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pubmed-1201

w ostatnich latach coraz czciej wprowadza si do leczenia chirurgicznego choroby wiecowej zabieg pomostowania aortalno-wiecowego bez uycia krenia pozaustrojowego (off-pump coronary artery bypass opcab). zabieg przeprowadzany na bijcym sercu uwaa si za mniej inwazyjny, poniewa pozwala unikn efektw ubocznych zwizanych ze stosowaniem krenia pozaustrojowego. celem badania byo porwnanie iloci przetaczanych preparatw krwi w dwch grupach pacjentw operowanych z powodu choroby niedokrwiennej serca przy uyciu techniki pomostowania aortalno-wiecowego bez uycia krenia pozaustrojowego lub z uyciem krenia pozaustrojowego (cardiopulmonary bypass cpb). grupa i skadaa si z 84 osb (64 mczyzn i 20 kobiet), w rednim wieku 63,74 7 lat, ktre poddano zabiegowi z uyciem techniki opcab, a grup ii tworzyo 60 osb (54 mczyzn i 14 kobiet), w rednim wieku 63,51 6 lat, ktre poddano zabiegowi z uyciem pucoserca (cpb). 2,27 0,3 (grupa opcab) i 2,63 0,6 (grupa operacji w kreniu pozaustrojowym/grupa cpb) (p<0,05). rednia liczba jednostek koncentratu krwinek czerwonych podanych w grupie opcab wyniosa 2,31 0,18 jednostek na pacjenta, a w grupie cpb rednia liczba jednostek wieo mroonego osocza wyniosa 1,13 0,13 w grupie opcab i 1,57 0,15 w grupie cpb (p<0,05). recent years have seen a growing interest in off-pump coronary artery bypass surgery. the majority of cardiac procedures are performed on cardiopulmonary bypass, with blood transfusions being part of the procedure. as the extracorporeal circulation causes many side effects involving blood components, the restoration of hemoglobin concentration by means of transfusion is almost always essential. systemic inflammatory response syndrome (sirs) is usually self-limiting and may involve most organs [5, 6]. hemostatic disturbances secondary to cpb may cause such serious complications as disseminated intravascular coagulation (dic) [7, 8]. there were 152 patients (118 men and 34 women) at the mean age of 63 14 years enrolled in the study. the opcab group included 84 patients (64 men and 20 women) at the mean age of 64 7 years and the cpb group included 68 patients (54 men and 14 women) at the mean age of 63 6 years. the mean preoperative left ventricular ejection fraction was 53 9% and 51 8%, respectively. demographic and clinical data are presented in table i. all procedures were performed as a result of stable angina pectoris. 1.03 mmol/l in the opcab group vs. 8.78 0.70 in the cpb group. the mean hematocrit values were 0.41 0.05 in the opcab group vs. 0.42 0.03 in the cpb group, ns. there was no difference in serum platelet concentration, which was 251.42 74.01 demographical and perioperative data heparin was administered at the dose of 2 mg/kg in the opcab group and 3 mg/kg in the cpb group, and the desired act was 350 s and 480 s, respectively. postoperatively, heparin was neutralized by protamine administered at the dose of 1 mg per 1 mg of heparin. the cpb group was subsequently subjected to the procedures of ascending aorta and right atrium cannulation. cardiopulmonary bypass was conducted in moderate hypothermia (27-29c) with cold, crystalloid cardioplegia administered antegrade in accordance with the st. the octopus iii (medtronic, usa) stabilization system was used and intraluminal shunts were applied during each distal anastomosis. the obtained data were entered and analyzed using the statview 5.0 software (sas institute, inc., continuous variables were described as mean values sd and compared using student's t-test or the mann-whitney u-test. the test or fisher's exact test was chosen to compare categorical variables. to evaluate changes over time, we used repeated measures analysis of variance (anova). the obtained data were entered and analyzed using the statview 5.0 software (sas institute, inc., cary, nc, usa). continuous variables were described as mean values sd and compared using student's t-test or the mann-whitney u-test. the test or fisher's exact test we used repeated measures analysis of variance (anova). values of p<0.05 were considered significant. there were no perioperative deaths and no case of postoperative low cardiac syndrome was found in the study groups. there were 12 patients (14%) in the opcab who did not require any blood product transfusions. two reoperations (2.4%) in the opcab group and three (4.4%) in the cpb group were performed due to excessive bleeding (p<0.05). the mean cardiopulmonary bypass time was 63 18 minutes and the mean cross clamping time was 43 11 minutes in the cpb group. the mean packed red blood cells, fresh frozen plasma and platelet units transfused in the opcab group were 2.31 0.18, 1.13 0.13 and 0.28 0.16, respectively. the mean packed red blood cells, fresh frozen plasma and platelet units transfused in the cpb group were 3.94 0.30, 1.57 0.15 and 0.23 0.16, respectively. there was a statistically significant difference in the mean packed red blood cells (2.31 0.18 vs. 3.94 0.30, p<0.05) and fresh frozen plasma (1.13 0.13 vs. 1.57 0.15, p<0.05) transfusion rate between the groups. there was a difference in the mean serum hemoglobin concentration between the groups (opcab 7.79 0.91 mmol/l vs. cpb 7.03 0.88 mmol/l six hours after surgery, p<0.05 and opcab 7.47 1.10 mmol/l vs. cpb 7.17 0.99 mmol/l one day after surgery). figure 1 presents the differences in the serum hemoglobin concentration between the groups after surgery. the platelet count was comparable before the procedure (199 61 10/l vs. 178 41 10/l in the opcab and cpb group, respectively). the platelet count after surgery decreased progressively from 205 56 10/l, 192 53 10/l one day after surgery to 169 57 10/l on the 7 postoperative day. on the other hand, the platelet count after surgery increased progressively in the cpb group from the initial 155 41 10/l, 165 43 10/l one day after surgery to 369 72 10/l on the 7 postoperative day. hemoglobin concentration after surgery in opcab and cpb group there was also a statistically significant difference in postoperative drainage between the two groups (opcab: 755.54 42.82 ml vs. cpb: 895.74 47.35 ml, p<0.05). the first successful use of the heart-lung machine on humans occurred in 1953, when john gibbon performed surgery on a 15-month-old girl, celia bavolek, at jefferson hospital in philadelphia. eleven years later, in 1964, the russian surgeon kolessov performed the first successful heart bypass surgery on a beating heart. surgical revascularization was soon demonstrated to provide excellent survival results and relief of symptoms [11, 12]. renewed interest in beating-heart bypass grafting in the mid-1990s resulted from the option of revascularization without the potential complications of extracorporeal support. although the theoretical advantages of opcab procedures are generally accepted, the use of this technique still remains sporadic. for example, off-pump surgery constitutes only 20 to 25% of all coronary artery bypass procedures performed in the united states. a debatable issue is that of graft patency rates, which seem to be at least equivalent to those observed in the case of conventional techniques. the advantage of off-pump coronary artery bypass can be supported by such important factors as reduced morbidity and mortality, rapid return to usual functional capacity, and economic benefits. unfortunately, a lot of data reported in the literature concerning the outcomes of off-pump bypass grafting have been inconclusive as to the overall benefit of the technique. although the opcab technique eliminates cardiopulmonary bypass and hypothermic cardiac arrest, the manipulation of the ascending aorta by partial clamping has for the most part not been eliminated. so far, there have been 37 randomized clinical trials published, comparing opcab versus conventional cabg. no randomized trials have shown a significant reduction in the occurrence of stroke or myocardial infarction, acute renal failure, intra-aortic balloon pump (iabp) requirement, mediastinitis or wound infection, the recurrence of angina, or the need for reintervention within 30 days of opcab, in comparison with conventional cabg. similar results were obtained at 1 and 3 years after surgery. in the present study, there was a reduction of blood cell product transfusions in the opcab group, as compared to the cpb group (table ii). opcab procedures make it possible not only to limit the number of transfusions but to eliminate transfusions altogether [16, 17]. the elimination of blood product transfusion can be essential in the case of patients with religious restrictions, such as jehovah witnesses. blood products transfusions the serum hemoglobin concentration in the opcab group remains stable throughout the postoperative period, as presented in table i. at the same time, we can observe a decline in the hemoglobin concentration in conventional cabg patients until the 12 postoperative hour. the restitution of the hematocrit level was achieved by administering packed red cell transfusions thereafter. off-pump surgery allows for reducing the rate of blood product transfusions or eliminating them altogether. patients undergoing conventional cabg surgery were characterized by higher postoperative drainage, presumably due to more serious coagulation disturbances.

introductionthere has been a growing interest in off-pump coronary artery bypass (opcab) grafting in recent years. beating-heart surgery is believed to be less invasive as it allows the side effects of extracorporeal circulation to be avoided.the aim of the studythe aim of the study was to compare blood product transfusion rates between two groups of patients undergoing surgery for ischemic heart disease with either the off-pump technique or using cardiopulmonary bypass (cpb). material and methodsthere were 152 patients enrolled in the prospective randomized study. all procedures were elective. there were 84 patients (62 men and 20 women) at the mean age of 63.74 7 years who underwent opcab (group i), and 68 patients (54 men and 14 women) at the mean age of 63.51 6 years who underwent cardiopulmonary bypass (group ii). resultsthere were no perioperative deaths. the mean number of grafts was 2.27 0.3 (opcab group) and 2.63 0.6 (cpb group) (p<0.05). the mean number of packed red blood cells transfused in the opcab group was 2.31 0.18 units/patient and 3.94 0.30 units/patient in the cpb group (p<0.05). the mean number of fresh frozen plasma units transfused was 1.13 0.13 in the opcab group vs. 1.57 0.15 in the cpb group (p<0.05). there were 12 patients (14%) in the opcab group who had no transfusion. conclusionsone of the most important advantages of the opcab technique is that it makes it possible to reduce the rate of blood product transfusions.

