrl/lpr mice, cx3cr1 and cx3cl1 expression in the kidney of nzb/w f1 mice do not change with lupus progression, suggesting differences between various lupus-prone mouse models [23, 24, 61, 96]. administration of nh2-terminally truncated cx3cl1 analogs blocked cx3cl1/cx3cr1 interaction and significantly ameliorates glomerular and vascular lesions in mrl/lpr mice, reducing the infiltration of macrophages and cx3cr1 cells to the glomerular, interstitial, and perivascular regions. t cells, however, were only reduced in the interstitial regions. with cx3cr1 blockade, the levels of circulating anti-dsdna igg and igg-containing ic were otherwise not affected, which, together with unchanged splenomegaly and lymphadenopathy, suggested a direct function of cx3cl1/cx3cr1 in the kidney that promotes ln progression in mrl/lpr mice. in sle patients, cx3cl1 expression is significantly increased in the glomeruli in class iv glomerulonephritis compared to other classes. in addition, glomerular cx3cl1 expression is positively correlated with the infiltration of glomerular cd16 cells that express cx3cr1, which deteriorates lupus disease, suggesting the clinical involvement of cx3cl1/cx3cr1 in ln development. aside from the t cell-related chemokine receptors discussed above, cxcr6, ccr4, and ccr6 that are associated with the recruitment of th1, th2, and th17/treg, respectively, have been also studied in sle [16, 97, 98]. cxcr6 and its ligand cxcl16 have been shown to be involved in autoimmune diseases such as rheumatoid arthritis. blockade of cxcl16 in mice also attenuates glomerulonephritis induced by antiglomerular basement membrane antibodies. in both mrl/lpr and nzb/w f1 mice, the expression of cxcr6 and cxcl16 while the lack of available blocking antibodies has hindered the investigation of the role of cxcr6/cxcl16 in ln, cxcr6 has been shown to facilitate the infiltration of activated cd8 t cells to the inflamed liver. it is thus possible that cxcr6 cd8 t cells may be recruited into the inflamed kidney in ln through a cxcl16-dependent mechanism. moreover, it has been shown that the level of soluble cxcl16 (scxcl16) in the serum of sle patients is significantly higher compared to hc and is positively correlated with sle disease activity index (sledai) of patients. in addition, the concentration of scxcl16 drops with disease remission. therefore, cxcl16 may be involved in ln development by recruiting cxcr6 t cells into the nephritic kidney. ccr4 has two chemokine ligands, ccl17 (thymus and activation-regulated chemokine, tarc) and ccl22 (macrophage-derived chemokine). the expression of renal ccr4 and its two ligands is increased in mrl/lpr mice as ln progresses. interestingly, blockade of ccl22, but not ccl17, in mrl/lpr mice led to reduced proteinuria and serum creatinine with improved renal function [56, 103]. moreover, the number of ccr4 t cells is reduced in the peripheral blood of sle patients compared to hc, suggesting increased migration of these cells into inflamed tissues. accordingly, ccr4 cells are found in the kidney of sle patients that colocalize with cd4 cells. thus, ccr4 t cells may selectively use ccl22 to migrate into the lupus nephritic kidney. ccr6 is the chemokine receptor for ccl20 (liver and activation-regulated chemokine, larc, or macrophage inflammatory protein 3, mip-3). ccr6 is expressed on t cells, preferentially on th17 and treg cells [40, 104110]. the interaction of ccr6 and ccl20 can recruit treg and th17 cells into the kidney in murine nephrotoxic nephritis [98, 111]. whether this interaction can recruit th17 cells into the kidney to promote ln, or recruit treg cells to attenuate ln, remains to be explored. in nzb/w f1 mice, the expression of ccr6 and ccl20 these results suggest that ccr6 and ccl20 may function to regulate ln by recruiting th17 and treg cells. as summarized above, in the kidney of both sle patients and lupus-prone mice, many chemokines rarely expressed at steady state are induced or significantly increased with ln progression, suggesting that local and/or systemic inflammatory factors may trigger the upregulation of these chemokines. as summarized in table 3, targeting both renal parenchymal cells and renal-infiltrating immune cells, nucleic acid-containing antigens and autoantibodies are considered to be the major inflammatory stimulators initiating and/or accelerating chemokine release in the lupus nephritic kidney. mesangial cells and other intrinsic renal cells like glomerular capillary endothelial and proximal tubular epithelial cells that express several toll-like receptors (tlrs) have the potential to be activated by different antigens to produce inflammatory factors including chemokines. polyi: c rna that mimics viral dsrna can induce mesangial cells from mrl/lpr mice to produce ccl2, whereas mesangial cells from humans can be activated by polyi: c to produce cxcl1 through the tlr3 signaling pathway [112, 113]. mesangial cells from lupus-prone mice, compared to nonlupus mice, were more sensitive to lipopolysaccharide (lps) stimulation as shown by the higher tlr4, myd88, and nfb expression and higher ccl2 production, suggesting a mechanism of how bacterial infections accelerate lupus disease. besides exogenous factors, primary mesangial cells isolated from nzb/w f1 mice, upon self-nucleosome or nucleosome-containing ic stimulation in vitro, have been shown to produce several chemokines including ccl2, ccl7, ccl20, cxcl2, and cxcl5, suggesting self-antigen and autoantibody-mediated mesangial activation. regarding autoantibody-induced mesangial activation, it has been shown that pathogenic anti-dsdna igg can upregulate cxcl1 and cx3cl1 transcripts and the secretion of cxcl1 from mesangial cells isolated from mrl/lpr mice through both fc receptor- (fcr-) dependent and independent pathways. further studies have shown that fcr-independent pathway is dependent on tlr2/4 and the receptor for advanced glycation end products (rage) but independent of dna/tlr9, as pathogenic anti-dsdna igg clone 1a3f can bind high mobility group box 1, an endogenous ligand for tlr2/4 and rage, through which 1a3f activates tlr2/rage-myd88-nfb pathway in mesangial cells, leading to the production of several chemokines including cxcl1, cxcl2, cxcl5, cxcl16, ccl7, and ccl20 [96, 116]. autoantibodies can also indirectly activate intrinsic renal cells. when incubated with immobilized igg mimicking ic deposition in the kidney, lymphocytes isolated from human pbmc can be activated in a fcr-dependent way to produce il-1 that in turn stimulates human mesangial cells, glomerular capillary endothelial cells, and proximal tubular epithelial cells to further produce ccl2. moreover, a transcription factor, fli1, has been shown to directly bind the promoter region of ccl2 and ccl5 genes to promote their expression in primary endothelial cells of the kidney in nzm2410 lupus-prone mice [118, 119]. in addition to the activation of renal parenchymal cells, renal-infiltrating macrophages and dendritic cells have been shown to produce several chemokines upon stimulation by tlr2/4, tlr3, tlr7, and tlr9 ligands. biglycan, an endogenous stimulator of tlr2/4, is increased in the serum and kidney of both sle patients and mrl/lpr mice. an in vitro study further shows that macrophages and dendritic cells produce cxcl13 upon biglycan activation of tlr2/4-ros signaling pathway that is independent of inflammasome. polyi: c rna mimicking viral dsrna and a tlr7 agonist mimicking viral ssrna can induce macrophages and dendritic cells isolated from mrl/lpr mice to produce ccl2 through the tlr3 and tlr7 signaling pathways, respectively [112, 121]. in addition, it has been shown that tlr7 and tlr9 are mostly detected in renal-infiltrating macrophages and dendritic cells rather than intrinsic renal cells of mrl/lpr mice [26, 121]. cpg, mimicking bacterial dna or self-dsdna, can induce ccl2, ccl5, and ccr5 in the kidney through the tlr9 pathway when injected into mrl/lpr mice. detailed studies have shown that exogenous cpg or bacterial dna particularly bind to infiltrating macrophages and dendritic cells in the glomerular and tubulointerstitial regions of the kidney in mrl/lpr mice. chloroquine-blocked tlr9 pathway abolishes ccl5 induction in spleen monocytes, further demonstrating tlr9-dependent chemokine induction in renal-infiltrating innate immune cells upon cpg-dna triggering. finally, mirna-125a has been shown to indirectly downregulate ccl5 production by activated t cells through targeting kruppel-like factor 13 (klf13). it has been demonstrated that mirna-125a is downregulated while klf13/ccl5 are upregulated in pbmc of sle patients compared to healthy controls, suggesting that dysregulation of ccl5 in sle patients is dependent on mirna-125a. to date, renal biopsy is still the gold standard for accurate diagnosis and classification of ln and for the prognosis of ln activity and chronicity in patients upon treatment. however, chemokines in the urine of lupus nephritic patients have been studied according to established diagnosis of ln classes that suggest their potential use as noninvasive biomarkers for ln activity monitoring, treatment responses, and remission/flare prediction after the biopsy diagnosis. urine chemokines can be used to supplement renal biopsy diagnosis of ln. in both adult and juvenile sle patients, urinary ccl2 (uccl2) concentration is significantly higher in nephritic patients than nonnephritic patients and healthy controls [123, 124]. moreover, both protein and mrna levels of uccl2 are significantly higher in sle patients with active ln compared to those with inactive ln [77, 125130]. further studies have shown that uccl2 alone or combined with other factors can distinguish different classes of ln, as uccl2 concentration is positively correlated to progressive ln classes and significantly increased in diffuse proliferative group compared to focal proliferative and mesangioproliferative groups [123, 131, 132]. ln, it is important to monitor uccl2 whose level is high during interstitial inflammation in moderate-severe sle patients. besides uccl2, urinary cxcl10 (ucxcl10) concentration is also significantly higher in nephritic patients than nonnephritic sle patients. a cut-off value 93 pg/dl of ucxcl10 has been proved to be a good prediction of nephritis with high sensitivity and specificity. also, ucxcl10 concentration is positively correlated with renal activity score and renal biopsy grade. in addition, ucxcl10 and cxcr3 mrna levels from class iv nephritic patients are increased compared to other classes. similarly, the urinary cxcl16 level can also distinguish inactive and active ln in sle patients. during the treatment of ln uccl2 has been shown to be a good biomarker to predict juvenile ln improvement. in this study, the cut-off uccl2 concentration is 343 pg/ml, with a value lower than that predicting an improved renal disease activity. two other studies have also shown that uccl2 is reduced in sle patients with complete or partial ln remission, while its level is maintained in nonremission patients, suggesting uccl2 as a good marker for prognosis [129, 132]. similarly, ucxcl10 is reduced in sle patients upon remission into inactive ln in a longitudinal follow-up study, suggesting ucxcl10 is also a good biomarker for monitoring ln improvement of sle patients following the treatment. after the remission, the elevation of uccl2 can be detected 2 to 4 months prior to another ln flare, and changes of uccl2 concentration can distinguish different levels of ln flare severity, suggesting that uccl2 may be a good marker for predicting recurring ln flares [129, 132]. as chemokines and chemokine receptors are important in the recruitment of leukocytes to the kidney in the development of ln, one would naturally think of developing new treatments for ln that target the interaction between chemokines and chemokine receptors. however, the design of such treatments should take into consideration the potential limitations (discussed below), as many commercial drugs designed to target chemokines/chemokine receptors in different diseases have unfortunately failed in clinical trials (summarized tables in references) [138, 139]. the complexity of chemokine and chemokine receptor system and possible redundancy are a challenge for the development of new drugs to block leukocyte infiltration [139, 140]. some chemokines, such as ccl5, can recognize several chemokine receptors (ccr1, ccr3, and ccr5), whereas some chemokine receptors, such as cxcr3, can interact with different chemokines (cxcl9, cxcl10, and cxcl11). current drugs including small chemical molecules and monoclonal antibodies are designed to simply block the interactions between chemokines and chemokine receptors by neutralizing either chemokines or chemokine receptors, which is insufficient to pinpoint the specific function of each chemokine/chemokine receptor pair. thus, detailed studies on the dynamic interactions and functions of each particular chemokine/chemokine receptor pair in the specific diseases are critical for successful drug development. in addition, the chemokine and chemokine receptor redundancy is reflected in a situation where one chemokine receptor may function in compensation of another if the other chemokine receptor is blocked. leukocytes always express more than one type of chemokine receptors on the surface, so blocking the ligation of one chemokine receptor may not completely or efficiently prevent the infiltration of leukocytes. for example, while both ccr5 and cxcr3 have been shown to promote organ transplantation rejection by inducing t cells infiltration in the transplanted organ, their functions seem to be redundant. ccr5 and cxcr3 double blocking compared to either single blocking makes a much greater prolonged allograft survival in a murine heterotopic heart transplantation model. another challenge for ln drug development that involves chemokines and chemokine receptors is to achieve cell-specific targeting. to this end, studies of chemokine receptor expression at the single cell level may help identify the cell type of interest. for example, renal-infiltrating t cells have been shown to express ccr1, ccr4, ccr5, cxcr3, and cxcr5 [24, 50, 67, 70, 87]. however, it is unknown whether the chemokine receptors are expressed on the same t cell subsets or differentially expressed on distinct t cell subsets. if we can define t cell subsets by using different combinations of chemokine receptors, we may be able to more specifically target pathogenic t cells by blocking the corresponding chemokine receptors. an additional limitation is the use of lupus-prone mice where most of the mechanistic studies are performed to better understand the pathogenesis of ln. the differences between human patients and mouse models make it difficult to translate the results of mouse studies to successful clinical trials. therefore, it is important to study the differences and similarities between sle patients and lupus-prone mice regarding their use of chemokines and chemokine receptors. an example is cxcl9, which is preferentially used in mice but not in humans, versus cxcl10, which appears to be the predominant chemokine in the kidney of sle patients [23, 67]. finally, how to specifically deliver chemokine-based drugs into the kidney is another important question. it is difficult to find a kidney-specific chemokine or chemokine receptor critical for the development of ln. systemically blocking a chemokine or chemokine receptor will likely lead to many outcomes other than attenuating ln, causing negative side effects such as an increased chance of infection or cancer. therefore, while targeting the interaction between chemokines and chemokine receptors is a promising avenue, further studies are required to dissect and better understand the mechanisms behind such interactions before a chemokine-based drug can be developed to treat ln. although many commercial chemokine receptor antagonists failed to reach expectations in treating different diseases, targeting chemokines/chemokine receptors may still be a promising strategy in ln. first, studies using sle patient cells/tissue and animal models summarized in this review have demonstrated the involvement of chemokines/chemokine receptors in ln progression, suggesting the potential of targeting this system in ln treatment. second, the failure of previously designed drugs is due to our insufficient understanding of the complicated chemokine/chemokine receptor system, which can be improved by further studies. third, with better understanding of chemokine/chemokine receptor system, future drugs designed to more specifically target particular chemokine and chemokine receptor interactions will minimize the off-target effects and side effects commonly observed for immunosuppressive drugs and monoclonal antibodies, which are nonspecific. finally, we may be able to learn from pathogens that are known to specifically target chemokine/chemokine receptor pairs to design better drugs with improved specificity.

lupus nephritis (ln) is a major cause of morbidity and mortality in the patients with systemic lupus erythematosus (sle), an autoimmune disease with damage to multiple organs. leukocyte recruitment into the inflamed kidney is a critical step to promote ln progression, and the chemokine/chemokine receptor system is necessary for leukocyte recruitment. in this review, we summarize recent studies on the roles of chemokines and chemokine receptors in the development of ln and discuss the potential and hurdles of developing novel, chemokine-based drugs to treat ln.

PMC4923605

pubmed-965

the treatment of hepatitis c virus (hcv) infection has evolved from peginterferon alfa (pegifn) plus ribavirin (rbv)based regimens to combinations of alloral, directacting antivirals (daa), including daclatasvir a pangenotypic ns5a inhibitor (hcv genotypes 16 in vitro) and the ns3 protease inhibitor, asunaprevir (active against hcv genotypes 1, 4, 5, and 6 in vitro). the efficacy and safety of daclatasvir and asunaprevir in combination has been assessed in multiple phase 3 studies, including the multicohort hallmarkdual study in treatmentnaive and pegifn/rbvineligible/intolerant hcv genotype 1binfected patients from 18 countries, including israel 1. the sustained virologic response rate at posttreatment week 12 (svr12) in the overall population of hallmarkdual following treatment with daclatasvir 60 mg once daily in combination with asunaprevir 100 mg twice daily administered orally for 24 weeks was 84% (542/643) and a comparable rate in patients from israel was observed (overall, 82% [9/11]; prior null responders, 3/3; pegifn/rbv ineligible/intolerant, 6/8). svr12 rates were enhanced in patients without ns5a resistanceassociated variants (ravs; l31 and/or y93) in both the overall population (92%) and patients from israel (7/8), and agree with similar findings from a pooled analysis of five studies in hcv genotype 1b infection 2. among the patients from hallmarkdual (ai447028; clinicaltrials.gov number nct01581203) who did not achieve svr12 was a cirrhotic 58yearold caucasian female from israel who had a null response to prior pegifn/rbv therapy and was ineligible for retreatment with pegifn/rbv for reasons of anemia and neutropenia. the patient had hcv rna at baseline of log10 5.43 iu/ml, no baseline ns5a or ns3 ravs shown to result in resistance to daclatasvir or asunaprevir, and 100% compliance with respect to daclatasvir and asunaprevir dosing and treatment duration. pretreatment clinical laboratory abnormalities were graded according to division of aids criteria v1.0. prior to initiating therapy with daclatasvir and asunaprevir, grade 3 elevations in total bilirubin were observed from pretreatment through ontreatment week 2, with subsequent fluctuations between grades 12 from week 4 to resolution at followup week 4. elevations in direct bilirubin were also observed from pretreatment (9.6 upper limit of normal [uln ]) that decreased or remained consistent at all ontreatment assessments (week 2, 7.0 uln; end of treatment, 3.3 uln) and at followup week 4 (3.0 uln). a pretreatment grade 2 aspartate aminotransferase elevation resolved by week 2, and a subsequent grade 1 elevation during week 4 resolved by week 6. a pretreatment grade 1 alanine aminotransferase elevation resolved prior to the initiation of therapy. alkaline phosphatase levels were elevated (grade 1) from pretreatment until followup week 4. during treatment with daclatasvir and asunaprevir, the patient experienced rapid viral decline and achieved detectable levels of hcv rna below the lower limit of quantification (25 iu/ml) at week 4. the patient then had undetectable hcv rna at week 6 that remained undetectable until relapse occurred at posttreatment week 12 (hcv rna was assessed at ontreatment weeks 1, 2, 4, 6, 8, 10, and 12; and posttreatment weeks 4, 12, and 24); hcv rna at posttreatment week 12 was log10 6.2 iu/ml (fig. 1). viral decline during initial treatment (daclatasvir plus asunaprevir) and retreatment (sofosbuvir plus simeprevir). initial treatment with daclatasvir and asunaprevir for 24 weeks; retreatment with sofosbuvir and simeprevir for 12 weeks. bl, baseline; eot, end of treatment; fu, followup week; hcv, hepatitis c virus. populationbased sequencing of the patientderived hcv ns5a region from plasma samples collected at followup week 12 revealed the emergence of signature ravs in ns5a (l31 m, y93h [with baseline polymorphisms r30q and p58s ]) and ns3 (d168v); these substitutions confer 44,000 and 357fold resistance to daclatasvir and asunaprevir in vitro, respectively. at followup week 24 (last available sample for testing), these ravs were still present. on july 15, 2014, approximately 6 months after initial treatment relapse, the patient was retreated with sofosbuvir 400 mg once daily administered in combination with simeprevir 150 mg once daily administered orally for 12 weeks. an assessment of ravs prior to retreatment was not conducted because plasma samples were not available. sofosbuvir and simeprevir is a combination therapy that is equipotent in wildtype hcv replicons representative of hcv genotype 1b and variants with signature ns5a (l31 m, y93h) and ns3 (d168v) ravs 3. retreatment baseline hcv rna was log10 5.39 iu/ml. retreatment with sofosbuvir in combination with simeprevir resulted in a rapid viral decline (detectable hcv rna below the lower limit of quantification at week 2) that resulted in undetectable hcv rna at retreatment week 4. the patient experienced an adverse event of mild rash between retreatment weeks 4 and 6 that resolved without sequelae. grade 3 elevations in total bilirubin were observed during retreatment weeks 4, 10, and 12; a grade 2 elevation was observed during postretreatment week 6. elevations above the uln of direct bilirubin were observed during treatment weeks 4 (3.9 uln), 10 (4.4 uln), 12 (3.6 uln), and postretreatment week 6 (2.7 uln). alanine and aspartate aminotransferase levels remained within normal limits through the treatment and followup periods. elevations in thyroidstimulating hormone were also observed during treatment week 4 (1.2 uln), at end of retreatment (1.4 uln), and during postretreatment week 6 (1.6 uln). overall, treatment with daclatasvir plus asunaprevir in the hallmarkdual study achieved high rates of svr12 in hcv genotype 1binfected patients without ns5a ravs and with or without ns3 ravs at baseline. this case demonstrates that in the infrequent cases of virologic failure, successful retreatment can be achieved with other alloral, daa regimens, even when the retreatment regimen contains the same class of drug. important considerations of this case report are the retreatment of a protease inhibitorexperienced patient with a second protease inhibitorcontaining regimen, the absence of rav testing prior to retreatment, and the timing of retreatment with respect to the persistence of ravs that emerged during initial treatment. current european recommendations for the retreatment of patients who have failed a regimen containing one or more secondwave daas are based on indirect evidence (hcv genotype, resistance profiles, the number of drugs administered, use of rbv, and treatment duration) and state intuitively, patients who failed on a daacontaining regimen should be retreated with an ifnfree combination including a drug with a high barrier to resistance (currently, sofosbuvir), plus one or two other drugs, ideally with no crossresistance with the drugs already administered. based on results in difficulttocure patient populations, retreatment should be for 12 weeks with ribavirin, or extended to 24 weeks with or without ribavirin (no data available comparing these approaches) 4. treatment guidelines from the american association for the study of liver disease suggest that ns5a and ns3 rav screening should be conducted prior to retreatment. it is recommended that patients without ns5a ravs should receive retreatment with ledipasvir/sofosbuvir with rbv for 24 weeks, those with ns5a ravs but without ns3 ravs receive simeprevir/sofosbuvir with rbv for 24 weeks, while sofosbuvir combined with either elbasvir/grazoprevir or paritaprevir/ritonavir/ombitasvir/dasabuvir may be efficacious in patients with ns5a and ns3 ravs (data are limited) 5. these recommendations are based, in part, on the observations that ns5a ravs are known to persist in the viral population for>1 year, whereas ns3 ravs are less stable and have been shown to be gradually replaced with pretreatment ns3 sequence over time 6. although guidelines from both the eu and usa generally do not recommend retreating protease inhibitorexperienced patients with a second protease inhibitorcontaining regimen, successful retreatment with such regimens has been reported. examples include daclatasvir/asunaprevir for the retreatment of ifn/rbv/telaprevirexperienced patients (svr, 95.5%) 7, grazoprevir/elbasvir/rbv for the retreatment of ifn/rbv plus either telaprevir, boceprevir, or simeprevirexperienced patients (svr, 96.2% [91.2% in patients with baseline ns3 ravs and 66.7% in patients with both ns3 and ns5a ravs at baseline ]) 8, and sofosbuvir/simeprevir with or without rbv for the retreatment of patients with experience of either telaprevir, boceprevir, paritaprevir, or gs9451 (svr 93%) 9. in the study of sofosbuvir/simeprevir with (n=10) or without (n=5) rbv for the treatment of protease inhibitorexperienced patients, an overall svr rate of 93% (n=14/15) was achieved after a 12week treatment duration in a patient population that was considered to have difficult to cure characteristics for this regimen (hcv genotype 1a, high baseline viral levels, and prior nonresponse to protease inhibitors). the single patient who did not achieve svr12 received sofosbuvir/simeprevir plus rbv and did not have ns3 ravs associated with resistance 9 months prior to treatment. of note, no patients had evidence of significant ns3 ravs (three patients had ns3q80k, all of whom, including one recipient of sofosbuvir/simeprevir without rbv, achieved svr12) and the majority of patients received prior protease inhibitorbased therapy>1 year prior to retreatment 9. this study and the current case report demonstrate that high rates of retreatment svr can be achieved without the use of rbv or extending retreatment duration to 24 weeks. the addition of rbv and extension of treatment duration to 24 weeks is recommended in treatment guidelines as mechanisms to increase response, and delay or prevent the emergence of ravs, particularly when low barrier daas (e.g., protease inhibitors) are used 4, 5. the studies described above show that retreatment of proteaseexperienced patients with a second protease inhibitorcontaining regimen can be successful in carefully considered cases, despite no clear guideline support for this approach. central factors in the success of this approach are the absence of significant crossresistance between the two regimens, and the timing of retreatment with respect to rav fitness and survivability. as previously noted, plasma samples prior to retreatment for rav screening were not available, and retreatment with sofosbuvir plus simeprevir occurred 7 months after the initial treatment failure with daclatasvir plus asunaprevir. this delay in retreatment may have allowed ns3 ravs to be replaced with pretreatment ns3 sequence over time 6; thus enabling a successful virologic outcome with a regimen containing the same hcv inhibitor class as asunaprevir (such as simeprevir). in hcv genotype 1b replicon in vitro assays, the presence of the ns3 d168v rav, which was detected at treatment failure, results in a 280fold loss of asunaprevir activity (ec50, 241 nm) 10 and 2591fold loss of simeprevir activity (ec50, 17,917 nm) 11, compared with wildtype hcv genotype 1b replicons; when tested in parallel using the same assay, the ec50 of asunaprevir and simeprevir in the hcv genotype 1b ns3d168v replicon were 200 nm and 5900 nm, respectively (unpublished data). the successful retreatment of this case study suggests that if ns3 ravs were present at the initiation of retreatment, the efficacy of sofosbuvir was sufficient to compensate for reduced simeprevir activity. in summary, this case study demonstrates that hcv genotype 1binfected patients experiencing virologic failure with daclatasvir plus asunaprevir therapy have potential retreatment options, including regimens containing a component with a shared mechanism of action as the initial treatment. r. safadi is an academic member at the hadassah medical organization and served as an advisory consultant of bristolmyers squibb.

key clinical messagethere is currently minimal clinical experience regarding retreatment options for patients failing directacting antiviral combination regimens. here, we report the outcomes of a hcv genotype 1binfected patient with virologic failure following treatment with daclatasvir and asunaprevir, who was successfully retreated with sofosbuvir plus simeprevir.

PMC4929798

pubmed-966

andropause, androgen decline in the aging male (adam), or testosterone deficiency syndrome (tds) is a true clinical entity characterized by symptoms of erectile dysfunction, decreased libido, osteoporosis, and general weakness. however, its occurrence is still poorly documented and most literature is from european or american continents. three questionnaires have been developed to detect tds in adult men: (1) st. louis university's adam; (2) the aging male survey (ams); and (3) massachusetts male aging study (mmas) questionnaire. we report a study in a small number of men to determine the prevalence of tds in healthy individuals above 40-years of age using the st. in order to assess by a standardized questionnaire and obtain blood sample for serum testosterone estimation in working men population, a general health check-up camp was advertised and mandated for all male workers (surgical technicians, peons, and ward assistants with regular working duration not exceeding 8 h) between 40 and 60 years of age for the departments of surgical sciences comprising of the departments of general surgery, department of urology, department of plastic surgery, department of neurosurgery, department of plastic surgery, department of pediatric surgery, and department of cardiothoracic surgery. ethical clearance for the study was obtained from the institutional ethics committee and in accordance with the declaration of helsinki. all participants were asked to fill a proforma in which the volunteers were asked to fill personal information like age, occupation, co-morbid conditions, drug intake, and past illnesses. after physical examination, some investigations like urine examination, hemogram, serum creatinine estimation, liver function tests, electrocardiogram, and ultrasonography of the abdomen were performed as part of the free camp on all volunteers. all the participants were also invited to participate in a study on andropause after signing an informed consent. serum testosterone (total and free) level estimation was performed simultaneously using elisa (drg international, inc., usa. fax: (908)233 0758, e-mail: corp@drg-international.com) in the department of pathology, the sample for which was withdrawn between 8 a.m. and 11 a.m. all tests were performed in duplicate and quality assessment was done with the help of a third party quality control sera. within-assay variability was determined and was found to be 6%. to calculate the sample size, the prevalence of tds was taken as 10%. to detect this prevalence in indian men with a precision of 5% and alfa level of 0.05, univariate analysis was done to assess the distribution of baseline variables. to assess association of tds or low-serum testosterone levels, chi-square test and t-test the androgen deficiency in aging male questionnaire: a positive answer represents yes to question 1 or question 7 or any 3 other questions of the 180 listed male workers between ages 40 and 60 years, 170 participated in the study. twenty-three volunteers had other co-morbid conditions like diabetes (12 cases), hypertension (12 cases), and tuberculosis (1 case). the mean body weight was 64.3 kg (range 5478) and the mean height was 167.4 cm (range 158180 cm). on the basis of st. louis questionnaire, of the 157 subjects, 106 (67.5%) tested positive for symptoms of tds (mean age 53.5 years; range 4060). of these 106 symptomatic cases, 41 and 32 subjects were found to have less than normal serum-free (mean serum-free testosterone 5.55 pg/ml; range 3.097.08) and total testosterone (mean serum-total testosterone 1.5 ng/ml; range 1.12.5) levels. the other 65 symptomatic subjects had normal total-and free-testosterone levels (mean age 51.8 years, range 4060). eleven asymptomatic subjects (mean age 55.1 years; range 4660) were found to have low free-serum testosterone levels (6.4 pg/ml; range 4.17.4) and in 6 of these cases low total-serum testosterone was identified (mean 1.4 ng/ml; range 1.22.5). the risk of tds was 70% significantly lower in the age group of 4050 years as compared to 5160 years (rr=0.3, 95% ci=0.10.8). the risk of tds was 2.9 times higher in those having at least one symptom as compared to those not having any symptom (rr=2.9, 95% ci=1.84.6) and this was statistically significant. the response to various questions in men with biochemically confirmed tds in shown in table 2. the association of different variables between andropause and non-andropause patients is shown in table 3 and table 4. a scatter plot for free-serum testosterone levels is shown in figure 1 and bar diagram of serum free testosterone [figure 2]. responses according to andropause/non-andropause association between different variables with andropause and non-andropause patients*significant, values in the parentheses are the percentage, rr=relative risk, ci=confidence interval mean sd of height, weight, and bmi of studied patients scatter plot of free testosterone for each patient bar diagram of serum free testosterone the massachusetts male aging study reported a crude incidence rate of 12.3 per 1000 person-years, leading to a prevalence of 481 000 new cases of adam per year in american men aged between 40 and 69 years. the prevalence of tds has not been reported from most asian countries.[24] to estimate the prevalence of this condition in india, we performed a pilot study in a small number of healthy volunteers working in the surgical departments of our hospital. the volunteers were asked to attend a free health checkup camp and before attending the camp they did not even know that they will be evaluated for tds too. however, when they reported for the checkup, they were invited to participate in tds study. in this way, we were able to avoid the bias that would have appeared had we advertised that it will be an so the chances of selection bias that volunteers with some sexual problems would attend the camp were minimized. louis university's questionnaire, the validity of which has been tested and reported previously.[57] the adam questionnaire in the original validation had a sensitivity and specificity of 88 and 60%, respectively, against the serum bioavailable testosterone levels. the sensitivity and specificity of ams has been reported to be 83 and 39%, respectively; however, in another study from europe none of the three ams domains correlated significantly with serum levels of total, bioavailable, or free testosterone in men older than 70 years. these questionnaires are useful as screening tools but fall short of expectation for diagnostic purposes. morley et al., compared the adam, ams, and mmas questionnaires using bioavailable testosterone as the biochemical gold standard for diagnosis of hypogonadism. the sensitivity for adam, ams, and mmas was found to be 97, 83, and 60%, respectively and the specificity for adam, ams, and mmas was 30, 39, and 59%, respectively. in our study, an unusually high number (67.5%) of volunteers reported symptoms of adam on the basis of st. louis university's questionnaire. this could be because of the nature of the questionnaire. the questionnaire was structured on a yes/no format and the volunteers did not have the scope of saying that they had only mild symptoms. many volunteers who had mild symptoms could have been picked up as symptomatic for androgen deficiency on the basis of this questionnaire. of interest, 58.5, 95 no answer to questions number 4, 5, 6, 8, 9, and 10, respectively. for possible explanation of this finding, a larger study in a more heterogenous group of people is required. some questions like have you noticed a recent deterioration in your ability to play sports? may not be relevant in some countries like india where most people above 40-years of age do not play sports. similarly, many of our volunteers reported that answers to questions 2, 3, 8, and 10 were quite close to each other [table 1]. some investigators have, therefore, developed shorter versions of adam questionnaire. due to the low specificity of the st. louis questionnaire, a substantial number of men tested positive for andropause symptoms based on this questionnaire but did have low-serum androgens. it is therefore likely that the population of men with symptoms of tds but who did not meet the criteria for the diagnosis had some other medical or psychological co-morbidity that was contributing to the symptoms. on the basis of biochemical evaluation, 52 subjects (33.1%) however, the total-serum testosterone levels were found to be low in 32 subjects only. this is because the normal total-serum testosterone levels have a wide normal range and do not decline as rapidly as do free and bioavailable (free and albumin-bound) testosterone.[1417] the more pronounced decrease in free than in total testosterone is explained by the age-dependent increase in the binding capacity of shbg (1.2% per year). many investigators recommend measurement of bioavailable or free testosterone as a better predictor for diagnosing andropause. the ideal test in men suspected of hypogonadism is the measurement of free testosterone by the equilibrium dialysis method. this method, however, is difficult to perform, not automated, and largely inaccessible to most clinicians. measurement of we have evaluated serum testosterone levels using the elisa, which is a reliable method. the reported prevalence of biochemical hypogonadism is about 7% of the age group less than 60 years old but increases to 20% in those over 60 years of age. our study in a small group of healthy men revealed symptoms of tds in 67.5% and biochemical hypogonadism in 33.1%. however, symptomatic hypogonadism was seen in only 26.1% (in 41 out of 157 cases). the prevalence of biochemical hypogonadism found in our study (26.1%) is similar to that found in a study of 316 canadian physicians aged 4062 years. low bioavailable testosterone levels (< 70 ng/dl) were present in 25% of these men; the questionnaire identified this group with a sensitivity of 88% and a specificity of 60%. however, our incidence is much higher than prevalence of 69.5% reported for community-dwelling men aged 4070 years from france. serum testosterone levels show diurnal variation and there is also substantial variation (~20%) from week-to-week. therefore, two testosterone measurements at least a week or two apart are recommended for diagnosing tds and starting treatment. we measured serum testosterone at a single point which may have overestimated or underestimated this condition. the relationship between symptoms of tds and hormonal levels in complex are still not fully understood and there is a lack of correlation between the clinical picture and the most commonly used biochemical confirmatory tests. additional questionnaires (ces-d, beck depression inventory, etc) for depression and additional biochemical data (like thyroid hormone levels) that may influence the response to adam questionnaire were not done. the population selected for this study was men working in hospital and is unlikely to be a representative sample of the general male population in india. another study using other validated questionnaires such as the ams scale or mmas and more detailed measurement of testosterone levels in a larger number of men is required to determine the prevalence of this condition in indian men. symptomatic hypogonadism was found to be at least as common as that reported in the western literature. the higher prevalence of patients with symptoms of hypogonadism but with normal serum testosterone found in our patients could be because of the nature of the adam questionnaire.

aim: to determine the prevalence of testosterone deficiency syndrome (tds) in healthy indian men employed in a hospital aged above 40 years. materials and methods: a general medical health check-up camp was organized for all male employees above 40 years age working in surgical departments. after clinical history and systemic inquiry, subjects were requested to fill the st. louis university's adam questionnaire based on which the total and free-serum testosterone estimation was then done. results:one hundred fifty seven healthy volunteers enrolled for the study (mean age 53.1 years; range 4060). the androgen decline in the aging male (adam) questionnaire detected 106 men (67.5%) to be symptomatic for tds. serum testosterone estimation in these subjects revealed 41/106 to have low free-serum testosterone levels and 32/106 to have low total-serum testosterone. in 11 and 6 cases, respectively, the serum free- and total-testosterone levels were found to be low although the subjects were asymptomatic for tds. conclusions:the prevalence of symptomatic biochemical hypogonadism was 26.1%. the higher prevalence of symptoms alone of tds was unusual. it could be because of the nature of the questionnaire. free-serum testosterone may be a better single test to diagnose symptomatic hypogonadism than total-serum testosterone.

PMC2710062

pubmed-967

the study was conducted at an intensive 4-day teaching program for final-year ophthalmology residents, held at hubli, karnataka state, south india, in january 2014. the questionnaire used in this survey was developed and validated in consultation with the division of research and patents of the parent institute of the chief author. the questionnaire contained 21 questions (appendix 1), eliciting information from the students about orientation and wet lab training received, operation theatre (ot) facilities for training, free surgical camps and detailed information about numbers and types of surgeries observed and performed; also, questions about what factors influenced the student to pursue ophthalmology residency, about future plans after finishing residency, and if considering fellowship training after residency, which sub-specialties were preferred. identity information like name was not solicited, and the students were free to not participate in the survey by simply not returning the form. to ensure increased response rate, the students were counseled about the objectives and importance of the survey during the training program. repeated announcements were made on each day of the training program requesting students to return completed forms. one hundred and thirteen residents participated in the survey by returning completed forms (72.9%). six respondents were found to be 1 year postgraduates (number of completed months of residency being<12) and were excluded from further analysis in the survey. the state-wise break-up of students was 76 from karnataka (71.02%), 16 maharashtra (14.95%), 8 tamil nadu (7.47%), 5 andhra pradesh (4.67%) and 2 kerala (1.86%). the results of this survey, therefore, present a picture mainly from karnataka state and southern india. twenty-four students were from a government medical college (22.42%), 38 from private medical colleges (35.51%), 22 from private eye hospitals (20.56%) and 23 from other hospitals (21.49%). residents (39.25%), 35 were dnb (32.71%) and 30 were diploma residents (28.03%). thirty-nine students (36.5%) chose got seat in the subject through entrance examination as the most important factor which influenced the decision to pursue ophthalmology residency. thirty-six students (33.6%) mentioned offers chance of combined medical and surgical practice as the most important factor. eleven students (10.3%) mentioned workload predictability/flexibility, while positive undergraduate experience made seven students (6.5%) choose ophthalmology. (5 students, 4.6%) and earning potential (2 students, 1.8%). forty-one students (38.31%) received orientation/training about ophthalmic surgery/ot. sixteen of the 41 students (39.02%) had received such orientation from their senior postgraduate/resident, 11 students (26.82%) from senior faculty and 4 students (9.75%) from junior faculty. sixty-one students (57.00%) had undergone wet lab/simulation lab training. fifty-six students (52.33%) had attended the wet lab training in their own college/hospital, and five had attended wet lab training in conferences. eighteen students (16.82%) mentioned that the ot in their center had no closed-circuit television facility for relaying surgical procedures while 37 students (34.57%) mentioned that their ot did not have a microscope with observer arm. seventy students (65.42%) did their first surgery in ophthalmic residency within the first 6 months, and 34 (31.77%) students did so before the end of the 1 year. twenty-six students (24.29%) mentioned that free ophthalmic surgical camps were not held in their center. on an average, 45 students (42.05%) went to the ot 2 times a week and 43 students (40.18%) went 3 times a week. the details of number of different types of surgeries done per week in the training center of the student, and median and range of number of surgeries done by the student so far are given in tables 1 and 2. the number of surgeries done per week in the training center of the students median and range of number of surgeries done per month at training centers and median and range of number of surgeries done by the students so far. median has been used as measure of central tendency instead of mean, as there are outlier values eighty-eight students (82.2%) mentioned join fellowship (or further training) as the future plan after finishing residency. six students (5.6%) wanted to join government service and 3 (2.8%) students wanted to join private service after finishing residency. joining medical college in the teaching profession and start own practice was chosen as the next step after residency by two students (1.8%) each. of the 88 students considering fellowship/further training, 37 students (42.5%) mentioned cataract surgery as the most preferred subject and 17 students (19.5%) mentioned cornea/anterior segment. thirteen students (14.9%) preferred vitreoretina/posterior segment and seven students (8.0%) were considering further training in general ophthalmology. glaucoma and orbit/oculoplasty were being considered by 6 (6.9%) and five students (5.7%) respectively. strabismus/pediatric ophthalmology was the first choice for two students (2.2%) while none mentioned uveitis as the preferred subject for further training. the final question of the survey regarded satisfaction of the student with the surgical training during residency. six students (5.6%) were very satisfied and 20 students (18.7%) were satisfied with their surgical training while 37 students (34.6%) found their surgical training during residency to be fair/average. totally, 29 students (27.1%) were not satisfied with their surgical training and 15 students (14.0%) rated their surgical training as poor. this survey with completed responses from 107 final-year residents provides us with a wealth of knowledge on the present status of surgical training in residency, especially in karnataka state and southern india, and on the immediate future plans of these residents. the residents surveyed in this study were from all types of training institutes in the region, including government and private medical colleges, and private eye hospitals and general hospitals. less than a quarter of the students had received formal orientation training about ophthalmic surgery from their faculty. teachers, in their personal capacity, may be teaching students and explaining surgical principles while in the ot, but a formal orientation/training before the student enters the ot will be of benefit and is also desired by the student, as documented by a survey published in indian journal of ophthalmology. ophthalmic conferences across india have been providing wet lab training for residents, but only a few students in this survey had attended one such. more students can be encouraged to undergo such training and training institutes can help improve ophthalmic surgical training by establishing wet labs in their centers. more than a third of the students were training in centers, where the ot did not have a microscope with observer arm (beam-splitter and assistant scope). the value of such equipment in the ot of a training institute would be immensely beneficial. the vast majority of training centers conducted free ophthalmic surgical camps for patients, as per this survey. most of the residents surveyed in this study went to the ot more than once a week. these can be termed as desirable statistics. another statistic which could be termed desirable would be that nearly every student performed his/her first ophthalmic surgery before the end of the 1 year of residency. this compares favorably with the results of a survey of 129 ophthalmology residency directors in the united states, which had reported that 40% of residents gained experience as primary surgeons during their 1 year and 43% performed only part of the surgery. when exactly a resident starts his first surgery is dependent on many factors, including personality, temperament and natural, surgical aptitude of the resident, as well as the trainer's considered judgment about the resident's readiness to operate. in this regard, both residents and trainers will find extremely informative kirby's excellent treatise on surgical technique for residents in ophthalmology, published in 1983, but still relevant in this era for its sheer wisdom and understanding of teaching and learning methods of ophthalmic surgical techniques. approximately a third of the respondents indicated that the total number of surgeries done per week in their center is<20. increasing the number of operations per week is as much an administrative and logistic issue as a medical issue and lies under the purview of the respective training institutions, but higher numbers would obviously be desirable for the broader experience and exposure that would be provided. in our country, conducting free ophthalmic camps for the needy can be a win-win situation for ophthalmic care-givers and patients. needy patients receive quality medical care at the hands of the teachers, which they may not otherwise be able to access, and they act as the providers of exposure and experience to the resident training under the teacher. conducting such camps will also help increase number of operations done per week in the training center. most of the students had performed manual small incision cataract surgery (msics), and this was also the surgery performed most commonly by the students table 2. an analysis of table 2 shows that the median value of students responses to performance of surgeries other than msics was zero for all surgeries excluding pterygium (5), dacryocystectomy (2) and trauma repair (5). increasing the number of noncataract surgeries done by residents questions 19 and 20 of the survey attempted to read the mind of the student regarding his/her immediate future. a huge majority planned to undergo further training, with cataract surgery and cornea/anterior segment being the most sought after subjects. it appears that most of the students would prefer to delay further the decision on practice till they are confident of their surgical skills. a quarter of the respondents in this survey were satisfied with their surgical training while a significant number, more than 40%, were not satisfied with the surgical training during their residency. a brazilian study published in 2013 on recent ophthalmology graduates reported 93.4% satisfaction with the acquisition of surgical skills during residency. improving satisfaction of residents with their surgical training should become an important goal of trainers. more than a third of the students in this survey indicated that the most important factor influencing their decision to pursue ophthalmology residency was that they were allotted to the subject through the entrance examination. a survey of junior residents in saudi arabia had found that the main factor influencing the decision to pursue ophthalmology training (97% of the respondents) was the ability to combine medicine and surgery. this meant that in our survey, a significant chunk of the students were doing residency in ophthalmology but had not come in desiring the subject specifically. if a student has come in because he had no perceived better choice, and subsequently, if he also feels that training being received is unsatisfactory, he may then find it difficult to involve himself in the subject, understand the subject and develop a liking for it. this can prevent the student from reaching his full potential, as he simply does not like what he is doing. training accomplished with modern standards with an aim to also satisfy the students expectations, positive under-graduate experience, family influence and earning potential. improving the experience of under-graduate medical students in their ophthalmology training may motivate more students to take up the field; such motivated students are also likely to strive to improve their own personal learning and satisfaction. finally, though the study achieved a fairly good response rate (72.9%), the nonresponse fraction may have an influence on the inferences and conclusions. the limitations of this study include selection bias owing to purposive sampling, which might influence the results and subsequent conclusions. ophthalmology residents in the region of karnataka and southern india are performing surgeries on a live human eye within the 1 year of residency and are entering the ot for surgery multiple times a week. further efforts from trainers can concentrate on adding to the quality of surgical training along with the quantity, by establishing more wet labs/simulation training programs, conducting formal orientation programs for residents before they start surgery and by promoting microscopes with observer arms and closed-circuit television facility. increasing the numbers of noncataract surgeries performed by residents and enhancing satisfaction levels of residents with their surgical training

settings and design: this study documents a survey of final-year ophthalmology postgraduates on the subject of their surgical training and their future plans after residency.purpose:this survey aimed to answer the question, what is the present status of surgical training in ophthalmic training centers? by obtaining information from students about (1) various methods used in surgical training (2) numbers and types of surgeries performed by them in the training centers (3) their plans after residency. materials and methods: a questionnaire containing 21 questions was distributed to 155 students attending an intensive 4-day teaching program. the questions related to orientation training, wet lab training, facilities for training, free surgical camps and detailed information about numbers and types of surgeries observed and performed. completed questionnaires were collected, and responses analyzed. results:one hundred and seven completed responses were analyzed. the majority had not received formal orientation training. more than half had undergone wet lab training. most residents performed their first ophthalmic surgery during the 1st year of residency and went to the operation theatre multiple times a week. most of the students planned to undergo further training after residency. more than half of the students found their surgical training to be fair or satisfactory. conclusions:the number and frequency of ophthalmic surgeries done by residents appear satisfactory, but further efforts from trainers on enhancing the quality and range of surgical training would benefit students and improve their satisfaction.

PMC4463553

pubmed-968

failure to achieve satisfactory weight loss has been reported in several series of morbidly obese patients who underwent bariatric procedures. the result of inferior weight loss after roux-en-y gastric bypass (rygb) in high body mass index (bmi) patients has led to the development of biliopancreatic diversion with duodenal switch (bpd/ds). the technical complexity of bpd/ds and its significant perioperative risks in the supermorbidly obese patients have resulted in a two-stage approach. in this method, a vertical sleeve gastrectomy (vsg) is initially performed, followed by a second-stage malabsorptive procedure after the initial weight loss and resolution of the obesity-related comorbidities. in the early era of minimally invasive surgery, laparoscopic vsg was proposed as a staged approach to the bpd/ds for high-risk super morbidly obese patients. in the last decade, however, laparoscopic vsg has been increasingly considered by many bariatric surgeons as definitive procedure because of its promising early and midterm outcomes. the technical simplicity and more modest learning curve of laparoscopic vsg have contributed to its rapid adoption among bariatric surgeons. as techniques in minimally invasive surgery improve, the previously high-risk bpd/ds is now performed as a single-stage operation with minimal complications and excellent outcomes. to our knowledge, there have been no reports that compare the complications and outcomes between laparoscopic vsg and laparoscopic single-stage bpd/ds. in this study, we investigated the outcomes of laparoscopic vsg as a stand-alone procedure and compare them with those of single-stage laparoscopic bpd/ds in morbidly obese patients. a prospectively maintained database of 100 consecutive patients who underwent laparoscopic vsg (group 1) and 100 consecutive patients who underwent laparoscopic bpd/ds (group 2) by two bariatric surgeons in a large independent teaching hospital was retrospectively reviewed. patient demographics (age, gender, bmi, and number of obesity-related comorbidities), intraoperative details, perioperative complications, length of hospital stay (los), weight loss outcome at 1-, 3-, 6-, 9-, 12-, 18- month intervals, and mortality were compared. major complications were defined as potentially life-threatening events that were directly related to the operation. these include anastomotic or staple line leak, hemorrhage, intestinal obstruction, inadvertent injury to other organs, venous thromboembolism, and all events that required return to the operating room. minor complications were defined as nonlife-threatening events that result in prolongation of the typical postoperative recovery course. these include superficial skin or soft tissue infection, minor incisional hematoma, urinary tract infection, or musculoskeletal problems. the standard criteria for bariatric surgery selection included bmi above 40 kg/m without comorbidities, or bmi above 35 kg/m with at least one obesity-related comorbidity. all patients underwent comprehensive preoperative medical evaluation, detailed psychological assessment, relevant laboratory, and radiologic testing, as well as esophagogastroduodenoscopy. a sleep apnea test was performed in patients with clinical suspicion of obstructive sleep apnea. all patients were counseled about other surgical options, including laparoscopic rygb and laparoscopic adjustable gastric banding. postoperatively, patients were encouraged to maintain an exercise program and regularly attend the bariatric patient-support group meetings. standard postoperative follow-up included visits to the outpatient clinic at 1-, 3-, 6-, 9-, 12-, 18- month intervals and then annually thereafter., statistical analysis was performed using student's t-test with p 0.05 considered statistically significant. patients in group 1 were older than those in group 2 (50.8 vs. 46.4 years; p<0.05), although gender distribution (female predominance), average bmi (48.8 vs. 51.9 kg/m; p>0.05), and number of obesity-related comorbidities (7.1 vs. 6.8; p>0.05) were comparable [table 1]. patients demographics one patient in each group required open conversion because of dense intraabdominal adhesions from prior laparotomy. compared to the bpd/ds group, the vsg group had a significantly shorter mean operative time (107 vs. 296.8 min, p<0.05). average blood loss was minimal (< 50 ml) in both groups, without intraoperative complications. in the bpd/ds group, one patient developed a staple line leak, which resulted in perigastric air-fluid collection 14 days postoperatively. this patient experienced significant postoperative nausea and hematemesis, which required placement of a decompressive nasogastric tube and blood transfusion. the bleeding resolved on postoperative day 2, without the need for endoscopic or surgical intervention [table 2]. 30-day perioperative outcomes and complications one patient in the vsg group was returned to the operating room for surgical hemostasis, related to a port-site hemorrhage. in the bpd/ds group, two patients were returned to the operating room, one on postoperative day 1 for an endoscopic release of a nasogastric tube that had been inadvertently sutured during the robotically-assisted creation of the duodenoileal anastomosis, and the other patient returned on postoperative day 2 because of port-site infection. the average los after vsg and bpd/ds was statistically comparable. in the vsg group, two patients had an extended hospital stay because of prosthetic heart valve complications, as well as urologic complications related to recurrent hematuria and urinary retention. in the bpd/ds group, two other patients stayed longer in the hospital because of the exacerbation of carpal tunnel syndrome and skin/soft tissue infection at one of the trocar insertion sites (9 and 13 days, respectively). in the first 3 months postoperatively, patients in the vsg group and bpd/ds group achieved a comparable percentage of excess weight loss. the weight loss, however, promptly reached statistical difference at the 6-month interval (45.4 vs. 52.4%, p<0.05), with the bpd/ds served as the more effective weight loss operation. the difference became even more significant at 9, 12, and 18 months, postoperatively [table 3]. percentage of patients who reached bmi<35 kg/m at 18-months postoperatively (final weight loss) was 60% in the vsg group and 85% in the bpd/ds group. at the end of study (18 months postoperatively), approximately 35% of vsg patients were lost to follow-up. in the bpd/ds group, it is a modification of the original biliopancreatic diversion operation described by scopinaro et al., in 1979 and the ds operation described by demeester et al., in 1987. ideally, bariatric procedure should be technically simple with acceptable low morbidity and mortality, sustained weight loss, as well as excellent resolution of obesity-related comorbidities. greater technical challenges and perioperative risks associated with the bpd/ds have led to the adoption of vsg as the initial and potentially definitive procedure for treatment of morbid obesity. patients with unsatisfactory weight loss and poor resolution of obesity-related comorbidities after vsg then proceeded with the ds during a second operation. many of the superobese and high-risk patients underwent the second stage within 2 years with improved comorbidities and surgical risk status. in contrast, patients with sufficient weight loss and excellent resolution of obesity-related comorbidities after vsg do not require the second stage of the bpd/ds. the primary goal of bariatric surgery is to find a technically simple, safe, and effective operation for the treatment of morbid obesity. when perioperative variables were compared between the two groups, the vsg group had a significantly shorter operative time, which correlated to its technical simplicity. literature showed that the operative mortality rate in the first reported laparoscopic bpd/ds series was 2.5% and specifically in a subgroup of patients with preoperative bmi greater than 60 kg/m, the mortality rate reached 6.5%. when compared to the 0% mortality rate reported by mukherjee et al., after laparoscopic vsg, bpd/ds was clearly a procedure with significant risks of death. additionally, in two large open bpd/ds series in 1993 and 2007 by marceau et al., the anastomotic leak rate was 2.7% and 3.75%, respectively. in our series of vsg and bpd/ds, however, the overall morbidity (1% staple line leak and 1% bleeding rate after bpd/ds, vs. 0% after vsg) and mortality (0% after both procedures) are significantly lower, compared with those reported in the literature. these findings suggested that in the current era of widely used minimally invasive approach, both vsg and single-stage bpd/ds can be performed laparoscopically, with a low complication rate and a relatively equal safety profile. our patients experienced excess weight loss of 18% and 31.4% at 1 and 3 months after the vsg, respectively. this early result was comparable with that achieved after the bpd/ds. at 6 months postoperatively, however, the bpd/ds group showed a significantly higher percentage of weight loss compared with the vsg. this difference may represent the role of malabsorptive component in medium and long-term weight reduction in postbariatric surgery patients. the third national health and nutrition examination survey 1999 reported that morbidly obese patients with bmi greater than 35 kg/m were found to have relative risks (rrs) of 1.97, 6.16, and 3.77 to experience heart diseases, diabetes mellitus, and hypertension, respectively, when compared to the nonobese population. in addition to a higher prevalence of obesity-related medical comorbidities, a significant increased risk of mortality was observed in patients with bmi>35 kg/m (rr=1.36, p<0.05). this information led us to conclude that although modest weight loss has been shown to improve many of the metabolic complications of obesity, greater degree of weight loss (target bmi<35 kg/m) is necessary to achieve all of the benefits of bariatric surgery. our study demonstrated that the vsg group experienced a 64% excess weight loss, while bpd/ds group experienced 79.6% excess weight loss at 18 months postoperatively. there have been different opinions among bariatric surgeons on the degree of postoperative excess weight loss to be considered adequate, but we believe that bmi should fall below 35 kg/m, in order to optimally benefit from a bariatric operation, based on the published third national health and nutrition survey data. hypothetically, if a morbidly obese male with bmi of 57 kg/m and body weight of 400 lbs were to undergo a laparoscopic vsg, his bmi would only fall to 36 kg/m at 18 months postoperatively. with this degree of weight loss (postoperative bmi still above 35 kg/m), the patient will continue to have an increased risk for developing heart disease, diabetes mellitus, and hypertension, compared to their nonobese counterparts, as mentioned above. when the same patient were to undergo laparoscopic bpd/ds, his bmi would fall to 31 kg/m postoperatively, which gives him the maximum benefit of the bariatric surgery. in this study, we found that after bpd/ds, a significantly higher percentage of patients were able to reach final bmi of<35 kg/m compared with the vsg group (85% vs. 60%, respectively). therefore, we believe a higher percentage of excess weight loss to achieve final bmi less than 35 kg/m using bpd/ds, should be sought in an appropriate clinical setting. there are several limitations in this study, such as its retrospective nature with relatively small sample size. our follow-up rate at 18 months postoperatively was only approximately 65%-70%, as described previously., where 22% of their patients were unavailable for medium-term follow-up, even in a country using socialized medical care system. laparoscopic vsg is a lower-risk and a technically simpler operation with promising weight loss outcome. the single-stage laparoscopic bpd/ds, however, produces superior weight loss with acceptable complication rates. the statistically significant difference in weight loss between the two operations became evident at 6 months postoperatively and persisted thereafter. we, therefore, recommend single-stage laparoscopic bpd/ds in patients with a high bmi, in an attempt to achieve the target bmi of less than 35 kg/m postoperatively.

background: vertical sleeve gastrectomy (vsg) was originally performed as the first-stage of biliopancreatic diversion with duodenal switch (bpd/ds) for superobesity as a strategy to reduce perioperative complications and morbidity. vsg is now considered a definitive procedure because of its technical simplicity and promising outcomes.aims:to analyze the outcomes of laparoscopic vsg and to compare them with those of single-stage laparoscopic bpd/ds. materials and methods: a retrospective review of 200 consecutive patients who underwent vsg and bpd/ds between 2008 and 2011. results:a total of 100 patients underwent laparoscopic vsg and 100 patients underwent laparoscopic bpd/ds. the patients in vsg group were older, but gender distribution and body mass index were comparable. mean operative time for vsg was significantly shorter compared with that of bpd/ds. a single patient in each groups required open conversion. staple line leak (n=1) and intraluminal hemorrhage into the newly-created sleeve (n=1) occurred in the bpd/ds group. mean length of stay was shorter after vsg (3.1 vs. 3.9 days). at 6 months postoperatively, excess weight loss between the two groups revealed statistically significant difference, favoring bpd/ds. conclusions: despite promising outcomes and technical simplicity of vsg, bpd/ds provides significantly superior excess weight loss in morbidly obese patients.

PMC3938871

pubmed-969

telomeres are specific sections of dna at the end of chromosomes comprised of tandem dna repeats (ttaggg). their biological role is to prevent dna shortening, protect chromosomes from inappropriate dna fusions, as well as dna breaks in order to maintain genomic integrity and stability (1). importantly, upon each cell division, telomere length (tl) decreases by 50200 base pairs, which together with cell aging, ultimately results in a crucial tl point which triggers chromosomal fusions and/or apoptosis (2). telomerase, consists of two subunits, an enzymatic protein component, telomerase reverse transcriptase (tert), that adds the telomeric dna repeats onto the end of chromosomes, and an telomerase rna template component (terc), that serves as a template for telomeric dna synthesis (3). due to the fact that tert is responsible for telomerase activity (ta), its expression is tightly regulated, being highly expressed only in periodically or continuously renewing tissues, such as in cells of the hematopoietic system, germ cells, the epidermis and tumors. contrary to tert, terc is widely expressed in all types of cells, but is unable to induce ta (4). the complex cell aging system regulates the longevity of cells, as well as senescence. without functional telomerase, a typical ' dividing ' cell will exhibit progressive telomere shortening, which upon reaching a ' critically ' short tl, results in telomere-dependent replicative senescence and in an inability to divide further (4). it is interesting to note that although the word ' aging ' is usually associated with old age, aging in the sense of telomeres is a life-time phenomenon that begins even before birth. age-related diseases manifest mostly in old age; however, the aging process at the cellular level can be viewed as a lifelong progression (5). importantly, premature aging phenotypes, collectively termed ' telomere syndromes ' (6), can be induced by rare mutations in human tert (htert). furthermore, htert overexpression in normal human fibroblasts has been shown to protect the cells from apoptosis and necrosis (7). nonetheless, some adverse factors which have been established to cause telomere dysfunction, telomere uncapping or other types of dna damage, such as oxidative stress, can induce premature cell senescence without the presence of critically short telomeres (8). telomerase and telomeres functions, due to their roles in apoptosis and senescence, have been examined in many aspects of reproduction biology, such as fertilization, placental development, stress and hypoxic conditions during pregnancy, as well as premature aging following intrauterine growth restriction (iugr). it is well established that female human fertility declines with increasing maternal age and that various adverse factors can contribute to aging-associated infertility in women (9). oocyte defects, such as chromosome abnormalities (aneuploidy), are a major cause of age-related decline in female fertility as they severely impair embryo implantation and development (10). numerous studies have focused on ta and its correlation with mammalian fertilization, as well as following the cleavage, and pre-implantation development processes. importantly, wright et al (11) demonstrated that ta exists in fetal, newborn, and adult testes and ovaries, but not in mature spermatozoa or oocytes. in human somatic cells grown in vitro or during replicative aging in vivo, telomeres erode to shorter and shorter lengths and continually decreasing levels of ta are detectable (12). according to liu et al (13), aberrant cleavage and increased cytofragmentation are significantly higher in homozygotic telomerase knockout (tr) eggs as compared to wild-type eggs. from both in vivo and in vitro fertilization (ivf) experiments, it appears that the absence of ta leads to telomere dysfunction, which in turn results in aberrant fertilization and the cleavage of tr gametes (13). luteinized granulosa cells (gcs) surround the oocyte and are major somatic cell components of the ovarian follicle. ta is further evidence of the stemness of normal, non-cancer cells in the ovaries (13,14). successful maturation, fertilization and pre-implantation embryonic development depends on a regulated programme of oocyte growth and differentiation coordinated with the development and differentiation of the surrounding gcs (15). importantly, it has been demonstrated that tert is expressed by gcs at all stages of ovarian follicle development (16,17). however, tert mrna expression and ta in gcs have been found to decrease with age and basal serum follicle stimulating hormone levels (18). indeed, the low level of ta in the human ovaries was found to be related to the age-related primordial follicle depletion and it was suggested that ta may be used as a marker of the ovarian functional age (19). importantly, studies have demonstrated that oocyte development is related to the ta of peripherally residing gcs. gcs play an important role in the maturation of oocytes and are closely associatd with their reproductive quality (20,21). interestingly, ta was found to be highest in the gcs of the small preantral follicles, and to decrease subsequently through different stages of antral development (22). moreover, it was shown that the relative tl was longer in gcs from mature oocytes compared with gcs from immature oocytes in humans (23). upon measuring ta in human gcs obtained from ivf and intracytoplasmic sperm injection cycles, it was demonstrated that the rates of oocyte maturation and good-quality embryo generation increased in a ta level-dependent manner (24). indeed, the same authors postulated that women with a high level of ta had a greater likelihood of becoming pregnant than those with non-detectable or low levels of ta (24). along the same lines, the lack of ta in gcs is associated with occult ovarian insufficiency (25). finally, in gcs obtained from the same individuals, it was shown that ta predicts ivf treatment outcomes better than tl (26). importantly, tl in human eggs was found to predict cytoplasmic fragmentation in embryos suggesting that telomere shortening induces apoptosis in human prei-mplantation embryos, well in accordance with a telomere theory of reproductive senescence in women (27). this data collectively suggest ta/tl of luteinized gcs is positively correlated with clinical pregnancy rate and, indicate that ta of ovarian luteinized gcs could help health workers to predict the clinical outcomes of ivf treatment. during normal pregnancies, a decrease in ta activity when comparing the early gestation period (< 10 weeks), with the late gestation period (> 10 weeks), is observed (28). thus, placenta and chorionic villi specimens from the first trimester of gestation exhibit significantly more ta than placenta and chorionic villi specimens from the second and third trimester during gestation (29,30). specifically, a high ta was identified in trophoblast cell fractions of the chorion, suggesting that this fraction is a major source of ta activity (31). additionally, the expression of htert was observed in chorions from early stages of gestation, but not in the placenta at the late stages of gestation, with a close correlation between ta and htert expression (31). iugr is a common pregnancy complication, which can be defined as the failure of the fetus to reach the size for which it is genetically programmed. delayed growth puts the fetus at risk during pregnancy and is associated with adverse outcomes during delivery and to neonate health problems. these may include a low birth weight, difficulty in handling the stress of vaginal delivery, decreased oxygen levels, hypoglycemia, low resistance to infection, low apgar scores (a test given immediately after birth to evaluate the newborn's physical condition and determine the need for special medical care), breathing problems associated with meconium aspiration (inhalation of stools passed while in the uterus), difficulties in maintaining body temperature, an abnormally high red blood cell count, long-term growth problems, and in the most severe cases, iugr can lead to still birth (33). interestingly, only weak ta was detected in the placenta and chorionic villi specimens from women with pregnancies complicated by iugr (34,35). indeed, htert mrna expression was detected in the placenta and chorionic villi specimens during the first, second and third trimester of gestation in normal pregnancies, whereas copy numbers of htert were significantly lower in placenta specimens from women with pregnancies complicated by iugr (21). the expression of hterc was observed in chorions obtained during early and late gestation and was not linked to ta (31). on the other hand (36), an analysis of the terc telomerase subunit gene copy number in placentas from pregnancies complicated by iugr revealed that the terc gene copy number was decreased in trophoblasts in placentas from women with pregnancies complicated by iugr, and it was thus suggested that this may promote senescence in trophoblasts in placentas from pregnancies complicated by iugr. in accordance with these data, a decreased tl was observed in placenta samples collected from women with pregnancies complicated by iugr (37). furthermore, a decreased ta in placentas from women with pregnancies complicated by iugr was associated with increased apoptosis (34). twins affected by growth discordance exhibit significant differences in their growth rate and size even though they develop in the same intrauterine environment (38). importantly, birth weight discordance amongst twins is closely correlated to perinatal morbidity and mortality (39). thus, specimens from placental tissues from twins affected by growth discordance (> 20% weight difference) or not (< 20% weight difference) were collected after birth, and ta was analyzed (40). the results revealed that in the growth discordant group, ta was significantly higher in the larger twin than in the smaller twin (40). oxygen is a necessity for life; yet, it is toxic to cells when dysregulated. thus, both high and low levels of oxygen are deleterious to developing embryos. in normal pregnancies, in early gestation, the fetus and placenta exist in a relatively hypoxic environment with an ambient po2<20 mm hg (41). the low oxygen tension favors cell proliferation and angiogenesis in the placenta, whereas it simultaneously supports vasculogenesis, hematopoiesis and chondrogenesis of the developing fetus (42,43). upon establishing intervillous circulation at approximately 1012 weeks of gestation, oxygen tension rises to 4080 mmhg and remains in this range throughout the second and third trimesters. in order to support normal placental function and fetal development, the placenta adapts to the alterations in oxygen levels by the modulation of hypoxia inducible factor-1 (hif-1) expression and by increasing cellular antioxidant defenses (44). thus, restricted oxygen availability is normal and necessary in utero; however, excessive fetal hypoxia leads to adverse outcomes, including fetal death, iugr and low birth weight (45). indeed, intrauterine hypoxia may be due to a variety of causes, including prolapse or occlusion of the umbilical cord, placental infarction, hypertension, anemia, pulmonary disease, preeclampsia, as well as maternal smoking (46). importantly, intrauterine hypoxia can cause cellular damage within the central nervous system and is associated with a reduced total brain volume and altered cortical volume and structure, a decrease in the total number of cells and myelination deficits (47). this results in an increased mortality rate, including an increased risk of sudden infant death syndrome (sids). moreover, oxygen deprivation in the fetus and neonate have been implicated as either a primary or as a contributing risk factor in numerous neurological and neuropsychiatric disorders such as epilepsy, attention deficit hyperactivity disorder (adhd), eating disorders and cerebral palsy, as well as in the later development of neurodegenerative diseases (48). hif-1 is a transcription factor of major importance in the cellular response to oxygen deficiency with specific roles during embryogenesis (49). hif-1 is a heterodimeric complex composed of the two basic helix-loop-helix pas domain (bhlh-pas) subunits hif-1 and hif-1 (50). the hif-1 gene is constitutively transcribed under hypoxic conditions and determines hif-1 biological activity (51), whereas the hif-1 subunit dimerizes with several different bhlh-pas proteins and is continuously expressed (50). hif-1 targets genes that encode proteins which regulate oxygen homeostasis and are critical for developmental and physiological processes in hypoxic environments (51). indeed, hif-1 activity is critical for normal fetal development, as it has been shown that murine embryos lacking functional hif-1 die on/or before e10.5 (52). an important issue to resolve is the existence of a correlation between hif-1 and ta. indeed, hif-1 expression was detectable, as demonstrated by western blot analysis, in first trimester placenta samples, but not in placental samples from the second and third trimester (53,54). accordingly, htert protein expression was increased in placental tissues before 10 weeks than in placental tissues after 10 weeks of gestation (54). in vitro studies thus, tl distribution in huvecs under hypoxic conditions seems to be regulated by a balance between telomere attrition by hypoxia and telomere elongation by enhanced ta acting on telomeres (55). indeed, coussens et al (56) demonstrated that hif-1 affects telomerase regulation in murine embryological stem cells and these authors suggested that hif-1a may have a physiologically relevant role in the maintenance of functional levels of telomerase in stem cells (56). furthermore, it was demonstrated that the htert promoter region contains two hif-1 consensus motifs, which are essential for htert transactivation by hif-1. the introduction of an antisense oligonucleotide for hif-1 diminishes htert expression during hypoxia, indicating that the upregulation of htert under hypoxic conditions is directly mediated through hif-1 (54). thus, nishi et al (54) suggested that the regulation of htert promoter activity by hif-1 represents a mechanism for trophoblast growth during hypoxia according to the previous data; there are two possibilities about the association between hypoxia induction and ta. dna damage in the telomere region can be induced by hypoxia, which would result in hif-1-induced telomerase expression in order to heal the damaged chromosome ends; or an anti-apoptotic response may be triggered by the hypoxic induction of telomerase (5658). a rapidly growing body of empirical evidence suggests that a major burden of disease can be traced back to the intrauterine period of life. importantly the sensitive biological functions of fetal cell proliferation, differentiation and maturation respond to, or are affected by conditions in the internal or external environment. the result of these responses are structural and/or functional changes in cells, tissues and organ systems that have important long-term consequences for subsequent health and disease susceptibility (59). exposure to psychosocial stress and/or biological stress mediators during gestation has been identified as a condition that may modulate the long-term programming effects of the intrauterine environment (60). indeed, exposure to psychosocial stress during gestation appears to be an important risk factor for the earlier onset of complex, common age-related diseases (60). studies on humans have demonstrated links between chronic or excessive psychosocial stress exposure and telomere biology (5,61,62). these data suggest that stress-related changes in telomere integrity may be a possible mechanism linking psychosocial stress and age-related disease (63). indeed, accelerated telomere shortening reflects stress-related oxidative damage to cells and increased aging (64). substantial evidence supports the hypothesis that depression creates abnormalities in stress-related biological outcomes. thus, individuals with mood disorders have a significantly shorter tl compared with stable individuals, representing as much as 10 years of accelerated aging (64). furthermore, it has been demonstrated that exposure to a major stress hormone, such as cortisol, is associated with the downregulation of ta in activated human t lymphocytes (65). this is highly relevant, as leukocyte tl is a predictor of age-related disease onset and mortality (66). there is also the question of whether cellular aging is related to patterns of allostasis (66). telomeric dna quantity, dna damage and heat shock protein gene expression have been used as physiological stress markers in chickens (67). exposure to maternal psychological stress during intrauterine life appears to induce not only adverse birth and neonatal outcomes, but also subsequent health and disease risk-related outcomes over the lifespan of an individual. the relevant outcomes include metabolic, endocrine, immune and cognitive processes (5). one important question that has yet to be addressed is whether exposure to stress during intrauterine development can produce variations in tl, thereby potentially setting up a long-term trajectory at birth that defines or contributes to individual susceptibility for complex and common age-related diseases. entringer et al (59) examined the tl in leukocytes of individuals whose mothers had experienced a high level of psychological stress during pregnancy. these authors demonstrated that exposure to maternal psychosocial stress during intrauterine life was associated with a significantly shorter tl in young adulthood (59). additionally, it has been suggested that maternal psychological stress during pregnancy may exert a ' programming ' effect on the developing telomere biology system that is already apparent at birth, as reflected by the setting of newborn tl (5). the association between ta and maternal stress exposure during pregnancy, has not yet been investigated and may shed further light on telomere biology. indeed, it was postulated by shalev et al (5) that a ' better understanding of the mechanisms that govern and regulate telomere biology throughout the lifespan may inform our understanding of etiology and the long-term consequences of stress and mental illnesses on aging processes in diverse populations and settings '. importantly, nutrition is a key factor supporting normal pregnancy and refers to the consumption of nutrients and the diet of the mother before, during and after pregnancy. thus, the nutritional status of the pregnant women is important as early as conception, and continues to be important throughout gestation and after birth during breastfeeding. it has been demonstrated that the nutrition of the mother is crucial as it may have an effect on the future health of the child; poor nutririon during pregrancy may lead to health complications in the later life of the child, and may lead to the development of cancer, cardiovascular disease, hypertension and diabetes (68). an inadequate or excessive amount of certain nutrients may lead to malformations or medical problems in the fetus and in the life of the developint child. indeed, neurological disorders and handicaps of the fetus are more common in pregnant women who are malnourished (69). as 23.8% of babies worldwide are estimated to be born with lower than optimal weights at birth due to the lack of proper and sufficient nutrition, malnutrition poses a major risk to the health of the fetus (70). in particular, personal habits, such as smoking, excessive alcohol and caffeine consumption, the use of certain medications and illegal drugs can negatively and irreversibly affect the development of the fetus, and these negative effects can take place during the early stages of pregnancy. specifically, a diet low in protein during gestation has no effect on placental weight, but results in a decreased weight of the offspring (72). importantly, malnutrition in female rats has been shown to increase the production of the superoxide free radical (o2) (73). it is well established that oxidative damage is a major feature of the aging process and can lead to telomere shortening (74), which is associated with cellular senescence in vitro and in vivo (75,76). these data lead to the conclusion that increased cell apoptosis in the offspring can be caused by maternal protein restriction (75). importantly, maternal diet was found to influence tl in the rat offspring (77,78). thus, the offspring of mothers who consumed a diet low in protein during gestation had significantly shorter telomeres compared to the controls. in addition, maternal protein restriction during lactation increased longevity and reduced renal telomere shortening compared with offspring that were maternally protein-restricted in utero and then suckled by normally fed dams (79). the nutritional programming of coenzyme q was found to be relevant to aortic tl in rats with different gestational regimes of this coenzyme (80). the functions of telomerase and telomeres have been examinedin many aspects of reproduction biology, such as fertilization, placental development, stress and hypoxia conditions during pregnancy, as well as premature aging following iugr. importantly, varying ta activity has been shown during the progression of normal pregnancies; ta activity being significantly higher in the early as compared to the late gestation period. it is noteworthy that under pregnancy-related pathological conditions, ta is decreased or absent, which results in significantly shorter telomeres. in depth studies on telomere biology during reproduction can improve our understanding of the significance of telomerase in fetal development and lifelong consequences on illnesses and aging processes in different populations. this understanding may lead to better prevention policies and may perhaps reveal novel therapeutic strategies.

telomeres are specific dna regions positioned at the ends of chromosomes and composed of functional non-coding repeats. upon cell division, the telomeres decrease in length by a preordained amount. when the telomeres become critically short, cells lose the ability to divide and enter a specific functioning mode designated as ' cellular senescence '. however, human tissues express an enzyme that deters the shrinking of the telomeres, the telomerase. due to its ability to maintain telomere length, the telomerase slows down and possibly suspends the aging of the cells. in regard to this, solid evidence demonstrates that female human fertility decreases with increased maternal age and that various adverse factors, including alterations in telomerase activity, can contribute to age-associated infertility in women. the fact that telomerase activity is regulated in a time- and location-dependent manner in both embryo and placental tissues, highlights it potential importance to the successful completion of pregnancy. since maternal age is a crucial determining factor for the success of in vitro and in vivo fertilization, numerous studies have focused on telomerase activity and its correlation with mammalian fertilization, as well as the following cleavage and pre-implantation developmental processes. associations between telomerase activity and pregnancy complications have been previously observed. our aim in this review was to summarize and critically discuss evidence correlating telomerase activity with pregnancy complications.

PMC4918539

pubmed-970

progressive neurologic deterioration or back and leg pain in children with a history of myelomeningocele (mmc) surgery may be due to cord tethering or growth of an intradural inclusion tumor. loss of motor function in the lower extremities, gait abnormality, back or leg pain, scoliosis, and foot deformity are common findings in postsurgical tethering. here, we report a child who developed progressive neurological deterioration and severe back and leg pain subsequent to large infected dermoid tumor extending underneath the skin to the spinal canal and intramedullary area. a 3-year-old boy was referred to neurosurgical department because of severe back and leg pain and motor regression. he was a known case of lumbar mmc and was operated at 8 months of age. he could sit at 11 months of age and stand at age of 2 years on his knees, but could not move the distal part of his legs at all. his back pain had started 3 months ago, which was severe at night and made him unable to sit. subsequently, he experienced severe leg pain, which prevented him from moving the proximal part of his lower extremities and his mother faced difficulty while cleaning him after urination or defecation. on physical examination, the child was afebrile, but very irritable, with good mental performance, and normal neurological examination of upper limbs. both lower extremities had lost any movement in proximal and distal parts (0/5). a small tender bulging was identified in his back under the mmc repair incision for one week before admission, but no dermal sinus was found inside or around the incision. spinal magnetic resonance imaging (mri) was performed one month before surgery, which revealed cord tethering (cord at l4- l5level). distal cord was attached to a mass, which was isointense in t1- and hypointense in t2-weighted images. it was extending from distal cord through the bony defect to the extraspinal canal space. the cord was dilated from the level of tethering to l1level with a lesion, which was isointense to hypointense in t1- and hyperintense in t2-weighted images, dissimilar to the intensity of cerebrospinal fluid [figures 1 and 2]. notice the hypointense signal inside the expanded cord compatible with infected dermoid tumor found during surgery hypointense mass in t2-weighted image is extending from distal cord through the bone defect to the extraspinal canal space and the prior mmc surgery field the child underwent surgery with the suspicion of tethered cord associated with an inclusion tumor. in the prone position, the prior midline vertical incision was opened. during the dissection of subcutaneous tissue, dura mater, cord and dermoid tumor were attached to each other at the depth of previous surgical field, and the tumor was extending from the extraspinal area to the intramedullary space between l4 and l1 vertebral levels. microsurgical release of all intradural adhesions was performed to achieve circumferential untethering of spinal cord associated with gross total resection of intramedullary mass. complete excision of the dermoid cyst and capsule were performed, which was followed by primary closure of the dura. on histopathological examination, specimen had the characteristic features of a dermoid cyst, with simple squamous epithelium, hair follicles, and cholesterol-containing debris. he could move proximal part of his lower extremities with score of 1-2/5, but was unable to stand on his knee as he could do before. children with mmc are born with a wide range of neurological deficits that will remain after primary repair of lesion. any new changes in the previous neurological, urological or orthopedics status warrant an extensive investigation to rule out the possible causes, especially tethered cord syndrome. tethered cord is a common complication following mmc surgery, which may be found in most patients when they are evaluated with spinal mri, but symptomatic tethering occurs only in 10-30% of them. tethered cord may present with subtle changes in prior neurophysical situation, which become progressive and disabling if neglected. spinal cord tethering due to dermoid cyst subsequent to mmc surgery has been reported so far, which can occur decades after closure.[58] all patients with symptomatic tethered cord have attachment of spinal cord to the prior closure site, which can be associated with inclusion tumors in 16% of instances. dermoid tumors following mmc surgery may result from inadequate excision of dermal elements during dissection of placode or dura mater from the skin or with implantation of dermal elements inside the repair site., this tumor can be an associated abnormality with mmc, which was undiagnosed or neglected because of very small size at the time of primary repair, especially during surgery without magnification. cases of dermoid tumor in mmc children who were operated during the intrauterine period have been reported, suggesting a higher potential for rapid growth of this tumor in lesions that were closed in utero rather than after birth. dermal sinus tract as a rare congenital abnormality is accompanied by inclusion tumors in 43% of instances, wherein most tumors are dermoids. intramedullary spinal abscess without sinus tract is extremely rare, but a devastating condition in children.[1216] intramedullary abscess and dermoid tumor without dermal sinus have been reported before; however, not following mmc repair and especially not associated with tethered cord. in order to explain infected dermoid tumor in this patient with prior mmc repair, we propose several explanations implantation of bacteria during closure of mmc sac can lead to abscess formation but occurence three years after the first surgery seems to be unusual. microscopic sinus tract through the preceding mmc surgery incision can be a way for bacteria to reach the subcutaneous space. hematogenous superinfection of the dermoid cyst during repeated urinary tract infections in mmc patient is another possiblity for abscess formation in this case. dermoid tumors, even infrequent, should be considered in the differential diagnosis of the causes of neurological deterioration in patients with a history of mmc repair. along with tethered cord syndrome, dermoid tumor can complicate the prior neurological and physical status of the child, and should be searched vigilantly during evaluation of these patients. because of rapid growth of dermoid tumors and associated infection, these can deteriorate the previous neurological status in a more aggressive and destructive manner as compared to tethered cord per se. awareness of such pathology in patients with previous history of myelomeningocele repair, early diagnosis and surgical intervention can provide better result with less disability. tethered cord syndrome should be investigated in any patient with a history of mmc repair if one finds neurological, orthopedics, or urological deterioration. dermoid tumor is considered as an important differential diagnosis during thorough evaluation of the causes of worsening. in order to minimize the risk of dermoid tumor development following mmc surgery, any residual epidermal or dermal elements the operative field should be explored accurately proximal and distal to the sac to find any associated anomaly or minute inclusion tumor and prevent late inclusion tumor formation and tethered cord.

intramedulary dermoid tumors are rare tumors mostly found associated with dermal sinus tracts. spinal dermoid tumor can occur after myelomeningocele repair. infected dermoid tumors are reported in patients with dermal sinus tracts as well, but have never been reported subsequent to myelomeningocele surgery. here, we report a rare association of infected dermoid tumor with tethered cord without dermal sinus tract in a child who had been operated for myelomeningocele during infancy.

PMC3401659

pubmed-971

radiotherapy has been proven to successfully treat local and regional neoplasic lesions but it may adversely impact on normal tissues. high vulnerability to irradiation was already documented in various bone tissues (pelvis, sternum, vertebra, clavicle, femoral head, and mandible) with subsequent deleterious effect on the bone metabolism and healing leading thereafter to infection, atrophy, pathological fractures, and osteoradionecrosis. for instance, the incidence of osteoradionecrosis after conventional radiotherapy ranges from 0.9% to 35%, with an increased risk when doses given to the mandible exceed 60 gy. thus, irradiation of the mandible represents the most devastating radiotherapy-induced complication and might sometimes lead to surgical resection. since vascular ischemia is one of predictors of postirradiation degeneration, the inception of angiogenesis by implantation of bone marrow mesenchymal stem cells (bmscs) might represent a therapeutic approach for rehabilitating the irradiated bone tissue. such potentiality was already documented in diverse ischemic pathologies such as hindlimb ischemia or myocardial infarction [7, 8]. previous data regarding the role of bmscs in the bone reconstruction have outlined their active contribution in the bone formation when seeded on various scaffolds [9, 10]. in a dog model of mandible segmental defect, the feasibility of bone reconstruction using morphologic and 3-d beta-tricalcium phosphate scaffold seeded with autologous bmscs was highlighted by both bone formation and bone vascularization. experiments with bmscs in the treatment or the prevention of radio-induced damage were reported on intestine [11, 12] and skin [1315] using systemic [1416] or local [11, 13] delivery. little is known however about the effect of bmscs in irradiated bone tissue, and especially, the bioavailability and biodistribution of these cells within the targeted areas since their in vivo monitoring is now mandatory to further understand their benefice. the study was designed to explore, in a rat model of hindlimb irradiation, the feasibility of rehabilitating irradiated tibial bone tissue by intramedullary implanted bmscs. the assessment of bmscs ' retention and distribution were conducted up to 7 days following transplantation using in-oxine-labeling technique. therapeutic effect on bone perfusion and metabolism this study was conducted in 14 wistar rats (initial body-weight of 410 g460 g). all experimental procedures were in accordance with our local ethical committee and with the regulations of the animal welfare act of the national institutes of health guide for the care and use of laboratory animals (nih publication no. three months after experiencing a hindlimb irradiation with a monodose of 30 gy a tc-hdp scintigraphy was performed. thereafter, animals were referred into 2 groups: a control sham-operated group (n=6) and a treated group (n=8) in which in-labelled bmscs (2 10 cells) were intramedullary injected in irradiated tibial diaphysis; bmscs being harvested before irradiation were cultured until passage 4, and their mesenchymal phenotypes were evidenced by flow cytometry. to evaluate changes in bone blood flow and metabolism, serial tc-hdp planar scintigraphy was scheduled at 3 months after irradiation and at 2 months after the cell therapy. the early cell retention after the cell therapy was assessed by additional dual recordings of tc/in activities done at 2 hours, 48 hours, and 168 hours after the cell injection. briefly, the animals were placed in prone position upon a thick polystyrene phantom and their hindlimb was immobilized by adhesive tape. the focus skin distance was 70 cm, and the field size was 20 30 cm. the collimating block was positioned on a 0.5 cm thick acrylic platform to shield the body and only irradiated the exposition of the left hindlimb without the pelvis. radiation with co was delivered in a vertical beam from a theratron 780c x-ray machine delivering gamma rays of 1.25 mev energy and dose rate of 1.4 gy/min. after the intravenous injection of 9 mci of tc-hdp and under general anesthesia, the acquisition was recorded using a single-head gamma camera (sopha dsx, smv-ge) equipped with a 1.5 mm pinhole collimator (165 mm focal length, 44 mm radius of rotation) and with the following parameters: 256 256 matrix, 1.14 zoom, and 140 (20%) kev energy window. two acquisitions were performed: a dynamic hdp uptake (blood flow) consisted of images obtained at 1 second intervals for 60 seconds reflecting vascularity and a delayed (3 hours after) acquisition of hdp uptake reflecting osteoblastic metabolism. changes in accumulation of the tracer in irradiated bone and surrounding tissues were evaluated by measuring uptake within regions of interest (roi) on the computer-processed images software (dysplay, console vision, general electric). planar scintigraphic images of the body distribution of in-labeled bmscs were provided by the same single-head gamma camera (sopha dsx, smv-ge) already described [8, 19]. two 20% energy windows centered on the 172 kev and 246 kev photopeaks of in were applied. the initial image was recorded 2 h after cell transplantation during a 15-min period and then at day 2 (48 h) and day 7 (168 h) during time periods of 20 and 40 min, respectively. in activity from each image was expressed relative to the total injected activity (total body activity determined at 2 h) and after additional corrections for the physical decay of in (2.9 days). autologous bone marrow cells, harvested from the left tibias by punction, were cultured and expanded as previously described in detail elsewhere [19, 20]. briefly, aspired whole bone marrow cells were suspended in iscove's modified dulbecco's culture medium (life technologies, cergy pontoise, france) containing 10% fetal bovine serum (life technologies, cergy pontoise, france), 0.1 mmol/l mercaptoethanol (sigma, france), 100 u/ml penicillin, and 100 g/ml streptomycin. the cells were grown in a 5% humidified co2 atmosphere at 37c, and the medium was changed every 2 days. to ascertain the mesenchymal phenotype of transplanted bmscs, the expression of cd34, cd44, cd45, and cd90 surface antigens of cells prior to implantation (passage 4) was analysed using flow cytometry method (facscalibur; becton dickinson, meylan, france) and the cellquest software (becton dickinson, meylan, france). as already described [7, 8], bmscs (2 10 cells/ml) were trypsinised and incubated at 37c with 15 mbq of in-oxine (mallinckrodt medical b.v., holland) during a 10-min period, the labelling process being stopped by 5-min centrifugation at 950 g. this 10-min incubation period was previously found to result in both a sufficiently high labeling efficiency (69%) and absence of significant deterioration of cell viability (96%). after stab incision, a 1 mm diameter drill hole was performed perpendicularly to the orientation of the tibial cortical bone. the in-labelled cells were conditioned in a 1 ml syringe (2 10 cells in 50 l) and were injected through the mini-invasive perforation into the bone marrow of the left tibia. to prevent leakage of transplanted cells in the surrounding tissues a biocompatible bandage (irm dentsply 78467 konstanz germany) the statistical analysis was carried out with the statistical package spss version 14.0 (spss, inc., we used mann-whitney tests for the unpaired comparisons and wilcoxon tests for the paired comparisons in each group. for each test, a p value<0.05 was considered to be indicative of a significant difference. the 30-gy irradiation induced 3-4 weeks later a persisting alopecia in the irradiated hindlimb (figure 1(a)) without affecting however the daily locomotor activities of those animals. at 2-mo scintigraphic imaging, radiation-induced bone defects appear as areas of attenuation of signal intensity covering the irradiated lower limb, with pronounced effect in the tibia (see figure 1(b), e.g.,). the pretherapeutic data of the group control and the cell-treated group were resumed in the table 1. in both groups, compared with the total body activity, irradiation of the hindlimb produced similar alteration in tibial values of bone perfusion blood flow (early uptake of tc-hdp) and bone osteoblastic metabolism (late uptake of tc-hdp). for example, bone perfusion blood flow was 3.2 0.8% at the irradiated tibia compared to 3.8 1.0% found in the healthy one (p<0.05). a slight decrease in bone metabolism of circa 10% was found in irradiated tibias, but values did not reach statistical significance (2.0 0.3% versus 2.3 0.6% found in healthy counterparts). flow cytometry analyses (figure 2(a)) showed that the engrafted bmscs of passage 4 expressed strong expression of cd44 and cd90 surface antigens (> 80%). thus, these cells were negative for cd45 and cd34 (percentage of positive cells were 2.41 2.47% for cd45 and 1.99 2.72% for cd34). these data were consistent with our previous studies and in accordance with criteria defined by the international society for cellular therapy (isct). 99mtc-hdp scintigraphic examinations performed after intramedullary implantation of bmscs have documented, especially in the tibial area, a significant rise in both bone blood flow and bone metabolism during the posttherapeutic first week (table 2 and figure 5). at 48 hours, the bone blood flow found in cell-treated tibias was 4.7 0.7% corresponding to an enhancement of 62% compared to basal pretherapeutic values (p<0.01). similarly, the bone metabolism was 35% higher than that measured before treatment, values were 2.7 0.5% (p<0.01 versus pretherapeutic data). these effects persisted at 7 days, bone blood flow was 4.5 1.0% (p<0.01 versus pretherapeutic data), and bone metabolism was 2.6 0.6% (p<0.05 versus pretherapeutic data). at 2-mo followup, these uptake values were found to be down to 3.1 1.4% for the bone blood flow and 1.7 0.3% for the bone metabolism. damage of normal tissue secondary to radiotherapy is still a major problem in cancer treatment. stem cell therapy seems to be a new treatment option in radio-induced tissue abnormalities [2224], providing a mean to reduce related side effects and to improve the quality of life of patients. in this study, we investigated the feasibility of bmscs when injected intramedullary in an experimental rat model of radio-induced degeneration recently described by our group. in the present study, in-oxine labelling of bmscs was successfully used to follow bone retention and body distribution of bmscs when injected intramedullary within irradiated bone. in-labelled cells have been widely used in humans in localizing areas of inflammation by imaging the leukocyte distribution. furthermore, in-labelling techniques have been applied in various experimental settings in animal to analyse the migration of dendritic cells, the biodistribution of transplanted hepatocytes, and of injected mscs in animals model of heart or lung disease [7, 28]. as previously described, the technique used here reached a high efficiency (69%) with a low toxicity (viability>95%). in addition, it has been previously demonstrated that the leakage of in from labelled cells resulted in a high in uptake in the liver and spleen and usually had hepatobiliary and renal excretion pathways [7, 29]. this is in accordance with our observations, and no in radioactivity was found in bones outside the area of injection. approximately 70% of grafted cells could be estimated to be retained within bone damaged area 2 hours after transplantation. the disappearance of radiolabeled grafted cell may be explained by the method used for bmscs injection which could be associated with a leakage of bmscs from the injection site before bandage and residual bmscs in the injection syringes. these data are fully consistent with those of the study of tran et al., where approximately 60% of in labeled bmscs were still present 2 hours after direct transplantation in a necrotic rat myocardium and were retained within the heart throughout the 7 days of followup. in the present study, after 48 hours, bmscs number decreased to approximately 40% and remained unchanged until the 7th day. the mechanism responsible for cell loss during the first two days remains to be explored. these results highlighted that at short term, the engrafted bmscs remain localized within the area of injection into irradiated bone. many studies of cell therapy using mesenchymal stem cells [14, 16], used the systemic injection as modality of administration. in comparison, using local injection, cells engraftment seems to be better. for example, in franois et al. 's study, rats were transplanted with a dose of 5 10 bmscs 24 hours after irradiation of the hindlimb at a single dose of 26.5 gy. fifteen days later, the implantation rates of bmscs in bone and bone marrow were, respectively, approximately 12.5% and less than 0.25%. the major limitation of this approach is constituted by the very low number of bmscs that home to the site of injury. a possible reason for the inefficient engraftment and homing could be the entrapment of bmscs in the lungs moreover, vascular ischemia and fibrosis, characteristic injury of irradiated tissue [1, 32], may prevent homing and engraftment of bmscs. after cell therapy, the bone blood flow and bone metabolism evolved similarly, and a significant increase of these values was observed during the seven days following the bmscs engraftment. the influence of the surgical procedure used in the present study would require further investigation, especially regarding the role of the inflammation cells response and the local recruitment of mesenchymal stem cells that should have been induced by the wound healing after drilling the cortical bone. however, the benefit obtained seems to be transient since 2 months after cell therapy, blood flow time and bone uptake of tc-hdp did not differ significantly from data measured in ungrafted animals irradiated at 30 gy. this result slightly differs from those achieved in our previous study, in which autologous fat was used as source of mesenchymal stem cell and grafted within irradiated area, inducing clinical benefit that appeared to be linked to the improvement of vascular network and disappearance of necrotic area. additional results demonstrating the potency of bmscs therapy in irradiated tissues were recently reported describing a case of regenerative reconstruction of a terminal stage of osteoradionecrosis with bmscs and progenitor cells. another explanation that stands for the short effect of engrafted bmscs might be related to the native hypoxic microenvironment of the medullar area target of the bone. the bmscs used here were expanded according to most of the conventional cell culture procedures, that is, in normoxic condition (21% o2). although they have mesenchymal characteristics, recent works from our group and others have suggested that bmsc, when cultured under 5% o2 rather than under 21% o2, had better growth advantage in long-term cell expansion. thus, the hypoxic bmsc expressed more adhesion and extracellular matrix molecules and displayed more plasticity features. it is then possible that different in vitro conditions during the cell selection and expansion might lower their ability to repair when reimplanted in native environment. in conclusion, the present study shows the feasibility of the intramedullary implantation of bmscs in the attempt to rehabilitate the irradiated bone. our data suggested that bmscs appear to remain around the injection site, without evident body redistribution, for at least a 7-day period along with a transient benefice on bone blood flow and bone metabolism.

background. we aimed to explore (i) the short-term retention of intramedullary implanted mesenchymal stem cells bmscs and (ii) their impact on the bone blood flow and metabolism in a rat model of hindlimb irradiation. methods. three months after 30 gy irradiation, fourteen animals were referred into 2 groups: a sham-operated group (n=6) and a treated group (n=8) in which 111in-labelled bmscs (2 106 cells) were injected in irradiated tibias. bone blood flow and metabolism were assessed by serial 99mtc-hdp scintigraphy and 1-wk cell retention by recordings of 99mtc/111 in activities. results. the amount of intramedullary implanted bmscs was of 70% at 2 h, 40% at 48 h, and 38% at 168 h. bone blood flow and bone metabolism were significantly increased during the first week after cell transplantation, but these effects were found to reduce at 2-mo followup. conclusion. short-term cell retention produced concomitant enhancement in irradiated bone blood flow and metabolism.

PMC3163406

pubmed-972

recent advances in obstetrical and neonatal intensive care management have increased the survival rates of very low birth weight infants. in these infants, artificial ventilation is related to potential iatrogenic lung damage and therefore reduced to a minimum. the introduction of new strategies of surfactant therapy includes a very short period of mechanical ventilation (insure) or even avoidance of endotracheal intubation in newborns with respiratory distress syndrome. as a consequence, noninvasive respiratory therapy has become increasingly important and is used in even the youngest neonates. in these infants, apnoea has emerged as a major clinical problem, manifested by an unstable respiratory rhythm reflecting the immaturity of the respiratory control systems. methylxanthines, such as theophylline and caffeine, are the mainstay pharmacological treatment for apnoea and have proven to reduce chronic lung disease and long-term outcome [5, 6]. in line with current international consensus in the 2 reporting nicus, caffeine base is given with a loading dose of 10 mg/kg and a maintenance dose of 5 mg/kg/day. published very reassuring data [7, 8] on short-and long-term outcome of high dose caffeine, more data are needed to change the existing caffeine dosing protocol. if apnoea persists under methylxanthines, doxapram may be considered as the therapy of last resort before endotracheal intubation and mechanical ventilation. doxapram is a respiratory stimulant that encourages breathing by stimulating both peripheral and central chemoreceptors [911]. doxapram was introduced more than 25 years ago into neonatology to treat neonates struggling with persistent idiopathic apnoea of prematurity [12, 13]. some randomized controlled trials studied doxapram, but none were placebo controlled [1719]. a number of observational trials suggested potential short-and long-term side effects such as hypertension and irritability. the most devastating, however, were the suggested negative effects of doxapram on cerebral oxygenation and long-term mental development [2022]. these concerns about long-term outcome and important improvements of neonatal care such as the introduction of surfactant therapy and maternal corticosteroids pushed the use of doxapram in preterm infants to the background. nevertheless, we recently noticed a reintroduction of doxapram use in our departments in premature infants with superficial breathing patterns and apnoeas that are unresponsive to methylxanthines. in this situation of persistent apnoea and imminent neonatal respiratory insufficiency, doxapram is used as rescue medication. doxapram is administered in order to prevent preterm neonates from endotracheal intubation and mechanical ventilation. however, doxapram is still off-label in premature infants and evidence about efficacy and safety is lacking. we therefore performed an audit on doxapram use and the effects of doxapram use in two dutch tertiary nicus during the last 5 years. due to the retrospective nature of our audit, the committee of medical ethics of lumc and the medical ethical committee of vu university medical center have declared that formal approval was not required. data of patients who received doxapram were analyzed from two tertiary neonatal intensive care units (leiden university medical center, leiden, center 1; vu university medical center amsterdam, center 2) in the netherlands. from prospectively collected drug databases all eligible patients from 2006 till 2011 were selected in both centers. data on total numbers of patients admitted per gestational age group in both centers were also collected. in both centers, neonates were treated with caffeine base formulations. a loading dose of 10 mg/kg was given, with daily subsequent doses of 5 caffeine was occasionally switched to theophylline with a dose of 68 mg/kg/day. although promising data regarding higher daily caffeine doses were published in 2004 and 2011, both nicus chose to use standard dosing regimens [7, 8]. the doxapram treatment policies of both centers were different during the study period. in center 1, intravenous and oral doxapram were used until 2011 and dosed based on personal experience of the attending physician. during doxapram treatment,, doxapram was incorporated into a written apnoea policy from 2010. before 2010, it was used and dosed based on personal experience of the attending physician. the written policy advised intravenous use of doxapram with a starting dose of 0.5 mg/kg/hr that could be increased every the protocol advised to consider the use of doxapram after persistent apnoea with maximal noninvasive ventilatory support and caffeine therapy. data about pregnancy, delivery, and baseline patient characteristics were analysed in order to determine which group of patients received doxapram. the use of doxapram was evaluated by its duration, dosage, route of administration and efficacy. doxapram therapy was defined as successful if no endotracheal intubation due to apnoea was necessary. no criteria for respiratory insufficiency or endotracheal intubation were included in the protocols of both centers. outcome of patients was analyzed by collecting data about mortality, short-term morbidity (incidence of necrotizing enterocolitis, intraventricular hemorrhage, periventricular leukomalacia, retinopathy of prematurity, and persistent ductus arteriosus) and pulmonary condition. background characteristics; doxapram data and morbidity of patients who were successfully and unsuccessfully treated with doxapram were compared using chi-square and fisher's exact tests as well as non-parametric mann-whitney u statistical tests wherever appropriate. from 2005 to 2010, 122 out of 1,501 admitted premature infants with a gestational age less than 32 weeks in the two centers received doxapram (8.13%). all neonates received caffeine with a loading dose of 10 mg/kg and daily maintenance doses of 5 mg/kg. until 2010, doxapram was most often used in the youngest neonates with 26.7%, 21.4%, and 26.6% of all preterms born after 24, 25, and 26 weeks of gestation, respectively. doxapram was more frequently used in center 1 (84/802; 10.5%) than in center 2 (38/699; 5.4%). there was a wide variation in the doxapram doses used and in the duration of therapy (table 2). median postconceptional age at the start of doxapram therapy was 27.3 weeks with the youngest infant being 24 4/7 weeks and the oldest being 31 3/7 weeks at the start of doxapram therapy. doxapram was initiated at a median postnatal age of 13.5 days (iqr 11 days) and patients received doxapram for a median duration of 108 hrs (iqr 147 hours). nine patients (7.4%) received a doxapram loading dose (median 2.5 mg/kg). in all other patients, the cumulative dose of doxapram ranged from 0.33 mg to 781.5 mg doxapram per kg bodyweight. twenty-nine newborns (24%) received 2 courses of doxapram; 2 patients (1,6%) received a third course of doxapram therapy. outcomes of the patients who received doxapram are shown in table 3. in 6 cases, it could not be determined if doxapram was successful, as it was unclear if the patient was intubated because of persistent apnoea or because of other reasons, such as infection. in 79 out of 116 neonates (64.8%), baseline characteristics were comparable between patients in whom doxapram was successful compared to those with unsuccessful treatment (table 1). if doxapram treatment was successful, it was used longer (median (iqr): 5.5 (5.7) days versus 1 (4.65) day; mann-whitney u 722, p<0.001) but in lower average dosages (median (iqr): 0.97 (0.62) mg/kg/hr versus 1.38 (0.94), mann-whitney u 952, p<0.003). patients who received doxapram therapy for a long period of time, incidence of necrotizing enterocolitis, intraventricular hemorrhage, periventricular leukomalacia, retinopathy of prematurity, persistent ductus arteriosus, or worsening of pulmonary condition did not increase. their stay at the neonatal intensive care unit was shorter (median (iqr): 33 (26) versus 42 (34) days; mann-whitney u 815, p=0.002). off all the included patients, 49.2% received doxapram intravenously, 41.5% received doxapram orally and 9.3% received doxapram via both routes. if intravenous doxapram was effective and the neonates did not need an intravenous route anymore, doxapram was given through the nasogastric tube in center 1 only. doxapram therapy was successful in 67% of intravenously treated and 70% of orally treated neonates. if both routes were used subsequently, the success rate was 55% (chi-square test: p=0.63). almost two third of all treated newborns did not progress to ventilatory support although they were suffering from frequent apnoea before treatment with doxapram. these data suggest a potential important role for doxapram in the treatment of apnoea of prematurity. however, it is remarkable that underlying evidence about the efficacy and safety from well-designed clinical trials is almost completely lacking. for 50 years, doxapram has been used as an emergency agent in adults suffering from life-threatening respiratory insufficiency. it was the anesthesiologist gupta who first reported the successful administration of doxapram in newborns with breathing difficulties after birth and from the 1980s on doxapram became popular for the treatment of neonates struggling with persistent idiopathic apnoea of prematurity [12, 13]. in the past 30 years, only three randomized controlled trials compared doxapram with other therapeutic strategies in a total of 86 neonates. peliowski and finer compared doxapram with theophylline and placebo in a crossover design in 31 preterm newborns with apnoea. they found a short-term doxapram treatment success rate of 64 percent in neonates with a mean gestation of 31 weeks. eyal et al. found success rates of, respectively, 53 and 55 percent comparing doxapram monotherapy with aminophylline monotherapy. if doxapram was used in addition to aminophylline, it was successful in 8 out of 10 patients compared to 0 out of 9 placebo treated newborns. studied the effects of doxapram after extubation and found a reintubation rate of 21% compared to 47% of placebo treated newborns. in analogy with our data, they suggested that doxapram is successful in treating apnoea of prematurity in at least a considerable part of the premature neonates. a wide variation in used doxapram dosages varying from 0.18 up to 4 milligram per kg bodyweight per hour was found in the current study. as suggested by barrington et al., most patients started with a dose of 0.5 mg/kg/hr. the same study also showed increasing success rates of doxapram with increasing dosages up to 2.5 mg/kg/hr. a dose effect relationship might be apparent but has until now not been studied sufficiently. a prospective doxapram dose finding study and randomised placebo controlled analysis of the effectiveness of doxapram with a sufficient number of patients is needed to determine the actual efficacy and effective dosages of doxapram on apnoea in premature newborns. as this was not a randomized controlled trial, hard conclusions can not be drawn. however, retrospectively, we found positive results in almost 65% of infants treated with doxapram. response to doxapram therapy seemed to be associated with a shorter stay in the neonatal intensive care unit without serious side effects. protecting newborns from reintubation and ventilation might theoretically protect them against bronchopulmonary dysplasia but the doxapram responders merely depict the neonates who initially had less severe lung problems. the most important and potentially dangerous suggested side effect consists of decreased cerebral oxygenation and blood flow velocity. this might result in decreased cerebral perfusion and damage to the developing brain leading to long-term developmental delay [20, 21]. as our study was not a randomised controlled trial doxapram is given to a specific category of preterm neonates with pulmonary problems, therefore no representative control could be found in the rest of the patients on our wards during the study period. routine follow-up of the doxapram treated premature infants has not shown long-term safety issues at this time. the reintroduction of doxapram can be explained by the introduction of new techniques aimed at minimizing invasive ventilation of premature neonates. techniques such as insure, cpap, nasal intermittent mandatory ventilation, and surfactant without intubation may ultimately lead to an increased use of doxapram as pointed out by kribs et al.. surfactant administration through a nasogastric tube resulted in doxapram use in 28 percent of the surfactant treated neonates via a nasogastric tube compared to 17 percent of patients receiving standard surfactant therapy. in the coming years therefore, this study shows that doxapram as off-label drug is frequently used in premature neonates in neonatal nurseries and especially in the youngest infants. as increased use of doxapram is to be expected, prospective well-designed studies are needed to address the issues of short- and long-term safety. until then, doxapram should be used with caution.

apnoea of prematurity is treated with noninvasive respiratory therapy and methylxanthines. for therapy unresponsive apnoea doxapram is often prescibed in preterm neonates. the duration, dosage and route of administration of doxapram together with its efficacy was evaluated in two dutch neonatal intensive care. outcome concerning short-term safety and neonatal morbidity were evaluated. during 5 years, 122 of 1,501 admitted newborns<32 weeks of gestational age received doxapram. 64.8% of patients did not need intubation after doxapram. 25% of treated neonates were<27 weeks of gestation. a positive response to doxapram therapy on apnoea was associated with longer duration of doxapram usage (p<0.001), lower mean doses (p<0.003), and less days of intensive care (median 33 versus 42 days; p<0.002). no patients died during doxapram therapy. incidence of necrotizing enterocolitis, intraventricular hemorrhage, periventricular leukomalacia, retinopathy of prematurity, persistent ductus arteriosus, or worsening of pulmonary condition did not increase during doxapram therapy. doxapram is frequently used for apnoea of prematurity, despite a lack of data on short-term efficacy and long-term safety. until efficacy and safety are confirmed in prospective trials, doxapram should be used with caution.