PMC4283870

pubmed-1202

myocardial infarction (mi), and subsequent reperfusion injury, is the most common and clinically significant form of acute cardiac injury and results in the ischemic death of cardiomyocytes.1,2 among the pathological mechanisms underlying myocardial ischemia/reperfusion (mi/r) injury, inflammation and inflammatory cell infiltration, together with the activation of innate and adaptive immune responses, are the hallmark of mi and reperfusion injury.3,4 ischemic cardiac injury activates the innate immune response via toll-like receptor (tlr)-mediated pathways and upregulates the chemokine and cytokine syntheses in the infarcted heart. tlrs, which are expressed by inflammatory cells and also on endothelial cells and cardiomyocytes, can recognize endogenous danger signals released during cell death following myocardial ischemia and reperfusion.5,6 a growing body of evidence suggests that modulating tlr activation may enhance the benefits and blunt the negative effects of the inflammatory response, providing new therapeutic options for preventing mi/r injury.6 chemokines stimulate the chemotactic recruitment of inflammatory cells into the infarct. one of the well-studied cc chemokines, cc chemokine ligand 2 (ccl2), is a potent chemoattractant for monocytes, macrophages, t cells, and nk cells; cc chemokine receptor 2 (ccr2), the receptor for ccl2, is mainly expressed by monocytes and macrophages. the ccl2/ccr2 signaling pathway has been implicated in postischemic inflammatory response, and pharmacological inhibition or genetic targeting of the ccl2/ccr2 pathway might represent an attractive approach to blunt excessive inflammation and prevent detrimental ventricular remodeling.712 various cytokines promote adhesive interactions between leukocytes and endothelial cells, resulting in the transmigration of inflammatory cells into the site of injury. the recruitment of inflammatory cells is a dynamic and superbly orchestrated process comprising sequential infiltration of the injured myocardium with neutrophils, mononuclear cells, dendritic cells (dcs), and lymphocytes.3,4,13 neutrophils migrate into the infarcted myocardium during the first hours after the onset of ischemia and peak after one day.1 thereafter, monocytes and their descendant macrophages dominate the cellular infiltration and release inflammatory mediators, reactive oxygen species, and proteolytic enzymes, contributing to the initiation and resolution of inflammation, phagocytosis, proteolysis, angiogenesis, infarct healing, and ventricular remodeling.1,3,14 meanwhile, dcs and t lymphocytes are recruited into the injured myocardium, contributing to wound healing and ventricular remodeling.1517 both detrimental effects and the beneficial role of these inflammatory cells have been documented in the pathophysiology of mi and reperfusion injury, and the diverse and seemingly conflicting roles may be attributable to the subset heterogeneity and functional diversity of the inflammatory cells.3,4 mi/r triggers a complex inflammatory reaction accompanied by cytokine release and inflammatory leukocyte infiltration into the endangered myocardial region.1,3,4,18 although the inflammatory response and cytokine elaboration after mi/r are integral to the healing process and contribute to left ventricular (lv) remodeling, excessive inflammatory responses after mi/r injury are detrimental for cell survival and extracellular matrix integrity via an enhanced activation of proapoptotic signaling pathways, with subsequent poor clinical outcome.3,19,20 these findings suggest that an inflammatory reaction is essential for the healing process, and thus, no effective therapeutic strategy against inflammation has been established.4 it has been widely accepted that myocardial infarct healing and post-mi remodeling are processes in which leukocytes, cytokines, and chemokines play both a beneficial role and a detrimental role.3,4,20 recent studies have demonstrated that recruited monocytes/macrophages persist for days in the infarct zone and contribute to inflammation, phagocytosis, proteolysis, angiogenesis, and collagen deposition. reduced macrophage infiltration resulted in decreased inflammation, diminished interstitial fibrosis, and attenuated lv remodeling and dysfunction. on the other hand, macrophage participation is integral to wound healing and tissue repair after mi.3,4,21 as shown in figure 1, these diverse and seemingly contrasting functions might be attributed to macrophage heterogeneity, which is characterized by differential activation, distinct phenotypes, and diverse functions. hence, the challenge lies in ameliorating the detrimental inflammatory response while not affecting the tissue repair response. the innate immune system is activated after various forms of tissue injury and triggers immune responses in the host.22 the role of innate immune responses in cardiac ischemic injury and tissue repair has been shown to be more pivotal than first thought. the innate immune system contributes importantly to the progression of myocarditis and the remodeling process after mi.2325 our understanding of the pathogenesis of mi/r injury became much clearer with the discovery of tlrs. tlrs are expressed by leukocytes and recognize pathogen-associated molecular patterns and endogenous danger signals released during cell death.22 tlrs are also expressed in cells with no direct role in host innate immune responses, such as endothelial cells and cardiomyocytes.26 on activation, tlrs exert their inflammatory response through nuclear factor kappa-light-chain-enhancer of activated b cells (nf-b) translocation to the nucleus.5,6,27 thus, tlrs hold great promise as a therapeutic target within the innate immune system, for cardiac ischemia and other conditions, without affecting host defense or proper scar formation after infarction. modulating tlr activation may enhance the benefits, blunt the negative effects of the inflammatory response, and provide new therapeutic options after mi/r injury. this concept is supported by observations in tlr knockout mice.2831 tlr4-deficient mice sustained smaller infarctions and exhibited less inflammation after mi/r injury.28 ex vivo experiments showed that tlr2 hearts performed better than wild-type hearts after mi/r injury,29 and tlr2 mice were protected against endothelial dysfunction after mi/r injury.30 circulating tlr2 was also demonstrated to mediate mi/r injury. administration of a tlr2 antagonist just five minutes before reperfusion reduced infarct size and improved cardiac performance and geometry. furthermore, antagonizing tlr2 reduced inflammation and cell death after infarction.31 thus, tlr2 has been established as a new therapeutic target for the treatment of acute ischemic and reperfusion injury, even when it is initiated in the late ischemic period. in the setting of mi, deficient tlr2 or tlr4 signaling in mice prevented adverse cardiac remodeling, resulting in preserved cardiac function and geometry after mi.24,32 in addition to tlr signaling, activation of the innate immune system through interleukin-1 receptor-associated kinase 4 (irak-4) signaling was important for bone marrow-derived dc mobilization and maturation, contributing to post-mi mortality and adverse remodeling. in irak-4 knockout mice, attenuation of toll/interleukin-1 receptor signaling resulted in lower expression of cytokines and decreased inflammation through blunted innate immune response.15 as the cause of inappropriate activation of the inflammatory response after mi, an autoimmune reaction is a possible mechanism of unnecessary inflammatory reactions induced secondary to myocardial injury.33,34 it has been demonstrated that cardiac myosin acts as an endogenous ligand for tlr2 and tlr8,35 and the presence of autoimmunity to cardiac myosin and troponin is associated with an adverse clinical outcome after mi.34,36,37 additionally, lymphocytes obtained from the spleen of rats that have suffered mi can injure normal cardiomyocytes,38 and heart failure was induced by adoptive transfer of splenic lymphocytes from rats after mi.33 furthermore, mice preimmunized with murine cardiac troponin i displayed greater infarct size, increased more significant fibrosis, higher inflammation score, and more cardiac dysfunction after mi.37 these findings indicate that myocardial damage results in the release of not only endogenous ligands of tlrs but also self-antigens. taken together, autoimmune responses against myocardial antigens may contribute to secondary myocardial injury after mi and may be a new mechanism of maladaptive lv remodeling after mi. chemokine expression is a prominent feature of the postinfarction inflammatory response, and as an essential player in inflammatory leukocyte trafficking, chemokines are involved in i/r injury, myocardial healing, infarct angiogenesis, and scar formation after mi.39 in addition, chemokines exert important effects on nonhematopoietic cells, such as endothelial cells, smooth muscle, and fibroblasts, and may modulate fibrous tissue deposition and wound angiogenesis.40 one of the extensively studied cc chemokines, ccl2, which was originally named monocyte chemoattractant 1, is a potent chemoattractant for monocytes, t cells, and nk cells and has been implicated in a wide variety of diseases characterized by monocyte-rich leukocyte infiltrates.40 ccl2 upregulation has been observed in murine, rat, and canine models of mi/r.7,41 in the canine model of reperfused infarction, induction of ccl2 mrna occurred only in ischemic segments within the first hour of reperfusion, peaked at three hours, and was localized by immunostaining on the venular endothelium.7 ccl2 mrna levels were increased by 40-fold in the noninfarcted lv one day after left coronary artery ligation, and increased levels persisted for 28 days.7 ccl2 expression was also increased in both experimental and clinical heart failures.42,43 enhanced myocardial ccl2 expression contributed to reperfusion injury, infarct healing, and ventricular remodeling through the following two mechanisms: ccl2-induced infiltration and activation of inflammatory cells, such as monocytes/macrophages and lymphocytes,7,9,10,39 and ccl2-induced transcription factor causing cardiac cell death and ventricular dysfunction.44 ccl2 also promotes the induction of other cytokines, matrix metalloproteinase, and transforming growth factor- through an autocrine/paracrine mechanism,40,45,46 thus modulating fibrous tissue deposition and wound healing. given the essential effects of ccl2 signaling on different cell types involved in the postischemia inflammatory response, the pharmacological inhibition or genetic targeting of ccl2/ccr2 signaling might represent an attractive approach to blunt excessive inflammation and decrease monocyte/macrophage infiltration, thereby promoting infarct healing and preventing detrimental ventricular remodeling. in agreement with this concept is the fact that ccl2-deficient mice display a decreased and delayed macrophage infiltration and myofibroblast accumulation associated with a diminished interstitial fibrosis, improvement of lv dysfunction and regional hypocontractility after mi/r.9,10 similarly, administering a ccl2 competitor reduced inflammatory monocyte recruitment, limited neointimal hyperplasia, and attenuated mi/r injury in mice.12 moreover, anti-ccl2 gene therapy improved the post-mi survival rate, which was associated with a decreased macrophage recruitment and an attenuated contractile dysfunction, interstitial fibrosis, and lv cavity dilatation.7 genetic deletion of ccr2 resulted in a decreased macrophage infiltration and a reduced tnf- and matrix metalloproteinase expressions, which might contribute to the attenuation of lv remodeling after mi.8 nahrendorf et al showed that pretreatment of mice for three days with a lipid nanoparticle that encapsulated a short interfering rna targeting ccr2 prior to the induction of mi/r injury resulted in reduced numbers of monocytes and macrophages in the heart and reduced the infarct size by 34%.14 the recruitment of inflammatory cells is a dynamic, well-organized process with sequential infiltration of the injured myocardium with neutrophils, mononuclear cells, dcs, and lymphocytes.3,4,20 a growing number of studies have demonstrated that recruited monocytes/macrophages persist for days in the infarct zone and contribute to inflammation, phagocytosis, proteolysis, angiogenesis, collagen deposition, and ventricular remodeling in the setting of myocardial reperfusion injury and postinfarction healing.710,19,20,4749 on one hand, excessive and prolonged infiltration of inflammatory macrophages into the infarct myocardium is harmful, contributing to excessive inflammatory response, tissue destruction, interstitial fibrosis, cardiac dysfunction, and adverse ventricular remodeling.710 on the other hand, a controlled recruitment of macrophages is essential to wound healing and tissue repair through phagocytosis of necrotic cells, facilitating angiogenesis and extracellular matrix reconstruction in the ischemia-injured myocardium and the infarct.48,49 these diverse and seemingly conflicting functions may be attributable to macrophage heterogeneity as characterized by differential activation, distinct phenotypes (m1: classically activated macrophages and m2: alternatively activated macrophages), and diverse functions, both pathogenic and protective.5052 the subset heterogeneity and function diversity also hold true for the monocytes. as reported by nahrendorf et al.14, infarcted hearts modulate their chemokine expression profile over time, and they sequentially and actively recruit ly-6c and ly-6c monocytes via ccr2 and cx3cr1, respectively. ly-6c monocytes digest damaged tissue, whereas ly-6c monocytes promote healing via myofibroblast accumulation, angiogenesis, and collagen deposition.3,4,14 thus, a therapeutic strategy targeting ccr2 monocyte/macrophage migration is a promising approach for treating numerous inflammatory diseases without disrupting inflammation resolution, which is associated with noninflammatory monocytes and alternatively activated macrophages. consistent with this concept, recent comprehensive analysis of mouse cardiac macrophage subsets in a steady state and during inflammation have revealed that proinflammatory macrophages, which comprise half of all ly-6c monocytes and mhcii cd11c ccr2 macrophages, specifically express ccr2.53 this finding might explain why short interfering rna, targeting the ccr2 axis that blocks the monocyte/macrophage lineage, was successful in preventing the ischemic cardiac injury.14 the role of monocytes/macrophages in mi/r injury and postinfarction healing is a double-edged sword, as illustrated in figure 1. monocyte/macrophage recruitment is integral to the infarct healing; on the other hand, an uncontrolled inflammatory cell infiltration may exacerbate reperfusion injury and compromise the reparative functions mediated by these cells. thus, the challenge is how to ameliorate the detrimental effects of proinflammatory monocytes/macrophages, while sparing the beneficial roles of the reparative or regulatory macrophages. the existence of monocyte/macrophage subset heterogeneity and their biphasic recruitment in response to ischemic injury provide a possible solution. a recent study has demonstrated that knocking down interferon regulatory factor 5, which is a critical transcription factor favoring m1 polarization, decreased infiltration of m1 and ameliorated inflammation following mi, thereby improving infarct healing.54 additionally, nuclear receptor subfamily 4, group a, member 1, was demonstrated to be essential to ly-6c monocyte production. in the absence of nuclear receptor subfamily 4, group a, member 1, ly-6c monocytes expressed increased levels of ccr2 on their surface, avidly infiltrated the myocardium, and differentiated to proinflammatory macrophages, resulting in defective healing and compromised heart function.55 these findings suggest that therapeutics targeting distinct macrophage lineages by genetic manipulation of polarity-determining genes may serve as new treatments for cardiovascular diseases. stem cell therapy has been considered as the promising therapeutic in treating cardiovascular diseases, and many preclinical studies and clinical trials reported beneficial effects of stem cell therapy on infarct healing, although the underlying mechanism remains poorly understood. a recent study has provided an insightful explanation that the cardioprotective effects of mesenchymal stromal cell treatment might be attributed to the cross talk between injected stem cells and macrophages.56 mesenchymal stromal cell treatment reshaped the macrophage response by favoring m2 polarization, resulting in increased numbers of m2 and changed cytokine profile of macrophages, thereby improving postinfarction healing.56 dcs and their precursors are considered sentinels of the immune system, and they circulate through the blood and nonlymphoid peripheral tissues, where they become resident cells over time.57 after pathogen invasion or tissue injury, dc precursors accumulate rapidly in the local infected or injured tissues.57 as early as 1993, it was demonstrated that dcs infiltrated the injured myocardium and participated in the activation of lymphocytes after mi.58 however, both detrimental effects and beneficial or regulatory roles of dcs in the postinfarction healing and remodeling were observed.15,16,59 it has been demonstrated that g-csf improved early postinfarction lv remodeling through decreased dcs infiltration and suppression of dc-mediated immunity.59 bone marrow-derived dcs mobilization, mediated via irak-4 signaling, contributed to postinfarction myocardium apoptosis, th1 cytokine expression, and interstitial fibrosis, leading to an increased mortality and an adverse lv remodeling.15 by contrast, in transgenic mice expressing diphtheria toxin receptor on dcs, which allowed the investigator to specifically deplete dcs by injecting diphtheria toxin, dcs were demonstrated to be a potent immunoprotective regulator during the postinfarction healing process via control of monocyte/macrophage homeostasis.16 in addition, dcs were suggested to regulate the development of autoimmune heart failure through the recognition of heart-specific peptides.60 in addition to neutrophils, monocytes/macrophages, and dcs, lymphocytes are also present in the ischemic and reperfused myocardia and the infarct. cd4 t lymphocytes accumulate in the infarct zone early during reperfusion, and the infarct-sparing effect of adenosine a2a receptor activation is primarily due to inhibition of cd4 t cells infiltration and activation in the reperfused heart.61 hofmann et al.17 demonstrated that cd4 t cells proliferated in draining lymph nodes shortly after ischemic injury, and cd4 t-cell-deficient mice displayed higher total numbers of leukocytes and proinflammatory monocytes and increased lv dilation as determined by serial echocardiography up to day 56 after mi. additionally, foxp3 cd4 regulatory t cells (treg) contributed to inflammation resolution and beneficially influenced infarct healing by modulating monocyte/macrophage differentiation. mechanistically, treg cell depletion was associated with m1-like polarization, characterized by decreased expression of inflammation-resolving and healing-promoting factors. therapeutic treg cell activation induced an m2-like differentiation within the healing myocardium, associated with myofibroblast activation and increased expression of monocyte/macrophage-derived proteins, fostering wound healing.62 apart from t lymphocytes, the interaction between mature b lymphocytes and monocytes was also involved in myocardial ischemic injury and maladaptive lv remodeling. mature b lymphocytes selectively produce ccl7 and induce ly6c monocyte mobilization and recruitment to the heart, leading to an enhanced tissue injury and deterioration of myocardial function. genetic or antibody-mediated depletion of mature b lymphocytes impeded ccl7 production and monocyte mobilization, attenuated myocardial injury, and improved cardiac function.63 collectively, these findings suggest that therapeutic modulation of lymphocytes constitutes a new approach to improve infarct healing post-mi. postinfarction immunoinflammation, as characterized by inflammatory cell infiltration and the activation of innate and adaptive immune responses, is essential to cardiac injury and repair. the inflammatory cascade may provide unique opportunities for interventions aimed at reducing cardiomyocyte injury while optimizing the healing response and attenuating adverse remodeling. timely resolution of the inflammatory infiltrate and spatial containment of the inflammatory and reparative response into the infarcted area are essential for optimal infarct healing. for instance, targeting proinflammatory leukocyte subsets, to dampen detrimental inflammation while sparing the wound healing roles of the anti-inflammatory monocytes/macrophages, may reduce cardiomyocyte injury and adverse remodeling. despite the challenges ahead, we are hopeful that new therapies for myocardial ischemia and reperfusion will soon be integrated into clinical practice.

myocardial infarction (mi) is the most common cause of cardiac injury, and subsequent reperfusion further enhances the activation of innate and adaptive immune responses and cell death programs. therefore, inflammation and inflammatory cell infiltration are the hallmarks of mi and reperfusion injury. ischemic cardiac injury activates the innate immune response via toll-like receptors and upregulates chemokine and cytokine expressions in the infarcted heart. the recruitment of inflammatory cells is a dynamic and superbly orchestrated process. sequential infiltration of the injured myocardium with neutrophils, monocytes and their descendant macrophages, dendritic cells, and lymphocytes contributes to the initiation and resolution of inflammation, infarct healing, angiogenesis, and ventricular remodeling. both detrimental effects and a beneficial role in the pathophysiology of mi and reperfusion injury may be attributed to the subset heterogeneity and functional diversity of these inflammatory cells.

PMC4892199

pubmed-1203

the frequency of chronic kidney disease (ckd) has been progressively increasing over the last two decades (1) and has become a worldwide public health problem. the prevalence of ckd is estimated to be 816% worldwide (2). kidney transplantation is the best alternative treatment for end-stage renal disease and health-related quality of life and survival of the patients are improved compared with dialysis (3, 4). worldwide, more than 1.4 million patients with ckd receive renal replacement therapy with incidence growing by approximately 8% annually (5). unfortunately, despite significant improvement in graft function, kidney transplants can still fail due to acute rejection and chronic allograft nephropathy (1, 3) that can lead to three fold greater risk of death compared to patients with functioning grafts (1, 6). due to the increasing demand for renal transplants, identifying potential risk factors implicated in graft failure is essential to improve patient survival and quality of life (1). to achieve this purpose, traditional statistical techniques such as cox proportional hazards (ph) model however, it relies on restrictive assumptions such as proportionality of hazards and linearity of effects on log hazard function (linearity assumption) (7). besides, the performance of traditional methods like cox regression is not reliable in the presence of high rate of censoring (8). potential prognostic factors affecting renal graft have also been investigated by several studies with cox ph model (3, 9, 10). ideally, it would be important to improve the predictive performance of the models identifying potential prognostic factors affecting renal graft via learning theory and data mining techniques for survival time that require no assumptions. machine learning methods such as tree-based approaches have recently been developed to handle right censored survival data and their effective performance has been confirmed in different areas (11). random survival forests (rsf), is a non-parametric tree-based ensemble learning method that can automatically handle the difficulties of cox model and can also be used to select and rank variables (7, 11). due to the limitations of the cox model, using rsf to identify effective risk factors for survival has been suggested (7). although, several studies have confirmed the promising performance of rsf compared to traditional cox model (8, 1214) in different disease, there is no attempt to use rsf in renal transplantation and compare its performance with cox model. this study aimed to identify prognostic factors affecting renal graft by rsf and compare its performance with cox proportional hazard model. the present study utilized a data set corresponds to a retrospective cohort study which was conducted in hamadan, western iran, from 1994 to 2011. the number of 475 patients underwent kidney transplantation in ekbatan or besaat hospitals and was eligible to enroll the study. to identify important risk factors, the patients who did not have any information about risk factors were eliminated from the analysis. in this regard, only 378 out of 475 patients were considered in the present study because the information about potential risk factors was not observed for the rest of the patients. the risk factors were age, sex of donors and recipients, type of donor (living-donor or deceased donor), familial relationship, hemoglobin level, blood groups of donors and recipients, duration of dialysis before transplantation (year), cold ischemic time (min), creatinine level at discharge, body mass index (bmi) of donor (kg/m2), left or right kidney, type of immunosuppressive drugs used (imuran, prednisolone, cyclosporine vs. cellcept, prednisolone, cyclosporine), duration of hospitalization (day), volume of urine excretion during the first 24 h after transplantation (ml/24 h), and occurrence of acute or hyperacute rejection. in this regard, acute rejection is related to formation of cellular immunity, which usually occurs to some degree in all grafts, except between identical twins and hyperacute rejection is initiated by preexisting humoral immunity and usually manifests within minutes after transplantation (3). the survival time was the time between kidney transplantation and episode of rejection (3). rsf is an extension of random forest rf to right-censored survival data with the same principles underlying rf, which enjoys all its important properties (7, 15). each tree consists of nodes (variables) in which classification or split was implemented. in survival settings, tree node splits according to maximizing survival differences between daughter nodes (new nodes). in this regard, in each tree, survival time and status of the patients were considered as response variables. then, the ensemble estimate for the cumulative hazard function (chf) is drawn by calculating the chf for each sample in a data set, and summing this ensemble over the observed survival times yields the predicted outcome referred to as ensemble mortality (a measure of mortality for a patient that has been shown to be an effective predictor of survival) (15). each run of rsf was performed for the kidney transplant data set based on 1000 trees under log-rank splitting rule. the importance of each model covariate was also determined by a rapidly computable internal measure of variable importance (vimp) that can be used to rank variables. the larger vimp, the more predictive the variable (the threshold value is 0.002) (11). moreover, multiple imputation strategy based on rf was utilized for treating missing data (7). five imputed data set were provided and then combining rules (16, 17) were applied to calculate evaluation criteria and vimp. in order to compare the performance of rsf and traditional cox ph, two criteria were used including integrated brier score (18) and c-index (19) using out-of-bag (oob) data. a perfect prediction rule would have a concordance of 1 (20). random-forestsrc, a freely available package from the comprehensive r archive network (cran). the mean survival time for 378 patients was 7.354.62 yr, the median survival time was 6.81 yr. out of 378 transplantations, 37 (10%) episodes of rejection occurred, and the remaining 341 patients (90%) were censored. mean and standard deviation of variable importance (vimp) for kidney transplant data over five imputed data set. each run based on 1000 trees under log-rank splitting the cold ischemic time, recipient s age, creatinine level at discharge and donors age are highly predictive, and duration of hospitalization is moderately predictive. however, type of donors, hemoglobin level, donor s sex, immunosuppressive drug usage, post-transplantation condition, recipient sex, familial relationship, donor and recipient blood group, side of the kidney, duration of dialysis and urine volume are unlikely to be predictive. according to cox ph model, three variables of recipient age, type of donor (living vs. deceased), and episode of post-transplantation acute and hyperacute rejection were identified as most important variables. rsf had lower prediction error based on integrated brier score (0.081) compared to cox model (0.088). in addition, the c-index of rsf was considerably higher (0.965) than that of the cox model (0.766). the effect on survival of the most five influential covariates found in the rsf analysis was displayed with 5-yr partial survival plots in fig. 1. the estimated partial survival for a covariate indicates estimated survival for different levels of the covariate when the effects of all other covariates are justified. it can be seen from figure that, as cold ischemic time increases up to about 35 minute, the five-year predicted survival increases as well and it tends to decline after 35 minute. partial 5-year predicted survival for five most influential covariates on survival in kidney transplant data. rsf identified cold ischemic time, recipient s age, creatinine level at discharge, donor s age and duration of hospitalization as the top five most important predictors of survival for graft failure patients in the present study. several authors estimated the survival rate of kidney transplantation and detected the risk factors of graft rejection (2124). our results showed that the cold ischemic time variable was the most important factor in the risk of graft rejection, which is consistent with the results of some other studies (25, 26). cold ischemic time is one of the risk factors that is involved in immediate anemia in renal transplant recipients (27). based on the results, as recipient s age increases predicted five-year survival time increases as well. this may be a result of stronger and more efficient immune system in younger recipients (3). previous studies have reported creatinine level at discharge as a risk factor in rejection of kidney transplantation (9, 10, 32). donor age was the fourth top risk factor, which had a negative correlation with graft rejection, i.e. kidney rejection is more likely among those recipients who receive kidney from older donors. this result is also similar to the result of other studies (3, 25, 26, 33, 34). the fifth top most important variable was duration of hospitalization, confirmed eralier (35, 36). this study focused on the performance of rsf method in identifying potential risk factors for survival of kidney graft failure patients compared to traditional cox model. the results demonstrated that the rsf model performed significantly better than the conventional cox-proportional hazard model. several studies also confirmed the promising performance of rsf compared to cox ph model in real data sets (8, 12, 14). rsf had better performance compared to cox ph model based on prediction error criterion (13). therefore, it can be applied successfully for identifying risk factors of the kidney transplantation survival. rsf deals with the traditional cox model issues such as proportionality assumption coherently and automatically (37) and analysts do not require knowing in advance the relationship (i.e. linear, nonlinear) of a variable over time (8). besides, the performance of cox regression is not reliable in the presence of high rate of censoring which was the case in the present study (about 90% censor rate). while, rsf is a robust extension of random forest a highly used machine learning method that has gained much interest in a variety of fields of application and generated a vast amount of computational literature in the last decade (8, 38). however, the performance of different methods is data dependent and conducting additional studies is needed to compare rsf to cox regression to document further its performance in clinical settings (8). there were some limitations in the present study. reliable sources of data obtained from prospective design were required for estimation of survival rate and associated prognostic factors, but the present study used a data set of a retrospective cohort study and medical records. quality and accuracy of estimates depends primarily on the quality of recorded data, but verifying the accuracy of data was not possible in the present study. besides, quality of the services and technology may vary over time, but we have no document to justify this issue. these issues might bias results. in addition, long-term follow-up duration results in losing some patients, which in turn may lead to biased results (3). rsf identified a different subset of risk factors in chronic nonreversible renal graft rejection than the cox ph model. the rsf is a promising method for intuitive variable selection and is a way to eliminate the doubt in the black box approach to statistical analysis that should be further investigated in survival analysis of other diseases (8). ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.