PMC3860126

pubmed-973

hemophagocytic lymphohistiocytosis (hlh) secondary to infectious disease is an important entity especially in tropics where infectious diseases are rampant and still pose a major threat. a timely diagnosis and prompt treatment can improve the clinical outcome of this otherwise potentially fatal condition! this article aims to alert the clinicians that in persistent unresolved fever especially in tropics, a diagnosis of secondary hlh should be given due consideration and we present 6 cases in this paper. all patients presented between march 2012 and march 2013 (details shown in table 1) and fulfilled the revised criteria of hlh as listed in table 2. of them, the mean age at diagnosis was 33.83 years (range: 20 to 64 years), with a male: female ratio of 2:1. three patients presented with evidence of hepatomegaly and/or splenomegaly. all of the patients had at least a bi- or trilineage cytopenia, elevated liver enzymes and hyperferritinemia. two of the cases were secondary to dengue fever and one secondary to disseminated tuberculosis, one secondary to pulmonary tuberculosis, one secondary to malaria (falciparum) and one secondary to leptospirosis. all patients had good recovery and none of them relapsed at a median follow up of 4 months. characteristics of secondary hlh patients m male; f female, y yes; n no; csa cyclosporine; na not available proposed hlh diagnostic criteria, 2009 ravenous macrophage-hemophagocytosis of rbcs by macrophage seen hlh is a hyper-inflammatory condition which may be familial or occur secondary to autoimmune diseases or infection, malignancy or other triggers. despite advances in the diagnostic work up of febrile patients, hlh remains elusive from the diagnostic capabilities of many clinicians and continues to be a potentially fatal disease entity. the underlying pathophysiology of the disease constitutes an unrestrained immune activation with defective macrophage function regulation. an excessive activation of macrophages leads to a cytokine storm in the host and leads to host tissue damage and organ dysfunction associated with the syndrome. excessive pro-inflammatory or defective anti-inflammatory responses leading to this cytokine storm can be triggered by host factors or environmental agents. accordingly, a hlh arising in the setting of an underlying genetic mutation is termed familial hlh, in the setting of an underlying rheumatologic disease like rheumatoid arthritis is termed as macrophage activation syndrome (mas) and in the setting of an underlying infection it is termed as reactive or secondary hemophagocytic syndrome (hps) or secondary hlh. case reports describing mas post bone marrow transplant in patients with juvenile rheumatoid arthritis, or secondary to sle or dermatomyositis or other autoimmune diseases have been reported. macrophage and neutrophil activation is a hallmark in conditions like stills disease which can lead to hyperinflammatory condition with hlh. overproduction of proinflammatory cytokines, uncontrolled activation of t cells, and macrophages associated with decreased natural killer cell and cytotoxic cell functions seem to be the hallmarks of the immunologic abnormalities in mas. recent human and murine investigations suggest that all hpss should be differentiated based on etiology and pathogenesis as treatment strategies for each may vary. however, all etiologies lead to a state of hyper ferritin levels. the precise mechanism of ferritin as a trigger or a bystander in pathogenesis needs to be explored. the reactive or infection-associated hlh remains a relatively important and yet unfortunately an underdiagnosed entity especially in the tropical world. various combinations of high grade fever sometimes a second spike of fever after a brief period of recovery which coincides with fresh cytopenias, unresponsiveness to broad-spectrum antibiotics, new onset organomegaly or sudden increase in size of organomegaly in the setting of an infectious disease are some of the diagnostic clues for this disease. in case of infections, a simple blood investigation that shows elevated levels of serum ferritin should raise the suspicion of a coexisting hlh. a tentative diagnosis of hlh for initiation of immunosuppressive therapy can be done when clinical and lab abnormalities exist as defined in revised 2009 hlh protocol. also, in the resource poor settings, a single value of ferritin more than 10,000 in the absence of iron overload conditions like hemochromatosis and thalassemia syndromes can act as a surrogate marker for hlh with a sensitivity of 90% and specificity of 96%. hps secondary to infections has been classified as a separate entity under international classification of diseases by world health organization. other viruses like dengue, herpes, cmv, hiv have been reported to have hps secondary to them. secondary hps has frequently been associated with intracellular pathogens that stimulate th1 immune response. of the bacterial infections, other reports with organisms like salmonella, leptospirosis, malaria, toxoplasmosis, leishmenia, rickettsia and other organisms have been postulated in secondary hlh. although case reports and case series have frequently reported the reactive hlh secondary to infectious causes especially in the tropical countries, it continues to remain as an under diagnosed and under-reported entity. the treatment of secondary hlh includes aggressive treatment of underlying condition along with immunosuppressive therapy. however, recent data suggest to a less intense immunosuppressive therapy. as opposed to the familial hlh, where allogenic stem cell transplant is the only curative treatment, most of the infection associated hlh cases respond to a course of corticosteroids. some patients may need additional treatment with drugs like etoposide and cyclosporine; however, a full course of hlh 2004 protocol is rarely required in them. in our case series, all patients were initiated with dexamethasone at a dose of 10 mg/m/day. three patients with hlh secondary to underlying tuberculosis, malaria and leptospirosis [case 2, 5, 6 in table 1] each responded to steroid monotherapy. within 2 days of starting steroids their fever reduced with improvement in cytopenias. one patient with dengue fever had a ferritin values more than 100,000 along with severe pancytopenia. she was given two doses of etoposide iv at a dose of 100 mg/m (reduced dose) at weekly intervals. other two patients were given one single dose of etoposide following which they became symptomatically better. one patient with dengue and one with tuberculosis however had delayed recovery of platelet count and they were started on oral cyclosporine. csa was given for duration of three months following which it was tapered and stopped. this is in tune with the other case reports and case series of secondary hlh. one possible explanation for this is removal of inciting agent by means of effective antibacterial therapy. because of its powerful proapoptotic activity describe their unique experience with stem cell transplant in treating secondary hlh in an adolescent. srinivas et al. in their systemic review of hemophagocytosis syndrome (hps) in tropics found infectious trigger as the cause in 51% of the adult patients. leishmenia was seen in 40.6%, rickettsia in 18.8% malaria in 15.6% and enteric fever in 9.4%. 56% patients were secondary to viruses, 26% secondary to dengue virus, 17.3% were secondary to ebv and 8.7% each to cmv and parvovirus b 19. most of the literature on hlh in tropics is centered on few hospitals and includes case reports and case series. larger studies and trials are required to throw more light on this potentially fatal condition and unfold the mysteries of ravenous macrophages. in india, hlh associated with dengue fever and malaria with a high parasite index has been documented. in one study from india, the dengue virus has been found to be the most common agent causing hlh in children. many of the previous case reports of hlh are reported in complicated dengue fever like dengue hemorrhagic syndrome. crohn's disease and immunosuppression are associated with an increased risk for developing secondary hlh, although none of our patients had underlying disease or immunosuppressed status .. however, cases with classical dengue syndrome have also been reported from indian subcontinent. in our series also, we report two cases of classical dengue fever with secondary hlh. our cases add to the existing literature of handful of cases of hlh in dengue. although more common in tropics a case of elderly female has been described by cdc, usa in which hlh secondary to possible dengue infection proved fatal and physicians in west need to be alerted about possible travel acquired dengue which can have fatal complications like secondary hlh. describe the time-lines of six cases of confirmed dengue with varying severities of hemophagocytosis. both our patients presented with fever, pancytopenia, organomegaly, high ferritin and fulfilled the criteria of hlh and responded to corticosteroids and etoposide. hlh secondary to malaria was reported in a young man (case number 5) who had persistent fever, falling counts despite treatment with antimalarials. had described one of the first cases of hemophagocytosis secondary to malaria (falciparum) which resolved with antimalarials. park et al. discusses four case reports of hlh secondary to vivax malaria all of which resolved with antimalarials. however, studies in the pediatric population show degree of parasitemia is associated with severity of disease. similarly our case was secondary to p falciparum malaria with severe parasitemia seen on peripheral smear. the patient responded to corticosteroids .. hlh secondary to disseminated tuberculosis has been described in past. pristilla et al. in their review analyzed 36 cases of tuberculosis with secondary hlh. they found fever to be the most common presenting symptom and evidence of extra-pulmonary tuberculosis was found in 83% of cases. in our series, case number 2 a young boy with fever, weight loss, military tuberculosis and choroid tubercles was diagnosed as a case of disseminated tuberculosis. no significant improvement with anti tubercular treatment prompted us to look for other causes and was diagnosed as secondary hlh based on clinical features, high ferritin and bone marrow evidence of hemophagocytosis. steroids were added to his treatment course and he showed significant improvement within 2 weeks. similarly an elderly female was diagnosed to have hlh secondary to pulmonary tuberculosis and was started on steroids. however, she did not improve and was further treated with iv etoposide and oral cyclosporine and showed complete remission of symptoms and evidence of hemophagocytosis on follow up after 4 week. this is consistent with earlier studies which showed negative tuberculin test should never preclude the possibility of overwhelming tubercular infection in hlh. leptospirosis is a spirochete which is commonly prevalent in coastal belt of south india. to the best of our knowledge, no case of hlh secondary to this disease in adult has been reported. the diagnostic challenge in making appropriate diagnosis has been discussed in one of the previous case reports from taiwan in a young male who presented with shock and had evidence of reactive hemophagocytosis. our patient was a fisherman from the endemic area who presented with fever and oliguria. in due course he developed hepatosplenomegaly, severe cytopenia, esr of 5, high ferritin and bone marrow evidence of hemophagocytosis. in conclusion with limited experience and lack of guidelines for treatment of tropical hlh, a high index of suspicion is something clinicians should bear in mind .

hemophagocytic lymphohistiocytosis (hlh) is a potentially fatal hyper inflammatory condition, if not recognized and treated in time. a high index of suspicion can help identify the condition early. this condition can occur in the primary or secondary form. secondary hlh or hemophagocytic syndrome (hps) secondary to infections is an important clinical entity especially in tropical world. in this article, we share our experience with this entity and make an attempt to explore literature about ravenous macrophages which occurs secondary to infections. it is a series of six cases of hlh secondary to infectious disease in our center in a coastal city in south india over last one year with follow up.

PMC4943449

pubmed-974

the management of patients with coagulopathic disorders undergoing orthopaedic surgery requires a dedicated, multi-disciplinary team with detailed perioperative planning. bernard-soulier syndrome (bss) is an extremely rare disorder, affecting 1 in 1 million individuals worldwide. it is caused by a deficiency in glycoprotein 1b-v-ix which is required for normal platelet-mediated clot formation. a 40 year old, female with known bss and developmental dysplasia of her left hip (ddh) was referred to us for consideration of left total hip arthroplasty (tha). consultation with her haematologist for pre-operative optimization of platelets and related clotting times together with detailed discussions of her intended anaesthesia protocol and surgery resulted in a successful operation with less than anticipated blood loss. bss is an extremely rare bleeding disorder that puts patients at very high risk of blood loss following surgery. this is the first report that we are aware of describing a bss patient undergoing a tha. a cohesive, highly specialized, multi-disciplinary team is crucial to the success of these patients. bernard-soulier syndrome (bss) is a rare, inheritable (autosomal recessive) platelet disorder affecting an estimated 1 in every 1 million individuals. a mutation involving glycoprotein complex 1b-v-ix renders platelets unable to bind von willebrand factor (vwf) and form clots. bss patients require rigorous perioperative planning, however, the literature is devoid of reports describing bss patients undergoing orthopaedic surgery. we present a young female patient with bss that underwent total hip arthroplasty (tha) for her dysplastic left hip. a 40 year old, 83 kg female with developmental dysplasia of her left hip (ddh) was referred to us for consideration of left tha having suffered increasing left hip pain over the past three years. her diagnosis of ddh was not made until adulthood despite her left leg being shorter than her right leg by 2.5 cm. she was diagnosed with bss at the age of 18, and her haematology reports confirm 2 prior hospital admissions related to delayed bleeding following wisdom- teeth extraction at the age of 20 and cervical cone biopsy; the latter of which required transfusion with platelets and packed red blood cells (prbc). prior to this, she had been taking birth control pills from the age 14 for heavy menstruation. pre-operative cross-match and group and screen of blood was conducted confirming blood type a+. blood work also confirmed that she was not factor xiii deficit as initially suspected (fxiii activity, 1.04 u/ml). her glycoprotein 1b level was 15% with an estimated platelet count of 12 (x109/l) and inr 0.9. we consulted our thrombosis, haemophilia and internal medicine teams for pre-operative optimization. having a clinical nurse specialist tranexamic acid (ta) 1 g po tid was initiated 24 hours prior to surgery and continued until 14 days post-operatively. 20 mcg of ddavp (desmopressin) diluted into 50 ml of normal saline was given intravenously over a period of 30 minutes, 1 hour prior to surgery. immediately following this, she received 1 adult dose (4 units/318 ml) of pooled, buffy-coat platelets which elevated her platelets to 79 (x109/l) along with 1 unit of prbc to bring her haemoglobin (hb) to 135 (table 1). intravenous antibiotics (ancef, 2 g) were given within 30 minutes of surgery. induction of general anaesthesia was conducted using a mixture of intravenous propofol (170 mg), sufentanil (20 mcg), versed (2 mg) and rocuronium (50 mcg). a glidescope (verathon inc., anaesthesia maintenance comprised isoflurane (1.8%) with 58% o2 at 1.2l/min and air at 1.3l/min. once sedate, our patient was positioned in a standard right lateral decubitus position, supported by a stulberg frame (innomed inc., particular attention was taken to avoid undue tension through soft tissues during dislocation of the hip. similarly, bone bleeding following transection of the femoral neck and reaming of the acetabulum and femoral canal was addressed immediately., ny, usa, 2 g in 10 ml sterile normal saline) was packed into the femoral canal and acetabulum for a period of 3-4 minutes prior to placement of implants. the femoral component (accolade ii; stryker canada, hamilton, on, ca) was impacted into the femoral canal in the typical fashion. to optimize functional range, we used an anatomic dual mobility (adm), bearing hip system from stryker (fig 1). bleeding was minimal following impaction of either the femoral or acetabular prostheses. muscle and surrounding fascial layers were infiltrated with 0.5% marcaine/epinephrine (1:1000; 40 ml). the incision was meticulously closed in multiple layers followed by steri- strips and mepore dressings. pre- (a) and post- (b) operative radiographs showing the new implant. post-operatively, daily transfusions of platelets were given up to post-operative day (pod) 14. transfusions comprised 2 single donor (232 ml), 4 plateletpharesis (208 44 ml) and 8 pooled, buffy coated platelets (345 15ml). platelet counts dropped by 40% post-operatively but recovered by pod 6 (see table 1). serum sodium levels remained stable; the lowest reading was 128 mmol/l on pod 2, recovering to 136 mmol/l by pod 6. systolic blood pressures were also stable at 103 14 mmhg, the lowest (98/59) occurring pod 1. her blood work was recorded daily, a summary of which is shown in table 1. her pain was controlled effectively with oral hydromorphone contin (6 mg, bid) and tylenol (975 mg, q4h). patient entered into rehabilitation pod 7 and was discharged home on pod 19 following complete review by our haematology and medicine teams. pre-operative blood work results; estimated counts; hb: haemoglobin; hct: haematocrit. the overall leg length discrepancy was improved from 2.5 cm to 1 cm without the need for additional soft tissue release. bernard-soulier syndrome, first described in 1948, is an inheritable bleeding disorder affecting 1:1,000,000 individuals [1-3]. patients have prolonged bleeding times and are at high risk for spontaneous bleeding with the membrane-bound glycoprotein 1b/v/ix found on platelets have been described to explain this phenomenon. transfusions of allogeneic platelets remain the definitive treatment for patients with bss although intravenous injection of recombinant activated factor vii (rfviia) is also effective. the potential for developing anti-platelet antibodies with repeat platelet transfusions remains true although mansour et al suggest that this risk may be lessened for patients with bss since the 1b/v/ix complex is not associated with a platelet-specific antigen site. total hip arthroplasty is a major surgery with significant risks of blood loss, deep vein thromboembolism (dvt), pulmonary embolism (pe) and infection. estimates of blood loss following tha in patient without a bleeding disorder vary considerably within the literature; from 300-1500 ml with 20-30% risk of dvt or pe [5-7]. tranexamic acid (ta) is an anti-fribinolytic that inhibits the activation of plasminogen to plasmin with an established role in joint arthroplasty and good evidence to support reduced blood loss with no increased risk of dvt or pe. we provided oral ta peri-operatively and topical ta through the open wound intra-operatively with doses equivalent to 12 mg/kg po tid and 24 mg/kg respectively. a prospective randomized control trial using topical ta (2g/100 ml 0.9% saline) ddavp binds v2 receptors found on endothelial cells to increase vwf release and promote factor viii. we saw a slight drop over the peri-operative period, however values quickly corrected after the ddavp was stopped. importantly, the inhibitory effect of propofol on platelets is not evident at clinically relevant doses and its ease of induction outweighs this theoretical risk. epidural or lumbar spine blocks and/or controlled hypotension can also reduce potential blood loss by 30-50%. nevertheless, the potential risk for bleeding in and around the spinal cord can not not be ignored. we strived for a systolic blood pressure of 100 mmhg intra- operatively and our total intra-operative blood loss was comparable to what we expect in patients without bss. furthermore, we only used 1l of 0.9% saline intra-operatively so avoided haemodilution which could potentially exacerbate our existing coagulopathy. we experienced no adverse transfusions reactions despite multiple platelet transfusions post-operatively and hb remained stable. we avoided use of non-steroidal anti-inflammatories which are known to increase peri-operative blood loss following tha. similarly, conservative use of narcotics is warranted to avoid extreme hypotensive episodes. we found long acting hydromorphone with extra-strength tylenol to be most effective for pain control allowing the patient to mobilize quickly after surgery. we made no special concessions regarding the patient s ddh other than use of the adm implant and minimal acetabular reaming. this is the first report of a successful tha on a young adult with bernard-soulier syndrome. strict intra-operative haemostasis with limited soft tissue perturbation, limited reaming of the acetabulum and tight, multi-layered closure are instrumental to limit blood loss. the utility of anti-fibrinolytics and ddavp in bss patients is clearly borne out in this study. the management of coagulopathic patients in orthopaedics is a very specialized area of medicine requiring an in depth understanding of the risks of the surgery inherent to these patients. specific algorithms continue to be refined with regards to the use of perioperative agents to minimize intraoperative blood loss just as surgical technique and technology continues to evolve to minimize perturbation of soft tissues and converse bone and in doing so limit the need for postoperative transfusion.

introduction: the management of patients with coagulopathic disorders undergoing orthopaedic surgery requires a dedicated, multi-disciplinary team with detailed perioperative planning. bernard-soulier syndrome (bss) is an extremely rare disorder, affecting 1 in 1 million individuals worldwide. it is caused by a deficiency in glycoprotein 1b-v-ix which is required for normal platelet-mediated clot formation. the deficiency results in prolonged bleeding time with high risk of spontaneous bleeds. few reports exist in the clinical literature of bss patients undergoing major surgery. case report: a 40 year old, female with known bss and developmental dysplasia of her left hip (ddh) was referred to us for consideration of left total hip arthroplasty (tha). consultation with her haematologist for pre-operative optimization of platelets and related clotting times together with detailed discussions of her intended anaesthesia protocol and surgery resulted in a successful operation with less than anticipated blood loss. she entered our rehabilitation program just one week after surgery. conclusion:bss is an extremely rare bleeding disorder that puts patients at very high risk of blood loss following surgery. this is the first report that we are aware of describing a bss patient undergoing a tha. a cohesive, highly specialized, multi-disciplinary team is crucial to the success of these patients.

PMC4719372

pubmed-975

prior to the antivascular endothelial growth factor (anti-vegf) era, age-related macular degeneration (amd) was considered the leading cause of severe visual loss and blindness in the developed world among people over the age of 50 years. various imaging methods are available for the diagnosis and classification of amd. until recently, color fundus photography (fp) was the gold standard for grading and staging in amd clinical trials [24]. by permitting visualisation of the choroidal and retinal microcirculation and providing detailed information about the presence of pathological vessels as well as the integrity of the blood retinal barrier, fluorescein angiography (fa) had become a central tool for detecting and classifying cnv as well as cnv activity in eyes with neovascular amd [5, 6]. during the past years, oct has dramatically gained importance for the diagnosis and management of patients with chorioretinal disease by noninvasively providing cross-sectional images of the neurosensory retina and the subretinal space, thus allowing a detailed characterization of structural changes. thus, oct is increasingly used to determine the presence and activity of cnv and the need for (re-) treatment [79]. new-generation spectral domain oct (sdoct) instruments provide even higher resolution and more dense coverage of the macular area compared with time domain oct. therefore, sdoct imaging is now widely used for the followup of patients with cnv undergoing anti-vegf therapy. this study aims to compare fp, fa, and sdoct imaging regarding their sensitivity and specificity for detecting amd, cnv, and cnv activity and to analyze whether sdoct may have the potential to replace the other imaging techniques. the european genetic database (eugenda), a database collecting amd patients as well as healthy controls, was retrospectively reviewed, and fp, fa, and sdoct images of 120 eyes of 66 consecutive patients were randomly collected. eyes with early, intermediate, or late amd as well as control cases were included. control eyes were required to show no signs for amd, but other chorioretinal diseases including cnv secondary to any other disease but amd was allowed. to be eligible for this study, all images had to be performed on the same day at the university of cologne, germany. fa images were performed using the spectralis hra system (heidelberg engineering, heidelberg, germany). the standard protocol included 30 stereo images of the transit phase, mid phase, and late phase up to 10 minutes following intravenous injection of fluorescein. sdoct images were acquired using the spectralis sdoct instrument (heidelberg engineering, heidelberg, germany). sdoct volume scans (15 20) composing of 37 parallel oct b-scans were used for analysis. for each oct b-scan, 20 images were averaged using the automated real-time (art) function. images were independently analyzed by reading center graders (tr, nfm, and sl) at the cologne image reading center (circl), which have been trained and certified in image interpretation of amd patients. discrepancies between graders have been solved by open adjudication. during analysis of one imaging technique, for all images, the presence of amd, cnv, and cnv activity was noted (table 1, figure 1). amd was defined as the presence of 10 small (63 m), hard drusen and pigmentary changes or at least 1 intermediate (64124 m) or large (125 m) drusen inside the 6 mm etdrs grid. cnv was considered present on fp, if subretinal or subrpe fibrosis and fibrovascular tissue or fibrin were seen; on sdoct, subretinal hyperreflective material or pigment epithelial detachments (peds) other than single drusen were considered signs for cnv. on fa, cnv lesions were graded according to the modified macular photocoagulation study (mps) grading protocol utilized in the treatment of amd with photodynamic therapy (tap) and verteporfin in photodynamic therapy (vip) studies [11, 12]. briefly, classic cnv was identified as an area of uniform early hyperfluorescence that showed extensive leakage in the mid and late phases. occult cnv was classified as areas of stippled hyperfluorescence that appeared in the mid and late phases of the fluorescein angiography. cnv was graded as present on fa, if classic or occult cnv lesion components or staining scar tissue was detected. cnv activity was noted, if fluid or hemorrhage was present on fp that was not related to any other retinal vascular disease but cnv, if classic or occult cnv leakage was detected on fa or if diffuse or cystoid intraretinal fluid or subretinal fluid accumulation was seen on sdoct. for each parameter to be evaluated, the following imaging modalities were defined as the gold standard: for presence of amd, fp was used as the gold standard. for the presence of cnv cnv activity was considered present if it was detected on either sdoct or fa (ground truth). signs for amd were detected on fa in 77 eyes with a sensitivity of 92% (69 out of 75) and a specificity of 82% (in 8 cases, amd was noted on fa but not on color fundus photographs). disagreement between fa and fp was mainly related to small drusen that have been noted on fa but not on fp, and rpe hyperpigmentation that has been seen on fps but not on fas. on sdoct, amd was considered present in 78 eyes with a sensitivity of 89% (67 out of 75) and a specificity of 76% (in 11 cases, signs for amd were noted on sdoct but not on fp). disagreement between sdoct and fp could mainly be explained by small or intermediate drusen and rpe changes that have been missed on sdoct and by intermediate drusen that have been noted on sdoct but not on fps. twenty out of those eyes showed pathologies other than amd, including high myopia, chorioretinitis, retinal vein occlusion, epiretinal membranes, diabetic retinopathy and central serous retinopathy, or idiopathic cnv. fifty-four out of those cases were diagnosed with amd based on fps. in 14 cases, cnv was seen in the control group (idiopathic or related to high myopia or chorioretinitis). fp showed a sensitivity of 78% (53 out of 68 eyes) for the detection of cnv, and a specificity of 100%. sdoct images showed signs for cnv in 64 out of the 68 cases (94%), specificity for detecting cnv was 98% (in one case, cnv was diagnosed based on sdoct but not on fa). in the 64 cases with agreement between fa and sdoct regarding the presence of cnv, a classic cnv lesion component was detected on fa in 25 eyes. ten out of those 15 cases showed staining scar as a lesion component on fa, and 5 cases demonstrated occult cnv leakage only. thirty-eight out of the 64 cases showed occult cnv lesion components on fa, with all of those demonstrating a ped on sdoct. in addition, a ped was seen in 22 eyes without occult cnv lesion components on fa, with 18 (82%) out of those cases demonstrating staining scar as a lesion component and 4 (18%) cases showing classic cnv leakage only. out of the 68 cases with cnv diagnosed based on fa, a total of 60 cases (88%) showed signs for active cnv either on sdoct (53 eyes) or fa (53 eyes), with an agreement between both imaging modalities in 79% out of all 68 cases (46 cases showed active cnv and 8 eyes no signs for cnv activity on both imaging modalities). in 7 cases, fluid was detected on sdoct without evidence for cnv leakage on fa and vice versa. if the ground truth for sdoct and fa was considered the gold standard for cnv activity, sensitivity was 88% for fa, 88% for sdoct, and 38% (23 out of 60) for fp, respectively. specificity for fp was 98% (in one case, cnv activity was suspected based on fp but not seen on fa or sdoct). sdoct is increasingly used in clinical trials as well as in clinical practice for the diagnosis and followup of patients with neovascular amd undergoing anti-vegf therapy. as a noninvasive imaging tool, it provides high-resolution cross-sectional images of retinal pathology, allowing to qualitatively and quantitatively analyze various parameters relevant for (re-) treatment decisions. our study confirms that sdoct is highly sensitive for detecting amd, cnv, and cnv activity; however, it may not yet fully replace the information provided by fa and fp. the presence of characteristic features of amd on fp such as drusen and rpe changes was missed on sdoct in 11% of cases in our study. this may be explained by the sdoct volume scan settings used, as the gap between two parallel oct b-scans was approximately 120 m; thus pathological changes may fall in between two adjacent scans and may be overlooked or may appear smaller than they truly are. on the other hand, amd was diagnosed on sdoct based on the presence of intermediate drusen in 24% of cases that were graded as control cases on fp. on fp, those pathological features have been either interpreted as rpe changes or small drusen, or they have been overlooked due to reduced image clarity. thus, sdoct may be helpful in identifying drusen and in differentiating drusen from hypopigmentation and thus may improve the quality of image interpretation in eyes with amd compared to fp imaging alone. however, care should be taken not to transfer size definitions for intermediate or large drusen from fps to sdoct, as due to their shape, drusen may appear larger on sdoct compared to fp, or they may appear smaller if they are captured at the border. further studies are needed to compare drusen sizes between those different imaging modalities before sdoct imaging can be reliably used for staging of early and intermediate amd in clinical trials. other imaging techniques such as autofluorescence imaging provide additional information concerning drusen and rpe changes and may thus be helpful to identify and classify those features. based on fa, cnv lesions components are categorized as classic or occult cnv leakage or staining scar tissue that may develop over time and indicate longstanding disease with poor visual function. reported a sensitivity of only 40% for the detection of new-onset cnv on time-domain oct. the low sensitivity may be explained by the use of time-domain oct, as pathological features may be overlooked more easily compared to sdoct due to the less dense scan pattern, lower image resolution, and higher rate of movement artifacts. occult cnv on fa is believed to correspond histologically to type 1 cnv, located between the rpe and bruch's membrane. in accordance with this, all eyes with occult cnv on fa demonstrated a ped on sdoct in our study. in contrast, classic cnv lesion components on fa histologically correspond to type 2 cnv, positioned in the subretinal space. thus, type 2 cnv lesion components are expected to present as hyperreflective material in the subretinal space on oct. this could be confirmed in our study as all cases with classic cnv lesion components on fa demonstrated subretinal hyperreflective material on sdoct. however, in order to correctly interpret oct images, it is crucial to consider that oct scans only represent pseudohistological images, created using information about the reflectivity and axial distribution of various structures. hence, subretinal hyperreflective material on oct scans may not only represent type 2 cnv, but may include, for example, subretinal hemorrhage, fibrinous material, or photoreceptor debris. this may explain why subretinal hyperreflective material was seen in our study on sdoct in 5 cases demonstrating occult cnv on fa without the presence of a classic cnv leakage or staining scar as lesion components. additionally, a ped was seen on sdoct in 22 eyes without the presence of occult cnv on fa. in those cases, other cnv lesion components such as staining scar or classic cnv may have covered the cnv membrane located in the sub-rpe space; thus occult cnv leakage was not detectable on fa. this finding indicates that high-resolution cross-sectional images provided by sdoct may add important information regarding subretinal and sub-rpe pathology compared to the two-dimensional en-face view of fa and fp imaging. agreement between sdoct and fa regarding the activity of cnv lesions in our study was seen in 79% of all 68 cases diagnosed with cnv on fa. seven eyes demonstrated cnv leakage on fa in the absence of intra- or subretinal fluid on sdoct, and 7 eyes showed signs for cnv activity on sdoct without evidence of cnv leakage on fa. reported a sensitivity of 90% and specificity of 47% for sdoct to detect cnv activity seen on fa. the disagreement between both imaging modalities may be explained by the fact that fa and sdoct imaging provides different information about retinal pathology. fa is used to obtain information about the perfusion and the growth of new vessels as well as the integrity of the blood-retinal barrier; thus fluorescein leakage over time can be seen during angiography. this information is missing on oct images; thus oct provides detailed information about pathological changes like, for example, the presence of cystoid spaces; however, it is not possible to detect whether they are caused by fluid accumulation from acute leakage from pathological vessels. thus, cystoid spaces on sdoct may not necessarily correspond to fluorescein leakage on fa, but may represent structural defects indicating chronic disease (figure 2). increase or decrease in the amount of fluid seen on oct may thus more reliably indicate cnv activity than the presence of fluid seen at one time point. in addition, care should be taken to not confuse intraretinal cystoid spaces or subretinal fluid with in their paper, the authors state that degenerating photoreceptors may become arranged in a circular or ovoid fashion in chronic diseases affecting the outer retina and rpe. in contrast, cnv activity seen on fa may be missed on sdoct if only intraretinal cystoid spaces and subretinal fluid accumulation are considered to represent cnv activity on sdoct. recently reported that intraretinal hyperreflective flecks and the inherent reflectivity and boundary definition of subretinal hyperreflective material may indicate active cnv even in the absence of intra- or subretinal fluid accumulation. additionally, fluid accumulation in the sub-rpe space such as serous components of a ped may indicate cnv activity. fa imaging at baseline in addition to sdoct is helpful to assess the cnv lesion subtype and the initial severity of cnv leakage; during followup, fa may confirm evidence of cnv activity whenever sdoct interpretation is challenging or inconsistent with retinal function. sdoct, fa, and fp imaging provide complementary information about pathological changes in chorioretinal diseases. our study indicates that drusen and rpe changes as signs for amd are best appreciated on fp. sdoct is highly sensitive to identify cnv and cnv activity; however, it can not fully replace fa in the management of patients with cnv. further studies are needed to evaluate which sdoct parameters (e.g., cystoid spaces, diffuse intraretinal fluid, subretinal or sub-rpe fluid, inherent boundary definition of subretinal hyperreflective material, or a change in the amount of fluid) best indicate cnv activity.

purpose. to compare color fundus photography (fp), fluorescein angiography (fa), and spectral domain optical coherence tomography (sdoct) for the detection of age-related macular degeneration (amd), choroidal neovascularisation (cnv), and cnv activity. methods. fps, fas, and sdoct volume scans from 120 eyes of 66 amd and control patients were randomly collected. control eyes were required to show no amd, but other retinal pathology was allowed. the presence of drusen, pigmentary changes, cnv, and signs for cnv activity was independently analyzed for all imaging modalities. results. amd was diagnosed based on fp in 75 eyes. sdoct and fa showed sensitivity (specificity) of 89% (76%) and 92% (82%), respectively. cnv was present on fa in 68 eyes. sensitivity (specificity) was 78% (100%) for fp and 94% (98%) for sdoct. cnv activity was detected by sdoct or fa in 60 eyes with an agreement in 46 eyes. sensitivity was 88% for sdoct and 88% for fa. fp showed sensitivity of 38% and specificity of 98%. conclusions. cnv lesions and activity may be missed by fp alone, but fp may help identifying drusen and pigmentary changes. sdoct is highly sensitive for the detection of amd, cnv, and cnv activity; however, it can not fully replace fa.

PMC3665260

pubmed-976

ovotesticular disorder of sex development (ot-dsd) is a rare disorder of sexual differentiation characterized by the presence of testicular tissue with distinct seminiferous tubules and ovarian tissue with mature ovarian follicles in the same gonad (ovotestis) or separately in a single individual (1, 2). ot-dsd it constitutes between 3 and 10% of the total dsd and presents significant diagnostic and management challenges (3, 4). typically, both mullerian and wolffian duct derivatives are seen, and most affected individuals commonly present with ambiguous external genitalia as neonates or infants. however, the phenotype of the external genitalia may range from normal male to normal female depending on the degree of testicular tissue present. the ovotestis is usually the most common gonad in individuals with ot-dsd, and such gonads are known to be at an increased risk of developing germ cell tumors (5). more than five hundred cases of ot-dsd including familial cases have been reported in medical literature (3, 6, 7). epidemiologically, the geographical distribution of ot-dsd shows a higher prevalence in the african continent, especially among south african blacks, with the number of published cases being 17 per 100 million people, followed by europe at 15.3. age and mode of presentation is often variable and usually comprises hypospadias, unilateral or bilateral cryptorchidism, inguinal hernia, urogenital sinus, gynecomastia at pubertal age or lower abdominal mass in adulthood (4). internally, the presence of an ovotestis is the most common finding followed by ovaries (4, 9). the chromosomal findings in ot-dsd were first reported in 1959 by hungerford, who demonstrated a 46,xx chromosomal complement in peripheral blood lymphocytes of an individual with this disorder (10). ot-dsd is a genetically heterogeneous condition with the predominant karyotype being 46,xx, while 46,xx/46,xy chimerism, 46,xy karyotype and x-y translocation are less frequent (4). the objective of this paper is to describe the diverse clinical, cytogenetic and histopathological features of five patients diagnosed with ot-dsd in sri lanka. this is a report of the clinical, cytogenetic and histopathological data of patients with ot-dsd who were referred to the human genetics unit for cytogenetic evaluation between 2005 and 2011. this facility serves as the main referral center for cytogenetic testing in sri lanka, and the majority of children with dsd in the country undergo karyotype testing at this center. the diagnosis of ot-dsd had been confirmed by histopathological examination of the gonads, which were biopsied either laparoscopically or at exploratory laparotomy. five milliliters of peripheral blood was obtained from each patient, and chromosomal analysis was performed on routinely cultured lymphocytes after gtg-banding. karyotyping was done according to guidelines of the international system for human cytogenetic nomenclature (iscn, 2005). at least 30 well-spread and well-banded metaphases were examined in each patient by an experienced cytogeneticist. the age, sex of rearing, mode of presentation, status of the external and internal genitalia and karyotypes of the patients are summarized in table 1. their ages ranged from 2 mo to 47 yr, and all were reared as males. in four of the patients (cases 1, 3, 4 and 5), two patients (cases 1 and 3) had perineal hypospadias with bilateral cryptorchidism, while the other two patients (cases 4 and 5), who were siblings, both had male external genitalia with a short, blind-ending vagina and bilateral inguinal lumps. case 2 was reared as a male but started feminizing and developed female secondary sexual characteristics at puberty with gradual atrophy of both testes. case 1 underwent surgical correction for perineal hypospadias during early childhood but remained undiagnosed until an exploratory laparotomy performed for an intra-abdominal lump led to the diagnosis of ot-dsd at the age of 47 yr. in addition to the lower abdominal mass, this patient was observed to have gynecomastia. two patients had bilateral ovotestis: one had an ovotestis on one side and an ovary on the other side, the other had an ovotestis on one side and a testis on the other side; the remaining patient had an ovary and testis separately on either side. four patients (cases 1, 2, 4 and 5) had a 46,xy chromosome complement, while 46,xx/46,xy chimerism was observed in the remaining patient (case 3). there were no cases of 46,xx karyotype. table 1 profiles of the five patients with ovotesticular disorder of sex developmentcaseagesex of rearingindication for investigationexternal genitaliainternal genitalia/gonadskaryotype147 yrmaleambiguous genitalia at birth/abdominal mass in adulthoodperineal hypospadias, bilateral cryptorchidismuterus with tubes, left ovotestis and right testis 46,xy223 yrmalefemale secondary sexual characteristics at puberty with gynecomastiamale external genitalia with bilateral atrophic testes uterus with tubes and bilateral streaky ovaries and atrophic testes46,xy32 momaleambiguous genitalia/inguinal lumpperineal hypospadias, bilateral cryptorchidismuterus with tubes, left ovary and right ovotestis46,xx/ 46,xy49 momaleambiguous genitalia/bilateral inguinal lumpsmale external genitalia with blind-ending vaginauterus with tubes and bilateral ovotestis46,xy55 yrmaleambiguous genitalia/bilateral inguinal lumps male external genitalia with blind-ending vaginauterus with tubes and bilateral ovotestis46,xy ot-dsd is a rare disorder of sexual differentiation that is phenotypically and genetically heterogeneous with wide-ranging manifestations. the presence of well-differentiated ovarian and testicular tissue in the same individual, whether as a single tissue-type gonad or an ovotestis, is the hallmark of this condition (9). most patients with ot-dsd have ambiguous genitalia and are often diagnosed within the first few months to years of life (1, 4, 9). in addition to ambiguous genitalia, patients may present with inguinal hernias, gynecomastia during adolescence or lower abdominal mass in adulthood (4, 11). four out of 5 patients in this series had ambiguous genitalia diagnosed in the first few months to years of life, and all were reared as phenotypic males. inguinal hernias and gynecomastia were also present in some patients. a small number of ot-dsd cases are incidentally picked up during laparotomy for unrelated causes. one of the patients in this series had a similar presentation; he presented with a lower abdominal mass in the fourth decade that was later discovered to be an 18-wk-size uterus at laparotomy. a similar case of ot-dsd in a 42 yr old was reported in north india (4). in ot-dsd, the degree of virilization of the external genitalia depends on the capacity of the testicular tissue to secrete testosterone. previous studies indicate that although approximately 70% of ot-dsd patients are raised as males, less than 10% have normal male external genitalia (1). it is generally believed that the leydig cell function of the dysgenetic testis is inadequate for normal virilization (4), and this is exemplified in this series by case 2, who had bilateral atrophic testes and started feminizing at puberty. previous reports have also indicated that testicular tissue in ot-dsd patients becomes dysgenetic and that germ cells begin to disappear with increasing age (12). the patients in this study had internal genital organs ranging from streaky ovaries and a uterus to atrophic testes, but the common presenting gonad was the ovotestis. it is known that gonadal tissue may be located at any level along the route of embryonic testicular descent and is frequently associated with an inguinal hernia. a sub-classification of ot-dsd based on the type and location of the gonads has been described (13). according to this classification, ot-dsd is considered to be lateral if a testis is present on one side and an ovary is present on the other side (case 2), unilateral if an ovotestis is present on one side and a testis or ovary is present on the other side (cases 1 and 3) and bilateral if an ovotestis is present on both sides (cases 4 and 5). (9) reported that the ovotestis was the most frequent gonad, accounting for 59% in their series of 16 patients with ot-dsd, and krob et al. (3) also reported that an ovotestis was found in 44.4% of 568 gonads examined. the same observation is reflected in this series, in which 4 out of 5 cases (1, 3, 4 and 5) had an ovotestis and 2 of the patients, who were siblings, had bilateral ot-dsd. with regard to the frequency of gonadal distribution, it is reported that the most common form is ovotestis plus ovary followed by bilateral ovotestis and ovary on one side and testis on the other side, representing 34%, 29% and 25% of cases, respectively (8). previous studies have reported that the risk of germ cell tumors, especially dysgerminomas, in patients with ot-dsd ranges from 4% among those with the 46,xx karyotype to up to 10% in those with 46,xy and 46,xx/xy chimerism (4, 5). analysis of the chromosomal distribution of ot-dsd shows that about 5065% cases have a 46,xx chromosomal constitution followed by 46,xx/46,xy chimerism, while less than 10% have a 46,xy karyotype (1, 8, 9). the 46,xy karyotype is believed to be extremely rare and equally distributed throughout asia, europe and north america (3). in contrast to published reports (3, 4, 8, 9, 14), the predominant karyotype was 46,xy in 80% of the patients in this series. there is no apparent reason for 46,xy being the most common karyotype in this study other than the small number of patients studied, with possible over-representation of the 46,xy karyotype in this study sample, or it could be reflective of the pattern seen in the sri lankan population. karyotypes such as 46,xx/46,xy, which was seen in only one patient, are thought to result from chimerism, possibly from double fertilization (involving two spermatocytes one x and one y) of either a binucleate ovum or of an ovum and its polar body (15). in some of the patients, proper investigation and accurate diagnosis had been delayed for many decades in spite of ambiguous genitalia being detected early in life, which led to clinical and psychological problems in their adult life. it is therefore recommended that in addition to cytogenetic evaluation, phenotypic males with ambiguous genitalia should be thoroughly investigated by means of either abdominal ultrasound scan and/or exploratory laparoscopy/laparotomy with histopathological examination to properly assess the exact nature of their internal genital system. although considered a rare presentation, the cases reported in this series suggest that abnormal inguinoscrotal or abdominal masses may occur in ot-dsd and should be considered in the differential diagnosis of patients, especially in those with cryptorchidism and inguinal hernia having the 46,xy karyotype.

ovotesticular disorder of sex development (ot-dsd) is a rare disorder of sexual differentiation in which the gonads of an individual are characterized by the presence of both mature ovarian and testicular tissues. the objective of this paper is to report the clinical, cytogenetic and histopathological findings in sri lankan patients diagnosed with ot-dsd who were referred to the human genetics unit for cytogenetic evaluation during 2005 to 2011. five patients had histopathologically confirmed ot-dsd. their ages at presentation ranged from 2 mo to 47 yr. clinical symptoms varied from ambiguous genitalia and inguinal hernias at birth to a lower abdominal mass presenting in adulthood. all 5 were reared as phenotypic males. an ovotestis was detected in all cases except one, and the predominant karyotype was 46,xy. the findings in this series of predominantly 46,xy karyotype are in contrast to previously published reports that have reported 46,xx as being the predominant karyotype. it is therefore recommended that individuals with ambiguous genitalia who have the 46,xy karyotype should be thoroughly investigated by ultrasonographic or laparoscopic assessment to determine the exact nature of their internal genital organs. ot-dsd should also be considered in the differential diagnosis of patients with cryptorchidism and inguinal hernia.

PMC3687650

pubmed-977

extracellular adenosine concentration increases under metabolically stressful conditions, notably in the tumor microenvironment, where hypoxia is frequently given [2, 3]. such accumulation of adenosine mediates, through four distinct receptors (a1, a2a, a2b, and a3), complex and diverse effects that lead to tumor immunoescape. this includes cytoprotection and growth promotion of tumor cells [5, 6], angiogenesis increase [7, 8], and suppression of effector (antitumor) t cells. although cells are provided with adenosine transporters, the main source of this nucleoside in the tumor interstitium is the hydrolysis of extracellular atp, which also accumulates in tumors, by membrane enzymes known as ecto-nucleotidases [6, 10]. different families of these enzymes, acting extracellularly, are responsible for the generation of adenosine from adenine nucleotides (i.e., atp, adp, or amp): (1) the ectonucleoside triphosphate diphosphohydrolase (e-ntpdase) family, that includes four plasma membrane-bound members: ntpdase1 (cd39), ntpdase2, ntpdase3, and ntpdase8; these enzymes are differentially expressed and hydrolyze with different affinities nucleoside triphosphates and diphosphates to their monophosphate derivatives (e.g., atp and adp to amp); (2) the ectonucleotide pyrophosphatase/phosphodiesterase (e-npp) family, capable of hydrolyzing nucleoside triphosphates to monophosphates and pyrophosphate (ppi), such as atp to amp and ppi; (3) the alkaline phosphatase (ap) family, that includes ubiquitous enzymes degrading broad range of substrates, such as adenine nucleotides and ppi, releasing inorganic phosphate (pi); (4) the 5-nucleotidase family, with only one member attached to the outer plasma membrane, the ecto-5-nucleotidase (cd73), a glycosyl phosphatidylinositol-linked membrane-bound glycoprotein that efficiently hydrolyses amp to adenosine [1113]. two members of ecto-nucleotidases families, the e-ntpdase cd39 and the 5-nucleotidase cd73, acting sequentially, seem to have a crucial role in tumor-immune cell interaction. they both are expressed not only by infiltrating immune cells but also by tumor cells, and their expression is regulated by hypoxia [10, 14]. increased cd39 and cd73 expression has been described in various cancer types, mostly in correlation with a poor prognosis [1517]. both molecules are considered promising therapeutic targets in oncology, and cd73 has already been proven to inhibit tumor growth and metastasis in a breast cancer model in mice [1820]. however, until now there were not available data concerning ecto-nucleotidases expression in endometrial cancer (ec). there are two clinicopathological variants: the estrogen-related, type i, endometrioid carcinoma, and the nonestrogen-related, type ii, nonendometrioid carcinoma. although there are different molecular alterations that have been already identified in ec, with different prevalence between tumors [22, 23], there is need to decipher the complete molecular profile of ec pathogenesis to improve diagnosis and favor the design of new therapeutic strategies. the aim of the present work was to study the expression of cd39 and cd73 in endometrioid (type i) and serous (type ii) ec when compared with nontumoral endometrium. to achieve this objective, protein and gene expression experiments, as well as in situ enzyme activity assays, the ethical principles of this study adhere to the declaration of helsinki, and all the procedures were approved by the ethics committee for clinical investigation of bellvitge hospital. endometrial samples from adenocarcinoma (endometrioid and serous types) and their corresponding nontumoral tissue (if present) were obtained from hysterectomy specimens at the service of gynecology of bellvitge hospital. fresh samples were cut, embedded in o.c.t. freezing media (tissue-tek; sakura finetek, zoeterwoude, the netherlands), snap-frozen in a shandon histobath 2 (neslab instruments inc., usa) at the service of pathology, and stored at 80c until used. alternatively, endometrial samples were obtained from the tumor bank of bellvitge biomedical research institute (idibell). fifteen endometrioid adenocarcinomas (13 grade 1, 2 grade 2; all figo stage i) (5682 years old, median 61) and fourteen serous adenocarcinomas (grade 3; 9 figo stage i, 2 figo stage ii, and 3 figo stage iv) (6386 years old, median 77), and their adjacent nontumoral endometrium were used in this study. touch preparations of endometrial cancer tissue samples were obtained by lightly pressing the freshly cut tumor surface on clean glass microscope slides, thus generating a tumor cell imprint. briefly, tissue sections of 10 m thick and touch preparations were fixed in 10% phosphate-buffered formalin mixed with cold acetone (merck, darmstadt, germany) for 2.5 minutes. fixed samples were rinsed with pbs and preincubated for 1 hour at room temperature (rt) with pbs containing 20% normal goat serum (gibco, paisley, uk) and 0.2% gelatin (merck). samples were then incubated overnight at 4c with the following primary antibodies: anti-human cd39 clone bu61 (ancell corporation, minnesota, mn, usa) at 1/500, mouse monoclonal anti-human ecto-5-nucleotidase (cd73) clone 4g4 (abcam, cambridge, uk) at 1/50, and rabbit monoclonal anti-human cytokeratin 19 (ck19) clone epr1579y (abcam) at 1/200. after three washes in pbs, samples were incubated for 1 hour at rt with the appropriate secondary antibodies: horseradish peroxidase-conjugated goat anti-mouse (envision+system; dako, carpinteria, usa), alexa fluor 488- or 555-goat anti-mouse or anti-rabbit (life technologies, paisley, uk). nuclei were counterstained with haematoxylin or, alternatively, in fluorescence assays, to-pro-3 or dapi (life technologies) were used to visualize the nuclei. samples were mounted with fluoromount aqueous mounting medium (sigma-aldrich, sant louis, missouri, mo, usa). the results were observed and photographed under a light leica dmd 108 microscope (leica microsystems, wetzlar, germany) or, in fluorescence assays, under a nikon eclipse e-800 microscope (nikon, tokyo, japan) or under a leica tcs-sl spectral confocal microscope (leica). results from both tissue samples and touch preparations were independently evaluated by at least two observers. label intensity was scored as negative (), intermediate (+), or strongly positive (+ +). for enzyme histochemistry, adpase and ecto-5-nucleotidase (ampase) activities were localized by using the wachstein/meisel lead phosphate method [24, 25] in tissue samples and in touch preparations. briefly, fixed samples were preincubated for 1 hour at rt in 50 mm tris-maleate buffer, ph 7.4 containing 2 mm cacl2 and 0.25 m sucrose. enzyme reaction was carried out for 1 hour at 37c in the same buffer supplemented with 5 mm mncl2, 2 mm pb(no3)2, 3% dextran t250, and 2.5 mm levamisole, as inhibitor of alkaline phosphatases, and in the presence of 200 m adp or 1 mm amp, as substrate. for cd39 and cd73 inhibition experiments, 1 mm nf279 (tocris bioscience, bristol, united kingdom) and 1 mm, -meadp (sigma-aldrich) were added, respectively, to both preincubation and enzyme reaction buffers. the reaction was revealed by incubating with 1% (nh4)2s v/v for exactly 1 minute. samples were counterstained with haematoxylin, mounted with fluoromount aqueous mounting medium (sigma-aldrich), and observed and photographed as described above. 50100 g of human tumor (endometrioid and serous endometrial adenocarcinoma) and nontumoral tissue samples were homogenized in a buffer containing 20 mm hepes, 250 mm sucrose, 0.3 mm pmsf, 1 mm dtt, 1 mm egta, and 1 mm mgcl2 (ph 7.4) using a glass homogenizer (vidrafoc, barcelona, spain). after homogenization, samples were centrifuged at 600 g for 10 minutes at 4c in a beckman ja-20 centrifuge. the pellet was discarded and supernatants were centrifuged at 48,000 g for 20 minutes at 4c in a beckman ti-70 centrifuge. the resulting pellets were resuspended in a buffer containing 20 mm hepes, 0.3 mm pmsf, and 1 mm dtt (ph 7.4). protein concentration was determined by the method of lowry et al. using bovine serum albumin as a standard. adpase and ampase activities were determined by measuring the amount of pi using the malachite green colorimetric assay, as previously described. total rna from endometrial tumor tissue samples was isolated using the rneasy plus mini kit (qiagen, hilden, germany), following the manufacturer's protocol. total isolated rna (2 g) was reversely transcribed into complementary dna (cdna) using the first strand cdna synthesis kit (fermentas, thermo scientific, chicago, il, usa). quantitative real-time pcr (qrt-pcr) was performed to examine the expression of cd39, ntpdase2, and cd73 genes. designed large-scale taqman low-density array (tlda) microfluidic cards (applied biosystems, foster city, ca, usa) were used. the 384 wells of each card were preloaded with predesigned fluorogenic taqman probes and primers for cd39, ntpdase2, and cd73. cdna (1 g) combined with taqman 2x universal pcr master mix (applied biosystems) were loaded into each sample-loading port. qrt-pcr reactions were carried out using the abi prism 7900ht real-time pcr system (applied biosystems). data were collected using the sds v2.1 software (applied biosystems) and analyzed by the comparative ct (ct) quantification method using the expression suite v1.0 software (applied biosystems). the relative expression levels of cd39, ntpdase2, and cd73 genes were determined using 18s mrna as an endogenous control for normalization. results are expressed as the mean of the relative quantification (rq) of the tested transcripts (n=7 serous adenocarcinoma samples; n=7 endometrioid adenocarcinoma samples) the standard error of the mean (sem). results were obtained from five independent experiments performed using 1 g of cdna, all with duplicate measurements. student's t-test was used to compare the means of two independent groups of normally distributed data. extracellular adenosine in tumors, mainly generated by the sequential action of ecto-nucleotidases, has immunosuppressive effects through a broad range of actions, including inhibition of antitumor t-cell function, modification of local interleukin levels, and inhibition of phagocytosis (reviewed in). in this section we show and discuss our results on the expression of the ecto-nucleotidases cd39 and cd73 in type i and type ii endometrial carcinomas. cd39 was immunolocalized in the stroma of both nontumoral and tumoral endometria (figure 1(a)). for the nonpathological endometrium the expression of cd39 has already been previously described in association with stromal cells and blood vessels. here we show that label score was significantly higher for both endometrioid and serous types of tumors when compared with the corresponding nontumoral coexisting endometrium (figure 1(b)). no cd39 labeling was found in endometrial adenocarcinoma epithelia, as demonstrated with the double staining performed with the anti-cd39 and anti-ck19 antibodies (see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/509027). strong in situ adpase activity was detected in the tumor stroma, coinciding with the cd39 immunolocalization (figure 2(a)). equivalent results were obtained using atp as substrate for the in situ activity assay (not shown). adpase activity measured in tumor tissue homogenates demonstrated that serous (grade 3) adenocarcinomas had significantly higher activity than endometrioid (grade 1) adenocarcinomas (figure 2(b)). cd73 expression was strongly immunodetected in both types of tumors, in epithelial structures and in the stroma (figure 3(a)), thus partially colocalizing with ck19 (supplementary figure 2). specific cd73 activity, demonstrated with the inhibitor, -meadp, matched the immunolabeled structures (figure 3(a)). we have already previously demonstrated that the expression and activity of cd73 are abundant in nonpathological endometrium [24, 25]. consequently, due to the high expression of cd73 in tumoral and nontumoral endometria, label score comparisons were not possible. moreover, no differences among tumors were observed with the enzyme assays either in tissue slices or in tissue homogenates (not shown). immunolabeling and in situ enzyme activity results obtained with the touch prep technique were equivalent to those obtained with tissue slices (figures 2(c) and 3(b)). the usefulness of the touch prep technique for diagnosis has already been demonstrated in breast cancer with 100% sensitivity and specificity in the evaluation of tumor margins at the time of the surgery. this technique has also been previously used to demonstrate by immunolabeling a decreased expression of p2x7 atp receptor in endometrial cancer cells and also, recently, the relationship between p53 expression and the tumor grade. however, to our knowledge, this is the first report validating the use of touch preps for enzyme activity studies, therefore opening the possibility of performing such studies in cytological samples. in order to determine if the differences in cd39 protein and adpase activity between tumor types also involved gene expression changes, quantitative real-time pcr analyses were performed (figure 4). cd39 gene expression was 2-fold higher in serous endometrial adenocarcinoma than in endometrioid, coinciding with the data obtained with the protein (figure 4(a)). these gene expression changes did not apply to ntpdase2 (figure 4(b)), indicating that cd39 upregulation is not a general feature of other members of ntpdase family. no changes were detected in cd73 gene expression between the two tumor types (figure 4(c)). these results on endometrial tumors add to the list of human cancers in which cd39 is overexpressed and support the growing body of evidence that cd39 is a potential therapeutic target for cancer immunotherapy. antibody-based therapy and pharmacological approaches against cd73 have been reported to significantly inhibit tumor growth and improve antitumor immunity in mouse models [28, 32]. this also coincides with the higher grade of these tumors, but further studies are needed to establish statistical correlations with the tumor grade in the case of type i endometrioid tumors. the consequences of this high cd39 activity in endometrial tumors are increased levels of amp, the substrate for cd73, and also highly expressed in these tumors, which will, in turn, generate increased immunosuppressive levels of extracellular adenosine.

one of the strategies used by tumors to evade immunosurveillance is the accumulation of extracellular adenosine, which has immunosupressive and tumor promoting effects. the study of the mechanisms leading to adenosine formation at the tumor interstitium are therefore of great interest in oncology. the dominant pathway generating extracellular adenosine in tumors is the dephosphorylation of atp by ecto-nucleotidases. two of these enzymes acting sequentially, cd39 and cd73, efficiently hydrolyze extracellular atp to adenosine. they have been found to play a crucial role in a variety of tumors, but there were no data concerning endometrial cancer, the most frequent of the invasive tumors of the female genital tract. the aim of the present work is to study the expression of cd39 and cd73 in human endometrial cancer. we have analyzed protein and gene expression, as well as enzyme activity, in type i endometrioid adenocarcinomas and type ii serous adenocarcinomas and their nonpathological endometrial counterparts. high levels of both enzymes were found in tumor samples, with significantly increased expression of cd39 in type ii serous tumors, which also coincided with the higher tumor grade. our results reinforce the involvement of the adenosinergic system in cancer, emphasizing the relevance of ecto-nucleotidases as emerging therapeutic targets in oncology.