background: kidney transplantation is the best alternative treatment for end-stage renal disease. several studies have been devoted to investigate predisposing factors of graft rejection. however, there is inconsistency between the results. the objective of the present study was to utilize an intuitive and robust approach for variable selection, random survival forests (rsf), and to identify important risk factors in kidney transplantation patients. methods:the data set included 378 patients with kidney transplantation obtained through a historical cohort study in hamadan, western iran, from 1994 to 2011. the event of interest was chronic nonreversible graft rejection and the duration between kidney transplantation and rejection was considered as the survival time. rsf method was used to identify important risk factors for survival of the patients among the potential predictors of graft rejection. results:the mean survival time was 7.354.62 yr. thirty-seven episodes of rejection were occurred. the most important predictors of survival were cold ischemic time, recipient s age, creatinine level at discharge, donors age and duration of hospitalization. rsf method predicted survival better than the conventional cox-proportional hazards model (out-of-bag c-index of 0.965 for rsf vs. 0.766 for cox model and integrated brier score of 0.081 for rsf vs. 0.088 for cox model). conclusion: a rsf model in the kidney transplantation patients outperformed traditional cox-proportional hazard model. rsf is a promising method that may serve as a more intuitive approach to identify important risk factors for graft rejection.

PMC4822390

pubmed-1204

slips, trips and falls (stfs) lead recurrently to injuries in occupational situations1. these accidents are triggered by a movement disturbance (a slip or a trip) when working, especially when walking. other movement disturbances (a wrench slipping, an arm colliding with a wall, etc.) can occur in occupational situations; moreover, these can arise when performing different types of tasks (tightening a bolt, moving an item alone or with a colleague, etc.). this paper considers ostfs and, more broadly, occupational accidents with movement disturbance (oamds); the latter composing a set of accidents operationally defined by leclercq et al.2, 3, which involve a heavy cost in both human and financial terms4. the literature often advances workplace design and upkeep5, 6, access system configuration7 or, again, human factors8, 9 for explaining ostfas. implementing actions that neutralise these factors to secure displacements, however, such actions frequently overlook not only task diversity, but also production requirements and they can therefore only offer a partial response to preventing all oamds. research into these accidents shows that, as in all occupational accidents, many accident-causing event configurations stem from arbitration between production and safety, which can not be overlooked if progress is to be achieved in the prevention field. production-safety arbitrations lead to controls applied under working conditions in order to perform the task while maintaining safety. the control most frequently referred to involves walking fast to try to absorb a delay or confront an emergency. these observations provide a partial explanation for worker difficulties in systematically applying certain recommendations based on common sense (e.g. do nt rush) that are aimed at preventing oamds. this paper describes initially the need for, and limits involved in, neutralising the environmental factors in play and subsequently the production-safety arbitrations prompted by the so-called organisational oamd factors referred to in the literature. some of these arbitrations imply control implementation by the worker performing the task, which is then reflected in his/her displacements or, more generally, in his/her movements that increase exposure to oamd risk. focusing on organisational factors allows us to integrate these controls into a set highlighted by a general work organisation model. such a model indicates areas of similarity between oamd genesis and other occupational injury geneses. movement disturbance factors may be permanent and visible (floor in poor condition, congestion, difficult access to parts of a machine, etc.) in some work situations and may expose many workers over relatively long periods. this is the case of slippery floors in food processing shops, for example. such factors, along with haste, carelessness and awkwardness are frequently advanced when explaining oamd occurrence. neutralising environmental factors often involves taking action on certain working conditions (installing a slip-resistant floor, reconfiguring an access system, etc.). it is commonplace for companies that decide to raise their oamd-related safety level primarily focus of this type of action. however, in common with instructions designed to change behaviour (e.g. instructions to workers to move carefully from place to place or to adopt a safe, unhurried displacement, avoiding short-cuts), this action can not meet the requirements for preventing all oamds. instructions of this type alone in fact disregard other aspects, which are sometimes more difficult to objectify and control, such as urgency of the situation, fatigue or certain task requirements. as in all occupational accidents (oas), an oamd will often be caused by a combination of factors, each of which is of different nature. a clearly visible obstacle is never sufficient to cause a trip: it may simply not be taken into account by a worker, when his/her visual attention is absorbed by his/her task during a displacement. on the other hand, an oamd can occur without involving a permanent, visible anomaly in the environment: a worker, late for his/her appointment, misses a step when running up stairs that are not subject to any design defect. finally, many situations are temporarily more susceptible to oamd occurrence: for example, when performing his/her activity, a worker collides with an element in his/her environment, which obstructs his/her movement; he/she had intended to move this element on completion of priority work. in the situation illustrated by fig. 1.two images of occupational situations taken from the napo in safe on site/champions of the world video produced by, a first level of prevention would involve disposing of unwanted material or organising storage areas. at a given moment, presence of elements required for performing a task can also represent an accident factor for a worker or his/her colleague. in fig. 1b, this would be the cinder blocks left near a mason, but could also be a batten left lying on the ground when stripping formwork, a wheelchair when transferring a patient or a toolbox when conducting maintenance work. two images of occupational situations taken from the napo in safe on site/champions of the world video produced by neutralising accident factors that are permanent and visible in the work environment is important, but analysing the part played by this factor in a more comprehensive accident genesis is in fact just as important. behaviours adopted in work situations also need to be understood since they often reflect the presence of organisational factors. the literature includes in-depth analyses of occupational slips, trips and/or other movement disturbances conducted at various companies. events leading to injury are integral to the relevant company operation and some are related to production-safety arbitrations referred to long ago in the general occupational accident field. for example, situations described in terms of recovery or momentary co-activity by faverge10 reflect arbitrations revealed during iron mine accident analysis, in particular. monteau11 refers to known organisational risks, when analysing occupational health and safety from an organisational perspective. it should be noted that few of these accidentology studies are considered in relation to understanding and preventing oamds. yet, the contribution of multiple organisational factors has been highlighted during analysis of such accidents. research reported in bentley&haslam12 and in leclercq&thouy13 questions the role of work preparation in stfa occurrence. bentley &haslam12 effectively describe the difficulties encountered in distributing mail on time during periods involving snow and ice. leclercq&thouy13 show that a number of accidents have involved field operators climbing up into and down from trucks, when checking equipment required during the day at various building sites. this phase of their activity called for all the more care since instances of missing equipment were frequent. a specific study of oamds sustained by train drivers14 has revealed problems involving task allocation as well as recovery situations, i.e. situations in which the normal task is interrupted by an incident, from which the worker has to recover, in other words strive to restore the usual course of work10. these accidents occurred when inspecting a train prior to departure: in the first case, the train started to brake during the operation; in the second case, an inexperienced driver detected a brake failure he had never before encountered and did not know how to remedy and, in the third case, the driver once again climbed down from the train because he had overlooked an inspection point. in each case, the driver gave his full attention to inspection in order to prevent the train being delayed and, when walking, tripped on a sleeper or a plate creating unevenness in the ground. in the first two cases, the driver was performing a recovery operation at the time of the accident to restore the train braking system operation. all recovery situations introduce or accentuate a time constraint, so resources mobilised to make the braking system operational as quickly as possible were partially lacking in terms of controlling displacement and this effectively caused the driver to trip. bentley et al.15 refer to a concurrent visual task when explaining the occurrence of certain oamds; these authors also emphasise that, at a given moment, resources dedicated to performing the task may be lacking in terms of controlling displacement. in many cases, existing obstructions to displacement are due to earlier or simultaneous work performed by workers other than the worker, who sustains an oamd; this reveals the part played by co-activity or a succession of activities in oamd occurrence. for example, a worker has to divert to avoid tools useful to other workers installing new equipment, but left on his/her displacement route. displacement diversion may be considered as a form of recovery activity intended to restore a normal course of work by returning to the initial route. co-activity, historically described by cuny16, represents task performance by persons pursuing different production objectives and required to share concurrently a common workplace. interim situations or those involving subcontracted work, in particular, can generate co-activity or a succession of activities. finally, bentley&haslam12 have shown that the job and finish policy implemented at the time in the united kingdom s mail distribution company, which allowed workers to go home as soon as the last mail had been distributed, could encourage workers to take risks by hurrying or taking short-cuts. these authors reported that workers explained that the accident risk raised by reading mail addresses while walking was more acceptable than the time wasted in stopping to read the addresses. working conditions (hence movement performance conditions) play a part in oamd occurrence since they make it more or less difficult to control displacement and, more generally, movement during task performance. organisational factors highlighted during oamd analysis reveal worker arbitration between production and safety in the work situation, in which he/she is exposed to a risk of movement disturbance. production-safety arbitrations relate particularly to the organisational activity implemented by the company. neutralisation of organisational factors therefore requires local and collective management of the oamd risk to ensure proximity to the company s specific characteristics and to compare existing logics and viewpoints. bentley&haslam12 state that, depending on the workers distributing mail, managers consider performance a priority over safety and that the workers themselves prefer rapid performance to safer performance of their work; their attitudes reflect those of the management in this respect. as in the presence of any oa risk, controls are implemented to perform the task, while ensuring safety with regard to movement disturbance, in other words while ensuring movement control., for example in the case of a collision when bolting because the spanner slipped. oamds can also involve more atypical movements, such as picking up an object or walking, and in some cases, being cut by an element in the environment or missing a step when running up stairs. controls implemented in work situations are therefore virtually permanent and the worker manages the available resources to perform his/her task, while controlling his/her movement. the resources required for movement control vary in time and with respect to the work situation. for example, derosier et al.17 report situations, in which metallurgists are sometimes required to move over template elements similar to beams. at these moments, the resources required to control their movements are more extensive than those required when walking on a level floor. likewise, resources required for walking on a floor with variable slip resistance are more extensive than resources required for walking on a surface with uniformly high slip resistance. resources needed to perform the task as a whole are also variable. at certain moments, a worker s visual attention can be taken up by a task and can thus be unavailable for movement control14, 15. task characteristics and requirements will therefore condition resources, which could be dedicated to movement control. 2.model of ostfa understanding based on the worker and his/her activity (adapted from the model developed by vezina (2001) for musculoskeletal disorders). contributes to our understanding of movement disturbance by illustrating a work situation model based on the worker and his/her activity. this has been adapted from the model developed by vzina18 in relation to work-related musculoskeletal disorders (wrmsds). wrmsds and oamds are invariably outcomes of occupational risks, which manifest themselves through worker movements. this is why oamds and wrmsds possess common characteristics with an impact on prevention. oamd prevention has been the subject of little research to date and could therefore benefit from studies in the wrmsd prevention field, at least from a theoretical and methodological standpoint. similarities between wrmsd and oamd and their consequences for prevention have been developed by leclercq et al4. model of ostfa understanding based on the worker and his/her activity (adapted from the model developed by vezina (2001) for musculoskeletal disorders). figure 2 shows that controls are implemented in work situations to ensure safety when performing a task. movements performed at work are subject to continuous adjustment with respect to the required task and individual, organisational and environmental constraints, as reported by chassaing19 when studying wrmsds. some of the implemented controls can be easily observed (rushing, moving round obstructions, etc.) and the individual strategies, to which these controls contribute, can be examined in detail using personal interviews. sometimes, they can not be visually observed and their detection requires a very fine observation grid: one that accurately describes movements such as heel strike angle when walking, distance provided as a safety margin between the foot and a low-level obstruction during a displacement, supports used, etc. to acquire a best possible understanding of worker controls implemented to perform a task in an occupational situation, while avoiding movement disturbances, we need to combine two levels of analysis: analysis of the activity and analysis of the movement performed within the activity. macroscopic developments such as technological advances or the advent of regulation influence the conditions under which an operator performs his movements and hence the resulting risks present in occupational situations. this model illustrates the outcome of macroscopic developments in the occupational situation through productive organisation choices. illustrates a work organisation model developed by the niosh20. the nature of the different factors involved in oa occurrence is displayed, along with the boundaries within which these factors are effectively harmful. organisation of the niosh work model taken from sauter et al., (2002). this model illustrates the outcome of macroscopic developments in the occupational situation through productive organisation choices. in general, productive organisation characteristics evolve constantly under the specific effects of technical progress (automation, introduction of new technologies, etc.), subsequent growth in productivity21, employment market developments (active population characteristics, etc.) and reorganisations22 (outsourcing, etc.). these macroscopic developments and choices made by productive organisations affect the work situation (level of prescription, time and spatial constraints, etc.) in ways that condition how worker movements are performed. controls are continually implemented not only by a worker performing his/her task, while avoiding injuries, but also at the different levels illustrated in fig. 3. analysing and combining these controls contributes to our understanding and prevention of occupational accidents, in particular oamds. despite scientific progress in the safety field, oamds are still commonly considered simple accidents resulting from a malfunction in a simple system; this might suggest that their prevention is also this paper aims to encourage changes in these perceptions by attempting to orient the reader s vision towards organisational factors, which often combine with other accident factors to cause movement disturbance and injury in work situations. these risks manifest themselves in the worker s movement but are none the less an unwanted consequence of productive organisational decisions. oamd organisational factors reveal the need for local and collective management of this risk and the importance of a better understanding of movement/displacement performed under working situations, i.e. in a context integrating specific task requirements and working conditions. while organisational measures implemented by the company represent a lever for oamd prevention, two points should be noted: on the one hand, organisational activity is restricted as illustrated by fig. 3 and, on the other hand, its lever is not unique. maximum possible neutralisation of factors close to the injury in the accident genesis and risk awareness also constitute major lines of prevention. awareness of the oamd risk, in particular, is an essential prerequisite to any progress in preventing these accidents. perception of the oamd risk and its more or less accepted nature are factors, which determine both consideration of this risk at every level of the company and controls implemented by workers.

workplace design and upkeep, or human factors, are frequently advanced for explaining so-called occupational slip, trip and fall accidents (ostfas). despite scientific progress, these accidents, and more broadly occupational accidents with movement disturbance (oamds), are also commonly considered to be simple. this paper aims to stimulate changes in such perceptions by focusing on organisational factors that often combine with other accident factors to cause movement disturbance and injury in work situations. these factors frequently lead to arbitration between production and safety, which involves implementation of controls by workers. these controls can lead to greater worker exposure to oamd risk. we propose a model that focuses on such controls to account specifically for the need to confront production and safety logics within a company and to enhance the potential for appropriate prevention action. these are then integrated into the set of controls highlighted by work organisation model developed by the niosh.

PMC4246534

pubmed-1205

the introduction of digital technologies in clinical pathology practice could produce great benefits in the form of improved patient care, better efficiency of health services, and novel diagnostic tools. at the same time, it is clear that these benefits can only be achieved if digital pathology solutions are carefully crafted for the clinical prerequisites. whereas low-volume and nonurgent situations such as research, teaching, and to some extent, consultations, are currently feasible with existing digital pathology systems, if digital pathology is to reach prime time status then more work is needed to enhance the suitability of the systems for clinical routine. clinical deployment also requires solving issues such as validation mechanisms, cost-efficient digital storage, and medico-legal demands, as well as redesigning work practices for the digital era. efforts to advance the field are taking place around the world. in the nordic countries (sweden, denmark, norway, finland, and iceland), there is a particular concentration of development work towards clinical use of whole slide imaging (wsi). for example, all routine histology slides are today scanned in the hospitals of linkping and kalmar and extensive digital primary review is performed, and>60 wsi scanners have been installed in sweden to date. against this backdrop, the nordic symposium on digital pathology (ndp) was created to promote knowledge exchange regarding the state-of-the-art in digital pathology. the specific focus of ndp is advances toward the clinical adoption of wsi and other digital technologies in pathology. as these advances require a concerted effort from health care, industry, and academia, ndp is intended as a forum where professionals from all domains can meet. the first ndp event was organized in november 2013 and attracted 125 attendees, whose feedback lead to an expanded ndp event in november 2014 as will be detailed below. judging from the history of digitization of radiology imaging, there is reason to believe that the nordics will continue to be a forerunner in clinical use of digital pathology. while perhaps not reflected by its regional name, ndp aims to be a venue of broad international interest where state-of-the-art in digital pathology is discussed and advanced. nordic symposium on digital pathology symposium 2014 took place november 56 in linkping, sweden. a total of 144 attendees gathered, of which 47% listed health care as the primary affiliation, 33% industry, and 19% academia. the health care representatives were dominated by pathologists, but also laboratory technologists and it staff were in significant numbers. the participants represented 14 different countries from europe, north america, and australia, with the nordic attendees being in a large majority (87%). central to the program was a series of invited talks and a collaborative workshop on clinical deployment issues. the contents of these sessions will be outlined in the sections below. in the science and innovation session, a double-blind review process was carried out by the symposium's international program committee with 15 senior researchers in the field and this resulted in three jpi papers published alongside this editorial: a comparative study of input devices for digital slide navigation (jesper molin et al.), randomspot: a web-based tool for systematic random sampling of virtual slides (alexander wright et al.), and histopathology in three-dimensional: from three-dimensional reconstruction to multi-stain and multi-modal analysis in addition, the ndp included an industrial exhibition consisting 13 vendors, ranging from large multinationals to recent startups, showing everything from wsi scanners through enterprise image management to desktop electron microscopy. figure 1 shows a session snapshot and the program details are available at the npd website http://www.liu.se/ndp?l=en. a key part of the ndp program was the workshop discussing clinical adoption of digital pathology. the workshop was organized as an open floor discussion where broad participation was encouraged and also achieved. as an input to the discussion, a survey was distributed among the health care attendees in advance of the symposium. some results from this survey will be presented next as it paints an interesting picture of the attitude toward digital pathology in nordic health care. it must be noted that the respondents of the survey represents an extremely biased selection among the pathology community. since only ndp participants were asked, this means that respondents are likely to be among the most positive to digital pathology and also among the most experienced. there is also strong geographical dominance from the nordics and in particular sweden. of 74 it is likely that the pathologist dominance were even higher for some questions that require deep knowledge of clinical practice. role distribution of survey respondents the survey first asked: today, to what degree do you use digital images of histology slides in your practice? (in% of all histology cases.) the results are shown in figure 3, showing moderate levels of adoption but no use in about 50% of respondents. another bias to note for these questions is that several people from the same institution may have responded. since the most digitized sites kalmar and linkping had several attendees, these numbers are likely to represent higher usage than numbers on a per site basis. current use of digital pathology among survey respondents the same question was asked for the predicted situation at the end of 2016, shown in figure 4, showing significant optimism for near-future use of digital pathology. predicted use of digital pathology at the end of 2016 the respondents were also asked to judge the impact of digitization: what effect do you foresee that digital pathology will have compared to traditional microscope practice, with regards to the following areas? the impact grading was given in a five-point scale, major negativeminor negative status quo minor positive major positive. the assessments with regards to pathologist work are in figure 5 whereas other laboratory aspects are in figure 6 and overall impact in figure 7. foreseen impact of digitization with regards to pathologist's work foreseen impact of digitization with regards to laboratory aspects foreseen impact of digitization with regards to overall effects broadly, these responses indicate a very positive attitude toward digital pathology in terms of its effects on pathologist and laboratory working. however, more negative responses were received in the effect on pathologists efficiency (time per case and speed of slide navigation) as well as perceived delays in slide arrival from the laboratory until starting review. overall effects of digitization were seen as very positive, especially for the quality of care. finally, the respondents also were asked to state the three main barriers for adoption of digital pathology in their clinical practice. it was clear that the cost of implementation is a major issue; 79% of the responses mentioned lack of sufficient funds as a barrier. technology limitations were listed in 71% of the responses, some referring to insufficient performance of commercial solutions and some referring to lacking it infrastructure in their organization. a conservative attitude among colleagues other responses concerned lack of organizational engagement at local, national and international levels to develop protocols, work practices, and standards. after the survey results had been presented, a guided discussion between all participants took place. advanced digital pathology implementation efforts were reported by many contributors, either established (e.g. skne, linkping) or in planning (e.g., gothenburg, karolinska/stockholm, oslo, copenhagen). the planned use cases covered similar areas such as retrieving archived cases, presenting at multi-disciplinary meetings, obtaining second opinions (either for individual cases or to share work between institutions). digital pathology was seen by many contributors as a key enabler of higher quality pathology services, by increasing specialist reporting of cases or supporting and supporting colleagues across distances. those who had implemented digital pathology on a large scale were positive about the effects it had on workflow and reported no untoward delay in slide arrival caused by the extra step of scanning (in fact, one pointed out that the cases arrived in a more continuous flow, rather than the batches of glass slides normally received). several contributors (linkping, skne, toronto) had experience of working with moderate or large volumes of the digital work. fatigue using dp systems was mentioned by a few, possibly a combination of older less effective slide viewing software and the known effect of computer displays on eye fatigue. several experienced commenters pointed out that fatigue was often an issue with the microscope and that digital pathology offered ergonomic benefits which could be beneficial in the long term for the pathologist's experience. laboratories produce a lot of glass slides, and wsis produce large amounts of storage space. it was felt that data storage was very often raised as a concern by it departments involved in discussions about digital pathology implementations. some contributors pointed out that projections of hundreds of terabytes of image data per year, while realistic for 100% digital practice, may serve to inhibit pathologists and it departments from trying the technology, and that in fact to start going digital-only a modest storage capacity of a few terabytes is needed. it contributors pointed out that mature information lifecycle management systems for digital pathology had yet to appear (some contributors reported being charged excessive monthly costs for even small amounts of data), but it was likely that tiered storage systems would ameliorate the daunting costs associated with large amounts of live online storage, as would the constant reduction in price per terabyte of storage media. some discussion around how long digital images should be kept for and what latency before image retrieval might be tolerated revealed that several contributors had envisaged such tiered data arrangements would be needed. centers with mature or second-generation digital pathology systems often had it staff who understood well the complexities of wsi data, and well-integrated systems. experienced users re-emphasized the need for digital pathology to be supported by knowledgeable and (preferably) designated it staff. standards were mentioned by only a few contributors-the it staff, who expressed surprise at the lack of standardization of digital pathology image formats and interoperability, and those pathologists on their second generation of scanners who had experienced incompatibility between two different vendors products (e.g. in viewers or in image analysis algorithms). some audience members had experience of the last digital revolution in medical imaging (in radiology) and saw many parallels in the it needs, questions about validation/safety and clinical acceptance of the technology. the room was understandably full of those keen to adopt digital pathology, many of whom reported colleagues with more conservative attitudes. some of the objections were seen, however, as entirely justifiable (e.g. concerns over speed of diagnosis and diagnostic accuracy with digital systems) and many in the room agreed that these were issues that needed to be addressed. for successful implementations, the need for champions in each department was mentioned, as well as the value in immersing trainees in a digital working pattern from an early stage. wiser (and older) heads pointed out the benefits of actively seeking out sceptics and involving them in digitization projects. dr. andrew evans from the university health network, toronto described a long-standing program of digitization including telepathology and wsi in a university hospital network. he provided a very detailed description of the assiduous planning involved in digital pathology adoption and his experiences of involving the entire department in projects. metin gurcan of ohio state university spoke about his work in image analysis and computer aided diagnosis, introducing many parallels from radiological imaging and emphasizing the importance of pathologist-computer scientist partnerships and validation in such work. jan baak from stavanger university hospital gave an expansive talk on his long career in pathology imaging, especially speaking about his role in the prognostication of breast cancer with morphology and image analysis, emphasizing the ongoing value of good pathological assessment even in a genomic era. dr. sten thorstenson from linkping university hospital explained his long experience of digital pathology at kalmar and linkping, starting at a time when a terabyte really was a large amount of data. after 9 years he reports being entirely comfortable working digitally and has been reporting 100% from home for almost a year now without access to a microscope or physical slides. derek magee from the university of leeds gave an overview of his work in image analysis research. a focus area has been digital three-dimensional pathology, and in particular tackling the inherent challenges of the slide registration, color normalization, and histology-radiology correlation. thomas miliander from vrmland county council, sweden, presented this health-care provider's strategy for imaging it infrastructure. the approach taken is an enterprise image management backbone for all medical images, relying on standards for tight integrations with other information systems, a context into which now also wsi is entering. johan lundin from the institute for molecular medicine finland, helsinki, provided an overview of his group's work in digital pathology. the portfolio presented spanned from web and touch-enabled wsi viewing applications to low-cost handheld microscopes utilizing smartphone camera components. in a special session, elin kindberg of sectra presented preliminary results from a national swedish effort to investigate key medico-legal issues arising when deploying digital pathology. regarding access to swedish patient data outside of sweden the legal situation is clear that this is possible provided those appropriate security measures are taken. legal directions regarding whether all wsi data must be stored does not, however, exist; the swedish law only mandates good and safe health care. the conclusion is that the pathology profession needs to define what the legal mandate means in this case. the swedish pathologist society is now finalizing an official guideline document describing different possible paths, all legal, and all with different advantages and drawbacks. the 2 ndp spanned across many areas of interest with regards to the emerging use of wsi and related it tools in clinical routine. feedback from attendees indicates that this sharing of knowledge and experiences across organizations, disciplines, and sectors is an important catalyst for development of best practices and overall progress. organizing ndp 2014 has been a very rewarding experience, and we welcome attendees from all over the globe to future gatherings of this group.