PMC3953595

pubmed-978

the incidence of oral cancer especially squamous cell carcinoma accounts for nearly 2.4% of all cancers. due to significant number of oral cancer cases raising rapidly in the developing regions this life style habits such as heavy smoking and alcoholism are the important risk factors for developing oral cancer that increases at least three- to fifteenfold especially in females and young people. in addition, marijuana, chewing beetle-leaf, human papilloma virus, ultraviolet radiations, iron deficiency anemia, candida infections, immunosuppression, and deletion or mutation of tumor suppressor genes are some of the other causes of oral cancer. lack of public awareness regarding oral health and low intake of fruits and vegetables, older age, and poor oral hygiene are some of the implications for oral cancer. majority of the oral cancer was detected at late stages (iii and iv) and early diagnosis is important to increase patient survivability and to delay its prognosis. in 2011, world health organization (who) reported the incidence of oral cancer deaths in malaysia to about 1.5% of the total deaths, with age adjusted death rate of 7.72 per 100,000 populations. malaysia ranked 14 in the world with annual oral cancer deaths of 1,587. increasing the public awareness and early diagnosis, it is important to have sufficient knowledge and awareness among dentists for detection and early diagnosis. initiatives were undertaken by university of malaya to increase the oral cancer awareness in malaysia such as malaysian oral cancer research initiative (mocri) and oral cancer research&coordinating centre (ocrcc). these publicity initiatives are crucial to improve the oral cancer awareness among general public and health professionals in malaysia. in addition, general dental practitioner's role is decisive in identifying the oral mucosal changes that may lead to oral cancer. assessing the knowledge of dental students paves the way towards understanding their level of awareness in the early detection and prevention of oral cancer. to the best of our knowledge, previous researches on dental students ' knowledge and awareness were conducted in the university of malaya (um) and universiti sains malaysia (usm) [9, 10]. since there is a paucity of information regarding oral cancer awareness in undergraduate dental students in different other regions of malaysia, therefore it is pertinent to assess these characteristics in senior dental students (third, fourth, and fifth year) at international islamic university, kuantan, pahang, malaysia. the aim of the current research was to assess the knowledge and awareness of oral cancer towards early identification of risk factors among undergraduate dental students. this is a descriptive cross-sectional study to assess the oral cancer knowledge and awareness of senior undergraduate dental students using a survey questionnaire, adopted by carter and ogden and brzak et al.. ethical permission to conduct the study was obtained from the respective deans, international islamic university, malaysia. the study was conducted via face-to-face interview at international islamic university malaysia during the period of february to march, 2015. sample size was determined using 95% confidence interval, with an accuracy of 60% for the total dental students being 300 studying in international islamic university given a confidence interval of 5.5; the recommended sample size is 155 or more. a systemic random sampling technique was used to select senior dental students which includes third, fourth, and fifth years. in general, as included in the curriculum, dental students receive information regarding oral cancer during their oral pathology and oral medicine sessions in their first and second year as well as oral examination during their clinical sessions. students of both gender studying third-, fourth-, and fifth-year dentistry were included in the study. a 9-item pretested questionnaire was employed after explaining the purpose of the study and verbal consent was obtained from each study participant. the questionnaire constitutes 7 close-ended (yes/no) questions such as (1) oral mucosal examination (2 items), (2) advising current and future patients regarding risk factors for oral cancer, (3) opportunity to examine oral lesions, (4) knowledge regarding prevention and detection of oral cancer, (5) point of referral selection, and (6) desire for further information or teaching regarding oral cancer. two open-ended questionnaires were asked to identify the risk factors for development of oral cancer and encouraged to select at least three to four options out of 10 options. in addition, interest of preferences for obtaining oral cancer information (1 out of 3 options). the wilcoxon rank-sum test and chi-square were used to identify the difference between groups. a total of 162 students were approached, and 114 questionnaires were returned with an overall response rate of 70.3%. eighty-eight were females and twenty-six were males with a mean age (standard deviation) of 24.36 (7.12). sex distribution with the number of respondents per year of course was shown in table 1. when asked about the examination of oral mucosa of the patients, all the students answered yes during their clinical training. of those who examine the oral mucosa routinely, a high majority of the students (97.3%) would not examine the oral mucosa of the patient with high risk of developing oral cancer. significantly, 67.5% of the students did not get opportunity to examine the oral lesions (= 15.892, df=2, and p=0.000) (table 2). more than ninety percent of the participants preferred to refer patients with oral lesion as a point of care to dental specialties rather than doctors. however, significantly, two-thirds (65.7%) of students felt that they did not have sufficient knowledge about prevention and early detection of oral cancer. this was much higher observed in third-year student participants (42/45) than others (= 28.598, df=2, and p=0.000). of note, most of the study participants (95.6%) requested further information regarding oral cancer prevention and early detection, with more than fifty percent preferred to obtain in the form of information package (52.6%), twenty-eight percent through seminars, and nearly twenty percent as lectures (figure 1). a majority of the dental students (93%) identified a number of different risk factors for oral cancer were shown in figure 2. all the participants identified poor oral hygiene as a major risk factor, whereas 70.7% identified diet with low vitamin c levels as a risk factor for oral cancer. in addition, only 14.5% of the participants identified alcohol and chewing beetle leaves as a risk factors. however, other oral cancer risk factors such as immunosuppression, viral infections, and occupational hazards were poorly reported by the final year students, and none of the other students identified these as a risk factors. squamous cell carcinoma accounts for 90% of oral cancer and it is a general practice of the dental students to examine the patients ' oral mucosa. providing opportunity to examine and early detection can reduce the morbidity of oral cancer especially in high risk patients. it is the prime responsibility of the dental schools to provide sufficient knowledge to students for early diagnose in asymptomatic patients and prevent prevalent oral diseases. hence, this study was carried out to determine the level of knowledge and awareness of oral cancer among dental students at international islamic university, selangor, malaysia. the response rate of the current study was 70.3% which is much lower than the studies conducted on dental students in croatia (95%), india (90.6%), iran (88%), and brazil (75.1%) but fairly higher than the similar studies conducted in other specialties. although the response rate was low, a comparable number of students from different academic years participated in the study. in the present study, further, it was identified that a large majority of the students had an opportunity to examine patients with oral lesions. but unfortunately, a high majority of these students claimed that they failed to screen the high risk patient groups, which implies the gaps in their knowledge regarding oral cancer risk factors. poor knowledge is directly related to lack of awareness, and emphasis should be taken to provide more opportunities engaging undergraduates to take oral health histories and examine oral lesions in patients during clinical attachments that should be undertaken. it is arguable that majority of the oral cancer patients are asymptomatic and identifying the changes in their cancerous and precancerous lesions in the oral cavity would help them to apply their critical knowledge into practice, importantly needed in high prevalent countries like malaysia. for such reasons, ogden et al. claimed to implement work-based assessments to know these gaps and specific test for oral cancer within the curriculum prior to dental students graduation. regarding referral pattern for oral cancer, more than ninety percent felt that it is the dentist's responsibility to diagnose the oral malignancies. these results are encouraging as they demonstrate the recognition of dentistry, and it is their responsibility of dentists to diagnose and evaluate oral cancer. these results were consistent with other studies conducted by ogden and mahboobi, awan et al., carter and ogden, and fotedar et al. but contradict with study by brzak et al. where majority of the undergraduate dental students chose to refer oral cancer patients to a plastic surgeon specialist. a recent meta-analysis concluded that diagnosis delay is a potential risk factor for developing advanced stage oral cancer. in our study, the majority reported that they would advise their patients about oral cancer and associated risk factors after graduation. these findings were similar to the previous study performed in malaysia, uk, and croatia. it is crucial role of dentists to take a strong responsibility to offer advice to the patients on high-risk habits like cessation of smoking cigarettes and self-examination of oral mucosa to improve the oral hygiene. these counseling techniques also enhance early detection of oral mucosal changes in the oral cavity. approximately, seventy percent felt that they are have insufficient knowledge (p<0.001) with regard to prevention and early detection of oral cancer. these numbers are higher in those who were in third year (36.8%). in mccready et al. 's study 77% of dental students from second year and fourth year reported that they were poorly informed regarding oral cancer, whereas in carter and ogden's study 93% of the final-year medical students also reported the same. a well-designed institutional-based clinical training by incorporating different dental specialties such as oral medicine, dental oncology, and oral and maxillofacial surgery to improve the knowledge about oral cancer however, almost all the students requesting further information regarding oral cancer which is similarly identified in studies by awan et al, brzak et al, and mccready et al where more than 90% of the students requested to receive more information regarding oral cancer. further, majority of the students are interested in receiving further information in the form of information package which is also most preferred in other studies [1, 9, 10, 12]. study assessing the oral cancer prevention and clinical attitude among spanish dentists highlighted that providing continuous education through scientific newsletters and journals can provide positive preventive attitude in oral cancer. in our study, 106 out of 114 participants identified the risk factors for oral cancer. of these, all the students felt poor oral hygiene as the single most important risk factor. only sixty percent of students identified tobacco smoking as a risk factor for development of oral cancer. although previous studies revealed that smoking tobacco and alcohol consumption increase the incidence of oral cancer, these were unidentified by our third-year dental students. these findings were contradictory with other studies in the literatures, which show that around 90% of the dental and medical students identified tobacco smoking as an important risk factor 12,58 [1, 9, 10, 1214, 17, 19]. however, alarmingly, none of the third and fourth years identified alcohol, beetle-chewing, and immunosuppression as a risk factor. thus the knowledge on risk factors was poor in both third and fourth years and also very minimal in final-year dental students. there was trend towards better identification of risk factors which was observed with progression of their academic years, which is similarly noticed in other studies [9, 11, 14]. all these findings identified different knowledge gaps in identification of risk factors among dental students, and there is a need of educational intervention by implementing training or workshop particularly focusing on oral cancer. we used a prevalidated questionnaire which is used in other surveys assessing oral cancer knowledge [9, 11] on dental students to reduce selection bias. further, recent research identified that nearly 2030% of the oropharyngeal squamous cell cancer did not have traditional risk factors of smoking and cancer which may be falsely interpreted in the light of respondents ' knowledge. the study was conducted on senior dental students in a single institution in malaysia and may not be generalized to other regions. in addition, the data presented here is self-reported, and some of the respondents may provide extreme responses than others, due to the motivations and beliefs of the participants, and might be subjected to recall bias. however, we believed that the participants were honest to provide appropriate responses conducted in a single institution, and national level multifaceted studies are further needed to assess dental students ' knowledge about oral cancer. lack of awareness about the risk factors initiates the need based educational interventions among future dental practitioners regarding early detection and prevention of oral cancer in malaysia.

objective. to assess knowledge and awareness of oral cancer in the early identification of risk factors among undergraduate dental students. methods. a total of 162 undergraduate (third, fourth, and fifth year) dental students at international islamic university, malaysia, were approached to participate in the study, and those who agreed were administered. a 9-item pretested questionnaire contains questions on oral examination, oral cancer risk factors, and requests for further information. descriptive statistics were conducted using chi-square testing. results. the response rate of the study was 70.3% (114/162), with 26 (22.8%) males and 88 (77.2%) females. all undergraduate dental students were familiar with examining the oral mucosa of their patients and most were likely to advise patients about the risk factors for developing oral cancer (98.2%). nearly one-third (32.4%) of students reported examining patients with oral lesions as early signs for oral cancer (p<0.001) and nearly 70% agreed that they did not have sufficient knowledge regarding the prevention and detection of oral cancer (p<0.001). in addition, more than 95.6% agreed that there is a need for additional information/teaching regarding oral cancer. further, 61.3% and 14.1% identified tobacco smoking and drinking alcohol as major risk factors for developing oral cancer. conclusion. this study demonstrated lack of awareness about risk factors among undergraduate dental students regarding oral cancer. reinforcing awareness and enhancing the benefits of early detection on prevention of oral cancer should be done through training and/or educational intervention.

PMC4709628

pubmed-979

globally, approximately 40% of households rely on solid fuels including wood, dung, grass, coal, and crop residues for cooking. the 2010 comparative risk assessment of the global burden of disease attributed 3.6 million deaths yearly to the harmful byproducts of solid fuel combustion for cooking and an additional 0.3 million deaths from contributions of household air pollution to ambient air quality. while the proportion of households using solid fuels appears to be declining, most efforts to mitigate this health burden have focused on providing biomass-burning stoves that vent pollution outdoors and/or improve combustion efficiency to reduce emission rates. increasingly, some are focused on providing access to clean energy for cooking including electricity or liquefied petroleum gas. several conditions must be met if household energy interventions are to improve health: continuous access to a low-emissions energy source for cooking, sustained usage of this energy source, and discarding of the more polluting traditional stoves. stacking is well-documented through surveys, though little objective continuous monitoring of usage of multiple cooking appliances during intervention studies has occurred to date. in palwal district, haryana, we provided a fan-assisted, advanced cookstove, with modifications to improve combustion efficiency (not just improve fuel efficiency or vent pollutants outdoors), to pregnant women via local antenatal healthcare system workers. preliminary research evaluating potential interventions and describing this community has been published. during this initial work the philips hd4012as suitable, despite requiring access to power for battery charging and the need to chop the biomass fuel into small pieces. among other goals, this study evaluated the use of the intervention and primary traditional stoves over time and investigated predictors of usage. monitoring usage and adoption of intervention stoves traditionally relied on simple metrics obtained through interviews or by a trained observer. due either to recall bias or to the influence of an outsider in the home (the hawthorne effect). recent work in rwanda, for example, highlighted that usage estimates obtained from surveys were biased upward relative to objective measures from electronic sensors. these biases have been well described in water and sanitation studies, including recent evidence showing significant effects of structured observation on behavior and attempts to address these issues using simple data-logging sensors. previous studies of household energy identified maxim ic s ibutton technology as an objective, field-validated stove use monitor (sum). ibuttons are small, coin-shaped thermometers that log time-resolved instantaneous temperatures at the surface upon which they are mounted. properly placed, ibuttons offer both an objective measure of stove usage and a relatively unobtrusive way to monitor interventions over time. this paper describes time-trends in usage of the intervention and primary traditional stoves in rural indian homes. we examine how well short-term measures (1, 2, and 7 day mean measurements) of stove use predict study means, with the goal of optimizing sampling times and strategies for monitoring household energy interventions. we believe the data set described in this paper is the longest and deepest data set of measured stove usage generated to date, spanning over 15 months of monitoring at 10 min intervals on both intervention and primary traditional stoves in 200 homes (21 million data points). measuring multiple stoves required creation of new metrics to characterize shifts in usage patterns over time. our secondary focus on reducing total monitoring duration for assessing use, without compromising data quality informs strategies to optimize the conflicting goals of precise measurements and efficient fieldwork. this study took place approximately 80 km south of new delhi at the international clinical epidemiology network (inclen) somaarth demographic, development, and environmental surveillance site in palwal district, haryana, india beginning in november of 2011 and ending in march of 2013. at the time of the study, inclen was carrying out demographic and environmental surveillance in 51 villages, covering a population of approximately 200 000. during the study, ambient temperatures varied widely by season, reaching a maximum of 45 c in may and a minimum of 4 c in january (supporting information, si, figure s1). temperature data were logged every minute by the project meteorological station (onset microstation, onset computer corporation) at the inclen field headquarters in palwal town, between 5 and 12 km from study villages. a metal sheet stamped with a unique identifier and machined with a hole was used to securely hold each stove use monitor. the current study focused on 7 rural villages, selected based on their use of biomass for cooking, total population, and their accessibility to the somaarth field headquarters. all households recruited into the study used dung, wood, and crop residues in a traditional hearth (figure 1a) as the primary means of cooking. nearly all homes (n=200) cooked outdoors. the philips hd4012 (figure 1b) is a top-loading, fan-assisted semigasifier stove fueled by small wood pieces 5 cm in length and up to 2.5 cm in diameter. it contains a rechargeable battery that powers a fan used to enhance combustion efficiency. initial selection of the philips stove was based on its performance in laboratory testing by the u.s. epa, which found it to be among the cleanest stoves evaluated using standard simulated cooking methods. field emissions from this stove were evaluated by other research projects in india and our research team validated this stove s acceptability in the community prior to this project. at the time of the study, the stove was produced in ghaziabad, india, and sold for approximately 60 usd. participants who received the philips stove were trained on proper stove use and maintenance by community health workers and inclen field staff. contact information for inclen s field office, which was equipped with spare parts and had access to trained technicians and electricians, was provided to participants in case of any stove malfunction, error, or other user complaint. complaints could be filed during regular household visits by inclen field staff, through calls to inclen, or by visiting the field headquarters. upon receipt of a complaint, repair attempts were undertaken first by inclen support staff and then, if necessary, by electricians. a supply of replacement stoves was available to avoid prolonged interruption in homes with stove failures. detailed logs of stove reliability, malfunction, and maintenance were maintained by inclen field staff (see the si). upon enrollment into the study, field staff obtained informed consent, administered a baseline questionnaire, and installed a sum on the primary traditional stove in each participant s household. sums were placed in a custom-made metal holder and plastered onto the traditional stove side wall with the same slurry of mud and water used to construct and repair stoves. the holder and a sum can be seen in the inset image in figure 1a. the selected sums placement location did not disturb standard cooking practices, was protected from overflow and spills, and captured variability in temperatures adequately. stoves varied in shape and size between households; sums were placed in approximately the same location on each stove throughout the study. within 4 weeks after preintervention monitoring began, the philips intervention stove, prefitted with a sum (visible in figure 1b), was delivered to the home. a custom-made metal bracket, stamped with a unique stove i d, was used to hold the sums in an identical location on all intervention stoves. field workers visited homes every 2 weeks to inspect stoves and download data from the sums. touch and hold probe connected to a usb to 1-wire rj11 adaptor (maxim integrated, san jose, ca, u.s.a.). data transfer took approximately 25 min per stove and involved holding the probe to the surface of the ibutton. stove usage files were transferred to the field office, where they were inspected for errors and minimally processed. filenames contained metadata, including stove type (philips or traditional), household i d, and download date. raw files were archived at the field site and at inclen headquarters in new delhi. cleaned files were transferred to a secure server in the school of public health at the university of california, berkeley, and analyzed using r 3.0. approximately 20.6 million sums data points were collected during the main study, representing 143 000 stove-days of data from 408 stoves. the number and duration of usage events, derived from raw sums temperature traces, were determined for each stove on each monitored day. algorithms for processing sums data were created using an iterative process, beginning with recommendations from the literature that identify events by setting thresholds for the rate of increase and decrease in temperature. due to the high variability in ambient temperatures in palwal, we took advantage of our continuous ambient temperature measurement to adjust for diurnal variation. to compensate for variability in temperatures between households and the field office, we calculated the mean and standard deviation of ambient temperature by each recorded hour during the study. these values were used to create thresholds for evaluating whether a stove was in use or not. for each stove, the daily recorded sums temperature range (drange) sums data were then merged with data for mean hourly ambient temperatures (hmean amb) and their standard deviations (hsd amb). a stove was considered in use when the sums temperature exceeded the mean ambient temperature plus 6 times its standard deviation. any period detected for which the drange was less than 20 c was marked as a period of nonuse. to count the total number of daily uses, periods of use that occurred less than 40 min apart this clustering threshold was based on manual observation of temperature traces. for each stove, summarized data were analyzed to understand trends in usage of both the traditional and intervention stoves. all analyses were restricted to households for which we had at least 2 days of preintervention data (n=177). analyses were performed separately (1) for the entire data set for these households and (2) for days on which data were successfully collected from both traditional and intervention stoves (see the si). the proportion of stove use-time spent using the philips intervention stove was defined as follows:1where prop is proportion and dur is duration. all durations were calculated in minutes. while the proportion of time spent using an intervention is useful to track adoption, it does not take into account gains in efficiency of heat transferred to the pot by the intervention stove, leading to shorter cooking times, and thus, we linked durations of cooking derived from the sums with cooking power from laboratory studies to determine the utilized cooking energy (uce) in megajoules (mj):2where st is the stovetype. calculation of uce allowed estimation of changes in total energy used before and after deployment of the intervention. laboratory cooking power estimates were derived from controlled burning for water boiling using uniform wood fuel and may not be representative of conditions in the field, where multiple biomass fuels of varying moisture contents may be used. the metrics described above were used to create a log of daily household usage, including the number of uses, duration of use, and estimated energy used by each stove. overall trends in use of the traditional stove before and after introduction of the intervention were compared using t tests. we evaluated the change in daily mean traditional stove use after introduction of the intervention using linear mixed models to partition the between- and within-household variance components and to calculate the intraclass correlation coefficient (icc, the proportion of variability explained by between subject differences). models took the following form:3where yij is the i duration of use in household j, 0 is the overall intercept, bi is the random effect for household i, and eij is the leftover error. this baseline model was run first for the combined data set and then separately by period (preintervention and post intervention) for the traditional stoves. we additionally evaluated how well short measures of usage predicted the study average during stable periods of usage. this analysis was restricted to the traditional stove, which exhibited stable use patterns, and was performed independently for the pre- and postintervention periods. we calculated means from varying lengths (1 day, two consecutive days, two random days, 1 week, and 1 day per month) of usage data selected randomly from each household and study period and compared it to the mean duration of use for the entire study period. for these shorter measures, we calculated the probability of a random measurement falling within a precision interval (for instance, within 20% in either direction of the period mean). during the preintervention period, usage of the traditional stove was measured in 177 homes for, on average, 34 days (sd=35, range=3103). in this period, households used their primary traditional stove 1.4 times (sd=0.8) for an average of 209 min (sd=105) per day. after introduction of the intervention, the traditional stove was monitored for, on average, 251 days (sd=97, range=52426); the philips stove was monitored for, on average, 358 days (sd=54, range=139433). during the postintervention period, households exhibited a significant mean decrease in the use of their primary traditional stove to 144 min per day (p<2.2e-16, sd=134) once daily. the intervention stove was used, on average, 0.6 times daily (sd=0.8) for 60 min (sd=87) after its introduction. panels show temperature traces for the traditional stove (blue dashed line) and for the philips stove (solid red line). figure 2 shows patterns of the transition between traditional stoves and the intervention stove, as illustrated by data from two study households. in both panels, the dotted blue line is the sums trace from the traditional stove; the solid red line is the trace from the philips. pre- and postintervention patterns of use are shown. in the upper panel (mixed use), the philips is used upon introduction repeatedly over the course of a week concurrently with traditional stove use. philips use declines and tapers off in the final week. in the lower panel (philips use), a third pattern, in which the philips was rarely or never used, was observed but is not displayed. these types of patterns were typical of the larger population during the first month after introduction of the stove. use patterns during the first through third months postintervention in homes with sums data available on both stoves for at least 15 days per month are described in table 1. during the first month with the philips, almost all homes used both stoves (n=152). 6% of homes used the philips exclusively (n=9); only one home did not use the philips. among the homes using both stoves, the philips accounted for greater than 80% of cooking events in 17% of homes subsequent months exhibited wide variability between and within homes (see the si). among the 9 homes that exclusively used the philips during the first month, average use of the philips decreased from 111 min daily during the first month postintervention to 78 min daily across the remaining months. additionally, all households exhibited multiple days during later months in which neither stove was in use, suggesting that food was obtained by other means (from relatives or purchased), cooked in alternate locations, or cooked using stoves not fitted with sums. similar trends were noted for homes exclusively using the philips in months two and three. no use of either stove recorded. the variability in usage of the intervention and the lack of displacement of cooking tasks from the traditional stove to the intervention is emphasized at the study population scale in figure 3. between introduction of the intervention and postintervention day 200, there is a significant and consistent decrease of 0.28 min/day in use of the philips (p<2e-16); between day 200 and the end of monitoring, usage stabilizes but continues to decrease by 0.04 min per day. similar trends were noted for daily use event counts over time (see the si). most of the total variability in usage across stove types was due to variability within homes: 66% across periods for traditional stoves and 78% for intervention stoves. the total variability was highest for traditional stoves in the postintervention period, perhaps indicative of either a shift first to and then from the philips or mixed use of both stoves. si table s3 shows the means of use duration overall and by stove type and period and presents the calculated iccs, the proportion of variability explained by differences between subjects. prior to the intervention, households utilized 15.5 mj of energy per day (sd=1.5) from cooking with their traditional stoves (figure 4). after introduction of the intervention stove, utilized cooking energy from the monitored traditional stove decreased significantly to 10.6 mj per day (sd=0.86, p<2.2 10). in the first month after introducing the intervention, however, total average utilized energy increased to 21 mj daily, due to use of both stoves. counterintuitively, perhaps, decreasing usage of the more efficient philips in subsequent weeks led to decreasing total energy use. assuming the rate of energy consumption of each stove remained constant throughout the study, the average daily utilized energy across the postintervention period increased to 16.3 mj (p =0.003). the utilized cooking energy is presented separately for the traditional and intervention stoves (blue and red, respectively) and pre- and postintervention periods. of cooking duration1 day, 1 day per study month, 2 random or 2 consecutive days, and one consecutive week to predict mean stove usage of the traditional stove during the pre- and postintervention periods. these periods for the traditional stove were selected because they exhibited relative stability over time, as compared to the philips. short measurements had a low probability of predicting the study-wide mean of stove usage. precision varied across the pre- and postintervention periods (si figure s5 and table s4). short-term measures adequately predicted preintervention means with traditional stoves. during this period, a consecutive week of sampling had the highest probability (75%) of being within 20% of the long-term mean. just 18% of random single days were within 20% of the long-term mean for the traditional stove. the mean of samples taken for 1 day per month postintervention had a 66% chance of being within 20% of the long-term average. we report on the usage of an intervention stove distributed to 177 pregnant woman and related changes in use of the traditional stove over approximately 60 weeks in rural india. the data set consists of one of the largest and longest objective measurement campaigns of stove usage to date. by deploying stove use sensors for over a year, we were able to track and report for the first time the changes in usage of an advanced cookstove intervention and the primary traditional stove over time. few algorithms for converting temperature traces to event counts and durations of use have been published. we offer a novel analysis method: usage events defined as periods that deviate from ambient temperatures. this method does not rely on any additional assumptions about the distribution of the data and facilitates relatively fast analysis of large volumes of data. it does, however, require local measurement of ambient temperature, which can introduce additional cost. we focus on durations of use, as we believe this to be a more health-relevant metric and a better indicator of potential risk than number of events, which can be easily obtained from duration data if needed (see si). further evaluation of this algorithm is ongoing on both previously collected and new sums data sets. we are additionally investigating the feasibility of household or village level ambient sums to aid with signal processing and to account for microclimatic variability not captured by a single, meteorological station. finally, we are monitoring usage on many different stove phenotypes globally; these activities will help optimize sums placement practices, and evaluate and hone the described algorithm to determine its broader applicability. we see a need for standard methodologies for interpretation of ibutton signals that cater to specific research or programmatic goals. daily time of use and number of uses are simple metrics obtainable from sums data through a number of methods. interstudy comparisons of usage may be complicated, however, by the algorithm design decisions used to generate these metrics. for instance, time-of-use is impacted by the threshold at which the stove is no longer considered to be on; the number of uses is similarly affected by decisions about clustering of temperature peaks. clear specification of algorithm parameters ideally in the form of open-source code and evaluation of algorithms in multiple studies can help clarify differences between methods. we found continuously decreasing population trends in usage of the intervention stove over time. this trend leveled off between 175 and 200 days postintervention. while usage of the intervention had not completely ceased at the end of data collection, the number of homes using the intervention stove regularly and the related durations of use were lower than immediately after stove distribution. our findings are supported by other studies that have (1) indicated stacking of devices throughout the adoption process and (2) acknowledged a trial period during which the household evaluates the suitability of the intervention. utilized cooking energy showed similar trends, with an increase in total uce following introduction of the philips followed by a leveling off and stabilization. future studies should focus on similar calculations to understand if there is a setting, addition of the advanced stove seemed to increase overall energy use, perhaps because the users took advantage of an additional stove to provide more cooking services rather than substituting the philips for the traditional stove. any future studies seeking to calculate uce should evaluate cooking power in the field, as laboratory and field stove performance parameters often vary widely. because we relied on these laboratory estimates and applied them uniformly over the study period, we may be mis-estimating the actual utilized cooking energy. our findings indicate that the philips may have temporarily offset a portion of measured traditional cookstove usage, albeit in a way that may have increased total energy use. despite this continued use of the philips, however, it failed to become the dominant stove used in the home, as would be necessary to maximize health protection. importantly, without measurement of usage of both the primary traditional and intervention stoves, we would have been unable to make any determinations about the role of the philips as an added cooking appliance in household cooking. finally, we would not have observed the initial uptick and subsequent decrease in uce after introduction of the intervention. the high within-household variability of daily usage of both stoves especially in the postintervention period indicates that care must be taken when using short-term measures of usage to predict long-term means. this stands in stark contrast to previous work in guatemala, where the majority of variability was found to be between households. most likely, this is due to the difference between the character of the intervention in guatemala, which was well-known to the community and locally created, and the intervention in india, which while vetted in the community was an engineered object brought in from elsewhere. continuous measurements allowed us to evaluate the ability of short-term measurements to predict the long-term mean. short-term measurements of one or two consecutive days did a poor job of predicting the long-term mean, with the majority of measurements deviating from the mean by over 20%. for instance, one 24 h measurement per month of the study were much closer to the long-term mean. these findings suggest that future intervention studies should measure stove usage regularly to capture inherent variability in household behavioral patterns and to best capture changes in usage over time. given that short-term measures fail to accurately predict long-term means in relatively stable situations, their value in dynamic situations, such as the days and weeks following intervention introduction, is limited. attempts to assess adoption and use must track behaviors consistently for longer periods of time. although promoted by village health workers, the stoves were given to participants free-of-charge, which has been shown to impact perceptions of value. participants were enrolled based on pregnancy during the initial phases of the study and may not represent the broader population. cultural cooking practices related to pregnancy may impact adoption of an intervention stove; initial and long-term usage in households without a pregnant woman may be more consistent or significantly different from the patterns we observed. however, as our study population represents a particularly vulnerable group, indications on how they use this free intervention can inform future studies targeted toward similar communities. second, we were unprepared to instrument the other traditional stove types found in many households. while we placed two sensors, one on the intervention stove and one on the participant-reported primary traditional cookstove, it is possible that other traditional stoves were also used during the study period. further, there is possibility of the hawthorne effect: instrumentation of the primary traditional cookstove may have shifted usage to other, unmonitored traditional stoves. among users who exclusively used the philips during month 1 or 2 of the study, we noted multiday periods of inactivity with both monitored stoves in subsequent months, indicative of cooking elsewhere or use of another stove. we believe either of these reasons may account for the higher levels, on average, of traditional stove usage in the preintervention period. as a result of these caveats, our study paints only a partial picture of the true usage patterns in the home. as these secondary and tertiary stoves were reported to be used only for simmering milk or cooking during inclement weather, we do not believe there were wide changes in their use as a result of introduction of the philips. we can not, however, discount the possibility of use of unobserved and unmonitored stoves. the fieldworker burden for this study was high, with a small team of fieldworkers visiting each household every 2 weeks. households were spread over a relatively wide area, leading to significant transit time and costs and fieldworker turnover. similarly, the volume of data proved to be a logistical challenge to manage, clean, and transfer. strict protocols and fieldworker assurances facilitated analyses but could not, inherently, decrease data transfer and processing times. sums on traditional stoves were especially difficult to maintain over long periods of time due to challenges with placement related to overheating and exposure to water (see si). we are exploring alternate measurement techniques including infrared thermometers, thermocouple-based data-loggers, and wireless transmission of data to improve data completeness and fidelity for traditional stoves. comparisons of data measured with sums to participant-reported stove use and perceptions of the philips as a replacement for the traditional stove are in preparation. such comparisons have, in some cases, revealed that reported stove use is similar to measured use, while in other cases reported use exceeds measured use. future intervention studies should focus on long-term objective measurement of stove use and seek a deeper understanding of the individual and community behaviors motivating use or nonuse of an intervention through qualitative methods from behavioral science. stove usage is a critical link between the potential and delivered benefits of intervention programs. monitoring of usage over time is necessary to fully understand the potential for delivery of those benefits; in this study, short-term measurements of benefits immediately after intervention distribution would have been misleading and potentially led to mistaken claims of benefits. the low long-term usage of the intervention stove, while disappointing, is informative. it indicates (1) that preliminary work, while valuable to assess initial feasibility of an intervention, will most likely not predict long-term viability; (2) that measurement of usage of both traditional and intervention stoves is required over time to fully understand and accurately characterize adoption of an intervention and changes in traditional habits; and (3) that a combination of more transformative, aspirational interventions that can fully displace the traditional stove and education and training, to sway participants away from the old stove, will be required to fully realize benefits.

household air pollution generated from solid fuel use for cooking is one of the leading risk factors for ill-health globally. deployment of advanced cookstoves to reduce emissions has been a major focus of intervention efforts. however, household usage of these stoves and resulting changes in usage of traditional polluting stoves is not well characterized. in palwal district, haryana, india, we carried out an intervention utilizing the philips hd4012 fan-assisted stove, one of the cleanest biomass stoves available. we placed small, unobtrusive data-logging ibutton thermometers on both the traditional and philips stoves to collect continuous data on use patterns in 200 homes over 60 weeks. intervention stove usage declined steadily over time and stabilized after approximately 200 days; use of the traditional stove remained relatively constant. we additionally evaluated how well short-duration usage measures predicted long-term use. measuring usage over time of both traditional and intervention stoves provides better understanding of cooking behaviors and can lead to more precise quantification of potential exposure reductions and consequent health benefits attributable to interventions.

PMC4270394

pubmed-980

a 66-year-old caucasian woman with a medical history significant for chronic kidney disease (ckd) stage 3, hypertension, and morbid obesity was followed regularly at our nephrology and hypertension clinic. she was being treated aggressively for her hypertension, proteinuria, hyperuricemia, and secondary hyperparathyroidism (shpt). her baseline creatinine fluctuated between 1.5 mg/dl and 2.0 mg/dl, and egfr (6 variable modification of diet in renal disease) between 24 and 34 ml/min/1.73 m. her home medications included paricalcitol, gemfibrozil, febuxostat, pantoprazole, furosemide, lisinopril, latanoprost ophthalmic, and ferrous sulfate. our patient had never taken warfarin, vitamin d, or any oral phosphate binders. in 2009, her intact parathyroid hormone (pth) levels were noted to be rising despite her receiving oral paricalcitol 1 mcg daily. the dose was eventually adjusted up to 2 mcg daily without controlling her pth which climbed as high as 216 pg/ml. her calcium levels climbed from 9.0 to 10.4 mg/dl but never higher. her serum phosphorous levels ranged from 2.9 to 3.7 mg/dl before, during, and after subsequent treatment. most importantly, the ca po4 solubility product was never greater than 33.7. in august 2010, she presented with painful eruptions and ulcerations along the medial aspects of her thighs and intertriginous spaces of the lower extremities, slightly more on her right side. initially, our patient was concerned that it represented a drug-related exanthem in response to a newly initiated uric acid-lowering agent. accordingly, she had tried to self-medicate using over-the-counter preparations without success. the presence of violaceous ulcerations were identified along the aforementioned distributions, some of which had already begun to drain purulent material. she was prescribed oral doxycycline as well as cleocin-t gel and cultures and biopsy were procured while at the dermatology clinic. complement levels were normal but serum protein electrophoresis found elevation of acute phase reactants suggesting acute inflammation. cultures were unrevealing, however, the biopsy from the right medial thigh lesion revealed dermal necrosis with severe neutrophilic inflammation of the local blood vessels. the biopsy was nonspecific as calcium deposition was not identified, but the clinical picture was strongly suggestive of calciphylaxis. a repeat biopsy was not obtained due to our fear of inciting another ulcerating lesion. furthermore, it has been shown that dermatopathology is most often nonspecific and often needs to be repeated several times to see the typical microvascular calcifications associated with calciphylaxis.1 for this reason, our nephrology clinic generally does not recommend biopsy of these ulcers. at this point she was sent for a three-phase technetium bone scan that was markedly abnormal on blood pool imaging (figure 1). it demonstrated widespread hyperintensities in the soft tissue of her thighs bilaterally corresponding to the suspect lesions, once again more so on her right side. the use of this imaging modality in calciphylaxis has been reported numerous times and has been shown to be abnormal with soft tissue uptake in up to 97% of patients with calciphylaxis, whether ulcerating or not.1,2 as such, she was referred for a technetium sestamibi parathyroid scan, which was suspicious for a parathyroid adenoma on the lower pole of the left lobe of the thyroid. this scan was repeated and confirmed by our surgeon who then performed a subtotal parathyroidectomy the following day. intraoperatively, an enlarged parathyroid gland was identified, corresponding to the abnormality seen on imaging. it was later confirmed to be a hyperplastic parathyroid adenoma by pathology. in the weeks following surgery, the drainage from her wounds begun to wane, margins began to heal, and her pth level fell to 136 pg/ml, though did not fall lower on serial follow-up. it was at this point that her primary hyperparathyroidism was consider to have been definitively been treated, but the patient s background shpt had not. we were reluctant to give her oral vitamin d, or vitamin d receptor antagonists; instead, she was given cinacalcet. within 3 months, her pth had fallen to 38 pg/ml and her wounds had entirely healed. administration of the cinacalcet was not associated with hypocalcemia or significant change to our patient s ca po4 solubility product, which was low to begin with. calciphylaxis (often referred to as calcific uremic arteriolopathy) is a disabling, and potentially life-threatening complication that has been reported to affect as many as 4% of patients with end-stage renal disease.3 it has been observed to occur primarily in patients receiving maintenance hemodialysis and peritoneal dialysis. however, syndromes resembling calciphylaxis have been reported in individuals with pre-end-stage renal disease-staged ckd and even more rarely in those without any evidence of renal dysfunction but with inflammatory bowel disease, malignancy, trauma, and primary hyperparathyroidism.47 this disease entity is characterized by calcification, intimal hypertrophy, and thrombosis of small vessels that slowly lead to tissue ischemia followed by infarction. predominantly affecting subcutaneous tissue, this microvasculopathic process leads to the formation of painful nodules or plaques, which are often described as erythematous or violaceous foci that later erupt as necrotizing, nonhealing ulcers. these lesions are typically distributed in highly vascular regions with thick overlying adipose tissue such as the breast, abdomen, and thighs, although acral and even visceral organ involvement has been reported.4 the formation of these ulcerating necrotic lesions portends a grim prognosis as they are often accompanied by severe infectious complications resulting in mortality rates that can exceed 60%.3,8 several strategies aimed at treating and preventing this affliction in the nondialysis population have been reported in the literature. these include early parathyroidectomy,5,7 sodium thiosulfate,8,9 vitamin d analogs,9 bisphosphonates,10 cinacalcet,10,11 and hyperbaric oxygen.12 unfortunately, none of these maneuvers have been shown to be universally beneficial and the outcome for most patients afflicted with calciphylaxis remains quite poor. we describe a unique case of a patient with stage 3 ckd who presented with calciphylaxis when she developed primary hyperparathyroidism superimposed on background shpt that was actively being treated with paricalcitol. she developed her lesions in the face of escalating dosages of the vitamin d analog despite normal ca po4 solubility products. what is interesting in this case is that her wounds did not begin to heal until her parathyroid adenoma was surgically removed and did not completely heal until her pth level was normalized with cinacalcet. this is demonstrated in a straightforward way as pth is secreted in a constitutive fashion from the parathyroid cells without regard for ambient serum calcium levels, often driving the remaining parathyroid glands into dormancy in the process., the kidney has a decreased ability to generate 1,25-dihydroxycholecalciferol (calcitriol) from its precursor ergocalciferol via the kidney 25(oh) 1--hydroxylase enzyme. phosphate retention gradually occurs and this, in conjunction with reduced calcitriol levels, results in reduced intestinal calcium absorption and decreased serum calcium levels. as the degree of ckd progresses, the cellular expression of vitamin d receptors and calcium-sensing receptors (casrs) decrease within the parathyroid cells. furthermore, the casrs are less sensitized to ambient calcium levels.13,14 these circumstances eventually lead to an increase in pth release and a resultant rise in calcium levels. in the long- term, this phenomenon is maladaptive as it leads to bone demineralization (renal osteodystrophy). in these scenarios, pth is believed by some investigators to enhance coronary artery calcifications15 and lead to the aforementioned microvascular calcification, intimal hypertrophy, and thrombosis, which in turn leads to ischemia and subsequent calciphylactic ulcers.16 more recently, fibroblast growth factor 23 (fgf-23) has been established as an important player in the regulation of phosphate-vitamin d homeostasis. elevations in fgf-23 expression lead to the downregulation of residual renal 25(oh) 1--hydroxylase enzyme activity. this unfortunately potentiates the previously mentioned deficiency of calcitriol production, resulting in enhanced pth synthesis and subsequent parathyroid hyperplasia.17 in our patient, both pathways were operating concurrently. it also explains why she had an excellent initial response to a surgical intervention in the form of a subtotal parathyroidectomy. however, it was not until the calcimimetic agent cinacalcet was added that the pth level normalized followed by complete wound healing. calcimimetic agents activate the casrs, thereby potentiating the effects of ambient extracellular calcium on parathyroid cell function.18 this, in effect, reengages a negative feedback loop and suppresses pth secretion. calcimimetics also upregulate casr- and vitamin d-receptor expression and, in animal models, reduce parathyroid gland hypertrophy.19 in our patient, the use of cinacalcet allowed us to lower her secondary elevations in pth without having to provide her with calcitriol or vitamin d analogs, which theoretically may have worsened her calciphylaxis. in conclusion, calciphylaxis is a disabling and potentially life-threatening complication most often accompanying advanced degrees of renal insufficiency. a variety of interventions have been proposed to treat and/or prevent this morbid condition; unfortunately, the outcome for most patients afflicted with calciphylaxis remains poor. to the best of our knowledge, this case represents the first reported case of an individual with stage 3 ckd who developed calciphylaxis amidst mixed hyperparathyroidism (both primary and secondary) and was successfully treated with subtotal parathyroidectomy (for pathologically confirmed parathyroid adenoma) followed by cinacalcet (for background shpt). we feel this case is extraordinary because of the patient s development of calciphylaxis in spite of her relatively mild stage of ckd, as well as clinical response to specific therapy targeted at the pathophysiology of both primary and shpt as detailed. based on our experience in conjunction with that reported by others, we feel that administration of calcimimetics should be considered as an important adjunct in the treatment of calciphylaxis.

calciphylaxis is a disabling and life-threatening complication that primarily affects patients who are dialysis dependent. reports have grown in the literature of cases occurring in those who have advanced chronic kidney disease (pre-end-stage renal disease) or in the setting of transplantation. there are also a few reports of cases occurring in those without any form of chronic kidney disease but with primary hyperparathyroidism. this disease entity is characterized by calcification, intimal hypertrophy, and thrombosis of small vessels that result in necrotizing, nonhealing ulcers many of which are life threatening. although several strategies aimed at treating and preventing this affliction have been reported in the literature, the outcome for most patients with calciphylaxis remains quite poor. we describe a patient with comparatively early stage-3 chronic kidney disease who developed calciphylaxis in the setting of both primary and secondary hyperparathyroidism. predictably, after subtotal parathyroidectomy, her wounds did not completely heal and her biochemical markers of hyperparathyroidism did not completely normalize until her underlying secondary hyperparathyroidism was treated medically. it was only after initiating cinacalcet that the patient experienced complete wound healing and resolution of her calciphylaxis. it also supports other authors findings that cinacalcet may be an important adjunct in the treatment of calciphylaxis.

PMC3259692

pubmed-981

the success of this therapy is evident between 19401970, where twenty novel classes of antibiotics were discovered. these antibiotics vary, concerning their structure and mechanism of action. today, many of these drugs are not so effective because bacteria develop resistance, revealing a major challenge for our society. these proteins cleave the antibiotic, so it can not reach and interact with its target site. this is seen in streptococcus pneumonia and is possible because the bacteria obtains dna from other bacteria via recombinational events. the third way resistance to antibiotics occurs is by targeting new sites, e.g., methicillin-resistant staphylococcus aureus (mrsa). instead of just relying on the original penicillin binding proteins to maintain bacterial membrane integrity, this strain of bacteria obtained dna from an unknown bacterial donor. it has a new gene called meca which codes for an alternative protein, called penicillin-binding protein 2a (pbp2a). beta-lactam antibiotics are not capable of targeting these alternative proteins and thus, mrsa infections can be lethal. the last method involves a decreased uptake of the antibiotic, and if it does get into the cell, it is pumped out at a faster rate. these types of resistance are now prevailing in many species and strains of bacteria, in part, because of our propensity to use these agents too frequently. in time, bacteria with these resistant processes will emerge as the predominant form of the bacteria and will be difficult to kill. as we respond to these bacterial survival mechanisms we also inadvertently create drugs, which have the potential to influence other processes, e.g., human behavior. this occurs because our drug discovery process fine tunes itself as resistance develops and we simultaneously develop stereo specific overlaps with naturally occurring biochemicals, altering their actions downstream. in addition to the above concerns in antibiotic development, many of the agents exhibit toxic effects on the host. in part, we surmise, this is due to unique evolutionary relationships that link selective biochemical and molecular aspects of mitochondrial biology to primordial processes in bacterial progenitors. the mitochondrion is an enslaved bacterium, normally producing significant amounts of atp in comparison to glycolysis. it has a similar structure and function to that found in bacteria, and it has a higher level of mutations compared to the nuclear rrna. antibiotics that are supposed to target pathogens will also bind to mitochondria with high affinity and cause side effects. the commonality of these antibiotic-induced side-effects lead physicians to create a term for this phenomenon called antimicrobial-induced mania, or antibiomania, since it can occur in neural tissues due to higher metabolic rates [1921]. we and others propose that mitochondria dysfunction may be part of the core problem for abnormal behaviors induced by antibiotic treatment, e.g., depression, autism, etc. [dysfunctional mitochondria have recently become a center of interest in explaining mental disorders [2833]. ciprofloxacin induces a small percent of treated patients to develop psychosis [3436]. in this regard, gamma-aminobutyric acid (gaba) receptor binding is inhibited by ciprofloxacin. importantly, the 18 kda translocator protein (tspo) localized to the outer mitochondrial membrane, previously designated as the peripheral-type benzodiazepine receptor, has been found to be temporally enhanced in the striatum and substantial nigra pars compacta in a neuro-inflammatory rat model of parkinson s disease or diffuse nerve injury. interestingly, a reversal of repeated social stress-induced anxiety-like behavioral outcomes in rodents has been linked to the off-target peripheral effects of the widely used benzodiazepine lorazepam on tspo activation. more precisely, in vivo positron emission tomography (pet) scanning using the tspo-specific ligand [c]dpa713 has demonstrated enhanced signal in select brain areas due to in vivo microglial activation as a result of aging and neuronal degeneration. interestingly, a subtype a of gaba receptor (gabaa) is regulated by the level of mitochondrial reactive oxygen species(mros) at inhibitory synapses of cerebellar stellate cells. behavioral changes are not limited just to ciprofloxacin, but also occurs with exposure to metronidazole, ofloxacin, trimethoprim-sulfamethoxazole, cotrimoxazole, procaine penicillin and clarithromycin. additional examples of mitochondrial dysfunction, which are antibiotic-induced, are extensive and not limited to psychiatric behavior. aminoglycosides have been used for decades, and they are still considered to be effective for treating bacterial infections. however, there is a high risk of damage to sensory cells inside the inner ear when exposed to this antibiotic due to reactive oxygen species (ros) being released from the mitochondria [15,5155]. another experiment demonstrated that binding of aminoglycosides to the human mitochondrial h69 hairpin is the most likely factor in causing the side effect. moreover, tetracycline also works by manipulating gene expression via the tet-on/tet-off system. in addition to gene manipulation, it will also induce unnecessary stress upon the mitochondria by disrupting translation. therefore, translation-targeted antibiotics must be used with extreme caution, especially in patients that have mitochondrial translation defects. antibiotic-induced mitochondrial damage can be pronounced on neurons, as noted earlier for behavior, especially given their metabolism, which requires 20% of the oxygen entering the body. this phenomenon suggests that aspects of antibiotic activity and cancers may be connected via energy processing. examples are erythromycin, tetracycline, and glycylcyclines, which have beneficial roles in eradicating some cancer stem cell lines while chloramphenicol, a broad spectrum antibiotic, exhibits conflicting results. this drug works through the jnk and pi3k pathways, which lead to a phosphorylated c-jun protein binding to the promoter region of the matrix metalloproteinase-13 region (mm-13). vancomycin is a very potent antibiotic and is prescribed against resistant staphylococcus aureus (mrsa) infections. hmox1, a gene that is associated with cellular oxidative damage is regulated upon vancomycin exposure. exacerbating this event is the fact that antioxidant genes are down regulated, indicating that this potent drug could be increasing oxidative stress in nephrons. despite the danger in administering this antibiotic to kill staphylococcus aureus, the benefit of this drug clearly outweighs the risk of damages that can occur. the long-term effect of this agent on mitochondria has yet to be determined. pseudomonas aeruginosa destroys the cell by releasing pyocyanin, a permeable pigment that targets the mitochondrial respiratory chain. activation of the sphingomyelinase acid and the release of cytochrome c from the mitochondria shortly follow. staphylococcus, on the other hand, secretes a toxin (pvl) that creates holes in the mitochondrial outer membrane of neutrophils and stimulates apoptosis via bax genes. the prokaryotic bacterial organism has evolved, over millions of years, the ability to subvert the innate immune response via mitochondrial processes. this strategy, in all probability, is based on conserved common molecular knowledge. clearly, a good part of the communication is within and external to the cell s organelles, whether it is the eukaryotic mitochondria or prokaryotic ribosomes, this occurs via conformational matching, providing the reason for the mechanism of action. antibiotics can bind to the bacteria cell and cause changes in the bacterial physiological responses, and the efficacy of these antibiotics is limited or enhanced by environmental factors. the relationship that exists between antibiotics and induced ros have been studied through biochemical, biophysical and enzymatic assays. to further prove that ros, e.g., h202, is being produced, the promoters for oxidative stress regulator were analyzed and showed that there was significant activation of these genes due to the treatment of norfloxacin and ampicillin. these results prove extensive ros production is stimulated by antibiotics and strongly suggest that mitochondria, in general, can be involved in the response. the action of bactericidal drugs can be via interfering with the tricarboxylic acid cycle, destabilizing iron-sulfur clusters, so that iron will participate in the fenton reaction that occurs in the mitochondria, producing harmful hydroxyl radicals. hydroxyl radicals from the fenton reaction also will damage nucleotides in bacteria and cause the mitochondria to undergo metabolic stress. a possible alternative to advance future antibiotic development involves targeting fatty acid biosynthesis because of differences found in eukaryotic and prokaryotic cells [8486]. key proteins that can be inhibited in bacteria are, for example, acps, accbcd, fabd, and coaa. these proteins assist in enzymatic activities in simple prokaryotes and inhibit fatty acid synthesis gene expression. platensimycin, platencin, and phomallenic acid appear to destroy gram-positive cocci, such as staphylococcus- aureus, -pneumonia, and enterococcus faecium. relatively recent work demonstrates that some gram-positive bacteria are resistant to agents targeting fatty acid synthesis pathways. problematically, studies show that certain bacterial strains grow better when they get an exogenous source of fatty acids. interestingly, the large microbe population in the enteric system has not been examined for this phenomenon. these microbes may affect the activation state of white blood cells, which can enter the brain compartment and communicate with neurons. since these agents are already in use, their approval status for fda evaluation can be either shortened or exempt. in this case this timely mini-review brings attention to the role that mitochondria play in establishing an environment for normal overall behavior to emerge. pathological perturbations of this process via antibiotics, demonstrate the role this enslaved bacterium performs. a large amount of oxygen consumed, e.g., in the brain, testifies to its moment by moment critical activity. in the shared commonality of chemical communication with bacteria, antibiotic-induced mitochondrial interactions represent a critical factor in micro-environmental and organismic survival. thus, an enhanced microbial presence or antibiotic level may alter the energy supply of a cell and thus enhance the occurrence of an induced behavior disorder. in this case, the potential to initiate mitochondrial dysfunction becomes clear, and this cascading type of action ends in stimulating abnormal behaviors. clearly, antibiotics have an important place in medicine; despite the risk of damage to the host. in this scenario, one may expect alterations in behavior since they will emerge from high-level energy nerve cells. we speculate that in susceptible individuals and ones using these agents for extended periods of time and non-recommended doses, antibiotics may turn an acute stress response into one that is chronic.

clinical usage of several classes of antibiotics is associated with moderate to severe side effects due to the promotion of mitochondrial dysfunction. we contend that this may be due to perturbation of unique evolutionary relationships that link selective biochemical and molecular aspects of mitochondrial biology to conserved enzymatic processes derived from bacterial progenitors. operationally, stereo-selective conformational matching between mitochondrial respiratory complexes, cytosolic and nuclear signaling complexes appears to support the conservation of a critically important set of chemical messengers required for existential regulation of homeostatic cellular processes. accordingly, perturbation of normative mitochondrial function by select classes of antibiotics is certainly reflective of the high degree of evolutionary pressure designed to maintain ongoing bidirectional signaling processes between cellular compartments. these issues are of critical importance in evaluating potentially severe side effects of antibiotics on complex behavioral functions mediated by cns neuronal groups. the cns is extremely dependent on delivery of molecular oxygen for maintaining a required level of metabolic activity, as reflected by the high concentration of neuronal mitochondria. thus, it is not surprising to find several distinct behavioral abnormalities conforming to established psychiatric criteria that are associated with antibiotic usage in humans. the manifestation of acute and/or chronic psychiatric conditions following antibiotic usage may provide unique insights into key etiological factors of major psychiatric syndromes that involve rundown of cellular bioenergetics via mitochondrial dysfunction. thus, a potential window of opportunity exists for development of novel therapeutic agents targeting diminished mitochondrial function as a factor in severe behavioral disorders.