techniques for digital pathology are envisioned to provide great benefits in clinical practice, but experiences also show that solutions must be carefully crafted. the nordic countries are far along the path toward the use of whole-slide imaging in clinical routine. the nordic symposium on digital pathology (ndp) was created to promote knowledge exchange in this area, between stakeholders in health care, industry, and academia. this article is a summary of the ndp 2014 symposium, including conclusions from a workshop on clinical adoption of digital pathology among the 144 attendees.

PMC4355840

pubmed-1206

gestational diabetes mellitus (gdm) is defined as glucose intolerance 1st recognized during pregnancy with a prevalence of 2% to 14% in all pregnant women. gdm is associated with maternal, fetal, and neonatal adverse outcomes such as cesarean delivery, preeclampsia, shoulder dystocia, macrosomia, neonatal hypoglycemia, and perinatal death. although yet to be proven, screening and treating gdm may contribute to prevent adverse outcomes. the most commonly used screening and diagnostic methods of gdm are flawed because they give relatively poor negative and positive predictive values. the 2-step diagnosis method, the 1-hour 50-g glucose challenge test (gct), and the 3-hour 100-g oral glucose tolerance test (ogtt) are currently used in the united states, and the single-step 2-hour 75-g ogtt is used in european countries. in korea, the 2-step diagnosis approach has been used predominantly as in the united states, but several hospitals have adopted new guidelines for the diagnosis of gdm. it is also necessary to change the current counseling and treatment approaches for gdm patients because perinatal outcomes tend to differ according to glucose levels. patients with 50-g gct within 140 to 199 mg/dl had 2.5-fold increasing risk of large for gestational age (lga) and 2.9-fold increasing risk of macrosomia. langer et al demonstrated that every 10 mg/dl increment in fasting blood glucose (fbg) resulted in 15% increase in adverse composite outcomes. the purpose of this study is to evaluate the association between perinatal outcomes and 50-g gct values as well as fbg among gdm women. a retrospective analysis of 3434 pregnant women who had 50-g gct was carried out between march 2001 and april 2013 at severance hospital, seoul, korea. women with fetal anomalies, multiple gestations, overt diabetes mellitus (dm), and hypertension were excluded from the study. a total of 2631 pregnant women received the normal 50-g gct and 803 received the100-g ogtt because their 50-g gct value was greater than 140 mg/dl. as a result, 307 patients were diagnosed with gdm (gdm group) and 496 showed false-positive result (impaired glucose tolerance group). a false-positive result was defined as showing positive in the 1-hour 50-g gct but negative in the 3-hour 100-g ogtt. gdm was defined as showing 2 or more abnormal duration (hours) of 100-g ogtt values: fbg of 95 mg/dl or more; 180 mg/dl or more for 1-hour; 155 mg/dl or more for 2-hours; and 140 mg/dl or more for 3-hours. a total of 171 patients had normal fbg (< 95 mg/dl) and 136 patients had abnormal fbg (95 mg/dl) (fig. the 1-hour 50-g gct result was also divided into 20-unit increments, and these subgroups were used to evaluate maternal and perinatal outcomes. a total of 307 pregnant women (the same number as gdm patients) were randomly selected from the normal 1-hour 50-g gct group (n=2631) to be the control. the risks of adverse maternal and perinatal outcomes for subgroups of the gdm group were then analyzed and compared against the control group. maternal composite adverse outcomes included cesarean delivery and preeclampsia, while fetal composite adverse outcomes included lga, apgar score, intensive care unit admission, neonatal hypoglycemia, and hyperbilirubinemia. preeclampsia was diagnosed according to the criteria of the acog practice bulletin: new onset of blood pressure of 140/90 mm hg or more on 2 separate readings taken 6 hours apart after 20 gestational weeks; and proteinuria of 300 mg/24 hours or more. lga was defined as birth weight greater than the 90th percentile compared with gestational age. neonatal hypoglycemia was defined as blood glucose level of less than 40 mg/dl, and hyperbilirubinemia was defined as bilirubin level of more than 5 mg/dl. for statistical processing, the chi-square test or fisher exact test was used for categorical variables and the 2-sample t test or the wilcoxon rank sum test was used for continuous variables. multiple logistic regression analysis was performed to estimate the odds ratios (ors) of adverse outcomes with adjustment for confounders. cary, nc) and statistical significance was considered for p-values <0.05. based on the results of the 50-g gct, the clinical characteristics and perinatal outcomes of the impaired glucose tolerance (igt) group and the gdm group (table 1) were compared. significant differences were observed between the 2 groups in terms of their maternal age, body mass index (bmi) before pregnancy and at delivery, and family history of dm. for perinatal outcomes, the gdm group showed higher incidence of cesarean delivery, macrosomia, lga, and neonatal hypoglycemia. comparison of characteristics of igt and gdm groups. among gdm patients, maternal characteristics and perinatal outcomes study findings show statistical significance in bmi before pregnancy and at delivery, rate of cesarean section, prevalence of gestational hypertension, gestational insulin therapy, and hba1c at diagnosis. incidence of macrosomic newborn (3.5% for normal glycemic vs 22.1% hyperglycemic group, p<0.001) and lga newborn (10.5% for normal glycemic vs 36.0% hyperglycemic group, p<0.001) was higher in the fasting hyperglycemic group, meeting the carpenter and coustan criteria. moreover, the prevalence of neonatal hypoglycemia was 9.4%, and 15.4% in the normal glycemic and hyperglycemic groups, respectively (p<0.001). to compare perinatal outcomes between the 2 groups, the odds ratio was calculated after controlling for confounding factors (table 3). the odds ratio for perinatal outcomes was 6.72 (95% ci: 2.5917.49, p<0.001) with macrosomia, 3.75 (95% ci: 1.977.12, p<0.001) with lga, and 1.65 (95% ci: 0.793.43, p=0.183) with neonatal hypoglycemia. the maternal and perinatal outcomes of pregnant women with 50-g gct result of over 140 mg/dl were further stratified into 20-unit increments (table 4). the findings showed significant differences in bmi before pregnancy and at delivery, incidence of gestational hypertension and gestational insulin therapy, hba1c at diagnosis of gdm, macrosomia, lga, and neonatal hypoglycemia. the 50-g gct results were categorized and the perinatal outcomes were compared with 50-g gct normal group after adjusting for confounders (table 5). increased 50-g gct values in subgroups showed relevance with higher risk of perinatal outcomes. the ors of macrosomia (up to 20.31-fold), lga (up to 6.15-fold), and neonatal hypoglycemia (up to 84.00-fold) were higher in subgroups with higher 50-g gct values. among gdm patients, the group with 50-g gct level of 140 to 159 mg/dl was found to be associated with macrosomia (or: 4.31, 95% ci: 1.4213.13, p=0.010) and neonatal hypoglycemia (or: 17.27, 95% ci: 2.03147.12, p=0.009) when compared against the normal group. however, lga (or: 0.84, 95% ci: 0.401.73, p=0.628) did not show statistical significance. the subgroup in the 160 to 179 mg/dl range showed significant or for macrosomia (or: 5.95, 95% ci: 1.9218.49, p=0.002) and neonatal hypoglycemia (or: 53.72, 95% ci: 6.78425.70, p<0.001). lga (or: 1.48, 95% ci: 0.733.03, p=0.279) showed the tendency to increase but there was no statistical significance. also, the subgroups in the 180 to 199 mg/dl and the 200 mg/dl ranges showed a strong association with all adverse perinatal outcomes. therefore, risks of adverse perinatal outcomes increased as the values of 50-g gct results increased. maternal characteristics and perinatal outcomes according to fasting glucose values in gdm patients. or of perinatal outcomes for fasting glucose level 95 mg/dl in gdm group. maternal and perinatal outcomes according to 50-g glucose challenge test in gdm patients. or of perinatal outcomes according to 50-g glucose challenge test values in gdm patients. this study investigated the maternal and perinatal outcomes according to fbg and 50-g gct values in gdm pregnant women. the risks of macrosomia, lga, and neonatal hypoglycemia increased with fasting hyperglycemia and higher 50-g gct values. study findings also showed that the association between fasting hyperglycemia and adverse perinatal outcomes remain to be significant after adjustment for potential confounders. previous studies have found that postprandial hyperglycemia was associated with excessive fetal growth in gdm patients requiring insulin therapy or pregestational diabetes patients. other studies have indicated that meal-related glucose threshold measurement did not increase the risk of adverse fetal outcomes. recently, other researches have demonstrated significant association between fasting and 1-hour 75-g ogtt glucose values with lga newborns among gdm women. it has been reported that strict glucose control in this risk group may be necessary in order to avoid lga newborns. it has been emphasized in many studies that fbg is an important factor in gdm screening as well as in predicting neonatal adverse outcomes. gdm women with an isolated abnormal fbg were more likely to need hypoglycemic agents to obtain good glycemic control. the significance and magnitude of this association was consistent with the results of this study. in the abnormal fbg group, 47.1% (64/136) of gdm patients needed gestational insulin therapy to control the blood glucose but in the normal fbg group (p<0.001), only 12.9% (22/171) of pregnant women who were diagnosed with gdm required insulin treatment. the hapo studies have demonstrated the continuously increasing relationship between maternal blood glucose levels and adverse perinatal outcomes such as frequency of lga, neonatal hypoglycemia, cord blood serum c-peptide level above the 90th percentile, and primary cesarean section delivery. however, these studies are based on the single step 75-g ogtt, and gdm was diagnosed using the international association of diabetes and pregnancy study groups (iadpsg) criteria. in the hapo studies, the outcomes were compared after categorizing fasting, 1-hour, and 2-hour glucose values. in contrast, this study is based on the 2-step diagnosis of gdm, and the outcomes of the 50-g gct values and the fbg of 100-g ogtt were compared against each other. the outcomes found in this study give a more accurate picture of the situation in korea, where the 2-step diagnosis is predominantly used. in addition, without adjusting for confounding factors, such as prepregnancy bmi and gestational weight gain, adverse outcome risks may have been overestimated in other earlier studies. however, in this study, the relation between maternal hyperglycemia and adverse perinatal outcomes were analyzed after adjusting for bmi before pregnancy and gestational weight gain, because bmi is also related to maternal and perinatal outcomes in gdm patients. fbg has been used as the most important indicator for the diagnosis of dm in nonpregnant adults because it reflects impaired insulin secretion and resistance. fbg values tend to stay constant throughout the entire period of pregnancy and this is also true for nonpregnant patients. fbg values have less individual variation compared to other glucose values; therefore, abnormal fbg level is a significant indicator in diagnosing gdm. at present, fbg is a good screening test for gdm with advantages such as simple procedure, reasonable cost, reproducibility, easy access, and wide acceptance. recently, other studies have reported that abnormal fbg alone is capable of detecting 50% of pregnant women with gdm from a pool of women who had already been diagnosed with gdm with another screening method. if combined with the 2-hour plasma glucose level, another 25% of pregnant women with gdm can be detected. in 2016, park et al developed a more practical and efficient screening tool using fbg and prepregnancy bmi for predicting adverse outcomes of gdm. this new screening tool focused on predicting the maternal and perinatal adverse outcomes of gdm patients. the findings of this study show that bmi before pregnancy, bmi at delivery, hba1c value at diagnosis, and the application of gestational insulin therapy were much higher in the abnormal fbg group. several studies have indicated that maternal prepregnancy bmi was associated with the risk of gdm. sacks et al confirmed that maternal bmi had a powerful impact upon fetal birth weight. therefore, gdm patients with both higher bmi and abnormal fbg values can have potentially worse perinatal outcomes. this study also showed that abnormal fbg values according to the carpenter and coustan criteria had significance association with higher incidence of lga, macrosomia, and neonatal hypoglycemia. in addition, adverse perinatal outcomes according to different 50-g gct values among gdm patients were evaluated in this study. the risks of macrosomia, lga, and neonatal hypoglycemia increased with increasing 50-g gct values. several studies have examined the relationship of 50-g gct and perinatal outcomes. in 1987, leikin et al reported that the false-positive gct group (gct values of 135 mg/dl or more but normal ogtt values) had higher incidence of macrosomia compared with the normal gct group (11.9% vs 6.4%, p=0.009). recently, other retrospective cohort studies also showed that false-positive gct is an independent risk factor for adverse perinatal outcomes (or: 5.96, 95% ci: 1.310.3). the findings of this study suggest that the risks of the macrosomia (up to 20.31-fold), lga (up to 6.15-fold), and neonatal hypoglycemia (up to 84.00-fold) increased with higher 50-g gct values. more importantly, 50-g gct values in the range of 140 to 159 mg/dl were not associated with lga compared to the normal 50-g gct group. these results should be interpreted with caution since confidence intervals were very wide due to a limited number of cases in the 50-g gct normal group. in 2013, figueroa et al evaluated the relationship between 50-g gct values and perinatal outcomes in mild gdm patients, and showed that gct values of 140 mg/dl or more were associated with an increase of composite perinatal outcomes, lga, and macrosomia. however, there was no evaluation for the group with gct value of 200 mg/dl or more. they only included mild gdm group with fasting glucose value less than 95 mg/dl. the strength of this study is that a full spectrum of gdm patients were examined using the carpenter and coustan criteria, including the group with 50-g gct higher than 200 mg/dl. therefore, it was possible to evaluate the continuous 50-g gct values in the lower or upper ranges. this study also included normal and abnormal fasting glucose groups and the abnormal fbg group tended to have a greater risk for adverse perinatal outcomes. a randomized clinical trial of a larger scale, using prospectively collected data from a well-characterized trial cohort, is ideal and necessary to validate the findings of this study. all gdm women were treated to achieve the recommended value of their glycemic profiles and 28.0% (86/307) of the pregnant women with gdm needed insulin therapy. even without the influence of gdm management in addition, long-term health complications such as childhood obesity, impaired insulin sensitivity, or type 2 diabetes mellitus were not considered for in this study. in conclusion, also, composite perinatal outcomes such as macrosomia, lga, and neonatal hypoglycemia are more frequent with increasing 50-g gct values. therefore, more attention and care should be given during prenatal counseling, as well as more active therapeutic intervention taken when necessary with closer fetal monitoring, with the objective to reduce adverse perinatal outcomes in gdm patients with abnormal fbg or high 50-g gct values.

abstractpregnancies complicated by gestational diabetes mellitus (gdm) are associated with increased risks of adverse maternal and fetal outcomes. the risks of adverse pregnancy outcomes differ depending on the glucose values among gdm patients. for accurate and effective prenatal counseling, it is necessary to understand the relationship between different maternal hyperglycemia values and the severity of adverse outcomes. with this objective, this study reexamines the relationship between maternal hyperglycemia versus maternal and perinatal outcomes in gdm patients. for this study, maternal hyperglycemia was diagnosed using the 2-step diagnostic approach.medical records of 3434 pregnant women, who received the 50-g glucose challenge test (gct) between march 2001 and april 2013, were reviewed. as a result, 307 patients were diagnosed with gdm, and they were divided into 2 groups according to their fasting glucose levels. a total of 171 patients had normal fasting glucose level (< 95 mg/dl), and 136 patients had abnormal fasting glucose level (95 mg/dl). the 50-g gct results were subdivided by 20-unit increments (140159, n=123; 160179, n=84; 180199, n=50; and 200, n=50), and the maternal and perinatal outcomes were compared against the normal 50-g gct group (n=307).maternal fasting blood glucose (fbg) level showed clear association with adverse perinatal outcomes. the odds ratio (or) of macrosomia was 6.72 (95% ci: 2.5917.49, p<0.001) between the 2 groups. the ors of large for gestational age (lga) and neonatal hypoglycemia were 3.75 (95% ci: 1.977.12, p<0.001) and 1.65 (95% ci: 0.793.43, p =0.183), respectively. also, the results of the 50-g gct for each category showed strong association with increased risks of adverse perinatal outcomes compared to the normal 50-g gct group. the or of macrosomia (up to 20.31-fold), lga (up to 6.15-fold), and neonatal hypoglycemia (up to 84.00-fold) increased with increasing 50-g gct result.