PMC5240889

pubmed-982

pain in the upper trapezius (ut) region may be caused by the performance of repetitive tasks or continuous weight on the shoulder1, 2. brassieres may be a factor contributing to ut pain in women because the weight of the breast causes the brassiere strap to press on the ut muscle4, 5. from their teenage years on, women continuously wear brassieres on a daily basis6, 7. in most cases, the brassiere has parallel straps that go over the shoulders from front to back8. the weight of the breasts causes the parallel straps to cut into the outer shoulder and lengthen the ut muscle, causing pain5. excessive ut muscle activation resulting from continuous weight on the ut region generates myofascial trigger points that cause pressure pain9. generally, clinicians concerned about upper trapezius region pain have overlooked whether or not wearing a brassiere leads to upper trapezius region pain. in addition, previous studies have suggested that wearing a brassiere may be a factor contributing to ut region pain due to the weight of the breasts4, 5, 9. however, the influence of wearing a brassiere on the muscle activity of ut has not been scientifically proven. therefore, the purpose of the present study was to examine the effect of wearing a brassiere on ut region pain and muscle activity during arm elevation by women. fourteen female with ut region pain aged 25 to 47 years volunteered for this study. inclusion criteria were history of ut region pain for at least 6 weeks and a visual analog scale (vas) score>5 (severe pain) at rest. exclusion criteria included past or present neurological pain, cervical spine fractures, radiating pain to an upper limb, and a history of unresolved cancer. the subject s mean age was 34.12 11.62 years, and their mean height and weight were 158.73 4.49 cm and 54.65 6.23 kg, respectively. all subjects read and signed an informed consent form approved by the inje university ethics committee for human investigations prior to their participation in this study. surface electromyography (emg) data were recorded using a delsys trigno wireless emg system (delsys, inc., emg data were collected from the right side ut muscle (approximately half the distance between the seventh cervical spinal process and the acromion). sampling was performed at 1,000 hz, with a bandwidth of 20450 hz, and the root mean square was calculated using emg works 4.0 analysis software (delsys, boston, ma, usa). each maximum isometric contraction maneuver was performed twice for 5 s, and the average muscle activity of the middle 3 s of the two trials was used to normalize the data. pressure pain in the ut region was measured using a baseline dolorimeter (pain diagnosis and treatment, inc., great neck, ny, usa). the instrument consists of a gauge attached to a hard rubber tip 1 cm in diameter. the dial gauge can be calibrated in kilograms (kg) or pounds (lb), with a range of 130 kg or 160 lb. at intervals of 0.25 kg or 0.5 lb. inter-examiner reliability for the baseline dolorimeter is good to excellent (interclass correlation coefficient= 0.750.89)11. prior to testing, the subjects were instructed to indicate when the pressure point was painful. to measure the pressure pain in the ut, the subjects were instructed to sit upright on a chair with their feet on the floor looking straight ahead. the examiner stood and measured pressure pain in the middle of the muscle belly between c7 and the acromion. the right and left sides were each measured three times, and the average was calculated. the subjects were instructed to perform shoulder flexion with scaption. during upper limb elevation, the scaption plane was controlled using a vertical bar. subjects were asked to hold the right side shoulder at an angle of 120 degrees for 5 s. the middle 3 s of muscle activity averaged over three trials was used in the analysis. the pressure pain and emg activity were measured with and without wearing of a brassiere in a randomized order. ut muscle activity and pressure pain between with and without wearing brassiere were compared using the paired t-test. statistical analyses were performed using spss (ver. 17.0; spss, chicago, il, usa). the emg signal amplitude (% mvic) of ut increased significantly (mean sd, 50.87 8.92 compared to 39.79 7.08) when wearing a brassiere compared to the no brassiere condition in the females with ut region pain (p<0.05). the ut pressure pain did not differ between two conditions (p>0.05). previous studies have suggested that wearing a brassiere may be a factor contributing to ut region pain in women9, 10. however, no studies have reported the influence of wearing a brassiere on the emg activity of ut. to the best of our knowledge, this is the first study to demonstrate that a brassiere can increase ut muscle activity during shoulder scaption in women with upper trapezius region pain. our results shows there was a significant 21.7% increase in ut muscle activity when wearing a brassiere. a brassiere transfers the weight of the breast from the pectoral fascia to the ut region, generating a downward force on the shoulder12. to counterbalance the downward force, greater ut contraction is required to elevate the shoulder, resulting in increased ut muscle activity moreover, shoulder scaption resulted in greater contraction of the ut muscle due to the increased leverage of the arm in this study. sustained and repeated ut muscle contraction would quickly generate ut muscle fatigue, resulting in ut tenderness10. therefore, when wearing a brassiere, the ut sustains the small weight of the breast with ischemia induced by the brassiere, which would lower the pressure pain threshold of ut. a previous study suggested that brassiere removal during a day (24 hours) was effective at reducing ut and pectoral girdle muscle pain12. however, in this study, the pressure pain threshold showed no significant difference between the brassiere and no brassiere conditions, because subjects were asked to remove the brassiere for just one minute for no brassiere condition. further study is needed to investigate the effects of brassiere removal during the day on the ut muscle activity and pressure pain threshold of the ut region in women with upper trapezius region pain. the investigation and comparison of various designs of brassiere on the ut muscle activity and pain threshold is also required. a limitation of this study was that we did not measure the breast weight or size of the subjects; different sizes of breast may affect the pain intensity of ut. also, we did not control for the fit of the brassiere that each participant wore. in conclusion, wearing a brassiere can increase ut muscle activity, so clinicians should consider recommending brassiere removal for as long as possible, or suggest wearing a well-fitting and supportive brassiere when managing women with upper trapezius region pain.

[ purpose] this study examined the effect of wearing a brassiere on upper trapezius (ut) region pain and emg activity during arm elevation by women. [subjects and methods] fourteen healthy women were recruited. surface emg data were collected from the ut muscles during arm elevation. pressure pain in the ut region was measured using a baseline dolorimeter. [results] the emg activity of the ut increased significantly when a brassiere was worn compared to without. ut region pain showed no significant difference between with and without wearing a brassiere. [conclusion] this suggests that wearing a brassiere increases the muscle activation of the ut in women.

PMC4210395

pubmed-983

hypertension is one of the most common if not the commonest medical complication in pregnancy.19 hyper-tensive disorders in pregnancy are found to be the greatest single cause of maternal mortality.911 it also causes a lot of prenatal mortality.12 most of the complications caused by this problem could be reduced by early detection and proper management.1314 studies on the incidence of hypertensive disease in pregnancy in most developing countries, including saudi arabia, are scarce.15 few of those studies, if any, have looked at the extent of knowledge, attitude or practice of doctors or nurses dealing with hypertensive pregnant mothers. hence, the objective of this study was to determine the status of knowledge, attitude and practice (kap) of doctors and nurses in phc centers with regard to hypertension in pregnancy and to analyze factors affecting kap in the al-khobar area of the eastern province of the kingdom of saudi arabia. terms used to describe hypertension and its complications in pregnancy differ according to its presentation, gestational age of discovery and the presence of previous history of hypertension before pregnancy. the one defined by the 1972 committee on terminology of the american obstetricians and gynecologists (acog)131519 was adopted for the purpose of this kap study. doctors and female nurses working at all 8 phc centers in al-khobar area were enrolled for the study. two self-administered questionnaires structured to examine the essential knowledge, attitude, and practice of doctors and nurses in dealing with hypertensive disorders during pregnancy. the first was intended to gather demographic characteristics and information on the respondent that might affect the kap. these variables included age, sex, nationality, year of graduation, certification, training and experience in obstetrics, duration of work in phc in the country and any in-service training received. the second part of the questionnaire consisted of 49 questions divided into three sections dealing with practice, attitude and knowledge. the practice sections included questions designed to test competence of doctors and nurses in the skills of measuring blood pressure, management of the hypertensive pregnant mother and practices of health education. the knowledge section was composed of 28 questions to test basic medical knowledge regarding blood pressure reading, management of the problem and its complications. both questionnaires were given to three consultant obstetricians and two nurse educators for consent validity and elimination of non-essential questions. both knowledge and practice areas were scored on a zero and one additive scoring system in which each correct answer or practice was given one score and no mark given for a wrong response. the attitude part included questions that measured attitudes of participants towards the seriousness and commonness of hypertension in pregnancy and their motivation to improve their knowledge on the subject. attitude questions were scored using the five-point likert scaling system. in this scale, a high attitude was assigned to the answer if the respondent's answer was to agree or strongly agree to the question in the scale and a low attitude level for not sure or final scoring of the knowledge and practice section was satisfactory if the participant scored at least 60% of the total marks in these two sections. this was an arbitrary cut-off point based on the pilot study and judgment of the experts. on attitude, the questionnaire was handed over to five postgraduate doctors in general practice and their responses to attitude questions were used as a reference standard. the questionnaire for nurses both versions of the questionnaire were pilot tested on a pilot group of 10 doctors and 22 nurses, selected randomly from phc centers in a nearby city, al-dammam to assess level of difficulty, clarity, suitability and time required for their completion. data were fed into a personal computer, cleaned and analyzed using epi info and spss statistical packages. frequency distribution tables were constructed and appropriate tests including the multiple regression analysis to identify significant independent factors were applied. the total number of phc doctors in al-khobar area was 44. among those 36 (81.4%) who were present at time of data collection their ages ranged between 25-47 with mean of 37.1 years (sd 6.4) and 56% were in the 35-44 years group. arab nationals other than saudis including egyptians, palestinian, and jordanian formed the majority of doctors (41.7%), while saudis constituted 22.2% of the group and the rest were from the indian subcontinent. the mean number of years in practice after graduation was 12.6 years (sd 7.38). distribution of phc doctors according to years of practice after graduation the mean number of years working in phc was 4.7 years (sd 3.4) and 17 doctors (47.2%) had worked for over 5 years in phc centers. only, 4 doctors (11.1%) had postgraduate qualifications; out of these only one held a diploma in obstetrics. five doctors (13.9%) had had 3-6 months post-internship training in obstetrics but no certification. of the entire group, only 2 doctors (5.6%) had had courses in hypertensive disorders in pregnancy in the course of their work. in describing their general attitude towards managing hypertension in pregnancy, a majority of doctors (80%) stated clearly that they did not feel confident in managing hypertensive pregnant mothers and would prefer to see children or adult male patients in their clinic. nevertheless, the attitude of 34 doctors (94.4%) toward learning more about hypertension in pregnancy was positive. out of the maximum attitude score of 45 marks, the mean score for the attitude questions for the group reached a reasonably high figure of 30.69 marks. doctors attitudes towards hypertension in pregnancy doctors management of hypertension in pregnancy was quite deficient as 16 doctors (44.4%) reported that they had not actually seen any hypertensive pregnant mother during last year. around 53% of the doctors depended on bp measurement taken by a competent staff and did not check it. the doctors scores for practice was generally low, as only 60% of them got the correct answers to the skill of blood pressure measurement, a basic skill necessary for any medical graduate. in addition, almost all doctors wrongly reported that they had advised their pregnant hypertensive patients to lose weight and reduce their salt intake. another major mistake noted among 25% of the doctors was that they said they would start treatment of hypertensive cases immediately after diagnosis in their clinic without considering referral. the scores on practice of 75% of the doctors were below the satisfactory cut-of point. again the doctors score of 58.3% on all areas of knowledge was unsatisfactory since this directly affected the diagnosis and quality of care for hypertensive mothers. some of the responses dealt with normal and abnormal readings of blood pressure and the presenting symptoms of preeclampsia. doctors mean score 12.1 marks out of a total score of 21 on knowledge was moderate. the total number of phc nurses in al-khobar area was 120. among these, the 91 (75.8%) who were present at the time of data collection answered the questionnaire. their mean age was 29.8 years (sd 6.3) ranging between 20-44 years, 74% of them fell within the 20-34 age group. indians formed the second largest group (37.4%) followed by filipinos (11%). according to their qualification, 14 nurses (15.4%) held bachelor degrees and 77 (84.6%) had diplomas. mean years of practice after registration was 8.7 (sd 5.4) and their distribution is shown in table 3. mean duration of work in phc was 4.1 years (sd 2.8) and most nurses (69.2%) had spent<5 years working in phc in the country. distribution of phc nurses according to years of practice after registration thirty-seven nurses (40.7%) had spent<6 months in obstetrics training and 10 nurses (11%) had spent>1 year. most of the group (48.3%), mainly the saudis, had minimal training of 3 weeks in obstetrics ward during their internship period as required by the saudi female health institution. a majority of the nurses 73 (80.2%) had not worked in a phc setting before their current positions. out of those who had had previous phc experience, nine of them had had courses in hypertensive disorders in pregnancy during their work in phc. in describing their general attitude towards hypertension in pregnancy, 86% of them reported that it was a common health problem and they were all positive about learning more. they were all keen to talk to pregnant women about their problem and advice them to take bed rest. out of the maximum attitude score of 35 marks, the highest mean score was 30.02 marks. table 4 shows some of the attitude questions and their responses. nurses attitude towards hypertension in pregnancy the nurses were quite good on practice in the area of hypertension in pregnancy, as 95.6% of them scored satisfactory. the mean score of this section on practice was quite high for nurses being 8.3 marks out of a total score of 12 marks. the nurses level of knowledge was very low since only five of them (5.5%) scored satisfactory on the questions in this section. the deficiencies were in areas dealing with diagnosis and quality of care for hypertensive mothers. most of them (96.7%) wrongly considered salt restriction and weight reduction as important in the health education of hypertensive pregnant women. mean scores on knowledge was 9.02 marks out of a total score of 20 marks which was quite low. in the regression analysis of independent factors associated with kap of doctors and nurses, all variables in the first part of the questionnaire such as age, nationality and certification were included in a series of multiple regression equations against each of the dependent variables, namely, scores on knowledge, attitude, and practice for doctors and nurses. there was a positive significant association of the doctors attitude with their sex i.e., male, nationality, being older and previous enrollment in a course on hypertension (r square=0.26 p=0.02). their knowledge was only associated with previous training in obstetrics (r square=0.14, p=0.02), while practices of both doctors and nurses were not associated with any of the factors under study. the attitude of nurses, on the other hand, showed different associations as non-saudi nationals and training in obstetrics had a positive significant effect on their attitude (r square=0.08, p=0.02). their knowledge was only associated significantly with being non-saudi (r square=0.19, p=0.001). hypertension in pregnancy is one of the major causes of prenatal morality and morbidity.20 it is responsible for about 18% of maternal mortality,102122 maternal mortality in hypertensive disorders of pregnancy is primarily due to low standard of care and delay in referral.14 antenatal care (anc) is a major part of maternal and child services in phc. one of the most important functions of anc is to detect high-risk pregnancies and to give them the necessary care. findings from this study will help to identify the status kap of workers in phc on hypertensive disorders in pregnancy. it was discovered in this study that the definition of raised blood pressure and its management was not standardized among doctors and nurses in phc. this is not surprising as previous study by bisson in bristol23 in which a large group of general practitioners, hospital doctors, hospital midwives, community midwives and student midwives who were questioned, gave variety of action plans according to their understanding of diagnostic criteria. they considered the reading of 90 mmhg the model value of diastolic bp at which further action would be taken, whether proteinuria was present or absent. edema was considered a useful indicator by 93% of the respondents and 49% would use ankle edema in their assessment. another questionnaire-based survey by hutton24 on the management of hypertension in pregnancy completed by 65 new zealand obstetricians found that 40 (61.5%) doctors considered the diastolic of 80-85 mmhg, the lowest abnormal reading, at 28 weeks, and by 18 (27.7%) doctors at 36-week gestation. however, 20 (30.8%) doctors considered the diastolic of 90 mmhg, at 28 weeks and 42 (64.6%) doctors at 36 weeks gestation, the lowest abnormal reading. around 47% of phc doctors had spent more than 5 years in phc service in ksa and only 14% of them had had post-internship training in obstetrics but being males had not been involved in anc activity in phc. only 2 had had courses in hypertensive disorders of pregnancy. about 44% of the doctors had not seen any cases of hypertensive pregnant mothers during the last year. questions asked were concerned with four aspects namely, techniques of blood pressure measurements, history and physical examination, health education practice and action management to be taken by the phc doctors on discovered cases. it is obvious that having such little contact with hypertensive cases and lacking the basic training or refresher courses, their management skills will be inadequate. since hypertension is one of the commonest medical complications in pregnancy, it is important to educate the pregnant mother about it. almost all doctors report that they advise hypertensive mothers on the need for bed rest. this is an important non-pharmacological measure in the management of the problem.25 on the other hand, almost all doctors report that they advise their patients to restrict their salt intake and to go on a weight-reducing diet. salt restriction in hypertensive non-pregnant women can be effective but in pregnancy may aggravate the condition. moreover, diet restriction in pregnancy can lead to delivery of small-for-date fetus.25 similar findings of wrong advice were reported in other studies by trudinger26 and bisson.23 around 85% of phc doctors reported that they would not refer hypertensive cases after diagnosis and 25% of them would start medication immediately after diagnosis. in fact, the cornerstone of management of hypertensive cases starts in the phc center with accurate diagnosis and undelayed referral of these cases from the phc to the specialist or hospital.1621 phc doctors should be aware of their limitation in the management of these cases and not to jeopardize the health of the mother and her fetus. around 31% did not recognize this is a common health problem in pregnancy, while 97% of them stressed the importance of taking blood pressure reading at each anc visit. that 53% of phc doctors depended on blood pressure measurement taken by clinical staff without confirming it, the reading themselves is an alarming negative attitude as well as wrong practice. the diagnosis of hypertension in general or in pregnancy in any patient is not an easy task and proper management and modifications depend on it. it is therefore, important that the person responsible for the management, namely, the doctor, should confirm the reading again. in looking for factors affecting the attitude of doctors, it was found that gender i.e., male, arab, older and previous training in a course on hypertension were factors associated with higher scores. longer experience in phc work, being male and older and the absence of a language barrier for arabic speakers, including saudis, might explain to some extent their better attitude scores as compared to those of the females and younger doctors. the knowledge of about 58% of doctors on questions that bare on the diagnosis and quality of care of hypertensive mothers as well as nurses provide most of the vital anc services in the phc. around 53% of the phc nurses were non-saudis and non-arabic speakers and the language barrier adversely affects the health education in hypertensive cases. it is obvious from the results that there is a shortage of specialized courses for nurses. none of the saudi nurses had more than a diploma in nursing and there did nt seem to be any arrangements for their further movement at universities. a period of 3 weeks in an obstetrics ward for new graduate nurses as part of an internship period is not enough to give clear, pragmatic information on anc services in a phc setting. about 31% of the nurses have spent>5 years in the phc centers in the ksa, but only 10% had taken courses in hypertension. this clearly, demonstrates the necessity of a better arrangement for on-job training classes for them. their responses to the question of history and physical examinations were 87% correct and about 90% of them responded correctly to the questions on health education. their erroneous responses were on the items on restriction on salt intake and weight reduction. these good scores indicate that except for their wrong ideas on salt and diet restriction, which should be corrected at refresher courses, the correct procedure have been learnt. although the phc nurses were very good with practice in general, around 95% of them had poor knowledge. knowledge and facts, on the other hand, need to be updated by continuous education. around 86% of them felt that it was a common health problem and reported that they needed to know more about it. the importance of a health education as a vital task for nurses can not be ignored. the presence of these cases in the clinic, therefore, provides a good opportunity for the performance of this task. there was a strong positive association of attitude of nurses to non-saudi nationality and training in obstetrics. while all saudi nurses hold diplomas, about 30% of the non-saudi nurses have bachelors degrees. there is a masked difference in the type, duration and content of curriculum at the pre-graduate level for both groups and non-saudi nurses had higher knowledge scores than saudis. the implication of these findings clearly points out the need to improve the knowledge and attitude of phc nurses through refresher courses. good in attitude questions but had low scores on the practice and knowledge component of the questionnaire. the nurses scored high on practice and attitude but had low scores on the knowledge component. it is recommended that appropriate regular refresher courses on common and serious problems like hypertension be organized for doctors and nurses in the phcs. there should be opportunities for effective training of reasonable duration with clearly defined objectives under proper supervision in good hospitals to improve their knowledge and practice. it would be also appropriate to offer saudi doctors extra incentives for postgraduate study in family medicine to deal with these common problems, and to institute a suitable program of continuing medical education within the health centers for both doctors and nurses. it is also vital to review the curriculum of the female nursing institutions to update both its theoretical and practical content, and extend the duration of training in such common problems as hypertension in obstetrics.

objective: to assess the status of knowledge, attitude and practice (kap) of doctors and nurses in primary health care (phc) centers with regard to hypertension in pregnancy and to identify factors associated with kap in al-khobar, saudi arabia. methodology:using a self-administered comprehensive questionnaire, all available doctors and nurses in phc centers of the al-khobar area were approached to determine their knowledge, attitude and practice in hypertension during pregnancy. questionnaires were validated and pilot tested. each section of the questionnaire was scored and the mean scores calculated. factors affecting each section were identified by means of multiple regression analysis. results:a total of 36 doctors and 91 nurses were enrolled in the study. saudis formed 22.2% of the doctors and 47.3% of nurses. mean years of practice after graduation were 12.6 and 8.7 years for doctors and nurses, respectively. saudi nurses spend only 3 weeks in the obstetrics ward during the whole period of their internship. all saudi nurses hold only diplomas and not many courses on the hypertensive disorder are offered to both doctors and nurses after graduation. the practice of doctors particularly in the management of patients after reaching a diagnosis and educating them on diet and salt intake was poor. furthermore, their knowledge was also poor. though their level of knowledge was poor, the nurses practice was satisfactory, particularly in taking history and physical examination. the attitude of both doctors and nurses towards hypertensive disorders was in general, positive and satisfactory towards health education. nurses nationality and duration of post-internship training were the factors that influenced their attitude and scores on knowledge. conclusion and recommendation: the study revealed that both doctors and nurses working in the phc lacked training and knowledge in this area of their work. it is therefore necessary to give phc doctors and nurses refresher courses on common and serious problems like hypertension. a longer period of training in action management is needed to improve the knowledge and practice of doctors and nurses working in antenatal clinics in this area.

PMC3437072

pubmed-984

the use of implantable cardioverter-defibrillators (icds) is important for preventing sudden cardiac arrest in patients at high risk of fatal ventricular arrhythmias.1) in the past decades, cardiologists paid attention primarily to medical problems and prolongation of the life expectancy of patients with heart failure. recently, the quality of life as well as survival of such patients has been emphasized. therefore, the demand for subpectoral implantation of cardiac implantable electronic devices (cieds) is increasing in young female patients concerned about their body image. we report a case of combined subpectoral implantation of icd and augmentation mammoplasty via the axillary approach in a young female patient with dilated cardiomyopathy and small breasts. a 20-year-old female patient presented to the emergency department because of dyspnea and chest discomfort. transthoracic echocardiogram revealed an enlarged left ventricular dimension and severe global hypokinesia of the left ventricle (ejection fraction, 20%; fig. 1). cardiac magnetic resonance images showed severely decreased left ventricular function and ill-defined delayed enhancement in the septum, both compatible with dilated cardiomyopathy. after 9 months of optimal medical treatment including perindopril, furosemide and spironolactone, cardiac function had not improved. the patient still complained of dyspnea on exertion of new york heart association functional class ii. non-sustained monomorphic ventricular tachycardia was detected on 24 h electrocardiogram monitoring (fig. we proposed two options for icd implantation: subcutaneous or subpectoral implantation via the axillary incision. the patient preferred the latter option, and also requested augmentation mammoplasty for her small breasts. after consulting the plastic surgeon, we performed a combined subpectoral icd implantation and augmentation mammoplasty procedure via the axillary incision. before augmentation mammoplasty, the volumes of right and left breasts were 46 and 56 ml, respectively, as measured anthropometrically. under general anesthesia, skin incisions were performed on both axillary creases, and the plane between the pectoralis major muscle and the rib cage were dissected under endoscopic guidance. two 185 g form-stable gel breast implants (natrelle; allergan, irvine, ca, usa) were implanted into the subpectoral plane of both breasts. because the patient's blood pressure decreased in the sitting position, norepinephrine was infused intravenously to maintain mean arterial blood pressure above 80 mmhg. after bilateral breast augmentation by the plastic surgery team, the left axillary vein was punctured via the seldinger technique. a defibrillating ventricular lead (durata 7120q-58 cm; screw type, st. jude medical, valley view court sylmar, ca, usa) was inserted into the right ventricle via the 9 fr guiding sheath and was stably anchored at the right ventricular apex. an atrial lead (tendril sts 2088tc-52 cm; screw type, st jude medical) the ventricular and atrial leads were connected to the icd generator (ellipse dr, st jude medical). the icd generator was implanted into the subpectoral plane immediately above the left breast implant., volumes of the right and left breasts were 163 and 169 ml, respectively (fig. we present herein a successful case of combined subpectoral icd implantation and augmentation mammoplasty via axillary incisions in a young female patient who was dissatisfied with her body image. cieds have shown to improve survival and symptoms of patients with heart disease, and their use has been increasing.1)2)3) the use of icd implantation in young patients due to congenital heart disease, cardiomyopathy, and genetic disorders, such as a long qt syndrome, has also increased.4) currently, quality of life as well as survival is important. as the number of young patients who require cied has increased, physicians should be concerned regarding not only patients ' medical problems but also esthetic aspects and psychological fitness. in particular, young or female patients are more concerned about their body image and psychosocial distress associated with shock or sudden death rather than older or male patients.5)6) despite the reduction in the size of cieds in the last few decades, routine subcutaneous device implantation in the pectoral area still results in a visible scar and protrusion. furthermore, protrusion at the anterior chest causes awareness of the device and discomfort with daily activities when using purse straps, bra straps, or seat belts. recently, in the united states, the application of subpectoral cied implantation has increased in young female patients having cosmetic concerns.7)8) although there are several methods for cosmetic cied implantation, no nomenclature for these methods has been suggested. we suggest a nomenclature for cosmetic cied implantation based on a combination of three components: incision for lead insertion, incision for generator insertion, and layer of generator implantation. in previous studies9)10) and real-world practice, four types of cosmetic cied implantation have been reported: axillary-axillary-subpectoral, axillary-inframammary-submammary, infraclavicular-axillary-subpectoral, and infraclavicular-inframammary-submammary implantation (fig. 6). the submammary layer lies beneath the mammary glandular tissue and above the pectoralis major muscle. in the present case, we used axillary-axillary-subpectoral implantation. implantation is superior at it results in minimal and invisible scarring. complications of subpectoral or submammary cied implantation are not higher than that of those implanted subcutaneously. obeyesekere et al.10) reported 20 cases of submammary icd implantation, and did not note complications related to the implantation site, such as infection and device migration. during a follow-up of 5 years, the incidence of appropriate and inappropriate shock due to the icd was similar to that reported in other studies. the overall risk of any pocket-related complications was not different between the two groups, and lead complications occurred more frequently in the subcutaneous group.11) patients ' satisfaction and acceptance rates were higher in the submammary or subpectoral group than subcutaneous group.7)9) a generator is implanted between the pectoralis major muscle and the rib cage via an axillary incision. although the device is implanted subpectorally or submammarily, esthetic concerns remain in female patients with low body weight and small breasts. a combined subpectoral icd implantation and breast augmentation surgery has been reported.12) breast augmentation helps conceal the remaining protuberant chest due to the device. a combined subpectoral cied implantation and augmentation mammoplasty procedure is feasible, because the layer of cied implantation is identical to that of breast implantation. this combined procedure is usually performed by cooperation between a cardiac electrophysiologist and a plastic surgeon under general anesthesia. therefore, multidisciplinary care by a cardiac electrophysiologist, a plastic surgeon, and an anesthesiologist is important for combined surgery. in particular, vital signs and heart function monitoring during the operation are essential in patients with heart failure. generator change, icd removal or repositioning from the subpectoral to subcutaneous area also requires general anesthesia. as mentioned above, the complication rates of cied implantation in the subpectoral area and other areas are similar. indeed, in some case series, subpectoral cied implantations not combined with breast augmentation via an axillary approach were performed successfully.13) however, later procedures should be carefully managed, because the icd generator and breast implant were implanted in the same subpectoral layer. cases of combined subpectoral icd implantation and augmentation mammoplasty in asians have not been reported previously. combined subpectoral icd implantation and augmentation mammoplasty via the axillary incision is feasible in young female patients with icd indications and small breasts.

subcutaneous implantation of a cardiac implantable electronic device is the standard method. occasionally, subpectoral cardiac implantable electronic device (cied) implantation via axillary incisions is performed in young female patients for cosmetic purposes. because subpectoral cied implantation and augmentation mammoplasty involve the same layer, it is feasible to perform both procedures simultaneously. we report a case of combined subpectoral implantation of an implantable cardioverter-defibrillator and augmentation mammoplasty via the axillary approach in a young female patient with dilated cardiomyopathy and small breasts.

PMC5054189

pubmed-985

cerebral palsy (cp) is a non-progressive disease with symptoms that include neurological disorders or developmental disabilities. children with cp have spasticity, musculoskeletal problems, mobility disturbances, and decreased pelvic movements that lead to awkward movement and sitting posture1, 2. symptoms also induce unstable posture control, imbalance, and aberrant control of movement. postural control is the ability to control the body position in space, and it has a relationship with the sense of balance3. hippotherapy and horseback riding have been suggested as interventions for correcting the balance problems of children with cp4. two types of horseback riding are available: hippotherapy and therapeutic horseback riding (thr). hippotherapy provides better pelvic, hip, and trunk movement and influences childrens posture, balance, and coordination5. hippotherapy has short-term beneficial effects on the muscle symmetry of the trunk and hip, whereas thr has no effect on muscle tone6. the effects of these two types of horseback riding have been studied in children with cp, and both types have been demonstrated to improve gross motor function and promote better standing and bipedal balance7, 8. a horseback riding simulator (joba, panasonic inc., it was developed to overcome the primary limitations of hippotherapy, such as unavailability and high cost. this device offers an indoor experience of horseback riding and mimics the rhythmic movement of horseback riding, thereby promoting muscular strength and improving sense of balance. the objective of this study was to compare hippotherapy to the use of a horseback riding simulator (joba, panasonic inc., japan) with respect to their effects on the static and dynamic balance of children with cp. our results indicate that a horseback riding simulator has beneficial effects as an intervention for children with cp. this study included 26 children with cp who were receiving physical therapy at the h horseback riding center and the n horseback riding center in kyung-ki in korea. the selection criteria for the subjects were a modified ashworth scale (mas) grade less than+1. children who could perform more than 10 m independent walking and were available for more than 30 min training per day were selected. the parents or guardians of all the participants provided their written informed consent in accordance with the ethical principles of the declaration of helsinki (table 1table 1. general characteristics of subjectshippotherapyhrsgender (m/f)8/59/4age (year)10.81.610.02.2height (cm)125.812.6122.614.3weight (kg)25.26.425.55.7meansd, hrs: horseback riding simulator). meansd, hrs: horseback riding simulator the children were randomly divided into two groups: a hippotherapy group that included 13 children, and a horseback riding simulator (joba, panasonic inc. the two groups participated in 1 hour of exercise per day, 3 times a week, for 12 weeks. the hippotherapy group carried out anterior-sitting, posterior-sitting, and side-sitting exercises for 10 min each while horseback riding at a walking pace (6 km/h). the course leader held the reins of the horse and two side-walkers held the child s legs to assist the child in sitting in the saddle in order to prevent the child from falling and to help the child to exercise during the horseback riding walk. the horseback riding simulator group exercised with the same protective equipment and were assisted by the same leader and side-walkers. both groups received 20 min of conventional physical therapy before hippotherapy and performed stretching on the horse or horseback riding simulator for 5 min before and after the exercise. three physical therapists, who had more than 3 years of experience in pediatric physical therapy and had received education in the experimental method, participated in this study. the subjects static balance ability was measured using bpm (software 5.3, sms healthcare inc., uk) as the center of pressure sway length while standing for 30 seconds with the eyes open and looking to the front. statistically significant differences between the measurements obtained before and after the intervention in each group were determined using the independent t-test. the children were divided into two groups and evaluated pre- and post-test using bpm (software 5.3, sms healthcare inc., uk) and the pbs (pediatric balance scale) (table 2table 2. comparison of measurement values at pre-test and post-testvariablegroupprepostpbs (score)hippotherapy35.63.841.24.7*hrs35.84.738.55.3*sway length (mm)hippotherapy220.127.6135.014.3*hrs219.931.7142.818.8**p<0.05 meansd, pbs: pediatric balance scale, hrs: horseback riding simulator).*p<0.05 meansd, pbs: pediatric balance scale, hrs: horseback riding simulator the hippotherapy and horseback riding simulator groups showed significantly decreased sway lengths in the static balance test. comparison of the pre- and post-test sway lengths showed that the hippotherapy group and the horseback riding simulator group showed significant decreases in sway length of 85 mm and 80 mm, respectively (p<0.05). both groups showed improved static balance, but no significant difference was found between the two groups. dynamic balance was also evaluated pre- and post-test using the pbs (pediatric balance scale). the hippotherapy group showed an increase in score of about 4 points, while the horseback riding simulator group showed an increase of 3 points. both groups showed significant improvements in dynamic balance, but no significant difference was found between the two groups (p<0.05). balance control is very important for children with cp, and maintaining balance requires three distinct sensory system inputs: visual, somatosensory, and vestibular9. generally, the dynamic sense of balance depends primarily on vestibular input on unstable surfaces; however, static balance primarily depends on somatosensory input. many studies have investigated balance and clinical tools for children with cp. a systematic review identified 22 tools that can assess balance10. hippotherapy and thr are types of exercises that affect balance, coordination, and posture, contributing to the development of sensory and perceptual motor skills6, 11. a horse s rhythmic movement, velocity, and variations can facilitate righting and equilibrium actions. during hippotherapy and thr, these facilitations improve muscular co-contraction and postural balance resulting in improvements in the gross motor function of children with cp12,13,14,15. many studies of hippotherapy and thr in children with cp have demonstrated they have positive effects on postural control and balance. the horseback riding simulator has also been reported to be beneficial for children with cp, improving their postural control and global function, and providing enjoyment16. haehl et al. found no significant improvement of in hippotherapy and thr but indicated that hippotherapy had positive effects on the postural control and balance of children with cp17. other studies confirmed that hippotherapy and thr are suitable interventions for children with cp. the horseback riding simulator used in the present study offers several advantages over hippotherapy. the simulator is free from space limitations, has a low price, is easy to handle, and is non-affected by weather conditions16. use of the horseback riding simulator resulted in improved muscle strength and contraction in elderly people18. a previous study of children with cp reported that use of a horseback riding simulator significantly improved their postural control and balance16. our data indicate that both hippotherapy and use of the horseback riding simulator improved the static and dynamic balance of children with cp. however, due to the fast learning characteristics of children, the long-term 12-week exercise program might have been responsible for the similar results seen in balance improvement. many factors remain to be considered regarding exercise for children with cp. therefore, if other tools or exercises were utilized, the results might not match the results presented here. overall, our results indicate that children with cp may respond equally well to the use of a horseback riding simulator as they do to hippotherapy in terms of balance improvement. first, the number of subjects was small, and second, only static and dynamic balance were evaluated. in spite of these limitations, our results show that the use of a horseback riding simulator can be a good intervention for children with cp. further studies incorporating larger sample sizes, various cp types, and full utilization of the programs of the horseback riding simulator might be needed. in conclusion, the horseback riding simulator could be a useful alternative to hippotherapy for improving of static and dynamic balance of children with cp.

[purpose] we with respect to their effects on the compared hippotherapy with a horseback riding simulator (joba, panasonic inc. jp) static and dynamic balance of children with cerebral palsy (cp). [subjects and methods] twenty-six children were randomly divided into two groups: a hippotherapy group that included 13 children, and a horseback riding simulator (joba, panasonic inc., japan) group, which was also composed of 13 children. the two groups participated in 1 hour of exercise per day, 3 times a week, for 12 weeks. the subjects static balance ability was measured using bpm (software 5.3, sms healthcare inc., uk) as the center of pressure sway length while standing for 30 seconds with their eyes open and looking to the front. dynamic balance ability was measured using the pbs (pediatric balance scale). [results] both groups showed significant improvements in static and dynamic balance but significant differences between the two groups were not found. [conclusion] the horseback riding simulator could be a useful alternative to hippotherapy for the improvement of static and dynamic balance of children with cp.

PMC3976017

pubmed-986

animals: all experiments were approved by the animal care and use committee of the faculty of agriculture at the university of tokyo, according to guidelines adapted from the consensus recommendations on effective institutional animal care and use committees by the scientists center for animal welfare. experimentally nave male wistar rats (aged 8 weeks) were purchased from charles river laboratories japan (yokohama, japan). they were housed with three animals per cage in a room with an ambient temperature of 24 1 c and a humidity of 45 5%. the room had a 12-hr light /12-hr dark cycle (lights were switched on at 8:00). all rats were housed separately and were handled for 3 min twice daily, commencing 3 days before the conditioning day. fear conditioning: fear conditioning was performed in an illuminated room between 9:00 and 18:00, as described in our previous studies [11, 23]. a subject from the conditioned group was placed in an acrylic conditioning box (28 20 27 cm) for 20 min, where it received 7 repetitions of a 3-sec tone (cs, 8 khz, 70 db) that terminated concurrently with a foot shock (0.5 sec, 0.75 ma). we also prepared the non-conditioned group by presenting the tone and foot shock separately during a 20-min period. fear-expression test: we performed a fear-expression test as described in our previous studies [11, 14]. a test box (25 25 35 cm) was placed in a dark room illuminated with dim red light. the box had three acrylic walls, one wire mesh wall and a wire mesh ceiling. the wire mesh wall was constructed with 1-cm gauge mesh in the lower section (20 cm) and vertical bars spaced 1 cm apart in the upper section (15 cm), which prevented the rats from climbing up to the ceiling. the subject was placed in the box and kept undisturbed during a 3-min acclimation period. then, a cs was presented five times for 3 sec each at 1-min intervals during the first half of the 10-min experimental period. the behavior of the subjects during the acclimation and experimental periods was recorded with a video camera (hdr-hc9; sony, tokyo, japan) and an hdd-bd recorder (dmr-bw770; panasonic, osaka, japan). sixty minutes after the beginning of the acclimation period, each subject was deeply anesthetized with sodium pentobarbital and intracardially perfused with saline, followed by 4% paraformaldehyde in 0.1 m phosphate buffer. the brain was removed and immersed overnight in the same fixative, and then placed in 30% sucrose/phosphate buffer for cryoprotection. we used the avidin-biotin-peroxidase immunohistochemistry method to detect fos expression, as described previously [15, 16, 19]. briefly, we collected successive 30-m sections from bregma 2.16 mm through bregma 3.24 mm. half of the sections were stained with cresyl violet to confirm the location of the nucleus. the remaining sections were incubated with a primary antibody to c-fos protein (1:7,500; pc38, merck millipore, billerica, ma, u.s.a.) for 65 hr and a biotinylated anti-rabbit secondary antibody (ba-1000, vector laboratories, burlingame, ca, u.s.a.) for 2 hr. the sections were then processed using the abc kit (vector laboratories), and staining was developed by incubating the tissue in a diaminobenzidine solution with nickel intensification. data analyses and statistical procedures: the data are expressed as means standard error of the means (sem). a researcher who was blind to the experimental conditions recorded the duration of freezing (immobile posture, with cessation of skeletal and vibrissae movement, except for that associated with respiration) and the frequency of walking (number of steps taken with the hind paws) using microsoft excel-based visual basic software to record the duration and number of key presses, as in our previous studies [17, 19, 24]. the behavioral data for the conditioned and non-conditioned subjects during the acclimation and experimental periods were compared using a manova followed by fisher s plsd post hoc test. for immunohistochemical analyses of the bla and cea, we counted the number of fos-immunoreactive cells in each bilateral nucleus in all sections. we then compared the total number of immunoreactive cells between the conditioned and non-conditioned groups using a student s t test. when we detected activation of the nucleus, we compared the total number of immunoreactive cells between the right and left sides in each group using a paired t test. in order to assess the relationship between fear and neural responses, the correlation between duration of freezing and number of fos-immunoreactive cells in the bla and cea was analyzed using pearson s correlation analysis. rats were either fear-conditioned (conditioned group: n=10) or non-conditioned (non-conditioned group: n=11) to an auditory cs on the conditioning day. then, 24 hr after the conditioning procedure, each subject was placed in the test box and kept undisturbed for 3 min as an acclimation period. then, during a subsequent 10-min experimental period, five cs tones were presented. as summarized in table 1table 1.behavioral responses during the acclimation periodgroupfreezing (sec)walking (steps)non-conditioned10.9 5.545.9 5.4conditioned3.1 1.254.6 6.0data are expressed as means standard error of the mean., we found no difference between the conditioned and non-conditioned groups during the initial acclimation period (f(2,18)=0.964, p=0.400). in contrast, behavioral responses during the experimental period were significantly affected by the conditioning procedure (f(2,18)=14.3, p<0.01). a post hoc test revealed that the conditioned group showed increased freezing (p<0.01) and decreased walking (p<0.01) compared with the non-conditioned group (fig. (a) duration of freezing and frequency of walking (mean+sem) and (b) the number of fos-immunoreactive cells (mean+sem) in the basolateral complex of the amygdala (bla) and central amygdala (cea) of fear-conditioned and non-conditioned subjects.*p<0.05 according to a manova followed by fisher s plsd post hoc test for behavioral results, and according to a student s t test for fos expression in the bla and cea.). along with these behavioral responses, the conditioned group showed increased fos expression in the bla (t19= 3.29, p<0.01) and cea (t19= 2.30, p<0.05) compared with the non-conditioned group (fig. (a) duration of freezing and frequency of walking (mean+sem) and (b) the number of fos-immunoreactive cells (mean+sem) in the basolateral complex of the amygdala (bla) and central amygdala (cea) of fear-conditioned and non-conditioned subjects.*p<0.05 according to a manova followed by fisher s plsd post hoc test for behavioral results, and according to a student s t test for fos expression in the bla and cea. in the bla (fig. 2.representative photomicrograph showing fos immunoreactive cells in the left (a) and right (b) basolateral complex of the amygdala and in the left (c) and right (d) central amygdala of fear-conditioned subjects.), fos expression was similar between the right and left hemispheres, both in the conditioned (t9= 1.25, p=0.244) and non-conditioned groups (t10= 0.698, p=0.501) (fig. 3.number of fos-immunoreactive cells (mean+sem) in the right and left basolateral complex of the amygdala (bla) and central amygdala (cea) of fear-conditioned and non-conditioned subjects.*p<0.05 according to a paired t test.). in the cea (fig. 2c and 2d), we found that fos expression in the conditioned group was greater in the right hemisphere compared with that in the left hemisphere (t9= 2.75, p<0.05) (fig. fos expression in the non-conditioned group was similar between the right and left hemispheres (t10= 1.25, p=0.241) (fig. representative photomicrograph showing fos immunoreactive cells in the left (a) and right (b) basolateral complex of the amygdala and in the left (c) and right (d) central amygdala of fear-conditioned subjects. number of fos-immunoreactive cells (mean+sem) in the right and left basolateral complex of the amygdala (bla) and central amygdala (cea) of fear-conditioned and non-conditioned subjects.*p<0.05 according to a paired t test. in the fear-conditioned group, we further assessed the correlation between the duration of freezing during the experimental period and fos expression in the bilateral bla (p=0.265), bilateral cea (p=0.683), right bla (p=0.445), left bla (p=0.160), right cea (p=0.557) and left cea (p=0.979). when fear-conditioned subjects were re-exposed to the auditory cs, they showed fear responses, such as increased freezing and decreased walking. along with these behavioral responses, fear-conditioned subjects exhibited activation of the bla and cea, as assessed by fos expression. these results confirmed that the fear-conditioning was successful in our subject group. when we compared fos expression between the hemispheres, fos expression in the cea, but not in the bla, was greater in the right hemisphere compared with the left hemisphere. these results suggest that the right cea is more strongly activated in response to the auditory cs. in the present study, fear-conditioned subjects showed fear responses and an increment of fos expression in the amygdala simultaneously. given that the amygdala plays a critical role in fear responses [21, 22], we assume a causal relationship between these 2 phenomena. however, we can not deny an alternative possibility that the observed difference in fos expression was ascribed to the activation of the neurons that are not related to fear responses, because we did not confirm the type of neurons that showed fos expression. to clarify this however, the correlation was not significant possibly due to the small number of the subjects. therefore, further analyses are crucial to clarify this point. based on the findings in the present and previous studies, we hypothesize that the cea is the region of the amygdala that plays a lateralized role in auditory fear conditioning. in the present study, we found stronger activation in the right hemisphere in the cea, but not the bla. in addition, in our previous study in which we obtained results suggesting a lateralized role of the amygdala, the lesioned brain area included the cea. this hypothesis may also explain why previous studies have not reported a lateralized role of the amygdala. in rats with pre-training or post-training [2, 20] electrical lesion of the unilateral amygdala, differences in fear responses did not occur according to the lesioned side. however, the lesions in these studies appear to have been placed mainly on the bla, such that the damage to the cea was likely minimal. given that the cea mediates fear responses to the auditory cs independently from the bla, it is possible that the residual cea mediated fear responses, which prevented the observation of a lateralized role of the amygdala in these studies. in contrast to auditory fear conditioning, contextual fear conditioning has been reported to be right amygdala dominant. this may be attributable to the facts that an intact basal amygdala is indispensable for contextual fear conditioning. in an above-mentioned study, freezing in response to a contextual cs was reduced when a post-training, but not pre-training, electrical lesion was placed mainly on the right as compared with the left bla. specifically, rats received an electrical lesion of the entire amygdala in one hemisphere and a small chemical lesion of each sub-nucleus of the amygdala in the contralateral hemisphere. then, these rats underwent fear conditioning and were re-exposed to the contextual cs. the authors found that rats showed reduced freezing when the left amygdala and right bla were lesioned as compared with when the right amygdala and left bla were lesioned. given that the cea requires an intact bla, specifically an intact basal amygdala, to mediate fear responses to a contextual cs, it is possible that lesioning the bla was sufficient to impair the function of the ipsilateral cea, which enabled us to observe the lateralized role of amygdala in the present study. consistent with this notion, fos expression in the basal amygdala and cea in response to the contextual cs was greater in the right hemisphere than in the left hemisphere. one possible explanation for the lateralized role of the amygdala is that the conditioning procedure induces more synaptic plasticity in the right cea than in the left cea. some of the neurons that induce fear responses receive both synapses transmitting foot shock information and synapses transmitting the cs information. the foot shock information and cs information strongly and weakly activate the neurons, respectively. when these activations occur simultaneously during fear conditioning, the synapses transmitting the cs information are strengthened, which enable the cs alone to strongly activate the neuron. as a result previous findings suggest that this synaptic plasticity can be more easily established in the right cea than in the left cea. for example, neurons in the right cea show greater activation to pain than those in the left cea. in addition, pain activates the extracellular signal-regulated kinases required for synaptic plasticity associated with auditory fear conditioning in the right, but not the left, cea. therefore, during fear conditioning, it is possible that the pain caused by foot shocks activates a greater number of neurons that induce fear responses in the right compared with the left cea, which increases the number of the neurons that receive cs information simultaneously. as a result, the number of strengthened synapses in the right cea becomes higher than that in the left cea, leading to right hemisphere dominance in fear responses to the cs. in summary, we found that fos expression in the cea, but not the bla, was greater in the right hemisphere compared with the left hemisphere. these results suggest that the right cea shows a greater degree of activation in response to an auditory cs. although lateralized brain function has been reported for both fear conditioning and other phenomena, the biological significance of lateralization has yet to be clarified.

pavlovian fear conditioning is an experimental procedure in which a conditioned stimulus (cs) acquires an ability to elicit fear responses. this type of conditioning depends on the basolateral complex of the amygdala (bla) and/or central amygdala (cea). we previously found that rats showed reduced fear responses to an auditory cs when they were subjected to a pre-training chemical lesion of the entire right amygdala as compared with the left amygdala. based on this finding, we hypothesize that the bla and/or cea in the right hemisphere will be more strongly activated by an auditory cs than those in the left hemisphere. to test this hypothesis, we re-exposed fear-conditioned and non-conditioned rats to an auditory cs 1 day after fear conditioning. we assessed fos expression in the bla and cea in each hemisphere. we found that fear-conditioned subjects showed fear responses, such as increased freezing and decreased walking, as well as increased fos expression in the bla and cea. when we compared fos expression between hemispheres, fos expression in the cea, but not the bla, was greater in the right hemisphere compared with the left hemisphere. these results suggest that the right cea is more strongly activated by the auditory cs.

PMC5095625

pubmed-987

we reviewed outbreak reports to identify outbreaks associated with an imported food from the inception of the surveillance system in 1973 through 2014, the most recent year for which data were available. we obtained additional data for some outbreaks (e.g., country of origin) from the us food and drug administration (fda) and the us department of agriculture food safety and inspection service. we categorized implicated foods by using the schema developed by the interagency food safety analytics collaboration (3). we conducted a descriptive analysis of the number of outbreaks over time, by food category, and by region of origin. during 19962014, a total of 195 outbreak investigations implicated an imported food, resulting in 10,685 illnesses, 1,017 hospitalizations, and 19 deaths. outbreaks associated with imported foods represented an increasing proportion of all foodborne disease outbreaks where a food was implicated and reported (1% during 19962000 vs. 5% during 20092014). the number of outbreaks associated with an imported food increased from an average of 3 per year during 19962000 to an average of 18 per year during 20092014 (figure). number of outbreaks caused by imported foods and total number of outbreaks with a food reported, united states, 19962014. reporting practices changed over time; 19731997, imported foods anecdotally noted in report comments; 19982008, contaminated food imported into u.s. included as a location where food was prepared; 20092014, reporting jurisdictions could indicate whether each food is imported (yes/no) and the country of origin. the most common agents reported in outbreaks associated with imported foods were scombroid toxin and salmonella; most illnesses were associated with salmonella and cyclospora (table). aquatic animals were responsible for 55% of outbreaks and 11% of outbreak-associated illnesses. outbreaks attributed to produce had a median of 40 illnesses compared with a median of 3 in outbreaks attributed to aquatic animals. most of the salmonella outbreaks (77%) were associated with produce, including fruits (n=14), seeded vegetables (n=10), sprouts (n=6), nuts and seeds (n=5), spices (n=4), and herbs (n=1). other agents implicated were tetrodotoxin (3 outbreaks) and campylobacter, chaconine, paragonimus, other virus, sulfite, and trichinella (1 outbreak each). foods implicated were a chicken dish, crab cake, creampuff, beer, and a wheat snack (1 outbreak each). latin america and the caribbean was the most common region implicated, followed by asia (technical appendix). thirty-one countries were implicated; mexico was most frequently implicated (42 outbreaks). other countries associated with>10 outbreaks were indonesia (n=17) and canada (n=11). fish and shellfish originated from all regions except europe but were most commonly imported from asia (65% of outbreaks associated with fish or shellfish). produce originated from all regions but was most commonly imported from latin america and the caribbean (64% of outbreaks associated with produce). all but 1 outbreak associated with dairy products involved products imported from latin america and the caribbean. outbreaks in this analysis were reported from 31 states, most commonly california (n=30), florida (n=25), and new york (n=16). the number of reported outbreaks associated with imported foods, although small, has increased as an absolute number and in proportion to the total number of outbreaks in which the implicated food was identified and reported. although many types of imported foods were associated with outbreaks, fish and produce were most common. many outbreaks, particularly outbreaks involving produce, were associated with foods imported from countries in latin america and the caribbean. because of their proximity, these countries are major sources of perishable items such as fresh fruits and vegetables; mexico is the source of about one quarter of the total value of fruit and nut imports and 45%50% of vegetable imports, followed by chile and costa rica. similarly, our finding that many outbreaks were associated with fish from asia is consistent with data on the sources of fish imports (6). one quarter of the outbreaks were multistate, reflecting the wide distribution of many imported foods. systems like pulsenet have helped to improve detection and investigation of multistate outbreaks, resulting in an increased number of multistate outbreaks (7,8). the increasing number of outbreaks involving globally distributed foods underscores the need to strengthen regional and global networks for outbreak detection and information sharing. the importance of having standard protocols for molecular characterization of isolates and systems for rapid traceability of implicated foods to their source was illustrated during the investigation of a listeriosis outbreak linked to italian cheese imported into the united states in 2012 (9). newer tools like whole genome sequencing can also help to generate hypothetical transmission networks and in some instances facilitate traceback of foods to their origin (10). moreover, new tools that aid visualization of supplier networks facilitate the investigation of outbreaks involving the increasingly complex global economy (11). only a small proportion of fda-regulated foods are inspected upon entry into the united states. new rules under the food safety modernization act of 2011, including the preventive controls rule for human food, produce safety rule, foreign supplier verification program, and accreditation of third party auditors, will help to strengthen the safety of imported foods by granting fda enhanced authorities to require that imported foods meet the same safety standards as foods produced domestically (12). although data collection has improved in recent years, these findings might underestimate the number of outbreaks associated with imported foods because the origin of only a small proportion of foods causing outbreaks is reported. similarly, because of how data are collected and reported, the relative safety of imported and domestically produced foods can not be compared. because of changes in surveillance and changing import patterns, changes over time should be interpreted cautiously. our findings reflect current patterns in food imports and provide information to help guide future outbreak investigations. prevention focused on the most common imported foods causing outbreaks, produce and seafood, could help prevent outbreaks. efforts to improve the safety of the food supply can include strengthening reporting by gathering better data on the origin of implicated food items, including whether imported and from what country. region and country of origin of imported foods implicated in outbreaks, by food category, united states, 19962014.

the proportion of us food that is imported is increasing; most seafood and half of fruits are imported. we identified a small but increasing number of foodborne disease outbreaks associated with imported foods, most commonly fish and produce. new outbreak investigation tools and federal regulatory authority are key to maintaining food safety.

PMC5382743

pubmed-988

factor v is a coagulation protein that is present in blood plasma as a single-chain polypeptide (approximately 80%) and in platelet alpha-granules (approximately 20%).1 factor v is cleaved after binding to activated platelets and serves as a cofactor for factor xa in the prothrombinase complex. this complex forms on the platelet surface and has limited proteolytic activity, converting prothrombin to thrombin to aid in blood clotting.2,3 acquired inhibitors to factor v were first reported in 195524 and develop in extremely rare cases (0.090.29 cases per million persons) via development of alloantibodies or autoantibodies against factor v. patients with acquired factor v inhibitors generally present with hemorrhagic manifestations. here, we report a unique case of acquired factor v inhibitor in a patient with mantle cell lymphoma presenting with hematuria followed by thrombosis. a 64-year-old man initially presented to us with complaints of fatigue and joint discomfort for several months prior to december 2008. he denied experiencing any fever, night sweats, and weight loss, and did not notice any swelling, recurrent infection, easy bleeding, or bruising. his past medical history included hernia repair, renal colic, gastroesophageal reflux, and osteoarthritis. upon examination, one left axillary lymph node was swollen to approximately 12 cm, and was firm and slightly tender. his spleen was palpable on inspiration, and castell s sign was positive; however, his liver was not enlarged. a complete blood count showed normal platelet and hemoglobin levels of 206 10/l and 155 g/l, respectively, but a high white blood cell count of 18 10/l. a leukocyte differential indicated the following: lymphocytes 10.3; neutrophils 6.75; monocytes 0.58; eosinophils 0.23; and basophils 0.05. peripheral blood flow cytometry revealed a cd19, cd20, cd5, cd23, cd10 clonal b-cell population. the proportion of this fmc7-positive population reduced to 17% during a second flow analysis one month later, suggesting the possibility of mantle cell lymphoma or cd23-negative chronic lymphoid leukemia. given that the patient was asymptomatic and showed no signs of bone marrow failure, a strategy of watchful waiting was implemented. in august 2009, 8 months after his initial visit, the patient was admitted to the emergency room with a 2-day history of hematuria. evaluation of his blood plasma revealed a prolonged international normalized ratio (inr) of 6 and an activated partial thromboplastin time of 160 seconds. except for some minor skin bruising on his face, the patient had no other bleeding or bruising, nor was any area abnormal on examination. further, he had no past history of bleeding, with uneventful surgical procedures and tooth extractions in the past. on careful investigation of his coagulopathy, a prothrombin time mixing study was abnormal at 43 partially corrected to 30 seconds only (normal 12.515.7 seconds), suggesting the presence of an inhibitor. the thrombin time was 15.8 seconds (normal 15.518.3 seconds), serum fibrinogen level was 5.8 g/l), factor v level was<0.01 u/ml (normal 0.51.5 u/ml), factor viii level was 3.23 u/ml (normal 0.51.5 u/ml), factor x level was 1.08 u/ml (normal 0.51.5 u/ml), and factor ix level was 1.17 u/ml (normal 0.51.5 u/ml). based on these results, a factor v inhibitor test was performed and indicated a factor v inhibitor titer of 80 bethesda units. the patient was started on prednisone (1 mg/kg, 80 mg daily), and after 2 weeks of treatment, the bleeding had stopped and serial measurement of factor v levels showed a dramatic increase; moreover, within 7 weeks, factor v levels were normal and factor v inhibitor was undetectable. surprisingly, 7 weeks after initiation of steroid treatment, the patient developed an unprovoked severe pain with swelling in his right leg. a week prior to this event, his prothrombin time was 17 seconds, activated partial thromboplastin time was 28 seconds, inr was 1.4, and the factor v assay value was 0.62 bethesda units/ml, but factor v inhibitor was undetectable. doppler ultrasound revealed deep venous thrombosis, and low-molecular-weight heparin (dalteparin, 12,500 u) and coumadin (7.5 mg) were administered orally once daily. the patient s condition continued to improve with no further bleeding or thrombotic events, and anticoagulation was successfully discontinued after 8 months. the use of prednisone was gradually decreased over several weeks after confirmation of the disappearance of the inhibitor, and was discontinued completely in december 2009 (figure 1). screening assays to rule out inherited thrombophilia were negative for antithrombin, protein c, protein s, factor v leiden, and prothrombin 20210 gene mutations. fluorescence in situ hybridization analysis of a peripheral blood sample showed igh/ccnd1 t(11;14), which confirmed the presence of a clonal b-cell population characteristic of mantle cell lymphoma. the clinical presentation of factor v inhibitors can range from asymptomatic hematological laboratory abnormalities to life-threatening hemorrhage. factor v inhibitor has been associated with a number of conditions, including antibiotic use, sepsis, malignancies,10 autoimmune illnesses, vasculitis, valproic acid use, systemic amyloidosis, cold agglutinin disease, human immunodeficiency virus infection, hematopoietic stem cell transplantation, and liver transplantation, as well as use of amiodarone and warfarin.2,1113 if bleeding occurs, it predominantly involves the mucous membranes of the gastrointestinal, airway, and urinary tracts.2 in contrast with other coagulation factor inhibitors, the level of factor v inhibitors does not correlate with the degree of clinical bleeding, and streiff et al noted that patients who developed spontaneous factor v inhibitors had more significant bleeding than those with bovine thrombin-related factor v inhibitors.14 in addition, the risk of bleeding does not seem to correlate with prolongation of prothrombin time or activated partial thromboplastin time, factor v activity, factor v inhibitor levels, or the duration of presence of factor v inhibitor.14 because factor v is an anticoagulant, reports of thrombosis in patients with acquired factor v inhibitor are extremely rare.59 activated factor v (fva) is subsequently inactivated by activated protein c (apc), which cleaves fva at arg506, arg306, and arg679. after fva is cleaved at arg506 (fvac), it further interacts with apc and protein s to enhance the inactivation of activated factor viii (fviiia) by the aps/protein s complex, thereby indirectly acting as an anticoagulant.15,16 factor v inhibitor can hinder this cascade by decreasing the level of fvac, resulting in sustained activation of factor viii and leading to procoagulant diathesis. interestingly, an anti-factor v antibody isolated from a patient inhibited the cofactor activity of factor v during the apc/protein s complex s fviiia inactivation cascade by impairing the proper cleavage of fva by apc and inhibiting the inactivation of fva.17 this confirms that factor v inhibitors can lead to procoagulant diathesis by both direct and indirect mechanisms. given that the factor v inhibitor was undetectable in our patient, it is unlikely that it elicited the development of thrombosis during the course of treatment, but we hypothesize that the significantly elevated factor viii might have played a role. additionally, underlying hematological malignancy is another possible provoking factor. the patient presented in this case report had a unique clinical manifestation of acquired factor v inhibitor in that thrombosis occurred after factor v was no longer detectable by an assay measuring bethesda units; however, the patient still exhibited coagulopathy with high prothrombin time and inr, suggesting the continued presence of a small amount of factor v inhibitor. at first presentation, his factor viii level was significantly higher than normal at 3.23 u/ml (normal 0.51.5 u/ml) which might have caused this thrombotic event. to our knowledge, this is the first report of acquired factor v inhibitor in a patient with mantle cell lymphoma simultaneously presenting with this unusual coagulopathy.

acquired factor v inhibitor is a rare hemostatic disorder that presents with hemorrhagic manifestations in the vast majority of patients. factor v inhibitor may develop through a variety of mechanisms involving development of alloantibodies or autoantibodies specific to factor v. autoantibodies, in particular, have been reported in a number of conditions. in this report, we describe a case of acquired factor v inhibitor in a patient with mantle cell lymphoma who presented with hematuria. seven weeks after diagnosis and successful management, the patient developed deep vein thrombosis in the right lower extremity. the patient s factor v levels were normalized, and the inhibitor was successfully eradicated using corticosteroids. here, we discuss this rare disorder, its unusual manifestation, and provide a mini-review of the current literature regarding factor v inhibitors.

PMC3938440

pubmed-989

the experimental works of macklin and macklin provided insights into its pathophysiology [4, 5]; alveolar rupture occurs because of a pressure gradient between the alveolus and the surrounding tissues. this gradient develops either through overinflation of the alveolus or a reduction of interstitial pressure. the air that subsequently leaks into the interstitial tissue diffuses toward the peribronchial and perivascular tissue, and then towards the mediastinum, the neck and into the subcutaneous tissue. however, due to pressure equalisation between the affected and adjacent alveoli in the lungs, the interalveolar walls remain intact and the lungs inflated. the diagnosis of pm is confirmed by frontal chest roentgenogram, including the cervical region. typical radiological signs of pm include the continuous diaphragm sign (interposition of air between the pericardium and the diaphragm, which becomes visible in the central mediastinal part) and linear bands of mediastinal air parallelling the left side of the heart and the descending aorta (pleura is shown as a fine opaque line) with extension superiorly along the great vessels into the neck. in infants, the spinnaker sign (an upwards and outwards deviation of thymic lobes) can be seen when the thymus is raised above the heart by pneumomediastinal air that elevates the thymus and separates it from the cardiac silhouette beneath. various causes of pm are found in the literature, such as airway obstruction (e.g. foreign body aspiration), iatrogenic (e.g. mechanical ventilation), infections (e.g. pneumonia), obstructive lung disease (e.g. asthma), toxic effects (e.g. smoking), trauma (e.g. chest trauma), valsalva manoeuvres (e.g. vomiting) and the weakness of tissue (e.g. anorexia nervosa). in spontaneous pm, the underlying lung is healthy and the air leak is thought to be atraumatic. in neonates, known predisposing factors are mixed lung diseases, such as pneumonia or meconium aspiration syndrome, with coexisting atelectasis and airway obstruction., we retrospectively analysed the incidence, severity and causalities of pm in neonates and children>4 weeks of life admitted to our intensive care unit, and we investigated the possible differences between the groups. we retrospectively reviewed all records of children diagnosed with pm who were hospitalised in the interdisciplinary neonatal and paediatric intensive care unit of the university children s hospital in zrich, switzerland, between january 2000 and september 2006. the patients were divided into two groups according to their age: neonates (under 4 weeks of age) and children (over 4 weeks of age). we were interested in the causes of pm as documented by the treating physicians, the types and results of radiologic investigations performed, any invasive interventions used to treat pm, the severity of the pm and the length of stay in the intensive care unit. the incidence of pm in our intensive care unit was 0.08% for children>4 weeks of age and 0.1% for neonates. in all patients, pm was diagnosed by chest x-ray and all had a positive outcome related to the pm. all five patients with pneumopericardium (pp) did not suffer from any complications (e.g. pericardial tamponade). in addition to pm, two children of this group had subcutaneous emphysema (se), two a pneumothorax (pt) and two a pp. different causes were found for the air trapping. there were two traumatic aetiologies (rib fracture after a severe car accident, lesion in the hypopharynx after a fall). two children were diagnosed with obstructive bronchitis and in one child, barotrauma occurred intraoperatively due to a clamped expiratory tube during mechanical ventilation (fig. 1). one child had exercised vigorously three days before hospitalisation, which may have caused the pm. in one adolescent, pm occurred spontaneously. all children were hospitalised in the intensive care unit for one to seven days, depending on the severity of their underlying disease. diagnostics for the pm other than chest x-rays were performed in four patients. all of these had received a thoracic ct scan. in one child, who also had a huge subcutaneous emphysema and dysphagia, the reason for the air trapping could only be found by means of a laryngotracheoscopy, which showed a traumatic lesion in the hypopharynx. the patient s history revealed that she had fallen onto a piece of wood by her neck. table 1results for the group of children>4 weeks of lifeair leakaetiologyage (years)intubation (after diagnosis)pleural drainagediagnostic testsdays in icupm, pp, sespontaneous/3 days earlier intensive sport15.8nonochest x-ray 32pm, pttraumatic7.53 daysyesct/chest x-ray 35pm, se traumatic (lesion in hypopharynx)1.3nonoct/chest x-ray 3, oesophagogram with contrast medium, laryngotracheoscopy4pm, ppiatrogenic: equipment failure with barotrauma during mechanical ventilation2.3nonochest x-ray 22pmspontaneous15.4nonochest x-ray/ct1pm obstructive bronchitis5.6nonochest x-ray 2/ct2pm, ptobstructive bronchitis1.97 daysyeschest x-ray 77fig. 1pneumomediastinum (pm), subcutaneous emphysema (se) and pneumopericardium (pp) in a 2-year-old intubated patient results for the group of children>4 weeks of life pneumomediastinum (pm), subcutaneous emphysema (se) and pneumopericardium (pp) in a 2-year-old intubated patient the group of children older than 4 weeks stayed in the intensive care unit for a mean of 3.2 days (range 17 days), depending on the severity of the pm and the underlying disease. compared to the neonatal group, the length of stay in the intensive care unit was shorter. however, most neonates stayed in the intensive care unit longer, primarily because of comorbid conditions and not because of the pm. we found nine neonates who were diagnosed with pm (table 2); two premature and seven term infants, all of whom presented with signs of respiratory distress. three neonates were also diagnosed with a pp, one with se and five with a pt. birth weight ranged from 2,150 g to 4,140 g (mean 3,340 g). all children were vigorous at birth and none required resuscitation with bag mask ventilation or surfactant. before arriving in the intensive care unit, where the diagnosis of pm was confirmed by chest x-ray, two infants had received ventilatory support by cpap and one of the premature infants had to be intubated for respiratory failure. during hospitalisation in the intensive care unit, two children deteriorated and required mechanical ventilation for three and four days, respectively, and two other children needed cpap for a few hours. the age at admission to the intensive care unit ranged from a few hours to four days. one neonate was admitted to the intensive care unit due to convulsions and developed a pm on day six of life. the treating physicians felt that the pm may have been associated with a valsalva manoeuvre, which occurred during the seizure. other causes of pm were a pulmonary infection due to maternal infection and a possible barotrauma due to peak inspiratory pressure of 25 cm h2o in a mechanically ventilated premature neonate. two newborns had pm related to cpap and four neonates were diagnosed with spontaneous pm. neonates stayed in the intensive care unit for 313 days (mean 5.6 days), depending on the severity of the underlying diseases. table 2results for the group of neonatesair leakaetiologybirth weightgestational age (weeks)mode of deliverymechanical ventilation before diagnosisduration of ventilatory support after diagnosispleural drainagedays in icupmspontaneous4,140 g40 0/7vaginalnonono4pm, pp, sepremature lungs, barotrauma2,150 g34 4/7caesarian section pip max. 25 cm h203 days (intubation)no4pm, pt, pppremature lungs, spontaneous or cpap2,480 g35 6/7vaginalcpap4 days (intubation)yes6pm, ptspontaneous3,485 g38 1/7caesarian sectionnonono3pm, ptspontaneous3,440 g37 5/7vaginalno6 hours (cpap)no3pm, ppspontaneous2,830 g39 1/7caesarian sectionnonono13pm, ptspontaneous3,970 g38 5/7caesarian sectionnonono9pm, ptpulmonary infection due to maternal infection3,440 g38 5/7vaginalcpap1 day (cpap)no5pmconvulsions or spontaneous4,130 g40 5/7vaginalnonono4 results for the group of neonates all children with pm had a good outcome without any complications due to air trapping. in the group of children older than 4 weeks, only two children developed a respiratory insufficiency, leading to mechanical ventilation. in both of them, respiratory failure was related to their underlying condition (polytrauma with haematothorax and severe obstructive bronchitis, respectively). all other children were treated with oxygen only and stayed in the intensive care unit until they improved clinically and radiographically. regarding radiologic diagnostics, four patients of the group of children>4 weeks of life had ct scans (three of them had been done in outside clinics from where the patients had been admitted to our intensive care unit). retrospectively, the utility of the ct scans was put into question, as these scans did not change the patient management. the only patient in whom the ct scan changed management was the child with polytrauma. in this patient, other intrathoracic injuries needed to be ruled out. in the group of neonates, it was much more difficult to find the aetiology of the pm, since all neonates had presented with respiratory disease, and radiologic investigations were partially performed only after the use of cpap or tracheal intubation. five of the nine neonates had a spontaneous pm without risk factors, such as mechanical respiratory support (bag mask ventilation after birth, cpap, mechanical ventilation) or restrictive lung disease. three of these five babies were delivered by caesarean section. in the remaining four newborns, possible mechanical incidents leading to the air leak could be revealed: mechanical ventilation with high inspiratory pressure, cpap, pulmonary infection and convulsion. further investigations are needed to find the aetiology of spontaneous pm in healthy, term neonates. in conclusion, it is diagnosed by chest x-ray alone. whereas in older children mechanical events leading to the airway rupture can be revealed in most cases, about half of the neonates in our series suffered from pm without obvious reason.

the incidence, aetiology and pathophysiology of pneumomediastinum (pm), an uncommon and potentially serious disease in neonates and children, were evaluated. a retrospective chart review of all patients diagnosed with pm who were hospitalised in the intensive care unit of the university children s hospital zrich, switzerland, from 2000 to 2006, was preformed. we analysed the incidence, severity and causes of pm and investigated the possible differences between neonatal and non-neonatal cases. seven children and nine neonates were identified with pm. all patients had a good outcome. six cases of pm in the group of children older than 4 weeks were deemed to be caused by trauma, infection and sports, whereas one case was idiopathic. all nine neonatal cases presented with symptoms of respiratory distress. we were able to attribute four cases of neonatal pm to pulmonary infection, immature lungs and ventilatory support. five neonatal cases remained unexplained after careful review of the hospital records. in conclusion, pm in children and neonates has a good prognosis. mostly, it is associated with extrapulmonary air at other sites. it is diagnosed by chest x-ray alone. we identified mechanical events leading to the airway rupture in most children>4 weeks of life, whereas we were unable to identify a cause in half of the neonates studied (idiopathic pm).

PMC2254655

pubmed-990

age-related maculopathy (arm), also known as age-related macular degeneration, affects approximately 30 million people worldwide. in the united states, there are an estimated 13 million people with some vision loss from arm, including two million with the most severe late-stage of the disease. fortunately, there are an increasing number of effective treatments for late-stage disease, such as pegaptabnib (macugen; pfizer) and ranibizumab (lucentis; genetech). however, treatment of advanced arm often results in less than optimal patient outcomes because moderate to severe irreversible vision loss has already occurred before stabilization. the treatment of the disease in its earliest stages is the best strategy to preserve the patient s vision, which may be only mildly disturbed as measured by high contrast visual acuity measured under high luminance conditions. the disease s slow progression hampers the feasibility of clinical trials because currently accepted endpoints of acuity and fundus appearance are relatively insensitive to the disease s progression through its earliest stages. these endpoints require clinical trials to have large sample sizes or long follow-up durations to evaluate efficacy of a potential treatment. clinical trials evaluating early treatments are impractical because of the constraints current endpoints impose on trial design. while patients with early arm typically have good best-corrected visual acuity, impaired night vision is a prominent self-reported problem [13]. anatomical studies of donor eyes have documented significant rod dysfunction and photoreceptor drop out in comparison to cone photoreceptors. histopathological studies have found arm-related lesions in the rpe and bruch s membrane, which are generally invisible to clinical inspection [5, 6]. not surprisingly, these insults to the photoreceptors and the rpe, which supports the photoreceptors, disrupt vision. early arm patients exhibit moderate to severe impairment of rod-mediated dark adaptation and scotopic visual sensitivity even in the absence of visual acuity loss [7, 8]. rod-mediated dark adaptation appears to be more impaired in early arm patients than photopic visual sensitivity, scotopic visual sensitivity, acuity, and contrast sensitivity. advanced arm patients exhibit impaired cone-mediated dark adaptation [912] and early arm patients exhibit photostress impairment [13, 14]. however, rod-mediated dark adaptation appears to be more affected than cone-mediated dark adaptation at least outside the fovea in early arm patients. rod-mediated dark adaptation impairment is modestly reversible with vitamin a supplementation, whereas cone-mediated dark adaptation impairment is not. a pilot retrospective study suggests that the rod-mediated dark adaptation impairment is detectable at least 4 years before the lesions associated with the disease are clinically apparent (unpublished data). these findings suggest that dark adaptation impairment is a sensitive marker of early arm and raises the possibility that it may used to predict progression. the utility of dark adaptometry as a clinical outcome measure or practical diagnostic tool is hampered by a long test duration, high participant burden, and lack of standardized dark adaptometers. dark adaptation protocols used in prior research require up to 90 minutes, and typically more than 100 threshold estimates are made. the long duration and number of thresholds measurements the goal of this study was to develop a short-duration dark adaptation protocol that minimized patient fatigue, increased operator ease of use, and maintained the high sensitivity and specificity of research protocols. the protocol evaluated in this study has three distinct differences compared with prior research protocols: (1) the bleaching light intensity was significantly reduced to shorten the duration necessary to measure sensitivity recovery; (2) the stimulus was located within the area of greatest rod dysfunction to enhance the sensitivity of the test for detection of early disease; and (3) the speed of dark adaptation was estimated using a parameter that is robust to the shape of the dark adaptation function, which can be variable with disease severity. this protocol was implemented using a new dark adaptometer, the adaptdx, developed in collaboration with apeliotus technologies, inc. the institutional review board of the university of alabama at birmingham approved this study. written informed consent was obtained from all participants after the nature and possible consequences of the study were explained. normal adults and early to intermediate arm patients were recruited from the comprehensive ophthalmology and retina services of the department of ophthalmology, university of alabama at birmingham. inclusion criteria were as follows: (1) participants were 20 to 45 years old for the young normal group and participants were at least 55 years of age for the old normal and arm groups; and (2) best-corrected distance visual acuity of 20/100 or better in at least one eye. if both eyes qualified, the eye with the better acuity was enrolled as the test eye. if the acuities were the same, the right eye was enrolled as the test eye. exclusion criteria were as follows: (1) medical record or a general health interview indication of glaucoma, optic neuropathy or any ocular conditions other than arm, or refractive error (spherical equivalent) having an absolute value>6 diopters; (2) neurological diseases such as alzheimer s disease, parkinson s disease, history of stroke or multiple sclerosis; (3) diabetes; and/or (4) inability to perform the psychophysical task used to measure dark adaptation. best-corrected distance visual acuity was measured for each eye using the edtrs chart and expressed as logmar. the patient s test eye is dilated to 6 mm diameter, and corrective lenses are introduced as appropriate for the 30-cm viewing distance to correct for blur. an infrared camera located behind the fixation light continuously monitors the patient s test eye and displays an image of the eye on a personal computer-based operator control screen. the operator centers the patient s test eye to the red fixation light with the help of a reticule displayed on the image of the eye. the patient s test eye is bleached by exposure to a photoflash (0.25 ms duration, 6.38 log scot td second intensity) while the patient is focused on the fixation light. the flash of light passes through a diffuser and 4-diameter aperture centered at 5 on the inferior visual meridian to provide a uniform focal bleach surrounding the area to be tested during sensitivity recovery measurements. the patient focuses on the fixation light, and responds that a stimulus light is present by pushing a button. the stimulus light is a 1.7 circular test spot located at 5 on the inferior visual meridian. to focus on rod-mediated function, a stimulus wavelength of 500 nm was used, which is near the peak of rod sensitivity. sensitivity is estimated using a three-down/one-up modified staircase threshold estimate procedure. starting at a relatively high intensity (5.00 cd/m), stimulus lights are presented every two or three seconds for a 200-ms duration. if the patient does not respond within 2 seconds of stimulus onset, the stimulus light intensity remains unchanged on successive stimulus presentations until the patient responds. if the patient indicates that the stimulus is visible, the intensity is decreased for each successive presentation in steps of 0.3 log units until the patient stops responding that the stimulus is visible. after the patient indicates that the stimulus light is invisible by not pressing the button while the stimulus light is present, the intensity of the target is increased for each successive presentation in 0.1 log unit steps until the patient responds that the stimulus light is once again visible. successive threshold measurements start with the stimulus intensity 0.2 log units brighter than the previous threshold measurement. threshold measurements are made about once a minute for the duration of the measurement protocol; thus about 20 threshold measurements are made during the 20-minute test. the bleaching light (not shown) is presented through an aperture co-localized with the stimulus light the patient s view of the adaptdx. the bleaching light (not shown) is presented through an aperture co-localized with the stimulus light because arm patients can exhibit several differently shaped dark adaptation functions, the rod intercept, the rod intercept is the amount of time after bleach offset required for the patient s sensitivity to recover to a stimulus intensity of 5 10 cd/m (i.e.; 4.0 log units). this light level was chosen because it typically is achieved late in the second component of rod-mediated dark adaptation and is therefore completely mediated by rod function. each subject s dark adaptation function is plotted and the rod intercept is estimated by linear interpolation. patients with normal rod recovery will have shorter rod intercepts than those with impaired dark adaptation. stereoscopic 30 photographs were taken with a ff450 plus zeiss fundus camera for all participants more than 54 years old. photographs were evaluated using the age-related eye disease study (areds) severity scale for age-related macular degeneration by two independent graders who were masked to the clinical and functional characteristics of the participants. group assignment to normal or arm was based on the areds severity score of the test eye. these steps are indicative of normal (step 1), borderline arm (our terminology) (step 2), early arm (steps 36), intermediate arm (steps 79), and advanced arm (steps 10 and 11). the study population consisted of eight normal young adults (mean age=32.6 years old), nine normal old adults (mean age=73.1 years old) and 17 arm patients (mean age=75.1 years old). the old normal and arm groups had similar ages (p=0.42) and test eye acuities (p=0.14) (table 1). fellow eye acuity was higher on average for old normal adults than arm patients (p=0.01). arm patients exhibited substantially slower dark adaptation compared with normal old adults; whereas the dark adaptation speed was essentially the same for the young and old normal participants. representative dark adaptation functions for a young normal adult, an early arm patient and an intermediate arm patient are shown in fig. 2. the rod intercept for arm patients (mean=17.20 min, sd=3.8) was on average twice as long as the rod intercept for normal old adults (mean=8.73 min, sd=1.9) (p<0.0001). nine of the 17 arm patients failed to obtain the criterion sensitivity within the 20-minute protocol, and were assigned a rod intercept of 20 min; thus the substantial difference between these averages actually understates the relative impairment between the two groups. the rod intercept for normal young adults (mean=8.2 min, sd=1.4) was statistically indistinguishable from normal old adults (p=0.76). 2representative dark adaptation curves of a normal adult (closed circles), early arm patient (open triangles), and intermediate arm patient (open circles)table 1participant demographics young normal (n=8)old normal (n=9)arm (n=17)age (years), mean (sd)32.6 (5.3)73.1 (4.8)75.1 (6.4)gender,% (n)female100 (8)66.7 (6)52.9 (9)male0 (0)33.3 (3)47.1 (8)race,% (n)white62.5 (5)88.9 (8)100 (17)african american25.0 (2)11.1 (1)0 (0)asian12.5 (1)0 (0)0 (0)visual acuity, logmar mean (sd)tested eye.02 (0.4).04 (.12).14 (.19)fellow eye.05 (0.1).14 (.21).52 (.40) representative dark adaptation curves of a normal adult (closed circles), early arm patient (open triangles), and intermediate arm patient (open circles) participant demographics each subject s dark adaptation was classified as normal or impaired based whether the participant s speed of dark adaptation as measured by the rod intercept fell within the normal reference range (mean 2 sd) of the normal old adults. the upper limit of the normal reference range was 12.5 minutes. individuals with rod intercepts participants with a rod intercept 12.5 were classified as having normal dark adaptation. using this criterion, 15 of the 17 arm patients all nine normal adults fell within the normal reference range for a diagnostic specificity of 100%. the sensitivity and specificity of our 20-minute study protocol compares favorably to that of a previously used 90-minute research protocol (85% sensitivity; 100% specificity), despite the considerable shortening of the test. furthermore, it is useful to examine the two false negative results (those arm patients classified with normal dark adaptation). both were for participants with an areds fundus grade of 2. one of these patients exhibited three drusen of 63 m scattered throughout the central 3,000 m of the macula. the sensitivity of the test is 100% for those arm patients with an areds grade 3 and higher. the rod intercept for borderline arm patients (areds fundus grade 2) was, on average, 5.1 minutes slower than the normal old adult s rod intercept. the rod intercept for early arm patients (areds fundus grades 3 to 5) was, on average, yet another 3.4 minutes slower than borderline arm patients. finally, all of the intermediate and late arm patients (areds fundus grades 6) failed to reach the rod intercept within the 20-minute test time. table 2dark adaptation impairment increased with disease severity young normalold normalborderline armearly armintermediate armadvanced armsample size895651areds fundus graden/a123,4,56,7,8,910rod intercept, min mean (sd)8.2 (1.4)8.7 (1.9)13.8 (4.4)17.2 (2.7)20 () 20 () fundus photography and grading was not performed on young normal group.all subjects failed to reach the rod intercept within the 20-minute test duration. dark adaptation impairment increased with disease severity fundus photography and grading was not performed on young normal group. using a 20-minute, rod-mediated dark adaptation protocol, arm patients exhibited a dramatic slowing of dark adaptation in comparison with normal old adults. the magnitude of the impairment was similar to that previously measured using a 90-minute research protocol. as expected, reducing the bleaching light shortened the duration of the test, and moving the stimulus to an area of greater rod dysfunction preserved the sensitivity and specificity of the protocol. the magnitude of the dark adaptation impairment exhibited by early arm patients suggests that a rapid diagnostic screening test having a duration of less than 10 minutes is feasible. retest reliability was found to be quite high (intraclass r=0.95) in a separate, preliminary study of 40 normal adults and arm patients (data not shown). perhaps more intriguing, the increase in the amount of rod-mediated dark adaptation impairment with increasing disease severity as assessed by the areds severity system for arm allows the possibility of using dark adaptation as a clinical outcome measure. previously, we evaluated another short-duration protocol developed for the sst-1 dark adaptometer (lkc technologies, inc) for the detection of early arm. the sst-1 is a manual dark adaptometer that uses an abbreviated protocol, typically 20 to 30 minutes. the adaptdx and the sst-1 exhibit markedly different response characteristics. whereas the adaptdx shows no difference with aging but is highly sensitive to arm, the sst-1 discriminates aging effects but is insensitive to arm. an important difference between the protocols is that the sst-1 measures the sensitivity recovery of the entire retina, instead of just the macula or a portion of the macula. we have developed a short duration protocol that can sensitively and specifically detect early arm. the impaired night vision encountered in early arm is a clinically significant problem similar to acuity impairment encountered in late arm. as such, we believe dark adaptation is a suitable primary endpoint to evaluate efficacy of treatments aimed at early arm .

dark adaptometry may be a useful diagnostic test and clinical trial endpoint for age-related maculopathy (arm) because impaired night vision is a hallmark of early arm. a novel dark adaptometer, the adaptdx, was evaluated for the detection of arm. the adaptdx incorporates a 20-minute protocol optimized for the detection of arm. arm patients (n=17) exhibited substantial dark adaptation impairment compared with normal adults (n=17). the diagnostic sensitivity was 88% and the specificity was 100%. the diagnostic test characteristics of the adaptdx are similar to previously reported studies using 60- to 120-minute protocols.

PMC2802416

pubmed-991

to increase awareness of the limitations of high-risk human papillomavirus (hrhpv) laboratory-developed testing (ldt) widely used in us cervical cancer screening. a young woman in her 30s was diagnosed and treated for stage 1b1 cervical squamous cell carcinoma in which hpv 16 dna was detected using polymerase chain reaction testing. both 1 month before and 42 months before cervical cancer diagnosis, the patient had highly abnormal cytology findings; however, residual surepath (becton, dickson and company, franklin lakes, nj) vial fluid yielded negative hybrid capture 2 (hc2; qiagen nv, hilden, germany) hrhpv ldt results from each of the two specimens. a review of the available data indicates that (1) purification of dna from surepath specimens requires complex sample preparation due to formaldehyde crosslinking of proteins and nucleic acids, (2) hc2surepath hrhpv testing had not been food and drug administration-approved after multiple premarket approval submissions, (3) detectible hrhpv dna in the surepath vial decreases over time, and (4) us laboratories performing hc2surepath hrhpv ldt testing are not using a standardized manufacturer-endorsed procedure. recently updated cervical screening guidelines in the us recommend against the use of hrhpv ldt in cervical screening, including widely used hc2 testing from the surepath vial. the manufacturer recently issued a technical bulletin specifically warning that use of surepath samples with the hc2 hrhpv test may provide false negative results and potentially compromise patient safety. co-collection using a food and drug administration-approved hrhpv test medium is recommended for hpv testing of patients undergoing cervical screening using surepath samples. since 2001, adjunctive high-risk human papillomavirus (hrhpv) testing has become increasingly integrated along with cytologic testing as a part of routine us cervical cancer screening, initially as a preferred reflex test after atypical cells of undetermined significance liquid-based cytology results and on a more widespread basis after 2003 food and drug administration (fda) approval for routine cytology and hpv cotesting of women 30 years and older.1,2 recently updated cervical screening guidelines from the american cancer society, the american society for colposcopy and cervical pathology, and the american society for clinical pathology have proposed significantly lengthened screening intervals, particularly for patients with negative hrhpv test results and either negative or equivocally abnormal (atypical cells of undetermined significance) cytology findings.3 for women 30 years and older with either hrhpv-negative atypical cells of undetermined significance or double negative results, a screening interval of 5 years has for the first time been recommended.3 the guidelines, however, emphasize that the new extended screening intervals following negative hrhpv test results are based on hpv tests with performance characteristics similar to hpv tests used in the supporting evidence.3 since at least one-third of all us hrhpv tests use laboratory-developed test (ldt) methodology, largely exempt from regulatory oversight by the fda and unlikely to have undergone rigorous evaluation using grade 3+or grade 2+cervical intraepithelial neoplasia clinical endpoints in properly designed trials,3,4 the guidelines publications specifically recommend against the use of hpv ldts for cervical cancer screening.3 the most common form of hrhpv ldt to date has been hybrid capture 2 (hc2; qiagen nv, hilden, germany) performed on residual surepath vial fluid (becton, dickinson and company, franklin lakes, nj).57 although hc2 hrhpv testing is fda-approved from both the digene (qiagen) specimen transport medium tube (qiagen) and the methanol-based preservcyt vial (hologic, inc, bedford, ma), hc2 hrhpv testing from the surepath vial to date has not been able to obtain fda approval, despite multiple premarket approval submissions, beginning in 2002.8 in a 2002 press release, the manufacturer stated: we remain hopeful that resolution of the fda s issues will not significantly alter our prior expectations for introduction in 2003.8 qiagen investigators have acknowledged that purification of dna from surepath specimens requires complex sample preparation due to the formaldehyde crosslinking of proteins and nucleic acids.9 three additional hrhpv tests have now also gained fda approval from either the preservcyt vial or also from proprietary manufacturer s collection media, but none of these newer fda-approved hrhpv tests have been approved using the surepath vial.1012 recently, the authors encountered a patient diagnosed with invasive cervical cancer with two prior significantly abnormal pap tests and two negative hrhpv ldt cotest results. since virtually all cervical cancers are now thought to be due to persistent carcinogenic hrhpv infections,13,14 this case was investigated to better understand the possible causes of negative hrhpv ldt in screened patients developing cervical cancer. the patient was a young woman in her 30s, a gravida 3, para 3 cigarette smoker with a long history of abnormal pap test results and inconsistent follow-up due to medical appointment cancellations which the patient attributed to intermittent lack of insurance coverage. forty-two months before her diagnosis of cervical cancer, the patient had a surepath pap test interpreted as atypical squamous cells, can not rule out a high grade squamous intraepithelial lesion (figure 1). because the patient s gynecologist had ordered routine cytology and hpv cotesting in a woman 30 years and older, the residual surepath vial fluid the hrhpv test result was reported as not detected. in that report, an additional comment stated that patients without hrhpv rarely have cervical cancer. a cervical biopsy obtained 4 months later reported koilocytosis and an endocervical curetting as benign. high-grade squamous intraepithelial lesion. the patient failed to return for scheduled colposcopic evaluation. eighteen months later, the patient presented with irregular painful periods that were getting worse. a third surepath pap test was obtained and reported as high-grade squamous intraepithelial lesion (figure 2). the patient s gynecologist had again ordered routine cytology and hrhpv cotesting in a woman 30 years or older, and therefore the residual surepath vial fluid was again sent to the regional laboratory that had previously performed hrhpv testing. the hrhpv test, which utilized hybrid capture with signal amplification, was as before reported as not detected. in this report, however, an additional comment stated that the analytical performance characteristics of this assay, when used to test surepath or vaginal specimens, have been determined by (the laboratory). a cold knife conization was performed 1 month later and the presence of a poorly differentiated squamous cell carcinoma (scc) measuring 1 1 0.5 cm and extending to multiple biopsy margins was documented. one month later at an outside cancer referral center, the patient underwent a radical hysterectomy, bilateral pelvic lymph node dissection, bilateral salpingectomy, and left oophorectomy. final pathologic diagnosis was of a cervical scc (1.5 cm maximum tumor dimension) invading the upper third of the cervix with no lymphovascular invasion identified, and negative lymph nodes twenty-six months later, at last follow-up, the patient was reported as alive and well with no evidence of disease. paraffin sections of scc samples from the patient s cold knife conization specimen were used for hrhpv testing by polymerase chain reaction methods.15 hpv in tumor sections was initially tested for using m09/m11 pcr primers, which amplify an approximately 450 base pair conserved region of the l1 gene of hpv. hpv type-specific pcr was also performed by pcr amplification of portions of e6 and e7 followed by automated dna sequencing of the amplified products. using these methods, presence of hpv 16 type-specific e6 fortunately, in the case of this patient, the false negative results did not affect her course or management. confirmation of the presence of hpv 16 dna by pcr in this patient s invasive cervical scc is consistent with the current understanding that persistent infections with a group of approximately a dozen carcinogenic hpv genotypes cause virtually all cases of cervical cancer worldwide. however, negative hc2 hrhpv test results from residual surepath vial fluid 42 months and 1 month before tumor diagnosis were unexpected. since both pap tests contained highly abnormal cells (atypical squamous cells, can not rule out a high-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion), the discrepancy can not be attributed to a failure to sample lesional cells. in fact, it has been argued that one advantage of hc2 cotesting is that hc2 may detect hrhpv dna in patients with occult invasive cervical cancer, even when lesional cells are not sampled.16 in the largest study of hc2 tests collected in fda-approved specimen transport medium, positive hrhpv hc2 results were reported in 185 of 198 (93.4%) samples collected from patients with simultaneous histopathologic diagnoses of invasive cervical cancer.17 in the same study, similar positive hrhpv hc2 test results were reported in 246 of 264 (93.2%) specimen transport medium tubes collected from patients with simultaneous histopathologic diagnoses of grade 3 cervical intraepithelial neoplasia.17 in contrast, the very limited data from the most widely cited us laboratory self-validation study of hpv testing from the surepath vial showed positive hrhpv hc2 results for patients with cancer in only 33% (one of three tests) or 50% (one of two patients). also in contrast to the above cited data, the authors puzzlingly asserted that false negative hrhpv screening test results in patients with invasive cervical cancer are not surprising.7 hc2 uses a positive cut point of 1.0 relative light units per positive control, a cut point which corresponds to greater than or equal to 5000 hpv dna copies per test well, based on a receiver operating characteristic curve analysis versus cervical intraepithelial neoplasia grade 2 +, to minimize the detection of lower viral load hpv infections that are mostly benign.1820 nevertheless, a subset of invasive cervical cancers associated with low viral load have been described,21 and low viral loads in patients with developing invasive cervical cancer may fall below the detection cut point of fda-approved hrhpv tests such as hc2.17,22 in one of the authors own laboratories (rma), three of 31 (10%) patients diagnosed with invasive cervical scc and tested within the prior 12 months for hrhpv by hc2 from fda-approved preservcyt vial fluid had negative hc2 results.23 all three patients had hpv 18 detected by pcr in scc sampled in paraffin sections, and two also had detectible hpv 16.23 hpv testing from the non-fda-approved surepath vial is thought to be more challenging, primarily due to the formaldehyde crosslinking of proteins and nucleic acids.9,24 although recovery of dna and ribonucleic acid is largely unaffected by long-term storage in preservcyt,25,26 storage in surepath preservative fluid (becton dickinson) has been shown to affect the recovery of both dna and ribonucleic acid.27,28 upon exposure to surepath media, recovery of both dna and ribonucleic acid rapidly diminished. this reduction was most apparent in the 0150 hours range (ie, up to around 6 days).27 the websites of four large national laboratories that offer hc2 testing of referred surepath samples all indicated that surepath samples are stable at room temperature for hc2 testing for 1 month compared to 90 days/3 months for the fda-approved thinprep vial (hologic) (table 1). interestingly, none of the laboratories, when queried by telephone, could produce independent surepath laboratories referred to the fda-approved becton dickinson package insert which states that surepath preservative fluid preserves cells (for cytologic testing) for up to 4 weeks at room temperature (15c30c).29 even in the most widely cited validation study mentioned previously, the authors referred to the digene package insert for specimen stability parameters.7 accurun 372 series 400; (seracare life sciences, inc, milford, ma),31 the hpv proficiency testing vendor for the college of american pathologists has reported that hpv 16-positive control samples shipped in surepath fluid degraded so rapidly that detectible hpv dna was lost after 1 day.30 the vendor concluded that only use of a two tube methodology, separating the hpv 16 sample from the surepath sample until the time of testing, could be used for laboratory hpv proficiency testing.31,32 there is at present no standardized surepath hpv protocol that all laboratories use and no literature with sufficient detail to represent an agreed upon standard.6,7,3336 furthermore, the manufacturer can not under current regulations recommend standardized procedures for non-fda-approved hrhpv testing. as a result of continued ongoing widespread off-label hpv ldt use and related patient safety concerns, on june 8, 2012 the manufacturer of surepath released a technical bulletin which stated: the becton dickinson surepath sample medium has not been approved by the fda for use with the hc2 test and use [...] may under certain conditions provide false negative results. the becton dickinson surepath sample medium has not been approved by the fda for use with the hc2 test and use [...] may under certain conditions provide false negative results. false negative results could lead to inappropriate patient management and potentially compromise patient safety. the authors echo the cautions of the new us screening guidelines that emphasize that extended screening intervals following negative hrhpv test results be based on hpv tests with performance characteristics similar to hpv tests used in the supporting evidence. given those new cervical screening guidelines and manufacturer communications that caution against non-fda-approved ldt hrhpv testing from the surepath vial, it is reasonable to view continued widespread use of this nonstandardized off-label testing as a patient safety issue. with new, extended 5-year screening intervals proposed for many women, an increasing number of women an avoidable increase in false negative hrhpv results in women with both precancer and early invasive cervical cancer will place patients at unnecessary risk. the use of screening methods that have not been validated should be strongly discouraged.37,38 with four fda-approved alternatives, it is difficult to justify the use of anything but rigorously clinically validated specimens. the college of american pathologists should discontinue offering its current form of laboratory proficiency testing for hpv testing out of the surepath vial, as it could mislead participants to believe that their methodology is currently safe and acceptable. for laboratories that use surepath for cytology, co-collection of a second sample for hrhpv testing in an fda-approved collection medium

objective: to increase awareness of the limitations of high-risk human papillomavirus (hrhpv) laboratory-developed testing (ldt) widely used in us cervical cancer screening. methods and results: a young woman in her 30s was diagnosed and treated for stage 1b1 cervical squamous cell carcinoma in which hpv 16 dna was detected using polymerase chain reaction testing. both 1 month before and 42 months before cervical cancer diagnosis, the patient had highly abnormal cytology findings; however, residual surepath (becton, dickson and company, franklin lakes, nj) vial fluid yielded negative hybrid capture 2 (hc2; qiagen nv, hilden, germany) hrhpv ldt results from each of the two specimens. this prompted questions to be asked concerning the performance characteristics of hrhpv ldt. a review of the available data indicates that (1) purification of dna from surepath specimens requires complex sample preparation due to formaldehyde crosslinking of proteins and nucleic acids, (2) hc2surepath hrhpv testing had not been food and drug administration-approved after multiple premarket approval submissions, (3) detectible hrhpv dna in the surepath vial decreases over time, and (4) us laboratories performing hc2surepath hrhpv ldt testing are not using a standardized manufacturer-endorsed procedure. conclusion:recently updated cervical screening guidelines in the us recommend against the use of hrhpv ldt in cervical screening, including widely used hc2 testing from the surepath vial. the manufacturer recently issued a technical bulletin specifically warning that use of surepath samples with the hc2 hrhpv test may provide false negative results and potentially compromise patient safety. co-collection using a food and drug administration-approved hrhpv test medium is recommended for hpv testing of patients undergoing cervical screening using surepath samples.

PMC3496968

pubmed-992

here, we describe a case of a hispanic male with thrombotic thrombocytopenic purpura induced by nonsteroidal anti-inflammatory drugs (nsaids). the patient was a 21-year-old latino male who presented to the emergency department reporting a five day history of nausea, vomiting, and decreased urine output. the patient reported that symptoms began shortly after a purposeful consumption of a large quantity of ibuprofen (600-mg pills) in a suicide attempt. the exact amount of ibuprofen is unclear, but it was predicted that he consumed approximately 18 grams of ibuprofen. the past medical history was only notable for hypertension diagnosed three years prior, for which he was not receiving any medical therapy. the patient noted a prior recreational use of alcohol, marijuana and cocaine use, but he denied any use in the past few months. family history was unremarkable for blood disorders and heart or renal disease. on initial exam, the patient had a temperature of 35.8c, a heart rate of 56 beats per minute, a blood pressure of 122/70 mmhg, a respiratory rate of 18 breaths per minute and his oxygen saturation was 100% on room air. the patient was noted to be alert and oriented despite appearing drowsy. on physical exam, pertinent findings included jaundice, icteric sclera, dry mucous membranes, and generalized petechiae. heart and lung examination were unremarkable. the abdomen was found to be soft but slightly tender to palpation at the right upper quadrant. no rebound or guarding was appreciated. mmol/l, potassium of 5.1 mmol/l, chloride of 83 mmol/l, bicarbonate of 21 mmol/l, blood urea nitrogen of 184 md/dl and creatinine of 19.1 mg/dl, and glucose of 95 mg/dl. serum osmolality was 323 mosm/kg, with an anion gap calculated at 26. white blood cell count 13.4 4 (10(3)/mcl), hemoglobin was 11.9 (10(6)/mcl) with an mcv of 73 fl, hematocrit was 34%, and platelets were 31,000/mcl. total bilirubin was 24.6 mg/dl (direct bilirubin, 20 mg/dl), liver function tests were elevated with aspartate aminotransferase was 105 units/l, alanine aminotransferase was 167 units/l, and alkaline phosphatase was 464 units/l. international normalized ratio and prothrombin time were within normal limits. lactate dehydrogenase (ldh) was 8747 units/l, and haptoglobin was less than 10 mg/dl. on the peripheral smear, schistocytes, helmet cells, and large platelets were noted. ultrasound of the abdomen was unremarkable and hepatomegaly and atrophy of kidneys was not appreciated. the patient immediately underwent plasma exchange for a total duration of 5 days and simultaneously started on oral glucocorticoids (prednisone 1 mg/kg). following treatment, platelet count recovered and remained above 150,000 after 2 days. the initiation of intermittent hemodialysis, creatinine and blood urea nitrogen improved significantly. additionally, the ldh returned to baseline. psychiatry also evaluated the patient and concluded that the suicidal incident was rooted to an adjustment disorder. a few weeks later, the patient was seen in the outpatient setting where he continued to report resolution of all symptoms and normal urination. thrombotic thrombocytic purpura is a rare blood disorder characterized by clotting of small blood vessels and low blood platelet counts. a pentad often characterizes ttp that includes neurological deficits (often in the form of altered mental status), thrombocytopenia, fever, renal dysfunction, and evidence of microangiopathic hemolytic anemia. not all features must be present for a diagnosis, but important primary criteria include thrombocytopenia and microangiopathy.3 the diagnosis of ttp is made after an exclusion of more prevalent multisystem diseases such as disseminated intravascular coagulation, sepsis, malignant hypertension, systemic lupus erythematosus, and hematologic malignancies.2 ttp is a rare disease with approximately 3.7 cases per million per year in the united states. in approximately 90% of patients, the cause of ttp is unknown.4,5 known conditions that are associated with ttp include a postpartum state, postinfection (escherichia coli 0157:h7, e. coli 0111, e. coli 0104:h4), pancreatitis, malignancy, post-surgical, hiv, post-pneumococcal infection, and drug-induced. for the association with drugs, quinine is the most commonly identified.6 other documented drugs include oral contraceptives, extended-release opioids, valacyclovir and chemotherapeutic agents such as mitomycin c, gemcitabine, cisplatin, oxaliplatin, pentostatin, bevacizumab, and sunitinib. additionally, the chemotherapeutic agents are typically dose-dependent in their involvement with ttp, and therapies such plasma exchange have been found to be ineffective.4,6 other widely used medications such as the anti-platelet agents ticlopidine and clopidogrel have also been associated with development of ttp. it is not clear if there is a direct effect of the drug or its metabolites on the vasculature or if a secondary immune response is responsible.3 however, the underlying mechanism for how platelet consumption occurs and the thrombotic microangiopathy that ensues is well-understood. ttp is associated with a deficiency of a protein known as adamts13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13). the gene for the adamts13 protein is located on chromosome 9q34. for the congenital form, this protein functions to cleave a large von willebrand factor (ul-vwf) multimer (derived from endothelial cells) into a smaller form.7 if the ul vwf-multimer can not be cleaved, it remains in circulation and activated platelets adhere to it. platelets are then consumed in the process and the microvascular is further injured from the high shear stress.7 the platelet-uf vwf complexes, if large enough, can also thrombose in the microvasculature of multiple organs. clinically, the adamts13 protein level is a useful tool in reaching the diagnosis of ttp. it is important to recognize that levels of this protein can be variable and even normal in several of the drugs associated with ttp.8 in one study, the variability in ttp ranged from 1370%.9 therefore, a decrease in adamts13 activity and the presence of its antibody is more complementary to the diagnosis and overall clinical picture. a decrease in activity can be associated with ttp if less than 510% (detection range 0.5100%). the overall consensus is that the presence of an adamts13 deficiency is a critical underlying risk factor, but requires a secondary trigger to develop ttp.4 in clinical practice, if a diagnosis is even suspected, it is imperative to consider initiating plasma exchange (plasmapheresis with infusions of fresh frozen plasma) immediately.3 plasma exchange therapy for acquired ttp is effective because it removes adamts13 autoantibodies and ul-vwf multimers, as well as prevents further supply of adamts13.7 disease activity can then be tracked by monitoring ldh and platelets. notably, prior to the availability of plasma exchange, patient survival was about 10%. with the advent of plasma exchange technology, the overall response rate is now estimated at 8090%.3,4 the therapy options include plasma exchange, and recent studies have also shown a benefit in using rituximab as a first line therapy of acute acquired ttp.7,10 the rituximab was used in conjunction with plasma exchange and corticosteroids and administered at 375 mg/m intravenously within the first three days of admission and diagnosis with shown clinical benefit.10 the chemical compound 2-(4-isobutylphenyl) propionic acid, also known as ibuprofen, is a nonsteroidal anti-inflammatory agent used in the management of pain, inflammation, fever, etc. in the the us alone about 30 billion doses are utilized each year.11 the drug inhibits both cyclooxygenase 1 and 2 that prevents the production of thromboxanes and prostaglandins upon inhibition induce an anti-inflammatory, analgesic, and antipyretic effect. it is well characterized that the inhibition of the cox-1 pathway is responsible for most of the unwanted side effects.12 these commonly include nausea, dyspepsia, gastrointestinal bleeding, transaminitis, and, less likely, jaundice, fatal fulminant hepatitis, liver necrosis, and renal papillary necrosis and occasionally hepatic failure. ibuprofen is plasma protein bound with a large volume of distribution (0.110.19 l/kg). a single 400-mg dose in adults produces a peak plasma level at 12 hours after ingestion and is eliminated via the kidneys within the next 8 h and then is practically undetectable by 12 h. its maximum recommended daily dose is 12003200 mg.1214 anemia is the only known hematological effect.15 more serious effects associated with long-term use and can manifest as esophageal ulceration and chronic renal failure and can worsen heart failure.16 nsaid overdose is generally a benign process. in 2010, the annual report of the american association of poison control centers national poison data system (npds): 28th annual report reported 65,699 cases of overdose with 9,169 of those characterized as intentional. forty-seven cases were classified as a serious toxicity that manifested as status epilepticus, respiratory failure, ventricular arrhythmias, or cardiac arrest. no deaths due to ibuprofen were reported in 2010. the high index of safety centers around ibuprofen s inability to accumulate in the body regardless of subsequent doses.17 there have been no recent cases attributing the development of ttp to patients exposed to nsaids. one case report from 1974 described a 55-year-old italian female who developed ttp after ingesting 900 mg of ibuprofen. no other alternative etiologies could be accounted for and the patient eventually expired as no plasma exchange therapy was available at that time.2 however, there have been some reports of nsaids causing hemolytic uremic syndrome (hus), a disease process very similar to ttp.18 paradoxically, antiplatelet therapy was at one point considered as potential treatment for ttp. the theory stipulated that by preventing platelet aggregation with antiplatelet agents, further endothelial injury and thrombosis could be prevented. data from this study demonstrated that at least in certain cases, antiplatelet drugs probably play only a limited role in the treatment of patients with ttp.19 in another trial, prostacyclin (pgi2) infusion, again with the same principle, failed to reverse platelet aggregation caused by ttp.20 our patient presented with clinical symptoms that included altered mental status, acute renal dysfunction, thrombocytopenia, and evidence of red blood cell fragmentation. all symptoms evolved a few hours after ingesting a large amount of ibuprofen. in our assessment, other possible disease entities were considered but excluded such as hemolytic uremic syndrome (no history of diarrhea and negative e. coli 0157h7 study), autoimmune hemolytic anemia (coombs test was negative), disseminated intravascular coagulation (coagulation factors were normal), hiv, and absence of other known drugs. adamts13 activity was less than 10% and solidified our assessment that ttp was the clear diagnosis. ibuprofen, its metabolites or even components of the drug formula may have caused this condition. nonetheless, because of the widespread use of nsaids and the high mortality associated with undiagnosed ttp, we present this case-report to shed light into a possible, unknown association between ibuprofen and ttp.

a 21-year-old male presented to the emergency department after a 5-day history of recurrent vomiting and decreased urine output. history revealed ingestion of ibuprofen. during the diagnostic workup, the following was identified: white blood cell count 13.4 (10(3)/mcl), hemoglobin 11.9 (10(6)/mcl) with an mcv of 73 fl, hematocrit 34% and platelets were 31,000/mcl, sodium of 130 mmol/l, potassium of 5.1 mmol/l, chloride of 83 mmol/l, bicarbonate of 21 mmol/l, blood urea nitrogen of 184 mg/dl and creatinine of 19.1 mg/dl. he was later diagnosed with thrombotic thrombocytopenic purpura (ttp) based on the fact that he presented with most components of the ttp pentad (except for fever), which included altered mental status, acute kidney injury, thrombocytopenia, and evidence of red cell fragmentation and his adamts13 level was found to be less than 10% prior to therapy. the patient then received plasma exchange, oral corticosteroids, and hemodialysis, which led to a full recovery of platelet count and renal function.

PMC4222303

pubmed-993

mscs can be extracted from adult fat and bone marrow, as well as peripheral blood,8 9 and can be induced to differentiate into various mesenchymal tissues including bone, cartilage, and muscle.10 11 in vitro induction can be accomplished by growth factor supplementation and creating culturing conditions that are favorable for the preferred differentiation. specifically, adipogenic differentiation from mscs has been successfully accomplished in dulbecco's modified eagle medium supplemented with isobutylmethylxanthine, indomethacin, and either dexamethasone and insulin or with hydrocortisone.11 12 successful induction is then verified by the identification of lipid vacuoles within the cell and various other adipose cell markers. in comparison, in vitro chondrogenic differentiation has been induced by centrifuging the mscs into micromass pellets and culturing in a medium containing dexamethasone and transforming growth factor -3.13 chrondrocytes are then detected by the presence of secreted extracellular matrix components such as type ii collagen and aggrecan, among others.14 osteogenic differentiation may be induced in a culture of dexamethasone, ascorbic acid phosphate, -glycerol phosphate;11 12 14 15 successful differentiation is identified by the expression of alkaline phosphate and using cell-specific antibodies. successful models used to expand the msc population and induce osteogenic differentiation have also included substances such as transforming growth factor- and fibroblast growth factor-2.16 17 bone morphogenetic protein has also been shown to successfully expand the osteogenic cell population;7 18 however, limitations exist regarding cost and safety. the authors refer the interested reader to some of the early studies inducing msc differentiation for additional explanations as to the nature and function of the specific growth factor supplements.11 12 13 14 19 20 21 in vivo induction occurs through site-specific differentiation and is often implanted within scaffolds.7 10 in addition to requiring sufficient numbers of mscs implanted for bone regeneration,22 an osteoconductive matrix is also necessary for osseous growth.18 the scaffold provides a structural support for cell cell interactions, extracellular matrix formation, and new tissue formation. the osteoconductive scaffold occupies the site of the fusion, but also provides an environment conducive for the osteoinductive factors and tissue growth. various biomaterials have been investigated as scaffolds including hydroxyapatite, tricalcium phosphate, calcium sulfate, metals, and biodegradable polymers.23 serving as the initial structure for tissue growth and blood vessel formation, the biodegradable types of scaffolds eventually resorb as new bone is formed.24 isolating human mscs has traditionally been accomplished by using their selective adherence to plastic surfaces, which the hematopoietic cells less commonly do.25 this method, however, typically leads to heterogeneous cell isolation, in which only ~30% of the cells are multipotent mscs.26 although the mscs do not express the cell surface markers normally found on hematopoietic cells, such as cd34 and cd45, there are cell markers that are unique to the mscs.27 stro-1 was one of the earlier monoclonal antibodies developed to isolate mscs,28 and this was followed by additional cell-surface markers such as cd146, cd200, and cd271.29 30 31 use of these markers alone may not be sufficient to isolate pure samples of mscs. gronthos and colleagues28 described a method whereby they used stro-1 monoclonal antibodies and then isolated the cells using immunomagnetic cell sorting (macs). this resulted in a milieu of cells with varying intensities of stro-1 fluorescence intensity, which was then further purified for the high-intensity fluorescing cells using fluorescence-activated cell sorting (facs). further purification was accomplished using dual-color facs to isolate those cells with the surface markers vam-1, which are also expressed on mscs.32 another recent study utilized macs to separate cells that were cd45 (as this marker is not expressed on mscs) and then subsequently used facs to isolate cells that were cd146.30 the most effect cell identification technique seems to be a combination of facs and macs, though the cell surface markers of choice vary between laboratories. in an effort to identify the most efficient marker of mscs, delorme and colleagues31 used microarrays and flow cytometry to culture a pure sample of mscs that expressed 113 transcripts and 17 proteins not found on other hematopoietic cells. they found that cd146 and cd200 were among the most efficient markers to purify mscs. following isolation, mscs can be cultured in either fetal calf serum or human serum, which show no difference in their effects on the cells to proliferate and differentiate.33 for bone formation, mscs are then directed to differentiate into osteoblast lineage cells via the aforementioned factors (e.g., transforming growth factor-) or via selective genetic expression (e.g., osx, zip1).34 35 allogeneic transplantation of mscs can be done in the site of fusion, due to their hypoimmunogenic and even immunosuppressive nature. flow cytometry experiments have shown that mscs express intermediate levels of hla class i and little to no hla class ii or costimulatory molecules (e.g., cd40).11 36 neither undifferentiated nor differentiated major histocompatibility complexes elicit lymphocyte proliferation when transplanted,36 37 and in fact, they tend to alter the cytokine profile to an anti-inflammatory state by decreasing tumor necrosis factor- and interferon-gamma and increasing interleukin-10, interleukin-4, and regulatory t cells.38 mscs have even been used for their immunosuppressive actions in treating acute graft-versus-host disease.39 for these reasons, host-versus-graft disease, or graft rejection, does not appear to be a problem with msc use in spine fusion. the use of msc therapy in bone regeneration has been and is currently investigated both in animal models and in the clinical setting. this is being done through both local implantation of the stem cells and via gene therapy, as well as through autologous transfer of engineered extracted mscs. following the original discoveries of mscs by friedenstein et al40 41 and then of owen in 1988 that mscs could differentiate into bone,42 43 several groups demonstrated successful autologous transfer of mscs in healing long bone lesions in various animal models.44 45 46 for example, arinzeh and colleagues used allogeneic mscs loaded in a ceramic hydroxyapatite-tricalcium phosphate scaffold to treat large femur defects in adult dogs. they showed that not only did a callus of lamellar bone fill the lesion within 8 weeks, with complete new bone form by 16 weeks, histologically, there was no immune response detected.46 in a rat posterior spinal fusion model, cui and colleagues showed that cloned osteoprogenitor cells implanted in the fusion bed led to successful spine fusion in all animals, compared with only 50% fusion success in animals that were implanted with mixed marrow stromal cells.47 similarly, muschler et al, in a canine model of dorsal spinal fusion, demonstrated the superiority of the marrow-derived osteoblastic progenitors in promoting spine fusion versus the growth factor and cellular milieu found in a bone marrow clot.48 the effectiveness of mscs in promoting spinal fusion has been shown in progressively larger animals, including rabbit,49 50 51 52 53 ovine,54 55 56 57 and primate models.58 59 specifically, wang and colleagues58 performed anterior lumbar interbody fusions in nine rhesus monkeys, with two fusion sites each, that either utilized autologous bone-marrow derived mscs on a calcium phosphate scaffold, icbg, or a control ceramic graft treatment. they found that the msc group had equivalent biomechanical strength as compared with the icbg group, and that they were both biomechanically and histologically superior to the control ceramic graft group. orii et al59 performed posterolateral lumbar spine fusions in nine macaque monkeys, which received either marrow-derived mscs with a -tricalcium phosphate graft, autogenous bone, or a control tricalcium phosphate graft treatment. using both x-ray and manual palpation to identify fusion status, they found that the group receiving the mscs had the highest fusion rate (83.3%) compared with the autogenous bone group (66.7%) and the control group (0% fused). the aforementioned studies used bone marrow-derived mscs; however, another approach with potentially fewer complications and less morbidity may be the use of adipose-derived mscs (ascs). ascs were first identified in 2001 by zuk and colleagues60 61 who showed that ascs can differentiate into multiple cell types including osteogenic and chondrogenic cells, thereby providing an potential therapeutic avenue for bone regeneration. these cell types were subsequently shown to be capable of adhering to a bioengineered scaffold, as well as remaining viable, proliferating, and differentiating under various conditions.62 rodbell and jones standardized the first protocol for asc isolation.63 64 65 cowan et al66 and later levi et al67 used ascs to regenerate bone in large mouse calvarial defects. lopez and colleagues68 performed dorsolateral spinal fusions in 56 fischer rats, with either no graft, only a scaffold, a scaffold with allogeneic ascs, or a scaffold with syngeneic ascs. similar to the studies investigating marrow-derived mscs, they found that when ascs were used there were fewer inflammatory infiltrates compared with the control groups, as well as superior bone formation and fusion when using the ascs. clinical trials are currently investigating ascs in treatments ranging from type i diabetes mellitus to liver disease69 70 and may soon be used for spine fusion treatments as well. although there has been much success demonstrated in the animal models, there remain barriers prior to this therapy's translation into the clinical setting. this includes identification of the optimal number and concentration of mscs, as well as the ideal preparation and implantation techniques needed.71 minamide and colleagues50 compared the use of differing number of marrow-derived mscs in a rabbit posterolateral spine fusion model. the low dose contained one million cells per milliliter and resulted in the fusion of zero of seven spines. in contrast, the high dose was one hundred million cells per milliliter and led to the fusion of five of seven rabbit spines. perhaps an even greater concentration would have led to 100% fusion. wang et al58 compared bone marrow-derived mscs to autogenous icbgs in rhesus monkey spine fusion models and found that although the fusions were equivalent in stiffness, the autograft produced greater bone volume. this can be explained by the fact that only three million cells per milliliter were used for the msc-treated group, in contrast to the 100 million that was used successfully in the study by minamide et al. finally, gan and colleagues,72 used bone-marrow-derived mscs for posterior spine fusion in 41 patients. using a slightly different quantification method, they recovered and implanted 16.1 million nucleated bone marrow cells per milliliter, of which they measured an average of 213 mscs per milliliter (mscs were defined as colony-forming units expressing alkaline phosphatase). despite the relatively small number of implanted cells, 95% of their patients achieved complete fusion by 34 months ' follow-up. it is clear that we have yet to determine the optimal number of mscs needed for complete fusion prior to the translation of this technology to the clinical setting. although some experimental data suggest that increased cell concentrations are required to repair bony defects,58 73 others have suggested that the cell concentration is not sufficient but rather the delivery type and biological environment of the graft will significantly affect success of the fusion.48 for example, the study by minamide et al50 used a three-dimensional culture, as compared with the more traditional two-dimensional monolayer culture, as they believed that it enhanced proliferation and differentiation. however, there is literature on the osteogenic potential of mscs in various other skeletal defects as well as ongoing clinical investigations of mscs in spine surgery with various commercial products such as neofuse (mesoblast ltd., melbourne, austrialia) and osteocel (nuvasive, san diego, ca the earliest clinical studies involving mscs involved small case studies of autologous mscs used for bone regeneration. quarto et al74 showed successful and abundant callus formation in three patients with tibial, ulnar, or humeral fractures using autologous mscs; lendeckel and colleagues75 reported a case study where autologous ascs were successfully used to treat a large calvarial injury. subsequent trials, including some larger ones, involved autologous bone marrow successfully used to promote bone fusion in tibial nonunions,76 77 autologous mscs for femoral head osteonecrosis,78 79 80 81 and allogeneic mscs to treat osteogenesis imperfect,82 all of which showed the clinical feasibility of therapeutic application of mscs to promote bone growth. the 2011 study by goldschlager and colleagues,55 which demonstrated superior bone formation in ewes with the use of allogeneic ovine mscs (mesoblast limited, melbourne, australia) compared with autograft or stand-alone scaffold, was the precursor study to the ongoing clinical trials (nct01290367, nct00996073, nct01097486, nct00810212, nct00549913, nct01106417) using this msc allograft product in both cervical and lumbar spine fusions. results on safety and preliminary efficacy in patients will likely be revealed in the coming years. much of the preliminary research done in animal models has shown potential for the use of mscs, both bone marrow and adipose derived, for bone regeneration. although there have been limited systematic clinical trials, small case studies have shown that msc use in humans may have successful bone growth and long-term durability.74 some current limitations include decreased bone growth compared with autograft,58 weaker mechanical stability of the implanted graft and poor resorption of the bioceramic constructs,74 and ambiguity surrounding the optimal cell concentration and delivery method. more studies examining optimal msc concentrations are needed in larger animals, which are more comparable to humans, considering the fact that there is decreased potential for bone growth as compared with smaller animals (e.g., rabbits, rats, etc.).58 83 84 this also demonstrates the need for methods to maximize the number of mscs collected, as well as techniques that can be feasible in the operating room setting.85 other options can include obtaining somatic cells and converting them into pluripotent cells,86 using minimally invasive approaches to collect and culture bone marrow- or adipose-derived mscs prior to surgery, and potentially even using recombinant forms of mscs. the present hurdles to clinical use include optimization of osteoinductive and osteoconductive properties of mscs in bone grafts. vascularization of the implant and integration of the vasculature with the host will prove to be important; additionally the long-term mechanical strength and durability, particularly at the load-bearing sites such as the lower lumbar spine regions will need to be comparable to native bone.87 allogeneic msc therapy for spine fusion and other skeletal treatments is still in its infancy. there has been a surge of interest in the various msc formulations commercially available for clinical use in spinal surgery. this enthusiasm, and clinical use, must be tempered with the understanding that there are no clinical data that had defined the efficacy or safety profiles in spine surgery patients. therefore, it is imperative that the spine surgery community carefully evaluate the use of msc in spine fusion through well-designed and executed studies. although more than a decade of preclinical animal research that has shown promising results, the safety and efficacy of these products in randomized controlled trials must be ascertained. with the rapidly growing number of spine fusion surgeries performed annually, further study into fusion-enhancing compounds becomes increasingly necessary.

more than 50% of patients complain of postoperative donor site morbidity following iliac crest bone graft harvest, and recent discoveries have identified adverse outcomes following bone morphogenetic protein use in spine fusion. this has led the spine community to turn toward alternative methods to promote fusion following spine surgery. the present article reviews numerous studies that have shown the osteogenic potential of mesenchymal stem cells (mscs). mscs have been used with both in vitro and in vivo models and have involved animal studies ranging from rats to macaque monkeys to successfully induce bone regeneration in lesions of the tibia and spine. there is no fear of graft rejection, as there may be with other allograft materials, because neither undifferentiated nor differentiated mscs elicit lymphocyte response when transplanted; they tend to alter the cytokine profile to an anti-inflammatory state. early clinical trials are underway with various commercially available msc formulations. although there is much enthusiasm, it is integral that the spine surgery community carefully evaluate the use of mscs in spine fusion through well-designed and executed studies to determine the efficacy and safety profiles in spine surgery patients.

PMC4813091

pubmed-994

the influenza viruses include three genera, a, b, and c within the family orthomyxoviridae. avian influenza viruses (aivs), all of which are contained in the genus influenza virus a, are an economically important cause of disease in fowl and occasionally affect humans, pigs, and horses [1, 2]. each genus of the virus is further subdivided into serotypes based on the surface proteins, consisting of 16 different hemagglutinin (h) and 9 neuraminidase (n) subtypes. while only a limited number of h and n subtypes are circulating in humans and other mammalian species, all the h and n subtypes are found in avian species [3, 4]. the genome of influenza a viruses consists of 8 unique segments of single-stranded negative sense rna. the viral rna segments encode 10 recognized gene products, pb1, pb2, and pa polymerases, h, np, n, m1, m2, ns1, and ns2 proteins. the h6 subtype is one of the most commonly recognized subtypes in domestic ducks in southern china [6, 7] and in migratory birds in north america and in europe [810]. h6 viruses have caused several outbreaks in commercial poultry worldwide that resulted in decreased egg production and increased mortality [1113]. during the hong kong h5n1 incident in 1997, an h6n1 avian influenza virus, teal/hong kong/w312/97 (w312), was isolated from birds in a live poultry market. genetic characterization of this virus revealed that except for the h gene, the remaining 7-gene segments were closely related to those of highly pathogenic avian influenza h5n1 viruses (hpaivs) found in both poultry and humans. the present study reports on the outbreak of avian influenza (ai) caused by an h6n1 subtype of aiv isolated during january 2009 in kibbutz gvulot, in the southern part of israel. the present isolate, a/turkey/israel/09 (h6n1), is the third h6 aiv obtained in israel since the year 2000. therefore, circulation of these viruses was concurrent with that of the highly prevalent h9n2 aiv in israel [15, 16]. while the former h6 aiv isolates, a/duck/israel/289/01 (h6n2) and a/mallard/israel/320/01 (h6n2), were isolated from water birds, the present isolate was obtained from domestic turkey poults. 2) located in one farm, kibbutz gvulot, exhibited an increased daily mortality of approximately 1% over a two-day period. the clinical signs were swollen infraorbital sinuses, nasal discharge, respiratory rales, rattles, and lethargy. birds in the affected poultry house had developed aiv antibodies, as detected by elisa and aiv h6 haemagglutination inhibition (hi) tests. the elisa test was performed with the flockchek avian influenza antibody test kit (idexx, usa) according to the manufacturer's instructions, and the hi test employed a panel of reference antisera to h5, h6, h7, and h9. blood samples were tested for antibodies by the hi test using 4 (ha) units of the h6 subtype antigen. turkey poults from the six houses were distributed to 4 secondary farms, mishmeret, ramon, ein zurim, and evron. tracheal and cloacal swabs of clinically-affected turkeys were assayed for the presence of aiv by pcr and virus isolation in 11-day-old embryonated eggs. the presence of aiv was initially detected in the allantoic fluids (af) of dead eggs after 2-3 days of incubation, by the hemagglutination (ha) assay. testing of ha-positive af was performed by reverse transcriptase polymerase chain reaction (rt-pcr). in parallel, rt-pcr was performed on rna extracted directly from tracheal and cloacal swabs. viral rna was extracted directly from the tracheal and cloacal swabs and from af using the qiaamp viral rna mini kit (qiagen, valencia, ca) according to the manufacturer's instructions. firstly, aiv was detected using a primer pair specific for the m gene of influenza a virus, mf (5-ttctaaccgaggtyraaacgt-3) and mr (5-ctgggcacggtgagcgt-3) to amplify a 200 bp product (panshin a., kimron veterinary institute, bet dagan, israel, unpublished). rt-pcr was performed using a one step rt-pcr kit, (qiagen ltd.). a 25 l mixture containing 5 l of buffer, 1 l of dntp mix, 1 l of each primer, 1 l of one step enzyme mix, 13 l nuclease-free water, and 3 l rna template was used in the assay as follows: 30 min at 50c, 10 min at 95c, 30 cycles at 94c for 30 sec, then 30 sec at 54c, 30 sec at 72c, 5 min at 72c, and 15 min at 4c using the dna engine thermal cycler (bio rad, waltham, ma, usa). secondly, molecular subtyping was performed with a set of specific primers for genes of h subtypes h1h4, h6, h9, and h10 and h5, h7, and n subtypes n1 n2, n3, n7, n9 (panshin a., kimron veterinary institute, bet dagan, israel, unpublished). the primers used to amplify a 623 bp segment of the n2 gene were n2f (5-ytgyacagtrgtaatgacbgatggr-3) and n2r (5-cratrctryttgadgtccaccabac-3); the primers to used to to amplify a 470 bp product of the n3 gene were n3f (5-aayagrccwtggrtgagrat-3) and n3r (5-cccgatccaggttcattgt-3). the primers used to amplify a 509 bp segment of the n7 gene were n7f (5-ttggrtggtcragyacaagctgcc-3) and n7r (5-accaggrctyccagtdatwggattg-3), the primers used to amplify a 520 bp segment of n9 gene were n9f (5-caaccaatgcaagccaaacaata-3) and n9r (5-tttcgggcccatgtgttgatttc-3). the amplification conditions for h and n genes were similar to those of the m gene. the pcr products were purified with the megaquick-spin pcr&agarose gel extraction system (intron biotechnology, inc, gyeonggi-do, korea) according to the manufacturer's instructions. sequencing reactions were performed by the weizmann institute of science, rehovot, israel, using a 3700 dna analyzer (perkin-elmer, applied biosystems, foster city, ca., u.s.a.) and analysed by capillary electrophoresis. the bioedit package, version 7.3, and dnastar system software were used for sequence analysis and alignment. nucleotide sequences of all gene segments and available sequence data from genbank were used to generate phylogenetic trees. the intravenous pathogenicity index (ivpi) of aiv isolates was determined as described in the oie manual of standards for diagnostic tests and vaccines. the test employs a standard volume of virus which is inoculated intravenously into spf chickens. the ivpi test yields an index of virulence valued from 0 to 3, which is calculated according to illness severity and the viability period following inoculation. the mishmeret farm received poults from the original poultry houses nos. 1 and 4 and developed a low level of hi antibodies (geometric mean titer (gmt)=1.8); the evron farm received poults from the original poultry houses nos. 5, and 6 and did not develop hi antibodies (gmt=0); the ramon farm received poults from the original poultry houses nos. 1, 4, 5 and 6, and did not develop hi antibodies (gmt=0); the ein zurim farm received poults from the original poultry house no. 2, that was infected with the a/turkey/israel/09 (h6n1) isolate, and from house no., birds on the ein zurim farm developed hi antibodies to h6 and showed clinical signs (gmt=6.3). to identify the aiv h and n gene, specificity amplifications and hi assays the virus was isolated and characterized by classical methods (oie) and genome was detected by pcr. a/turkey/israel/09 (h6n1) was characterized as low pathogenic avian influenza virus (lpai) with an ivpi of 0.00. infection in the chickens used in the ivpi assay was confirmed by the hi test. the amino acid sequence of the h gene cleavage site was examined to support the virulence determination of the a/turkey/israel/09/(h6n1) isolate. the amino acid sequence at the cleavage site was pqietr*glf, indicating that the isolate was of low pathogenicity, since it did not contain multiple basic amino acids, characteristic of hpaivs. most lpaivs have a single arginine at the cleavage site, whereas hpaivs usually exhibit a multibasic amino acid motif (r and k) flanking the cleavage site [22, 23]. the genome of influenza a viruses consists of eight unique segments of single-stranded rna, which are of negative polarity (i.e., complementary to the mrna sense). the 8 gene segments of the a/turkey/israel/09 (h6n1) isolate were sequenced. the nucleotide sequences obtained in the present study were compared to those of aivs from the genebank (figures 18). figure 1 shows the phylogenetic tree of the complete h gene, subtype h6, of representative recent aiv h6 isolates reported worldwide, including the previous two israeli h6 isolates, which were not reported to the genebank. it seems that the h gene of the present isolate, a/turkey/israel/09 (h6n1), differed from the h genes of the two previous israeli isolates, as they belonged to different clades, although the differences in the nucleotide content were about 3.7-3.8%. it is notable that the h6 aiv, that shaped the clade to which the present isolate belonged, originated from waterbirds. figure 2 shows the phylogenetic tree of the n gene of n1 subtypes, based on the analysis of nucleotides 204 to 1149. the analysis includes representative aiv n1 genes compared on the respective gene fragment. while the compared sequences differed up to 31%, the present isolate, a/turkey/israel/09 (h6n1), was closest to the n1 from a/mute swan/aktau/06 (h5n1) and a/swan/mangystau/06 (h5n1). it is notable to mention that the hong kong avian a/teal/hong kong/w312/97 (h6n1) and the human influenza virus a/hong kong/156/97 (h5n1) showed a very high nucleotide homology in the 6 aiv internal genes, and especially in the n1 gene sequence [14, 24]. that similarity might indicate a common precursor and that the present virus could become a potential source of novel pathogenic aiv strains. figures 3, 4, 5, 6, 7, and 8 show the phylogenetic comparison of the a/turkey/israel/09 (h6n1) isolate complete internal genes, ns, m, np, pa, pb1 and pb2, respectively, to genes of representative aivs. the main feature revealed from these phylogenetic trees was the close similarity between the former two h6n2 aiv isolates, a/duck/israel/289/01 and a/mallard/israel/320/01, as compared to the phylogenetic divergence of the present h6n1 isolate, a/turkey/israel/09. the phylogenetic distances between the 2009 h6n1 isolate and the two previous h6n2 isolates were calculated. while the two previously described h6 isolates, a/duck/israel/289/01 (h6n2) and a/mallard/israel/320/01 (h6n2), were identical in their ns, m, np, pa, pb1, and pb2 genes, the a/turkey/israel/09 (h6n1) isolate differed from the former two isolates by 3%, 2.3%, 6%, 2.5%, 3.6%, and 5%, respectively. in conclusion, the present paper describes a third h6 aiv isolate (h6n1) in israel, which unlike the two previous h6n2 isolates that were obtained from ducks and mallard, was now detected in turkeys. this h6n1 lpaiv was shown to possess a low-pathogenic amino acid sequence at the cleavage site and had a low ivpi. a/turkey/israel/09 (h6n1) was not highly transmissible among commercial flocks. that phenomenon was supported by two observations; (a) the absence of clinically-affected cases in adjacent poultry houses and (b) the detection of only three cases of aiv h6 in israel.

an avian influenza virus (aiv), a/turkey/israel/09 subtype h6n1, was isolated from turkey poults exhibiting typical pathology associated with aiv infection. the virus was characterized by rt-pcr using aiv subtype-specific primers and by the haemagglutination inhibition test using aiv subtype-specific antisera. the virus has an intravenous pathogenicity index of 0 and possessed a nucleotide sequence at the cleavage site of the hemagglutinin gene, pqietr*glf, associated with avian influenza viruses of low pathogenicity. unlike the two previous h6n2 isolates originating from domestic ducks and mallard, the a/turkey/israel/09 (h6n1) was isolated from turkeys. the gene sequences of the a/turkey/israel/09 (h6n1) virus show divergence from the former israeli h6 isolates.

PMC3447296

pubmed-995

the association of pancreatic disease with fat necrosis at distant foci was first described by chiari in 1883 (1). the most common pancreatic disorders associated with pancreatic panniculitis are acute or chronic pancreatitis, especially the alcohol-related types, and pancreatic carcinoma. other pancreatic disorders have been infrequently reported to be associated with pancreatic panniculitis, and these include post-traumatic pancreatitis, pancreatic pseudocyst, pancreas divisum, and hemophagocytic syndrome (2). to the best of our knowledge, one case was associated with pancreatic adenocarcinoma, and the other two were associated with acute and chronic pancreatitis, respectively (1, 3, 4). we report here on a case of pancreatic panniculitis associated with acute pancreatitis that had a fatal outcome. physicians should be aware of this disease entity because this rare cutaneous manifestation may be associated with major morbidity and significant mortality. a 50-yr-old man with a history of alcohol abuse presented with increasing fatigue, generalized weakness, decreased appetite, and abdominal distension and discomfort for the past 2 weeks. he was admitted to the department of internal medicine under the impression of acute pancreatitis, and he was referred to the department of dermatology for the multiple painful subcutaneous nodules on his legs, which had suddenly developed 3 weeks before (fig. his hemoglobin value was 11.4 g/dl (reference range: 13-18 g/dl), the total count of white blood cells was 31,300/l (reference range: 4,000-10,000/l) with 90.7% segment neutrophils, but the coagulation profiles and platelet counts were normal. the serum amylase was 1,909 u/l (reference range: 20-120 u/l), and the lipase was 2,306 u/l (reference range: 5-51 u/l), an alanine aminotransferase level of 24 u/l (reference range: 7-40 u/l), and a lactate dehydrogenase level of 665 u/l (reference range: 200-400 the fasting glucose level was 133.9 mg/dl (reference range of 70-110 mg/dl) and the electrolytes were unbalanced. the calcium level was 7 mg/dl (reference range: 8.6-10 mg/dl), and the sodium level was 125 the blood urea nitrogen and creatinine were 40.1 mg/dl and 2.1 mg/dl, respectively. after 48 hr, the blood urea nitrogen was increased to 62.7 mg/dl after intravenous fluid administration. as his leukocytosis, elevated serum ldh at admission, and hypocalcemia, hypoalbuminemia, increase in blood urea nitrogen during initial 48 hr were poor prognostic factors in ranson criteria, increased risk of complications was predicted. the chest radiograpy showed mild pleural effusion. on the abdominal computed tomography scan and magnetic resonance (mr) imaging taken on the second day of admission, a swollen pancreas with an dilated pancreatic duct, a loculated fluid collection in the left anterior perirenal space, multiple hepatic cysts, and massive ascites u/l, the serum-ascites albumin gradient was calculated to be 1.07, and there was no evidence of malignancy. a skin biopsy performed on the 4th day of admission from the nodule on the left lower leg showed a diffuse subcutaneous fat necrosis and ghost-like cells with thick shadowy walls and no nuclei. there was a fine granular basophilic material deposited within and around the necrotic fat cells (fig. after 10 days of intensive medical care for the pancreatic disease, the patient's condition began to worsen; he and his family began to refuse any further treatment. despite a strong warning by physicians the pathogenesis of pancreatic panniculitis is still unknown, but the released pancreatic enzymes such as trypsin may increase the permeability of the microcirculation. lipase or amylase is then involved in the process of fat degradation, which results in the liberation of free fatty acids that combine with calcium to form soap (2). although elevation of the pancreatic enzymes is common in pancreatitis patients, pancreatic panniculitis is a very rare malady. mullin et al. (5) identified only one case in a retrospective review of 893 patients who had suffered with pancreatic disease from various causes. furthermore, well documented cases of fat necrosis with normal serum lipase levels have also been described (6). these reports, suggest that there would be some other factors that allow the pancreatic enzymes to escape from the circulation and act on the subcutaneous fat. zellman (7) suggested that some damage to the blood vessels via inflammation, edema, or altered immunity may act as the initiating factor. as for the two cases in the korean literature, one case was associated with chronic pancreatisis, in which the cutaneous lesions had occurred 3 months later than the other systemic symptoms, and they involuted as the underlying pancreatitis was ameliorated (4). the other case was associated with acute pancreatitis with pancreas divisum, in which his skin lesions had appeared 2-3 days after his admission due to acute pancreatitis; the skin lesions also resolved as the underlying pancratitis subsided with conservative treatment (1). on the contrary, in our patient, the skin lesions preceded the other systemic symptoms. although the underlying pancreatic pathologic conditions can vary, the clinical features of pancreatic panniculitis are similar. the legs were the most commonly affected area, but the lesions can also occur on the arms, thighs, and trunk. the lesions usually began as erythematous or red-brown subcutaneous nodules with a tendency to show central softening. in the mild form if the fat necrosis is severe, individual nodules may break down and extrude necrotic material (8). although patients with other panniculitides such as erythema nodosum, erythema induratum, lupus panniculitis, weber-christian pannicultitis, or alpha-1 antitrypsin deficiency-associated panniculitis can have similar clinical lesions, the diagnosis of pancreatic panniculitis is suggested by the presence of pancreatic disease and this the pathognomonic findings are collections of ' ghost cells ' and anucleated adipocytes containing intracytoplasmic fine basophilic granular material from the saponification of fat. the resistance of the fat cell membrane to lipase produces the shadowy walls and the fatty acids combine with calcium to form calcium soap (10). sometimes conservative care can ameliorate the pancreatitis, and this then results in the resolution of the skin lesions. in isolated cases, surgical correction of anatomic ductal anomaly or pancreatic pseudocyst, or the removal of gallstones has resulted in complete resolution (8). however, disseminated fat necrosis is associated with major morbidity and mortality. in a review of 27 patients with pancreatic panniculitis, all 8 patients with pancreatic carcinoma and 42% of the 19 patients with pancreatitis died of their disease, as was seen in our case (9). pancreatic panniculitis is a pathognomic finding of pancreatic disease, and as in our case, the cutaneous lesions may precede the usual manifestations of underlying pancreatic disease by several weeks to months (11).

pancreatic panniculitis is a rare disease in which necrosis of fat in the panniculus and other distant foci occurs in the setting of pancreatic diseases; these diseases include acute and chronic pancreatitis, pancreatic carcinoma, pseudocyst, and other pancreatic diseases. this malady is manifested as tender erythematous nodules on the legs, buttock, or trunk. histopathologically, it shows the pathognomonic findings of focal subcutaneous fat necrosis and ghost-like anucleated cells with a thick shadowy wall. we herein report a case of fatal pancreatic panniculitis that was associated with acute pancreatitis in a 50-yr-old man. he presented with a 3-week history of multiple tender skin nodules, abdominal pain and distension. laboratory and radiologic findings revealed acute pancreatitis, and skin biopsy showed pancreatic panniculitis. despite intensive medical care, he died of multi-organ failure 3 weeks after presentation.

PMC2693864

pubmed-996

the acute respiratory distress syndrome (ards), a clinically important complication of severe acute lung injury (ali) in humans, is a significant cause of morbidity and mortality in critically ill patients [15]. infectious etiologies, such as sepsis and pneumonia, are leading causes of ali [1, 2, 5]. histologically, ali in humans is characterized by a severe acute inflammatory response in the lungs and neutrophilic alveolitis [1, 5]. the physiological hallmark of ards is disruption of the alveolar-capillary membrane barrier, leading to development of noncardiogenic pulmonary edema, in which a proteinaceous exudate floods the alveolar spaces, impairs gas exchange, and precipitates respiratory failure [1, 57]. ali can result in persistent respiratory failure and prolonged dependence on mechanical ventilation, increasing susceptibility to multiorgan dysfunction and mortality. despite extensive investigation aimed at early diagnostic and pathogenetic factors of ali, current management is mainly supportive, as specific therapies have not been identified [5, 913]. animal models focused on ali pathogenesis have yielded insights into mechanisms that initiate injury; however, little is known about potential determinants of resolution. thus, new strategies are still required for achieving effective treatment of ali, which might ultimately aid the clinical therapy for ali patients. progranulin (pgrn), also known as granulin epithelin precursor (gep), pc-cell-derived growth factor (pcdgf), proepithelin, and acrogranin, is an evolutionarily conserved, secreted glycoprotein with 7 granulin (grn) repeats [14, 15]. pgrn played a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing, host defense, and tumorigenesis [1520]. of interest, recent findings suggested that pgrn was a key regulator of inflammation and that pgrn might mediate its anti-inflammatory effects, at least in part, by blocking tnf- binding to its receptors. however, whether pgrn could inhibit the lung inflammation and ultimately ameliorate the ali was still unclear. recent evidence showed that elevated soluble tumor necrosis factor- receptor levels in bal fluid were found to be associated with poor patient outcome in ali, implying that blockade of pgrn by the soluble tumor necrosis factor- receptor might contribute to the development of ali. thus, we hypothesized that pgrn might exert as a promising molecule for treatment of inflammation in ali. to address this issue, here we carefully evaluate the potential role of pgrn in treatment of ali using the murine model of lps-induced ali. we found that administration of pgrn significantly reduced lps-induced pulmonary inflammation and resulted in remarkable reversal of lps-induced increases in lung permeability, accompanied by a significant reduction of histopathology changes of lung. our findings strongly demonstrated that pgrn could effectively ameliorate the lps-induced acute lung injury in mice, suggesting a potential role for pgrn-based therapy to treat clinical ards. female balb/c mice at 6 weeks old were purchased from the center of experimental animals of tongji university. all mice were housed in the pathogen-free animal facilities of tongji university school of medicine. all animal experiments were performed according to the guide for the ethical guidelines of the shanghai medical laboratory animal care and use committee and the ethical guidelines of the tongji university laboratory animal care and use committee. briefly, female balb/c mice (n=6 per group) were anaesthetized and orally intubated with a sterile plastic catheter, and challenged with intratracheal instillation of 800 g of lps (e. coli 055:b5; sigma) dissolved in 50 l of normal pbs. naive mice (without lps instillation) were injected with the same volume of pyrogen-free pbs to serve as controls. mice were humanely killed at 3 d after lps challenge to collect tissues for analysis. tnfr1 (cd120a) and tnfr2 (cd120b) antibodies for neutralizing studies were purchased from ebioscience. our initial experiment showed that 200 g of tnfr2 antibody was effective to significantly inhibit the protective effect of pgrn on the lps-induced ali. thus, 200 g of tnfr1 antibody or tnfr2 antibody was used for neutralization experiment in this study. groups of mice were treated with pgrn via intratracheal instillation 30 min after their challenge with lps. the administration dose of pgrn was 2 g per mouse which was based on our initial experiments. the level of pgrn in bal fluid was determined by western blot using the murine pgrn affinity purified polyclonal antibody (r&d systems) or by elisa using the commercial murine pgrn elisa kit (r&d systems). according to previously described, bal was performed by instilling 0.9% nacl containing 0.6 mmol/l ethylenediaminetetraacetic acids in two separate 0.5 ml aliquots. an aliquot of the bal fluid was processed immediately for total and differential cell counts. the remainder of the lavage fluid was centrifuged and the supernatant was removed aseptically and stored in individual aliquots at 70c. number of neutrophils was calculated as the percentage of neutrophils multiplied by the total number of cells in the bal fluid sample. all analyses were performed in a blinded fashion. in line with previously described, bal fluid collected was centrifuged at 800 g for 10 min, and supernatant was collected for analysis of total protein, albumin, igm, and cytokine/chemokine levels. proinflammatory cytokine levels including tnf-, il-1, and il-6 in bal fluid were measured with murine cytokine-specific quantikine elisa kits (r&d systems). chemokine levels including cxcl2, je (the murine homolog of human ccl2) and kc (the murine homolog of human il-8) in bal fluid were measured using cytokine-specific bead kits (r&d systems). albumin and igm levels in bal fluid samples were measured using with a murine-specific albumin elisa kit (alpco diagnostics) and a murine-specific igm elisa kit (bethyl laboratories), respectively. lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 m thick sections. sections were stained with hematoxylin and eosin, and images were taken with a nikon eclipse e800 microscope (200x). for the lung injury score, images were evaluated by an investigator who was blinded to the identity of the slides as previously described [5, 22]. in brief, the extent of the pathological lesions was graded from 0 to 3 as shown in table 1. the score for each animal was calculated by dividing the total score for the number of sections observed. differences between the treated groups versus the injured group were assessed using a one-way anova with statistic software (graphpad prism version 4.00). to assess the potential role of pgrn in lps-induced ali, we determined the level of pgrn protein in bronchoalveolar lavage (bal) fluid of lps-induced ali mice using western blot at day 3 after lps challenge. we found that the level of pgrn in bal fluid was significantly decreased on day 3 in mice challenged with lps compared with the control groups (figures 1(a) and 1(b), p<0.05). to further confirm this result, we further performed elisa assay to detect the level of pgrn in the bal fluid. similarly, we revealed that the protein level of pgrn was downregulated in bal fluid on day 3 in lps-induced ali mice (figure 1(c), p<0.05). further, we evaluated the time course of pgrn levels in bal fluid in lps-induced ali mice and the control mice. as shown in the figure 1(d), we found a substantial increase of pgrn protein on day 1 and then decreased since day 2, which indicated that pgrn might be subjected to proteolysis during inflammation in lung. consistently, we indeed revealed an elevated expression of granulin, which were the units of pgrn, in bal fluid in lps-induced ali mice (figure 1(e)). combing to access the potential role of pgrn in the development of ali, we evaluated the effect of pgrn administration in the maintenance of body weight and mortality of lps-induced ali mice. as shown in figure 2(a), we revealed that the loss of body weight was about 20% in lps-induced ali mice. notably, we found that administration of pgrn effectively abrogated the loss of body weight of lps-induced ali mice (p<0.05). interestingly, when pgrn was administered twice at 40 h intervals, it could further maintained the body weight of lps-induced ali mice to a level similar to the control mice (figure 2(a), p<0.05). furthermore, we found that the mortality was approximately 40% in lps-induced ali mice, while administration of pgrn in lps-induced ali mice effectively maintained their survival, which was more apparent in ali mice received pgrn twice at 40 h intervals (figure 2(b), p<0.05). these findings suggested that pgrn was an effective candidate for preventing the development of ali. to investigate the possible mechanism underlying the protective effect of pgrn on lps-induced ali, we detected the total cell and neutrophil counts in bal fluid from mice treated with lps with or without pgen. as shown in figure 3(a), the total inflammatory cell count in the bal fluid was increased dramatically at day 3 after administration of lps (p<0.05). we revealed that neutrophils accounted for about 80% of the increased inflammatory cells and was significantly elevated in bal fluid (figure 3(b), p<0.05). notably, we found that administration of pgrn could significantly reduce the total cell and neutrophil counts in bal fluid (figures 3(a) and 3(b), p<0.05). when pgrn was administered twice at 40 h intervals, it could further reduce the total cell and neutrophil counts in bal fluid to a significant lower level (figures 3(a) and 3(b), p<0.05). to further assess the anti-inflammatory effect of pgrn we found that proinflammatory cytokines, including tnf-, il-1, and il-6, as well as chemokines including cxcl2, je (the murine homolog of human ccl2), and kc (the murine homolog of human il-8), were all significantly elevated in bal fluid in response to lps challenge (figures 3(c) and 3(d), p<0.05). in contrast, administration of pgrn effectively decreased the levels of proinflammatory cytokines and chemokines (figures 3(c) and 3(d), p<0.05). consistent to the above findings, administration of pgrn twice at 40 h intervals further reduced the proinflammatory cytokines and chemokines to a significant lower level in bal fluid (figures 3(c) and 3(d), p<0.05). we next determined the concentrations of total protein, albumin, and igm in bal fluid to evaluate the integrity of the alveolar-capillary membrane barrier and assess pulmonary vascular leakage as a marker for ali. as shown in figures 4(a)4(c), we found that the levels of total protein, albumin, and igm in bal fluid were all significantly increased in mice challenged with lps compared with that in the control mice (p<0.05). whereas treatment with pgrn effectively reduced total protein, albumin, and igm levels (figures 4(a)4(c), p<0.05). notably, administration of pgrn twice at 40 h intervals restored these lung injury indicators to levels similar to the control mice (figures 4(a)4(c)). to evaluate the potential role of pgrn in the histopathology changes of lung in lps-induced ali mice, histological assessment of lung sections 3 days after the administration of lps with or without treatment was performed. we revealed the marked inflammatory infiltrates, interalveolar septal thickening, and interstitial edema in lps-induced ali mice (figure 5(a)). administration of pgrn effectively reduced the airspace inflammation, which was more apparent in mice treated with pgrn twice at 40 h intervals (figure 5(a)). furthermore, severity of lung injury was also scored using a semiquantitative histopathology score system [5, 21], which evaluates lung injury in four categories: alveolar septae, alveolar hemorrhage, intra-alveolar fibrin, and intra-alveolar infiltrates. we found that treatment with pgrn could significantly reduce lung injury scores, which was more apparent in mice treated with pgrn twice at 40 h intervals (figure 5(b), p<0.05). recent findings suggested that pgrn could bind to tnfr and thus mediate its anti-inflammatory effects in collagen antibody-induced arthritis and collagen-induced arthritis. therefore, we next assessed the possible role of pgrn/tnfr interaction in the protective effect of pgrn on lps-induced ali. groups of mice were pretreated with neutralizing antibodies to tnfr1 or tnfr2, respectively, and then challenged with lps with or without pgrn treatment. as shown in figures 6(a)6(d), we found that neutralization of tnfr1 had no significant influence on the protective effect of pgrn on the lps-induced ali as evidenced by similar levels of total inflammatory cell count, proinflammatory cytokines, albumin, and igm in bal fluid. in contrast, blockade of tnfr2 significantly abrogated the protective effect of pgrn on the lps-induced ali as evidenced by elevated levels of total inflammatory cell count, proinflammatory cytokines, albumin and igm in bal fluid (figures 6(a)6(d), p<0.05). consistently, we found that blockade of tnfr2 but not tnfr1 could effectively inhibit the protective effect of pgrn on the histopathology changes of lung in lps-induced ali mice (figures 6(e) and 6(f), p<0.05). similar results were also obtained in mice treated with pgrn twice at 40 h intervals (data not shown). these findings suggested that pgrn/tnfr2 interaction was crucial for the protective effect of pgrn on lps-induced ali. ards is a complex clinical syndrome that is initiated by injury to the lung, often in the setting of pneumonia or sepsis. here we carefully evaluated the potential role of pgrn in treatment of ali using the murine model of lps-induced ali. we found that administration of pgrn effectively maintained the body weight and survival of lps-induced ali mice. furthermore, pgrn administration significantly reduced lps-induced pulmonary inflammation and resulted in remarkable reversal of lps-induced increases in lung permeability. moreover, administration of pgrn contributed to a significant reduction of histopathology changes in lung of lps-induced ali mice. our results provided clues for developing pgrn-based therapies to treat with ali. accumulating data suggested that pgrn played an important role in inflammatory response [15, 23, 24]. here we evaluated the expression of pgrn protein in bal fluid of lps-induced ali mice. we found that the level of pgrn protein in bal fluid was significantly downregulated 3 days after lps challenge in lps-induced ali mice. previous study showed that during inflammation, neutrophils, and macrophages released proteases which digested pgrn into individual 6 kda granulin units, which were actually proinflammatory and could neutralize the anti-inflammatory effects of intact pgrn [23, 24], which might partly explain the decreased level of pgrn protein in bal fluid of lps-induced ali mice. consistently, we indeed revealed an elevated expression of granulin, which was the units of pgrn, in bal fluid in lps-induced ali mice. however, the precise mechanism underlies the downregulation of pgrn in bal fluid of lps-induced ali still remains to be elucidated. in the present study our findings suggested that pgrn was a key regulator of inflammation and exerted an anti-inflammatory effect, which were in line with previous studies. as the half-life time for pgrn is about 40 hours, we further performed the second injection of ali mice with pgrn at 40 h intervals, and found that this strategy resulted in a more apparent reduction of the development of lps-induced ali. it should be pointed out that we did not observe any significant effect of pgrn alone on the lung injury of nave mice in this study (data not shown). our data strongly suggested that pgrn was an optimistic candidate for the treatment of ali. however, the lps-induced model of ali can not fully reproduce the complexity of clinical ali/ards in human patients. therefore, it is necessary to reproduce these findings in more clinically relevant models. besides, it is important to define the therapeutic window of pgrn intervention for ali at different dose and time points. in addition, it is also important to explore the possible effect of pgrn administration on host immune response in ali. the translation of our results into an effective new therapy for ards in patients will require, at the very least, that these issues be addressed. tnf-/tnfr signaling has received great attention due to its position at the apex of the proinflammatory cytokine cascade and its dominance in the pathogenesis of various disease processes [2528]. previous study showed that pgrn could bind to tnfr and then block the tnf- binding to its receptors. in this study, we evaluated the potential role of pgrn/tnfr interaction in the protective effect of pgrn on lps-induced ali. we demonstrated that blockade of tnfr2 but not tnfr1 could significantly inhibit the protective effect of pgrn on the lps-induced ali. in addition, we found that neutralization of tnfr1 or tnfr2 had no significant effect on the total cell response of ali mice (data not shown). our findings were consistent with previous study which showed that tnfr2 seemed to play an important role in ards. one is that tnfr1 is expressed ubiquitously, whereas tnfr2 expression is tightly regulated and found predominantly in hematopoietic cells [30, 31]. however, the precise mechanism for the effect of pgrn on the development of lps-induced ali undoubtedly needed successive studies. in the present study, we demonstrated a murine model of ali that administration of pgrn effectively prevented the development of lps-induced ali, at least in part, through their interaction with tnfr2. these findings might have potentially important implications for the treatment of ards, a clinical syndrome resulting from ali in human.

the acute respiratory distress syndrome (ards), a clinical complication of severe acute lung injury (ali) in humans, is a leading cause of morbidity and mortality in critically ill patients. despite decades of research, few therapeutic strategies for clinical ards have emerged. here we carefully evaluated the effect of progranulin (pgrn) in treatment of ards using the murine model of lipopolysaccharide (lps)-induced ali. we reported that administration of pgrn maintained the body weight and survival of ali mice. we revealed that administration of pgrn significantly reduced lps-induced pulmonary inflammation, as reflected by reductions in total cell and neutrophil counts, proinflammatory cytokines, as well as chemokines in bronchoalveolar lavage (bal) fluid. furthermore, administration of pgrn resulted in remarkable reversal of lps-induced increases in lung permeability as assessed by reductions in total protein, albumin, and igm in bal fluid. consistently, we revealed a significant reduction of histopathology changes of lung in mice received pgrn treatment. finally, we showed that pgrn/tnfr2 interaction was crucial for the protective effect of pgrn on the lps-induced ali. our findings strongly demonstrated that pgrn could effectively ameliorate the lps-induced ali in mice, suggesting a potential application for pgrn-based therapy to treat clinical ards.

PMC3431103

pubmed-997

patients who present to the emergency department with chest pain require rapid triage, evaluation and management. myocardial infarction with st segment elevation (ste) on electrocardiography (ecg) is a common presentation in emergency rooms across the world. acute coronary syndrome (acs) marked by ste on the ecg warrants consideration for emergent cardiac catheterization and possible percutaneous coronary intervention (pci). in fact, there are now core measures and quality metrics in place that grade a hospital's efficiency at caring for such patients. however, the time pressure to optimize such quality metrics may lead to an unintended rush to treatment prior to adequate evaluation. in this report, we present a rare case where myocardial infarction was seen as a presenting feature of an underlying hematologic disease, thrombotic thrombocytopenic purpura (ttp). this case highlights the importance of a thorough, yet efficient, clinical evaluation in which the history, physical exam, ecg and laboratory data were needed to make the appropriate triage decision and not miss an unusual diagnosis. a 48-year-old woman with no known coronary risk factors was transferred from an outside facility to the cardiac catheterization laboratory of our hospital with the diagnosis of ste myocardial infarction for primary pci. on arrival to our catheterization laboratory, the ecg from the referring hospital showed sinus tachycardia with normal axis and intervals. there was ste in leads i, ii, avl, v46 and reciprocal st segment depression in lead iii [figure 1]. laboratory data were not yet available. however, the patient's history of present illness was significant for malaise, fever, chills and lethargy that began 3 days prior to hospitalization. further questioning established that she had mild generalized abdominal pain and one episode of non-bloody diarrhea. the family also noted that she had been intermittently confused and was talking gibberish. on the morning of admission, she had severe chest pain associated with nausea, vomiting and dyspnea on exertion, which led her to seek medical care. sinus tachycardia at 121 beats per minute with st segment elevation in lead i, ii, avl, v46 on examination, she appeared toxic and in respiratory distress. vital signs revealed a blood pressure of 126/70 mmhg with a heart rate of 121 beats per minute. in addition, mottling of her skin and livedo reticularis over the thighs was noted. there were a few purpuric skin lesions observed in her antecubital fossa and upper arms. her jugular venous pressure was elevated up to the angle of the jaw. cardiac exam revealed a normal first and second heart sound along with a fourth heart sound. peripheral pulses were not palpable in the feet and were only faintly palpable in the arms. neurologically, she was somewhat confused, but the sensory and motor exam was essentially normal. given that the patient was not having active chest pain, the history was inconsistent with acs and she appeared more toxic than expected for a lateral wall myocardial infarction; cardiac catheterization was deferred and emergent laboratory studies were obtained. initial laboratory studies revealed a white blood cell count of 13.5 10/mm; hematocrit of 24%; mean corpuscular volume of 88.4 fl and platelet count of 6 10/l. her lactate dehydrogenase was elevated at 2820 units/l and haptoglobin was low at less than 10 mg/dl. coagulation profile showed international normalized ratio of 1.2, prothrombin time of 12.6 seconds, fibrinogen 199 mg/ l and d-dimer 1.27 feu electrolytes were within normal limits; acute kidney injury was noted with blood urea nitrogen 51 mg/ dl and creatinine 1.9 mg/dl. total bilirubin was markedly elevated at 32 mg% with an indirect bilirubin of 2.1 mg%. cardiac biomarkers were elevated with creatinine kinase of 487 units/l and mb fraction of 28.8 ng/ml. troponin-t was 0.86 ng/ml. urinalysis showed ph of 6.0, 3+albumin, 3+hemoglobin, eight wbcs and greater than two rbcs with some amorphous crystals. echocardiography demonstrated an ejection fraction of 4045% with severe hypokinesis of the inferior and basal anteroseptal wall. no significant valvular lesions were noted. in view of the acute onset of symptoms associated with microangiopathic hemolytic anemia, thrombocytopenia, acute kidney injury and waxing and waning mental status, the presumptive diagnosis of ttp was made. a disintegrin and metalloproteinase with thrombospondin motifs (adamst-13) activity was found to be low with presence of adamst-13 inhibitors in the plasma. she improved clinically with this treatment and normalized her platelets and lactate dehydrogenase, and ste in her ecg resolved [figure 2]. outpatient cardiovascular follow-up demonstrated normalization of the left ventricular ejection fraction by echocardiography and a completely normal nuclear myocardial perfusion stress study. further testing with invasive catheterization was not pursued as the entire episode was considered to be transient, secondary to metabolic derangement. ttp is defined as a severe, thrombotic microangiopathy that is characterized primarily by systemic platelet-von willebrand factor aggregation, organ ischemia, profound thrombocytopenia and fragmentation of erythrocytes. pathologically, focal areas of necrosis and hemorrhage may be seen in the pancreas, adrenals, heart, brain and kidneys.[35] although myocardial injury and necrosis are observed in a large number of patients with ttp, it is infrequently the initial presentation, and most likely thought to be due to microthrombi from massive platelet aggregation than plaque rupture thrombosis cascade. various studies have determined the incidence of myocardial infarction in ttp to range from 1541%.[69] however, the heart is one of the most frequently involved organs at autopsy examination of patients with ttp. mortality is considerably higher in patients with ttp who have positive cardiac biomarkers, necessitating closer monitoring in this subgroup. early recognition of myocardial injury in a case of ttp is crucial as it identifies higher risk. however, invasive therapy in the form of cardiac catheterization and pci may be fraught with complications and is precluded by acute kidney injury and low platelet count. thrombocytopenia also prevents the use of usual medical management in acs such as antiplatelet and anticoagulant therapy. beta blockers and hmg coa reductase inhibitors may be used although their role is questionable. in acute bouts of ttp, such as this case, the treatment of choice is rapid initiation of plasmapheresis. in addition, immunosuppressive therapy including steroid therapy is helpful, especially in the setting of auto-antibodies against adamst-13 factor. relapsing cases of ttp have been treated with rituximab, a monoclonal antibody against cd20 on memory b cells with good effect. although cardiac involvement is common in ttp, as an index event it can be misleading. as swift protocols are activated in the emergency room and catheterization laboratories to ensure quality control, it is equally important to integrate all aspects of patient's clinical and objective data to detect unusual disease entities

myocardial infarction with st segment elevation (ste) on electrocardiography (ecg) is a common presentation in emergency rooms across the world. myocardial injury and necrosis are infrequently the initial presentation in patients with thrombotic thrombocytopenic purpura (ttp). a 48-year-old woman presented with ste myocardial infarction from outside hospital for primary percutaneous coronary intervention. however, her clinical picture was not consistent. rapid evaluation revealed symptoms associated with microangiopathic hemolytic anemia, thrombocytopenia, acute kidney injury with waxing and waning mental status. a diagnosis of ttp was made with low adamst-13 activity. plasmapheresis was initiated along with intravenous steroid therapy. the patient had a full recovery and went home after full recovery of left ventricular ejection fraction and normal myocardial perfusion studies. rapid evaluation is needed to identify infrequent causes of ste myocardial infarction. as swift protocols are activated in the emergency room and catheterization laboratories to ensure quality control, it is equally important to integrate all aspects of the patient's clinical and objective data to detect unusual disease entities.

PMC3354466

pubmed-998

microalbuminuria (ma) was delineated many years ago to describe the appearance of small quantities of albumin, too little to detect by standard dipstick methods, in urine samples from subjects with diabetes [1, 2]. according to recent guidelines, ma may be defined as the urinary excretion of 30300 mg/d in a timed urine collection in adults. when spot urine samples are used, the recommended definitions of ma are the excretion of 17250 mg/g of creatinine in men and 25355 mg/g of creatinine in women. the gender-associated differences in definition result from variation in daily creatinine generation females have lower creatinine generation than males. in addition, variations in the quantification of albuminuria for these definitions of ma may need to be applied to the elderly (who have greatly decreased creatinine generation) or to excessively muscular individuals (who have high creatinine generation). urinary albumin concentration in untimed urinary collections may also be used to define ma values of 330 mg/dl generally are regarded as fulfilling the definition. albumin in urine is most often measured by immuno-turbidimetry using antibodies reactive with intact albumin. alternatively, high-pressure liquid chromatography can be used, but this method also measures nonimmunologically reactive albumin and albumin fragments, which yields higher values than immuno-turbidimetry. in general, ma corresponds to a trace reading on dipstick testing of urine, but results depend on the degree of urine concentration. dye-impregnated strips that have enhanced sensitivity for albumin are also available. simple, portable, point-of-service instruments that measure small amounts of albumin make assessment of ma readily available, reliable, and inexpensive. the dominant viewpoint is that albumin is normally filtered by the glomeruli in small but significant amounts, about 1 to 2 mg/min (~ 2 g/d), but the great majority (99%) of filtered albumin is reabsorbed and degraded by the proximal tubule, leaving less than about 5 g/min (7 mg/d) to be excreted. a minority view is that larger amounts of albumin are filtered, as much as 200 g/d, but a retrieval pathway in the proximal tubule reclaims the filtered albumin intact and delivers it to the circulation, except for small amounts of intact and partially or fully degraded albumin. in pathologic states, the glomeruli may become increasingly permeable to circulating albumin by virtue of disturbances in endothelial cell function, basement membrane abnormalities, or podocyte (visceral epithelial cell) disorders. currently, the focus is on disturbances of endothelial or podocyte function as causes of excessive albumin glomerular permeability, rather than disturbances of basement membrane physiology. albumin is negatively charged at physiologic ph and may be impeded in transglomerular passage by the anionic residues in endothelial cell, glomerular basement membrane, or podocyte. however, the existence of a charge-selective permeability barrier has been challenged. a reduction in proximal tubule reclamation or reabsorption of filtered albumin. finally, physicochemical alterations in the circulating albumin molecule can affect its permeation through the glomerular capillary wall, by altering the shape or possibly the electrical charge. thus, the mechanisms underlying increased urinary albumin excretion are complex and it is often difficult to ascribe ma in specific patients to a unique pathophysiologic process; however, endothelial cell dysfunction, a podocytopathy, or both seem to underlie most instances. nevertheless, it remains possible that alterations in proximal tubular function may contribute to albuminuria in some circumstances, such as during poorly controlled glycemia in diabetes mellitus. these considerations are important when one attempts to relate albumin excretion rates to the definition of kidney disease, because albuminuria arising from diffuse endothelial cell injury (eg, in atherosclerosis and hypertension) might best be regarded as a sign of a systemic disease process, affecting many organs and systems, rather than one originating in the kidneys. alternatively, albuminuria arising from a podocyte dysfunction could legitimately be regarded as a manifestation of a kidney disease. regardless of the underlying mechanisms and interpretation of the pathophysiologic meaning of albuminuria, important associations exist between the quantities of albumin excreted in the urine and progressive chronic kidney disease (ckd) and cardiovascular (cv) events. microalbuminuria, as defined above, has been consistently associated with an increased likelihood of progression of generic ckd to more advanced stages or even to end-stage renal disease (esrd) in large epidemiologic studies [12, 13, 14, 15]. these observations have been used to justify the inclusion of ma in the definition of generic ckd in the original classification schema of the kidney disease outcome quality initiative (kdoqi) of the national kidney foundation (nkf) in 2002. indeed, the presence of ma (defined as uacr of 30299 mg/g creatinine without gender or age adjustments) as the sole reason for diagnosing generic ckd accounted for 90% of those with stage 1 ckd and 87% of those with stage 2 ckd in the us national health and nutrition examination survey (nhanes) conducted from 1999 to 2004. in this cohort (n=13,233), 6.8% had self-reported diabetes and 27% had diagnosed hypertension. thus, the great majority of subjects designated as having stage 1 or 2 ckd in nhanes had isolated ma (ma without a clear reduction in estimated glomerular filtration rate [egfr], urine sediment changes, or structural kidney disease) as the defining abnormality. it is natural to ask whether such isolated ma is sufficient reason to designate a person as having generic ckd. as shown by a recent, very large, collaborative meta-analysis (chronic kidney disease prognosis consortium; n=105,872 subjects), the hazard ratio (hr) for all-cause mortality and cardiovascular mortality (adjusted for the effects of age, ancestry, history of cv disease, systolic blood pressure, diabetes, smoking, and total cholesterol concentration) rises progressively above a uacr of about 10 mg/g [17]. in subjects with normal renal function (egfr=90104 ml/min/1.73 m), the hr for cv mortality is 1.63 at a uacr of 1029 mg/g, 1.82 at 30299 mg/g, and 4.77 at greater than 300 mg/g. a decrease in egfr magnifies the effect of an increased uacr on both all-cause and cv mortality, especially at an egfr of 45 ml/min/1.73 m or lower. thus, there is little or no doubt that increased albumin exertion, even below the conventional ma range, is associated with an increased likelihood for mortality, both all-cause and cv-related. is elevated albumin excretion a biologic marker of underlying systemic disease (eg, cancer, atherosclerosis), or is it somehow involved in the causal pathway for fatal events, including cv disease? similarly, the risk of developing a progressive form of ckd is linked somehow to the presence of elevated albumin exertion. a large, community-based study from alberta, canada, (n=920,875) showed that subjects with normal or nearly normal renal function (egfr 60 ml/min/1.73 m, average age 46 years) and ma had event rates of esrd of 1.5 and doubling of serum creatinine of 2.8 times those of subjects with absolute events rates were low: 0.06 per 1000 person-years of follow-up for esrd in the normo-albuminuric group versus 0.09 per 1000 patient-years for the ma group. a lowered egfr magnified the effect of ma on the occurrence of esrd and doubling of the serum creatinine, especially at levels below 45 ml/min/1.73 m. in another community-based study (n=65,589 adults; 3.3% diabetic; average age, 50.1 years), researchers noted that the likelihood of developing (and surviving long enough) to receive treatment for esrd among subjects with ma and egfr 60 ml/min/1.73 m was 27 times that of subjects with normo-albuminuria and similar egfr levels [13]. subjects with an egfr less than 60 ml/min/1.73 m and ma had a 5.4-fold to 81-fold increased risk of esrd compared to those with an egfr 60 ml/min/1.73 m, depending on the degree of decrease in egfr. thus, the addition of ma to egfr greatly enhances the ability to detect and quantify risk of progressive ckd, particularly when egfr is less than 60 ml/min/1.73 m. these observations call for a revision of the 2002 version of the kdoqi-nkf classification schema for ckd that does not include albuminuria for diagnosis of stage 3 or above ckd taken together, these findings from epidemiologic studies using large databases strongly support the view that ma needs to be considered as a biomarker of adverse outcomes, even among those subjects with normal or nearly normal renal function. however, the strength of this association between ma and outcomes may vary by subject age and underlying disease (eg, diabetic or nondiabetic) or by concomitant illness known to influence albumin excretion rates (eg, obesity). for example, in the aforementioned meta-analysis conducted by the chronic kidney disease prognosis consortium [17], the pooled estimates of fully adjusted hr for cv mortality tended to be higher in those younger than 65 years of age compared to those older than 65 years with apparently equivalent degrees of ma. an analysis of the impact of ma on outcomes in the old-old (> 75 years of age) has not been conducted; however, as stated above, uacr has a tendency to increase with normal aging, predominantly because of the effects of sarcopenia on creatinine generation, not necessarily because of an absolute increase in albumin excretion with aging. furthermore, the association of ma with adverse outcomes in subjects with normal or nearly normal renal function (egfr 60 ml/min/1.73 m), after adjustment for the effects of aging may simply reflect the overall burden of atherosclerotic vascular disease in this group of subjects. as recently pointed out by kalaitzidis and bakris, untangling the influences of metabolic disturbances and hypertension in diabetic and nondiabetic subjects with concomitant ma can be problematical. it is still uncertain if ma is an integral part of the pathophysiologic pathways for cv disease or simply a bystander. the sine qua non of specific forms of ckd in which ma is present is that of diabetes mellitus. indeed, the concept of ma arose from studies of the natural history of diabetic nephropathy during the period 1981 to 1982 [1, 2]. shortly after its initial description in type 1 diabetes, ma we now recognize that ma in type 1 diabetes is a dynamic process with frequent spontaneous regression to normo-albuminuria [19, 20]. we also recognize the wide array of extra-renal factors that can influence albumin excretion, such as obesity, age, gender, distant inflammation, and certain drugs (eg, rosuvastatin) [21, 22, 23]. furthermore, studies done at the joslin clinic over many years indicate that more advanced stages of ckd are not uniformly associated with progression of ma to overt microalbuminuria [19, 24]. pathologic abnormalities indicative of diabetic nephropathy (eg, increased mesangial fractional volume) precede the development of ma, and very clearly renal functional decline can occur in the absence of ma, at least in type 1 diabetes. type 2 diabetes may be another story, because the relationship of ma and renal functional decline is not as well studied or understood as in type 1 diabetes, and this relationship is confounded by concomitant accelerated atherosclerotic macrovascular disease. nevertheless, several studies showed that progression of type 2 diabetes can occur in the absence of ma [21, 2226]. interventions designed specifically to reduce ma have not consistently shown a reduction of later progression to advanced ckd or esrd. some recent interventional trials (eg, the avoiding cardiovascular events through combination therapy in patients living with systolic hypertension [accomplish] trial) involving type 2 diabetic subjects showed that the change in albumin excretion rates can be disassociated from risk of progression to esrd under the influence of combined renin-angiotensin system blockade and calcium channel antagonism [21, 27]. taken together, these observations in type 1 and type 2 diabetes suggest, but do not prove, that ma may not be a reliable marker of kidney disease or its progression. among patients with well-established kidney disease (ie, egfr<4550 ml/min/1.73 m), the magnitude of albuminuria can be viewed as a marker of a more progressive course and a more rapid loss of renal function (a risk marker rather than a risk factor) for both diabetic and nondiabetic renal disease [28, 29]. this finding may well be a manifestation of lead-time bias, in that those with lower levels of albumin excretion have disease in its earlier stages and thus a greater reserve of functioning nephrons, whereas those with higher levels of albuminuria have no reserves and with each loss of nephrons, a corresponding decline in renal function becomes evident. in this sense, the level of albumin excretion is a relevant marker of kidney disease and risk of its progression. a conundrum exists when renal function is entirely or nearly normal: no overt structural renal disease is evident, yet albumin excretion is elevated into the ma range. no doubt this circumstance somehow predisposes to, or is a marker of a predisposition to, cv disease. for example, albuminuria correlates with parameters of aortic stiffness and carotid plaque formation but not with carotid intima-media thickness as a surrogate for widespread atherosclerosis. on the other hand, ma does correlate with signs of altered endothelial function, such as endothelium-dependent vascular dilatation [3133]; however, it is often difficult to disentangle the effects of alterations in renal function and concomitant metabolic and dyslipidemia states in identifying direct relationships between ma and endothelial dysfunction. isolated ma may well be a biomarker of widespread vascular injury and atherosclerotic burden. in this sense, it is not measuring a kidney disease per se, but only a secondary and indirect effect of a distant disease process on kidney physiology. although this conclusion may appear to be semantic rather than reality-based, it has immediate relevance for the diagnosis of ckd using current classification schema (ie, kdoqi-ckd). stage 1 and 2 ckd in this schema can not be reliably distinguished on the basis of egfr alone because current creatinine-based egfr formulas are imprecise. whether newer cystatin c based formulas will correct this deficiency remains to be seen; preliminary findings are encouraging, but not uniformly so [34, 35]. most cases of stage 1 and 2 ckd are currently defined on the basis of ma, typically in an isolated form. this definition is useful to stratify the eventual risk of cv events and all-cause mortality; however, does it have the same utility for defining the presence of and risk for progressive ckd and eventual esrd at all levels of egfr and in all ages? current evidence suggests that the utility of ma in predicting renal outcomes is most pronounced in those with well-established kidney disease (eg, egfr<4550 ml/min/1.73 m) and in younger subjects (eg,<65 years of age). in my opinion, whether it is appropriate to regard isolated ma as equivalent to kidney disease is still an open question. a compromise position would be to regard this laboratory finding as an indication of kidney damage rather than giving it the more ominous label of disease. this issue is not trivial about 30% of those diagnosed as having ckd in epidemiologic studies have their kidney disease identified by the finding of ma and egfr 60 ml/min/1.73 m in the absence of a self-reported diagnosis of diabetes.

levels of urinary albumin excretion that are below the usual limit of detection by qualitative testing, but are above normal levels (microalbuminuria; ma), can be readily identified by simple measures, such as the urinary albumin to creatinine ratio in untimed urine samples. such measurements, particularly when combined with assessment of estimated glomerular filtration rate (egfr), have utility as biomarkers for enhanced risk of all-cause mortality, cardiovascular events, progressive chronic kidney disease, and end-stage renal disease in diabetic and nondiabetic subjects. however, it is controversial whether isolated ma (ma in the absence of a clear reduction in egfr, urine sediment abnormalities, or structural renal disease) should be regarded as kidney disease. such ma could also be regarded as a manifestation of a diffuse endothelial (microvascular) injury and thereby collateral kidney damage. this article reviews the current evidence concerning ma as a marker of kidney disease or kidney damage.

PMC2941636

pubmed-999

diabetes mellitus (dm) is a metabolic disorder characterised by an increase in plasma glucose level due to insulin deficiency and/or resistance that can lead to damage to multiple organs. currently, approximate 347 million people are suffering from dm worldwide and the number will continue to increase. there are two types of dm: type 1 diabetes mellitus (t1 dm) and type 2 diabetes mellitus (t2 dm). t1 dm is caused by the destruction of the pancreatic -cells due to an autoimmune reaction, leading to absolute insulin deficiency, whilst t2 dm is characterised by insulin resistance, where the body fails to produce an appropriate physiological response to circulating insulin. t2 dm accounts for approximately 90% of all dm cases with the prevalence increasing with age. tremendous effort has been invested to understand the complications of dm and its impact on vision loss, neuropathy, and cardiovascular diseases; however, dm-induced cognitive dysfunction is seldom addressed and is not as well understood. since life expectancy has been markedly prolonged with advances in medicine, it has been suggested that the incidence of t2 dm and dementia would increase as the population ages [1, 2]. indeed, several epidemiological studies have shown that people with t2 dm have a significantly higher risk of developing cognitive impairments and dementia when compared to those with normal blood glucose levels [35]. a recent population-based longitudinal study has shown that the relative risk of alzheimer's diseases (ad) and vascular dementia (vad) in the dm population was 1.46 (95% ci: 1.201.77) and 2.5 (95% ci: 2.13.0), respectively, when compared to people without dm. moreover, dm has been suggested to be an individual risk factor for dementia [7, 8], independent of other established risk factors, such as hypertension and atherosclerosis [8, 9]. traditionally, ad is considered as the major diagnosis of dementia; however numerous clinical-pathological studies have suggested a significant contribution of cerebrovascular diseases to cognitive decline [11, 12]. although the exact pathophysiology of dm-mediated dementia has not been fully elucidated, existing evidence has shown that both cerebrovascular changes and neurodegeneration are implicated in the development and progression of dm-mediated cognitive dysfunction. to date nevertheless, numerous reports have highlighted the therapeutic potential of antidiabetic therapies in the treatment and prevention of cognitive dysfunction [1315]. in the largest randomized controlled trial to date, the accord-mind study, it was shown that the decline in total brain volume was significantly reduced in the intensive glycemic control group, compared to the standard glycemic control group. although the cognitive outcomes were not different, the effect of glycemic control in preserving cerebral structure can not be denied. findings from these studies have clearly indicated that treatments targeting dm could be a novel strategy to prevent dementia development and potentially to slow down the progression of cognitive dysfunction. through the advances in pharmacological therapy, many oral antidiabetic agents have become available. interestingly, oral antidiabetic drugs such as thiazolidinedione and metformin have been shown to have beneficial effects to slow the progression of dementia in both clinical and animal studies [17, 18]. however, many of these pharmaceutical agents are associated with various undesirable side-effects, such as weight gain, fluid retention, and increased risk for heart failure, limiting their compliance and utility in clinical practice. traditional chinese medicine (tcm), including chinese herbal medicines (chm) and acupuncture, has been used for thousands of years for the management of disease, maintenance of health, and prolongation of life expectancy. accumulated evidence suggests that many chms and their active ingredients possess hypoglycemic properties and that some tcm interventions have beneficial effects in the treatment and prevention of dm and its complications, with minimal toxicity and fewer adverse reactions. in addition, existing evidence has demonstrated the therapeutic potential of tcms in dm-mediated cognitive dysfunction. in this paper, we present a comprehensive review of current understanding of dm-mediated dementia and the scientific research on the use of tcms for the management of cognitive dysfunction in dm. an increasing number of studies have reported an acceleration of cognitive decline in patients with dm, independent of common cardiovascular risk factors [8, 22]. to date, dm is recognised as an independent risk factor for the development of cognitive dysfunction. in a meta-analysis based on twenty-five studies, it was estimated that t2 dm patients have 1.5-fold greater risk of cognitive dysfunction and 1.6-fold increased risk of dementia, when compared to people without diabetes. similarly, a recent report has shown a 1.5-fold higher risk of ad in people with diabetes than those without diabetes. most reports so far have suggested an increased risk of global cognitive dysfunction in diabetes [6, 23, 24], while some reports showed more selective cognitive impairment, mainly affecting learning, mental speed, and visuospatial process [2527]. it is important to point out that the discrepancy between these studies may simply be due to the variation of neurocognitive testing, such as age, education, sex, history of other illnesses, and the duration/severity of diabetes [28, 29]. many complications of diabetes, such as retinopathy, lower limb ulcers, and atherosclerosis, usually take years to develop before becoming clinically apparent. however, cognitive function decrement has been observed in the early stage of t2 dm. in children with t1 dm, deficits in cognitive development, including vocabulary, block design, general intelligence, speed of processing, and learning, have been observed as early as 2 years after the onset of t1 dm. these findings suggested that deficits in regulation of blood sugar level, even at an early stage, would have detrimental effects on cognitive function. interestingly, recent studies have demonstrated that elevated blood glucose levels may be a risk factor for impaired cognitive function leading to dementia, even among people without diabetes [32, 33] highlighting the relationship between high blood glucose level and dementia outcome. there is ample evidence from neuropsychological studies reporting that people who have dm also suffer from mild cognitive impairment (mci). longitudinal studies have shown that approximate 55% of patients with mci developed probable alzheimer's dementia over 3 years [34, 35] and the progression rate reached 100% after 9.5 years. it has been suggested that dm patients have 50% higher chance of developing alzheimer's disease than those without dm. progression of mci to dementia has been shown to be markedly accelerated by diabetes in elderly subjects who were either cognitively intact or diagnosed with mci at baseline. brain imaging studies have provided direct evidence to support dm-mediated mci and dementia [39, 40]. resting-state functional magnetic resonance imaging (rs-fmri) studies have revealed abnormalities in amplitude of low-frequency fluctuations (alff) in t2 dm patients in multiple brain regions. these present as decreased alff in the bilateral middle temporal gyrus and left fusiform gyrus and increased alff in bilateral cerebellum posterior lobe and right cerebellum culmen. moreover, recent studies have shown that alternation of alff and reduced connectivity of the hippocampus are associated with the presence of diabetic vascular disease and poor cognitive performance in t2 dm patients [42, 43]. although the mechanism between mci and the increased risk of dementia under dm is not fully understood, it has been suggested that the dm-mediated mci and dementia are not likely to form a continuum, given the difference in etiologies and risk factors between mci and dementia [44, 45]. people suffering from dm over a long period have been shown to express an elevated level of dementia [46, 47]. there are an extensive number of studies examining the effect of dm on cognitive functions in elder population [4850]. it has been shown that the prevalence of dementia in t2 dm patients increased over age, from 2.4% in the age group of 6576 and 5% in 7685 to 8.3% for patients over 85 years of age. several studies have also reported an increased incidence of dementia in individuals who were diagnosed with dm in midlife after an extended follow-up of 2535 years. however, the exact effect of midlife against late-life dm onset on cognitive impairment and dementia remains to be clarified. the mechanisms underlying the development of cognitive dysfunction in diabetes have not been fully elucidated. many hypotheses have been suggested based on the pathophysiological mechanisms through which diabetes might affect the initiation and progression of the pathology of dementia. these proposed mechanisms include various diabetic-specific factors or signalling pathways that may influence cognitive functioning, such as hyperglycemia, insulin deficiency, microvascular complications, and inflammation. in this section, we will highlight some of the risk factors and possible mechanisms related to cognitive dysfunction in diabetes (figure 1). vascular complications, including atherosclerosis, hypertension, stroke, and vascular comorbidity, are closely associated with dm. recent studies have reported that vascular complications are likely to be an important determinant of cognitive dysfunction and dementia. increased cerebral infarcts and reduction of amyloid-beta load a meta-analysis of longitudinal studies suggested that there is a stronger association of vascular-related cognitive impairment than ad with dm patients. interestingly, less alzheimer's-like pathology has been observed, but more ischemic lesions in t2 dm patients with a clinical diagnosis of dementia have been observed. indeed, an increasing number of studies are suggesting that the reduction of cerebral perfusion plays a significant role in the development of ad, supporting the hypothesis that cerebrovascular pathology such as stroke predisposes cognitive decline and dementia development. the detrimental effects of dm on cognitive function in vascular dementia have been demonstrated in a recent preclinical study. showed that exacerbated cognitive functions caused by diabetes were mediated via augmentation of neuronal cell death in the hippocampus through creb/bdnf signalling pathway in an animal model of vascular dementia. hypertension has been shown to be a significant risk factor for poor cognitive performance in both t1 dm and t2 dm patients [57, 58]. in addition to hypertension, atherogenic dyslipidemia is another common vascular risk factor in dm. dyslipidemia contributes to atherosclerosis development and has been found to increase risk of dementia in diabetes. moreover, reduced cerebral blood flow, upregulation of inflammatory cytokines [63, 64], endothelial dysfunction, and abnormalities in cerebral capillaries have been demonstrated in patients with diabetes. changes in cerebral vasculature by these factors are closely associated with stroke and brain damage, including brain infarct and white matter lesions, contributing to cognition deterioration in diabetes. given the complex pathophysiology of vascular complications in diabetes, more research is required to explore the exact mechanisms around how these dm-related vascular risks contribute to cognitive decline and dementia. however, majority of current findings have confirmed the association of vascular risk factors and cognitive decrements in diabetes and support the belief that predominant cerebrovascular pathology in diabetes could aggravate cognitive functioning. blood glucose levels are regulated by the endocrine system involving multiple organs and signalling molecules and pathways. upset of this precisely regulated process could lead to imbalance of blood glucose level, resulting in organ damage. although the exact mechanisms behind the association between dm and cognitive impairment or dementia are unclear, studies have shown that it is a multifactorial process where metabolic condition plays a significant role. chronic high blood glucose levels have been shown to have negative effects on cognitive functions and brain structure. numerous studies have demonstrated a close relationship between glucose intolerance and cognitive decrements and dementia [24, 69, 70]. it has been shown that people with poor glycemic control, with glycosylated hemoglobin (hba1c) higher than 7.0%, have a 4-fold higher risk of developing cognitive impairment. similarly, an inverse association of hba1c and cognitive function such as working memory, learning, and executive functioning has been observed in t2 dm patients. the results of these studies highlight the contribution of poor glycemic control in cognitive function deterioration process. multiple toxic effects of hyperglycemia on the brain, such as formation of advanced glycated end products (ages), generation of reactive oxygen species (ros), and activation of polyol, diacylglycerol, and hexosamine pathways, have been suggested. ages have been shown to contribute to microvascular complications, accelerated amyloid-beta deposition and senile plaque formation [9, 71]. a preclinical study has demonstrated that increased cerebral ages expression is associated with cognitive dysfunction in diabetic mice. similarly, increased ages levels have been observed in ad patients with t2 dm, when compared to nondiabetic ad patients. moreover, ages lead to ros generation via activation of a rage cell surface receptor for ages, which in turns leads to neuronal injury [76, 77]. it is well established that oxidative stress is implicated in both the onset and progression of diabetes and its complications. it has been shown that cognitive deficit caused by hyperglycemia in diabetic rat is associated with an increase in ros levels and reduction of antioxidant levels [78, 79]. in addition, increased ros generation has been shown to activate various cellular signalling pathways, such as the polypol pathway, protein kinase c activation, and increase of glucose shunting via the hexosamine pathway, all of which are related to neuronal injury and cerebral damage. interestingly, it was shown that administration of antioxidants could reverse the cognitive dysfunction in the diabetic rats [78, 79], suggesting a potential therapeutic target for dm-mediated cognitive impairment. sufficient glucose supply is vital for normal brain function and it is well established that hypoglycemia has detrimental effects on the brain [81, 82]. repeated hypoglycemic episodes are a common side-effect in patients who receive intensive insulin therapy for diabetes. in animal studies, it has been shown that exposure to low blood glucose levels can cause cerebral energy failure, neuronal necrosis, and brain damage leading to a flat electroencephalograph and cognition dysfunction. in human autopsy studies, multifocal or diffuse necrosis of the cerebral cortex, basal ganglia, and hippocampus was observed in patients who died of hypoglycemia. a dose-response relationship between the occurrence of severe hypoglycemic episodes and risk of dementia development has been reported in a retrospective study involving 16,667 t2 dm patients. although contradicting results have been reported by some studies [85, 86], arguing that tolerance to a hypoglycemic state can be developed in patients exposed to hypoglycemia chronically, the effect of hypoglycemia on some high-risk groups can not be ignored. for example, it has been shown that impairment of memory functioning is strongly correlated with severe hypoglycemia in t1 dm patients. the blood glucose level is regulated by insulin, a hormone generated by the beta cells in the pancreas. traditionally, it was believed that the brain is an insulin independent organ; however, recent studies have suggested otherwise. it has been shown that insulin is actively transported across the blood brain barrier and is also produced locally in the brain. furthermore, insulin receptors are expressed in the hippocampus and the cortex, indicating its functional role in the brain. in addition, being a regulator of food intake and energy homeostasis, insulin also plays a role in memory and learning. changes in insulin levels and receptor sensitivity could lead to deficits in cognitive function. in ad, impairments of cerebral insulin receptors activation and elevated insulin level in the csf have been reported, indicating the contribution of insulin in cognitive decline and dementia development. hyperinsulinemia is a common characteristic of t2 dm and has been identified as a risk factor for cognitive dysfunction and dementia progression [95, 96]. it has been suggested that hyperinsulinemia is associated with reduction of amyloid metabolism, due to downregulation of insulin-degrading enzyme (ide) levels in the brain. therefore reduced ide levels would lead to a accumulation in the brain, contributing to ad and cognitive impairment. it has been suggested that inhibition of insulin-mediated pathways can lead to hyperphosphorylation of tau and a production, via activation of the glycogen synthase kinase 3 (gsk3) signalling [99, 100]. it has been suggested that people suffering from dm are under a state of subclinical chronic inflammation [101, 102]. numerous proinflammatory markers and cytokines, such as c-reactive protein (crp), tumour necrosis factor- (tnf-), interleukin- (il-) 1, and il-6, have been shown to be upregulated in both t1 dm and t2 dm. given the fact that many of these inflammatory markers found in dm patients are closely associated with the pathogenesis of ad, there is an increasing interest in the link between dm and dementia. yaffe et al. reported that impaired cognitive functions were observed in dm patients with elevated crp and il-6 levels, but not in patients with normal levels of these markers. firstly, it has been shown that chronic inflammation in dm can induce changes in blood brain barrier (bbb) permeability. moreover, increased bbb permeability can also allow access of toxic substances and metabolites into the brain, leading to cerebral damage. secondly, neuroinflammation is a well-established factor in the development of cognitive decline, dementia, and other neurodegenerative diseases [109, 110]. it has been demonstrated that inflammatory cytokines can cause activation of glia cells leading to neuronal damage. for example, tnf- has been shown to induce hippocampal dysfunction, via activation of the jnk and the ib kinase/nfb signalling pathway [111113]. finally, inflammation plays a central role in the development of complications in vasculature, including stroke, contributing to cognitive impairment and dementia development as discussed in section 3.1. given the fact that dm is now an established risk factor for cognitive dysfunction and dementia, there is an increasing interest in targeting dm for the treatment of cognitive decline and dementia. several studies suggested that oral antidiabetic drugs such as thiazolidinedione and metformin could offer therapeutic benefits to reduce dm-related cognitive dysfunction in both patients and animal models [17, 18]. a recent study demonstrated that glycemic control using empagliflozin significantly prevented cognitive impairment via attenuation of cerebral oxidative stress and increase in cerebral brain-derived neurotropic factor in a t2 dm mouse model. this highlighted the potential of antihyperglycemic agents in the treatment of t2dm-related cognitive dysfunction. however, some clinical studies have demonstrated rather limited and inconsistent benefits of these glucose lowering agents in limiting cognitive decline and dementia development [1416]. furthermore, the risks associated with the use of antiglycemic/insulin therapies have raised the concerns regarding the long-term safety and effectiveness of these interventions for the management of dm-induced cognitive dysfunction and dementia [84, 85]. for example, hypoglycemia is commonly observed in t1 dm patients with tight glycemic control and in advanced t2 dm patients [116, 117]. although intensive insulin therapy has shown to successfully control glycemia and reduce vascular complications in dm patients, several reports have highlighted possible neuronal damage and cognitive impairment due to the incidence of hypoglycemia associated with the insulin therapy. traditional chinese medicine (tcm) has been used to treat dm for over thousands of years. a large number of tcm interventions belonging to several key modalities (such as herbal medicine, acupuncture, and taichi) have been used for the management of dm and its complications (table 1). according to traditional chinese medical system, chinese practitioners, who often adopt a holistic approach in treating their patients, manage diabetes through integrated care: nourishing and strengthening the body's functions rather than focusing solely on blood glucose control [119, 120]. despite a lack of scientific validation of the tcm interventions for diabetes, the accumulated evidence has demonstrated some promising results in relieving the symptoms and complications of diabetes [121, 122]. in this section, we will firstly review the current clinical findings of tcms used for dm-related cognitive dysfunction and then highlight some of the potential mechanisms underlying the tcm effects on the condition. numerous clinical studies have demonstrated the beneficial effects of tcm interventions on cognitive dysfunction and dementia. data from a meta-analysis suggest that tcm interventions appear to be a safer and more effective treatment for vascular dementia, based on 31 randomised clinical trials comparing 1605 patients on tcm treatments with 1263 patients on western medicine or placebo. it has been suggested that sleep apnea hypopnea syndrome (sahs) patients with t2 dm have a higher risk of cognitive decline than the nondiabetic sahs patients. interestingly, in a clinical study, a 2-week treatment with danhong injection, consisting of extracts of salvia miltiorrhiza and carthamus tinctorius l., significantly improved the montreal cognitive assessment (moca) score, especially in the executive function and memory domains, in 86 sahs patients with t2 dm. although blood glucose levels were not examined in this study, the results indicate that danhong injection could improve cognitive function in t2 dm patients. another clinical study in 36 t2 dm patients has demonstrated that combined huang qi (radix astragali) and chuanxiong (ligusticum wallichii) injections over 30 days significantly reduced blood glucose levels and improved cognitive function, whilst the standard pharmaceutical care with antidiabetic agents in the control group only reduced the blood glucose levels, indicating that the herbal formula may provide therapeutic benefits to dm patients beyond its blood glucose lowering property. in a clinical trial involving 164 diabetic patients with complicated coronary heart disease (chd), it was shown that while both isosorbide mononitrate (20 mg, twice a day) and fufangdanshendiwan (consisting of salvia miltiorrhiza, panax pseudoginseng var. f.) (270 mg, once daily) treatments for 16 weeks significantly improved the cardiac ischemia burden (as measured by 24-hour ambulatory ecg monitoring), improvements in insulin sensitivity and lipid metabolism indexes were only observed in patients received fufangdanshendiwan. moreover, the herbal formula was also shown to reduce a formation and improve cognitive function in the dm patients. as mentioned in section 3, increased oxidative stress and inflammation li and yeung have demonstrated that an 8-week treatment with zhi nao capsule consisting of extracts of codonopsis pilosula, polygonatum sibiricum, ligusticum wallichii, and acorus tatarinowii significantly increased serum superoxide dismutase (sod), reduced crp level, and limited cognitive decline and dementia development in t2 dm patients. in china, it is not an uncommon practice to use integrative strategies, combining tcm and western medicine interventions, in the treatment of dm and its complications. numerous studies have assessed the efficacy of the combined therapies to treat cognitive dysfunction in dm patients. nao xin tong, a complex herbal formula (consisting of radix astragali, salvia miltiorrhizae, angelicae sinensis, ligusticum wallichii, paeoniae rubra, flos carthami tinctorii, gummi olibanum, resina commiphorae myrrhae, ramulus cinnamomi cassiae, buthus martensi, lumbricus, and hirudo seu whitmaniae) has been shown to improve cognitive function in 32 stroke patients with t2 dm. in patients who received a combined therapy of alprostadil with lao xin tong, a greater cognitive enhancing effect was observed when compared to the alprostadil only group. in a clinical trial, 48 dm patients with vad were randomised to receive piracetam or piracetam plus oral shengmaidingzhi decoction (consisting of pseudostellaria heterophylla (miq.) pax, ophiopogon japonicus, schisandra chinensis, wolfiporia extensa, polygala tenuifolia willd., acorus tatarinowii, pinellia ternata (thunb.) breit, semen persicae, panax pseudoginseng var. the combined therapy group demonstrated greater improvements in the activities of daily living scale (adls) and the scale of elderly cognitive functions (secf) when compared to that of the piracetam only group. several studies have demonstrated that a combined therapy of bushenquyuyizhi decoction (consisting of cistanche deserticola, acorus tatarinowii, and panax pseudoginseng var. notoginseng) with nimodipine produced a significantly greater effect than nimodipine alone on cognitive function [155, 156]. in a more recent study conducted by zhao et al., 85 patients with dm-mediated vascular cognitive dysfunction were allocated to receive a 6-month treatment of aspirin (100 mg, once daily) or aspirin (100 mg, once daily) plus bushenquyuyizhi decoction (2.5 g per day, orally), over 6 months. at the completion of the 6-month treatment, cognitive dysfunction was improved in both treatment groups. however, the cognitive improvement was only maintained in the combined therapy group 12 months after the treatment, indicating a potential long-term effect of the herbal intervention, possibly via enhancement of general health and limiting disease progression in the patients. electroacupuncture is a form of acupuncture by way of applying a small electric current between pairs of fine needles that are inserted into acupoints selected according to the tcm theory. several recent studies have also demonstrated that electroacupuncture improved cognitive function and quality of life in diabetic patients [159, 160]. although the sample sizes were small (ranging from 25 to 32 patients) in these studies, the therapeutic potential of electroacupuncture should not be ignored and further investigations with a rigorous design are warranted. a large number of in vitro and in vivo preclinical studies have been conducted to assess the underlying mechanisms of tcm interventions in diabetes-related cognitive dysfunctions. reduced antioxidative levels and increased ros generation are closely associated with the pathogenesis of diabetes and its complications. antioxidant properties of chms have been demonstrated in numerous studies [161, 162]. for example, green tea, which is commonly consumed in eastern and asian countries, contains of a mixture of plant polyphenols that possess antioxidative and radical-scavenging activities. in obese kk-ay mice, green tea catechins reduced blood glucose levels and insulin resistance via inhibition of the tnf--induced ros generation. it has also been shown that green tea catechins markedly suppressed memory regression in samp10 mice, a mouse model of brain senescence with cerebral atrophy and cognitive dysfunctions. daily consumption of green tea catechins significantly reduced brain atrophy and suppressed dna oxidative damage. these effects were associated with the improvement of plasma antioxidative activity caused by daily green tea consumption. a recent study showed that green tea catechins remarkably ameliorated learning and memory impairments in a diabetic rat model, via the reduction of oxidative stress and nitric oxide modulation. in this study, green tea catechins also significantly reduced the blood glucose levels, indicating that green tea can suppress diabetes-mediated cognitive dysfunction via both hypoglycemic and antioxidative effects. indeed, numerous other chms, such as berberine and ginsenoside, have been shown to reduce diabetes-mediated cognitive decline via reduction of oxidative stress [167, 168]. tanshinol (tsl), a bioactive component of danshen (salvia miltiorrhiza), widely used for vascular disease, was shown to improve spatial working memory and attenuated vascular dementia in rats, via an increase in acetylcholine levels and reduction of acetylcholinesterase activity. tanshinone iia (tan iia), another bioactive component of danshen, was also shown to restore diabetes-induced nerve deficiency. the regulation of the cholinergic neurotransmission in the brain plays a vital role in memory and cognitive function. similar to tsl, several other herbal constituents, including lycopene, berberine, and curcumin, have been shown to ameliorate diabetes-related cognitive dysfunction, via protection of the cholinergic neurotransmission [167, 172, 173]. in addition, several other mechanisms have been proposed as underlying the effect of chms on dm-mediated cognitive dysfunction, such as through reduction of ages-mediated neuroinflammation and the downregulation of cerebral amyloid-beta (a). for example, danshensu, a bioactive component of salvia miltiorrhiza, has been shown to improve learning and memory in diabetic mice via suppression of age-mediated neuroinflammation. these effects were independent of blood glucose, insulin, and glycosylated hemoglobin levels, indicating a direct neuroprotective and anti-inflammatory effect of danshensu. liuwei dihuang decoction (lwdhd), a well-established tcm formulation, consisting of six herbs (rehmannia glutinosa libosch., cornus officinalis sieb., dioscorea oppositifolia l., paeonia ostii, alisma orientale (g. samuelsson) juz., and poria cocos (schw.) wolf), has been shown to attenuate neural apoptosis and a deposition in the hippocampus and cerebral cortex in a streptozotocin-induced diabetic rat model. in addition, lwdhd also reduced blood glucose levels, decreased oxidative stress, and suppressed inflammation in hippocampus of the animals. interestingly, chen et al. showed that administration of zibupiyin recipe (zbpyr), a modification of the zicheng decoction (consisting of 12 herbs: panax ginseng c. a. meyer, dioscorea opposita thunb. tomentosa, and glycyrrhiza uralensis fisch.), over a 6-week treatment, prevented dm-associated cognitive decline in db/db mice. the observed effect was possibly due to improving dendritic spin density and attenuating brain leptin and insulin signalling pathway injury. although these findings need to be confirmed in humans, it provides important preclinical data to support the potential benefits of tcm in preventing and slowing the development and progression of dm-associated cognitive dysfunction. data from the above studies have clearly indicated that the positive effects of chms could be mediated by multiple pathways and mechanisms. combination therapy underpins the philosophy of chms, where patients are generally treated with multiherb formulations. complex chemical mixtures of chms enhance therapeutic efficacy by facilitating synergistic action and/or ameliorating/preventing potential side-effects. the multicomponent and multitarget approach of chms makes them ideal therapies for disorders such as dm-mediated cognitive dysfunction and dementia, which have multifactorial/multisystem pathophysiological components. substantial effort has been invested to understand the effects of diabetes on cognitive decline and dementia in the past decade. recent studies have identified the risk factors and possible mechanisms underlying the pathogenesis of the dm-mediated cognitive dysfunction. by taking advantage of recent advancements in these processes, it is possible to develop better therapies for dm-related cognitive complications, including ad, vascular dementia, and cognitive decline. numerous clinical studies have highlighted the potential of tcms in the treatment of dm-related cognitive decline. despite the fact that most of these trials have shown positive outcomes, significant methodological issues such as small sample sizes and poor randomization exist in many of these studies. therefore, more rigorously designed randomized controlled trials are required to further validate these findings. finally, most of the preclinical studies assessing the effects of chms on dm-mediated cognitive dysfunction were performed using single herbs or isolated active ingredients. given that the traditional use of chms is based on complex formulation, more research on the synergic effects of herbal combinations is required for a more comprehensive understanding of the mechanisms underlying their effect on the diseases.

diabetes mellitus (dm) is a metabolic disorder affecting a large number of people worldwide. numerous studies have demonstrated that dm can cause damage to multiple systems, leading to complications such as heart disease, cancer, and cerebrovascular disorders. numerous epidemiological studies have shown that dm is closely associated with dementia and cognition dysfunction, with recent research focusing on the role of dm-mediated cerebrovascular damage in dementia. despite the therapeutic benefits of antidiabetic agents for the treatment of dm-mediated cognitive dysfunction, most of these pharmaceutical agents are associated with various undesirable side-effects and their long-term benefits are therefore in doubt. early evidence exists to support the use of traditional chinese medicine (tcm) interventions, which tend to have minimal toxicity and side-effects. more importantly, these tcm interventions appear to offer significant effects in reducing dm-related complications beyond blood glucose control. however, more research is needed to further validate these claims and to explore their relevant mechanisms of action. the aims of this paper are (1) to provide an updated overview on the association between dm and cognitive dysfunction and (2) to review the scientific evidence underpinning the use of tcm interventions for the treatment and prevention of dm-induced cognitive dysfunction and dementia.

PMC4427766

pubmed-1000

we sampled ants in three regions of morocco: first in the north-west, in the region of tangiers-tetouan where we focused on several areas: (1) mediona and cap spartel within the province of tangiers, (2) al hamra and balota within the province of tetouan, and (3) dardara, fifi, talassemtane national park, and bouhachem natural park within the province of chefchaouen. second, we surveyed the eastern region where we focused on two areas: (1) site of bni snassen within the province of berkan and (2) temsamane within the province of driouch. the third region of interest was marrakech-tensift-el haouz situated in the south-west of morocco, where the okaimden mountain in the province of al-haouz, was surveyed (fig. 1.the provinces and localities where sampling was undertaken for this study. the provinces and localities where sampling was undertaken for this study. for collections of ants from nests, specimens were collected under direct vision using an aspirator and forceps. a berlese funnel technique was also utilized for extracting ants from soil and leaf litter and at a few sites we used pitfall traps. specimens were studied under a leica s4d stereomicroscope and identified using available keys (cagniant 1996, 2009) taxonomic nomenclature follows bolton (2014). below, our collections are organized as follows: a2808: (3w, 1q); where a2808 refers to the collection code of reyes-lpez, and where (3w, 1q) refers to 3 workers and 1 queen. this information is followed by the date, locality, gps coordinates, and altitude in meters above sea level. plagiolepis pygmaea, formicinae material and data: a2816: nest; 11 june 2011; talassemtane national park, tazaout; 35 7.95 n, 05 06.76 w; 1,680 m; a1627: (14w); 15 july 2010; talassemtane national park, jbel bouslimame, spanish fir, abies pinsapo, forest; pitfall trap; 35 07.05 n, 05 08.16 w; 1,500 m. this species was found in rabat and casablanca by cagniant (1962). although it was described as a rare exotic of european origin, introduced to morocco through human activities, cagniant has never mentioned this species again. the workers that we examined, correspond to the latest description of this species (sharaf et al. 2011), in having funicular segments 2 and 3 of equal length, and distinctly broader than long, and 9 ommatidia in the longest row of the eyes. as jbel bouslimame is in a protected area, very remote from human settlements, we suspect p. pygmaea is indigenous to morocco. ponera testacea, ponerinae material and data: a2808: (3w, 1q); 16 may 2011; talassemtane national park, akchour; 35 15.88 n, 05 08.83 w; 566 m; a2807: (18w); 05 june 2011; talassemtane national park, tazaout; 35 15.90 n, 05 08.19 w; 1,475 m; a2743: (1w); 11 september 2011; bouhachem natural park; 34 59.51 n, 04 49.21 w; 1,390 m; a2676: (4w, 3q); 5 june 2011; talassemtane national park, tazaout; 35 15.90 n, 05 08.19 w; 1,475 m; a2673: (2w); 29 may 2011; talassemtane national park; 35 04.78 n, 05 10.10 w; 890 m. a2618: (10w); 12 july 2011; bouhachem natural park; 35 16.33 n, 05 29.47 w; 1,152 m. it seems this species is widespread in the north of morocco. we found a multitude of them, including whole nests, in leaf litter and soil samples, and under rocks and moss. the main place where we found this species is the talassemtane national park (chefchaouen). x. espadaler (unpublished data) previously collected it in morocco at: (1) bab-bou-idir, 25 may 1986, 1,500 m, q. canariensis, q. ilex; espadaler leg.; and, (2) tazzeka, taza, 29 march 1997, 1,400 m, c. hernando leg. strumigenys tenuipilis, myrmicinae material and data: a2596: (1w); 13 july 2011; chefchaouen, quercus suber forest; 35 06.10 n, 05 18.20 w; 488 m; a2598: (4w); 14 june 2011; dardara, quercus suber forest; 35 07.24 n, 05 17.60 w; 374 m. extracted from leaf litter and soil samples using the berlese funnel method. at dardara, it was given species status by brown (1953, p. 132) as s. tenuipilis. after further changes in generic placement, however, baroni urbani and de andrade (2007) have argued that it, along with many dacetines, should revert to the original strumigenys. in morocco, s. baudueri, p. argiola (cagniant 2006) and recently p. membranifera (taheri andreyes-lpez 2011), all of which are now placed in strumigenys, have been recorded from morocco. temnothorax pardoi, myrmicinae material and data: a2822: (14w); 04 september 2011; tangers, mediona, cap spartel; 35 46.57 n, 05 54.42 w; 214 m; a2821: (1w); 13 september 2011; chefchaouen, fifi; 35 01.39 n, 05 12.25 w; 1,190 m. extracted from samples of soil and leaf litter using the berlese funnel method. this species was described from workers captured in the south of the iberian peninsula (tinaut 1987). there are other records of this species being found in different areas of the iberian peninsula, including portugal (tinaut 1987). the present finding is important because up to now this species has been considered as endemic to the iberian peninsula. we intend to survey the coastal areas of the tingitane peninsula (north of the rif) in order to precisely delimit the area of its distribution. tetramorium parvioculum, myrmicinae material and data: a2599: (19w); 14 june 2011; chefchoauen, dardara, cork oak forest; 35 07.24 n, 05 17.60 w; 374 m; a2597: (6w); 13 july 2011; bouhachem, quercus suber woodland; 35 06.10 n, 05 18.20 w; 488 m; a2605: (2w); 11 july 2011; chefchaouen, dardara, akerat, cork oak forest; 35 06.10 n, 05 18.20 w; 488 m. t. parvioculum was described only recently from gibraltar, on the southern tip of the iberian peninsula (guillem and bensusan 2009). it has since been found on both sides of gibraltar, namely in spain and in morocco (guillem et al. aenictus vaucheri, dorylinae material and data: a2602: (10 w) nest; 13 july 2011; chefchaouen, akerrat forest, cork oak forest; 35 06.10 n, 05 18.20 w; 488 m; a2197: nest; tetouan, al hamra; 35 23.83 n, 05 22.27 w; 139 m; a2866: nest; 30 april 2011; near balota; mosaic of scrub and open woodland with pistacia lentiscus, quercus suber, calicotome villosa, cistus spp.; 34 57.13 n, 05 31.66 w, 140 m. this species is common in three separate locations in morocco. two are from the atlantic coast area of the tingitane peninsula and the other from the atlas of beni mellal (cagniant 2006). despite its hypogaeic habitat, cagniant considered this species to be very common in koumch (the region of beni mellal). aphaenogaster pallida group, myrmicinae in morocco, the pallida group (see schulz 1994) is represented by two species given below: foreli and leveillei. according to cagniant (1996), however, due to their subterranean existence, records are scarce and those which have been cited (from elsewhere in morocco) are not georeferenced. material and data: a2721: (6w); 03 april 2011; berkan, sibe of bni snassen; 34 48.91 n, 02 24.30 w; 729 m. aphaenogaster leveillei material and data: a2084: (11w); 12 february 2011; driouch, temsanane, abelkhache, open area surrounded by fields of crops; 35 12.86 n, 003 41.23 w; 385 m; a2656: (1w); 2 april 2011; pitfall traps; 34 48.90 n, 02 24.30 w; 732 m. material and data: a2802: nest; 22 september 2011; chefchaouen, dardara, cork oak forest; 35 06.24 n, 05 17.65 w; 433 m; a2848: nests; 26 february 2012; chefchaouen, akarat, forest of hayouna; 35 06.64 n, 07 19.43 w; 722 m. a2850: nest; 20 february 2012; marrakech, jbel okaimdem; 31 11.75 n, 07 51.35 w; 2,170 m. this species has been found in the tingitane peninsula and in the middle atlas (cagniant 1964, de haro and collingwood 1994), as well as in tunisia and algeria. our record from marrakech indictes that this species exists at much higher altitude (2,170 m) than previously recorded (200800 m). linepithema humile, dolichoderinae material and data: a2867: 4 january 2012; chefchaouen city; 35 10.07 n, 05 16.13 w; 591 m; 15 september 2012; rabat city, hassan; 34 01.045 n, 6 49.522 w; 15 september 2012; temara, harhoura (road to skhirat); 33 54.520 n, 6 58.672 w. l. humile is an invasive species that has been recorded many times in morocco, principally in some coastal areas of the tingitane peninsula (tangiers and cape spartel, cagniant 1964; martil and cabo negro, de haro and collingwood 1994). we can now confirm the widespread presence of this species in rabat and temara (especially in public gardens).. we have found it in various places in the town of chefchaouen, especially in gardens and in the walls of buildings. proceratium algiricum, proceratiinae material and data: a2395: (2w); 14 june 2011; chefchaouen, dardara, quercus suber forest; 35 07.24 n, 05 17.60 w; 374 m. this species has been recorded in morocco from the central zone, the middle atlas, and chefchaouen (cagniant, 2006). the ant fauna of morocco is particularly diverse with 233 species, when compared to only 180 species in both algeria and tunisia (cagniant 2006), but it remains less than the recorded 295 species in the iberian peninsula (roig and espadaler 2010). the rate at which ant species are added to the list of the moroccan fauna would suggest that there remain more to be discovered. our investigations also suggest that several species previously thought to be restricted in distribution or in choice of habitat are in fact more widespread and less selective (such as t. pardoi or t. parvioculum). it is difficult to speculate how climate change and an array of anthropogenic factors including farming practice will influence the ant fauna. for this reason, frequent and regular field work is required, with accurate data particularly of the macro and micro habitats where ant species are thriving, if we are to understand ant ecology in morocco.

a recent catalogue of the rich ant fauna of morocco included 214 species, with later studies adding an additional 12 species. following recent fieldwork in the north of morocco, we report five new records for the country (plagiolepis pygmaea latreille, 1798, ponera testacea emery, 1895, strumigenys tenuipilis emery, 1915, temnothorax pardoi tinaut, 1987, and tetramorium parvioculum guillem&bensusan, 2009) and we present new data on the distribution and natural history of six additional species. this work brings the total number of ants known from morocco to 233, taking into account two species which were omitted in the list of cagniant.

PMC4535474