PMC5023889

pubmed-1207

crohn s disease (cd) is a chronic inflammatory disorder of the gastrointestinal tract characterized by focal, asymmetric, transmural inflammation of uncertain etiology and of an unpredictable course. the clinical presentation of cd is characteristically manifested by repeated cycles of active and quiescent disease of definable patterns according to disease location and types (inflammatory, fibrostenotic and fistulizing).1,2 the prevalence and incidence of cd in the united states is estimated to be 50 per 100,000 and 5 per 100,000 annually, respectively.3 the treatment regimen is individualized based on disease activity, location and behavior taking into the account the balance between medications and their side effects and prevention of complications. the treatment of cd remains empirical and the disease is not curable since no clear etiology of cd disease has been yet elucidated. munkholm et al in their population-based cohort study from scandinavia4 demonstrated that among all patients treated with 5-aminosalicylic acid (5-asa) agents and corticosteroids (cs), 13% of patients will achieve complete remission, 20% of patients will experience annual relapse and 67% will have a combination of relapse and remission within the first 8 years after initial diagnosis. less than 5% of patients will have a continuous course of active disease. another population-based cohort based in olmsted county, minnesota, which was conducted prior to the routine use of anti-tumor necrosis factor (anti-tnf) agents, confirmed that a representative patient with cd would be expected to spend 24% of the time in medical remission without medications, 41% of the time in post-surgical remission without medications, 27% of the time in medical treatment with 5-asa derivatives and 7% of the time having disease activity requiring treatment with corticosteroids or immunomodulators.5 although cd has been recognized as having a chronic relapsing course, it is evident that the majority of patients remain in clinical remission at any particular time. however it is recognized that the majority of patients will progress from inflammatory to complicated fistulizing or penetrating disease over time. the cumulative risk for the development of a cd-related fistula has been estimated to be 33% at 10 years and 50% after 20 years based on a population based cohort study form olmsted county, mn.6 therefore the treatment strategies for cd must target lifelong management, addressing both short-term and long-term aspects of the disease and are guided by the disease location, severity, associated complications and concurrent therapy taken by the patients. these treatment strategies consist of a sequential (step up) approaches ranging from the first line agents such as 5-asa, controlled released corticosteroids (budesonide) and antibiotics all used to treat mild to moderate active cd to the second line with oral prednisone and the third line with conservative use of immunomodulators (azathioprine (aza), 6-mercaptopurine and methotrexate) and further biological therapy (infliximab, adalimumab, czp and natalizumab). recently, 5-asa has been critically analyzed for treatment of patients with cd and is suggested to be no more effective than placebo.7 however, recent data suggest that early initiation of combined treatment with immunomodulators and anti-tnf agents (infliximab) (top down) was more efficacious than conventional management with cs followed by aza and then infliximab8 in prior anti-tnf and anti-metabolite nave patients. therapy with anti-tnf antibodies has become a mainstay of treatment for patients with cd who are unresponsive to conventional medical management. currently there are three anti-tnf agents that have been approved by the u.s. food and drug administration (fda) in the treatment of cd, namely infliximab, adalimumab and czp. infliximab (remicade) is administered intravenously whereas adalimumab (humira) and czp (czp) (cimzia) are administered subcutaneously. treatment with infliximab consists of an initial loading regimen with three initial infusions at the dose of 5 mg/kg at week 0, 2 and 6 followed by every 8 week maintenance schedule.9 treatment with adalimumab (administered subcutaneously) consists of initial loading dose of 160 mg (given either in four doses of 40 mg each within 1 day or in two daily doses of 40 mg each over two consecutive days) followed by 80 mg dose given two weeks later with initiation of maintenance treatment after additional 2 weeks at dose 40 mg every 2 weeks.10 treatment with czp is initiated with an initial loading dose of subcutaneous injection of 400 mg at weeks 0, 2 and 4 followed by maintenance treatment every 4 weeks.11 czp was approved in april 2008 by the us food and drug administration and is currently approved for reducing signs and symptoms of cd and for maintaining clinical response in adult patients with moderate to severe activity of the disease with inadequate response to conventional therapy.12 anti-tnf therapy (infliximab, adalimumab and czp) was found to be significantly more efficacious than placebo in inducing remission at week 4 with a total mean difference in effect between anti-tnf agents and placebo of 11% (95% ci 6%16%, p<0.001) based on the results of a meta-analysis of fourteen randomized placebo controlled trials that included a total of 3995 patients with cd.13 these biologic medications were also significantly superior over placebo in maintaining remission at weeks 20 through 30 with total mean difference in effect between active and placebo arms of 23% (95% ci 18%28%, p<0.001) among patients who responded to an open-label induction with either infliximab, adalimumab or czp followed by randomized placebo controlled maintenance treatment.13 this review discusses the efficacy of czp and adherence to anti-tnf therapy with a particular focus on czp in patients with cd. the efficacy of czp was evaluated in two dose-response phase ii (n=384)14,15 and two phase iii (the pegylated antibody fragment evaluation in cd disease: safety and efficacy 1 (precise 1) and precise 2)16,17 (n=1330) randomized placebo controlled trials in adult patients with moderate to severe cd (table 1). the first phase ii trial (n=92) assessed efficacy of czp over 12 weeks period after single 30-minute intravenous infusion of either czp (cdp870) (1.25 mg/kg, 5 mg/kg, 10 mg/kg or 20 mg/kg) versus placebo.14 the primary endpoint was clinical response (decrease in crohn s disease activity index (cdai) score at least by 100 points when compared to baseline) at week 4.14 czp demonstrated similar efficacy to placebo in achieving primary endpoint (60% for 5 mg/kg, 58.8% for 10 mg/kg, 47.8% for 20 mg/kg vs. 56% for placebo).14 similarly, no difference between any dose of czp and placebo was observed with respect to secondary endpoints (clinical response, remission, cdai score decrease by 70 points, c-reactive protein (crp) levels at weeks 2, 8, 12).14 on the other hand, czp given at 10 mg/kg was significantly more efficacious than placebo in inducing remission at week 2 (47.1% vs. 16%, p=0.041).14 the second phase ii 12-week trial (n=292) evaluated efficacy of czp administered subcutaneously (100 mg, 200 mg or 400 mg) or placebo.15 treatment with czp was not superior over placebo as assessed at the primary study endpoint (clinical response at week 12) with the response rates among active drug arm of 36.5% (100 mg), 36.1% (200 mg) and 44.4% (400 mg) and in the placebo arm of 35.6%.15 however, czp was superior to placebo in achieving clinical response at week 2 (100 mg, 200 mg and 400 mg), 4 (200 mg and 400 mg), 8 (100 mg and 400 mg)and 10 (400 mg) with the highest rates for 400 mg dose at any analyzed.15 in addition, czp was superior to placebo in inducing clinical remission at week 4 (100 mg, 200 mg and 400 mg) and week 8 (100 mg and 400 mg) but not week 12.15 treatment with the highest dose of czp (400 mg) was superior to placebo in achieving primary (53.1% vs. 17.9%, p=0.005) and secondary endpoints in patients with high baseline levels of crp (10 mg/l) but not in those with low baseline crp levels in post-hoc analysis.15 based on these findings it was proposed that the efficacy of czp over placebo might not have been demonstrated due to the high placebo response rate in the large cohort of patients with low baseline level of crp.15 the latter findings were taken into consideration in the phase iii 26-week precise 1 trial (table 1).16 patients in this trial (n=660) were stratified based on low (< 10 mg/l) or high (10 mg/l) baseline crp levels and then randomized to subcutaneous injections of either czp 400 mg or placebo given every 2 weeks through week 4 and then every 4 weeks through week 26.16 czp was found to be significantly more efficacious in achieving the primary endpoint (at least 100 point decrease in cdai score at week 6 and both weeks 6 and 26 in a cohort of 302 patients with high baseline crp levels) with rates of 37% vs. 26% (wk 6, p=0.04) and 22% vs. 12% (both wk 6 and 26, p=0.05).16 in an overall cohort treatment czp was superior to placebo in achieving clinical response at week 6 (35% vs. 27%, p=0.02) and both weeks 6 and 26 (23% vs. 16%, p=0.02).16 on the other hand, no significant difference was found between the active arm and placebo arm with respect to achieving remission at any time point among all patients (wk 6: 22% vs. 17%, p=0.17; both wk 6 and 26: 14% vs. 10%, p=0.07) or patients with high baseline crp level (wk 6: 22% vs. 17%, p=0.29; both wk 6 and 26: 13% vs. 8%, p=0.24).16 concomitant use of immunosuppressants or cs, previous infliximab therapy or smoking status did not influence the response rates at the aforementioned time points.16 the presence of antibodies to czp were detected in 8% of czp-treated patients with a 4% rate in czp-treated patients who also received immunosuppressive agents and with 10% rate in czp-treated patients who did not receive concomitant immunosuppressive agents.16 the subsequent precise 2 trial (table 1) observed that czp was superior to placebo in maintaining response and clinical remission in responders to induction therapy with czp.17 among patients who received an open-label induction therapy with 3 single doses of 400 mg czp given subcutaneously every 2 weeks 64% (428/668) responded (at least 100 point decrease in cdai score vs. baseline score).17 these patients were stratified according to baseline crp levels (10 vs.<10 mg/l) and randomized to receive subcutaneously either czp 400 mg or placebo administered every 4 weeks through week 24 with follow-up through week 26.17 therapy with czp was superior to placebo in maintaining response to treatment through week 26 in patients with baseline high c-reactive protein levels (primary end point) (n=213, 62% vs. 34%, p<0.001) and in the intention to treat population (n=425, 63% vs. 36%, p<0.001).17 czp was also significantly superior to placebo in achieving clinical remission at week 26 in the cohort with high baseline crp (42% vs. 26%, p=0.01) and in all patients in the intention to treat population (48% vs. 29%, p<0.001).17 antibodies to czp were detected in 18% of patients receiving placebo maintenance therapy and in 8% of patients receiving continuous czp treatment (p-value not reported).17 among those who received concomitant immunosuppressive agents the rates of detectable antibodies to czp were 2% in czp maintenance arm and 8% in placebo maintenance arm.17 on the other hand, the rates of patients with detectable antibodies to czp in those not treated with immunosuppressants were 12% in czp arm and 24% in placebo arm.17 a recent detailed analysis of a cohort of 108 patients with fistulizing cd that participated in the precise 2 trial17 showed that 58 (53.7%) of them achieved clinical response to induction therapy with czp at week 6 and were subsequently randomized to receive further maintenance with either czp (n=28) or placebo (n=30).18 the complete fistula healing rate was achieved at week 26 in 36% of czp-treated and in 17% of placebo-treated patients (p=0.038).18 however, using the precise 2 trial17 pre-specified definition of fistula closure (closure of 50% of fistulas at any two consecutive post-baseline visits at least 3 weeks apart) there was no difference between czp and placebo arms with respect to percentage of patients achieving fistula closure (54% vs. 43%, p=0.069).18 an additional analysis of randomized maintenance trial data from precise 2 highlighted that czp-treated patients with shorter duration of cd (less than 1 year) had higher response rates at week 26 when compared to those czp-treated subjects with longer duration of cd (5 years) (89.5% vs. 57.3%, p<0.05).19 however, czp-treated patients with both short (< 1 year) and long (5 years) duration of cd had significantly higher response (89.5% vs. 37.1%, p<0.01 and 57.3% vs. 32.7%, p<0.001, respectively) and remission (68.4% vs. 37.1%, p<0.05 and 44.3% vs. 23.5%, p<0.001, respectively) rates than placebo recipients at week 26.19 the factors that independently predicted maintenance of response to czp at week 26 identified by logistic regression were shorter (< 2 years) vs. longer duration of cd (82.1% vs. 58.5% p<0.006), absence vs. presence of prior intestinal resection (67.5 vs. 51.6%, p<0.027), infliximab-nave status vs. prior exposure to infliximab (68.7% vs. 44.2%, p<0.002), and no corticosteroid use vs. corticosteroid use at baseline (65.7% vs. 57.3%, p<0.001).19 although the precise 2 trial demonstrated that the efficacy of czp is higher in patients receiving this agent as the first-line biologic when compared to infliximab-exposed individuals a post-hoc analysis of precise 2 data showed that patients with cd may benefit from treatment with czp regardless of prior use of infliximab when compared to those treated with placebo.20 czp was significantly more effective than placebo as maintenance therapy at week 26 in both infliximab-exposed (response: 44.2% vs. 25.5%, p=0.018; remission: 32.7% vs. 13.7%, p=0.008) and in infliximab nave patients (response: 68.7% vs. 39.6%, p<it should be noted however that the superiority of czp over placebo was more distinct in infliximab nave patients.20 these interesting observations however require future validation in a large prospective placebo controlled trials. all patients who completed 26-week precise 2 trial were offered open label extension treatment with czp given at 400 mg s.c. dose every 4 weeks for 54 weeks (precise 3 trial).21 the precise 3 trial included 141 patients who received czp (precise continuous group) and 100 patients who received placebo (precise 3 drug interruption group) in prior precise 2 trial.21 among patients who were either in response or remission at precise 3 baseline (week 26 of precise 2) the rates of sustained response (reduction in harvey-bradshaw index score 3 from baseline for all visits) and remission (hbi score 4 for all visits) were similar between continuous and drug interruption arms at 26 weeks (sustained response: 74.4% vs. 79.7%, respectively; sustained remission: 72.8% vs. 73.5%, respectively) and 54 weeks (sustained response: 66.1% vs. 63.3%, respectively; sustained remission: 62.1% vs. 63.2%, respectively) of precise 3 trial.21 on the other hand, patients in the drug interruption arm had a greater incidence of adverse events related to czp than those receiving czp continuously (32% vs. 23.4%, p-value not reported).21 therefore it has been suggested that continuous administration of czp should be recommended due to its effectiveness and more favorable safety profile.21 all patients who relapsed (increase in cdai 70 points above baseline or higher than baseline at week 6 of precise 2 trial with an absolute cdai score 350 points) and decided to withdraw before week 26 of precise 2 trial were offered an open- label extension study (precise 4).22 patients (n=124) enrolled in the precise 4 study were separated into either the continuous (n=49) or drug interruption (n=75) arm based on whether they received czp or placebo maintenance following czp induction during the precise 2 trial.22 patients who relapsed on czp maintenance therapy received a single dose of czp 400 mg s.c. whereas those who relapsed after czp interruption received three reinduction doses of czp 400 mg s.c. 2 weeks apart with subsequent maintenance administration every 4 weeks up to week 52.22 czp was equally efficacious in continuous and drug interruption treatment arms with 63.3% and 65.3% response (reduction in harvey-bradshaw index score 3 from baseline) rates at week 4, respectively and 54.8% and 59.2% further maintaining this response at week 52, respectively.22 the remission rates (hbi score 4) at week 4 were 28.6% and 44% among those treated with czp continuously and with prior intermission, respectively with sustained respective remission rates at week 52 of 64.3% and 54.5%.22 patients with cd experiencing disease relapse on czp maintenance therapy following initial response to induction with czp may benefit from administration of an additional dose of czp. similarly, those with recurrence of cd after czp discontinuation may achieve improvement in their symptoms after reinduction of czp.22 a phase iiib welcome trial (26-week open-label- induction, double-blind-maintenance, placebo-controlled trial evaluating the clinical benefit and tolerability of czp induction and maintenance in patients suffering from cd with prior loss of response or intolerance to infliximab) assessed the efficacy of czp in treatment of patients with active cd and prior loss of response or hypersensitivity to infliximab. 23 an open-label induction with czp administered s.c. at the dose 400 mg at weeks 0, 2 and 4 in 539 patients resulted in 62% response (decrease in cdai 100 points from baseline) and 39.3% r emission (cdai 150 points) rates at week 6.23 there were 329 patients who responded to czp induction at week 6 who entered a double-blind maintenance part of the welcome trial that compared the efficacy of czp 400 mg maintenance treatment administered either every 2 weeks (n=161) or every 4 weeks (n=168) from week 6 through week 26.23 both maintenance regimens displayed comparable rates of sustained response (36.6% vs. 39.9%, respectively p=0.55) and remission (30.4% vs. 29.2%, respectively, p=0.81) at the end of the trial (week 26).23 a recent randomized double blind placebo controlled 6-week trial (table 1) evaluated the efficacy of czp in 439 adult patients with active cd and no prior exposure to anti-tnf therapy.24 patients were randomly assigned to either a single s.c. dose of czp 400 mg or placebo given at 0,2 and 4 weeks with subsequent assessment of clinical remission (cdai 150 points) at week 6.24 overall, there was no statistical difference in remission rates at week 6 between czp and placebo groups (32% vs. 25%, p=0.174).24 however, among patients with baseline crp 5 mg/l treatment with czp resulted in a statistically significant difference in remission rates at week 6 when compared to placebo (p=0.031).24 certain demographic and baseline disease characteristics such as age 40 years, male sex, crp 10 mg/l, disease located in colon or in ileum and colon, no prior surgical resection, disease duration less than baseline mean, increased clinical disease activity (cdai 300 points) were associated with a statistically significant 23 fold higher rates of clinical remission with czp versus placebo.24 recent data from a swiss, prospective, questionnaire-based phase iv study of 60 clinical practice based-patients who received induction and maintenance treatment with czp 400 mg demonstrated 70% and 67% response (decrease of hbi score 3 points vs. baseline) rates and 40% and 36% remission (hbi 4 points) rates at week 6 and week 26, respectively.25 in addition, 36% and 55% of patients had complete fistula closure at week 6 and 26, respectively.25 among treated patients 88% and 67% continued czp beyond week 6 and week 26, respectively.25 adherence has been defined as the degree to which the patient follows medication intake and other doctor s recommendations.26,27 the term adherence is currently preferred over the term compliance since it underlines the equal role of both patient and the doctor in their relationship whereas the term compliance underlines only the greater power of the doctor.26,27 according to the world health organization patient s adherence to treatment determines the success of given therapy.28 on the other hand, the adherence to treatment in patients with chronic disorders in developed countries has been estimated to be at 50%.28 levy and feld grouped patients reasons of patients non-adherence to gastroenterology medical management into either lack of adequate skills or knowledge to comply with prescribed treatment (inadequate or poor information about prescribed medications), lack of patients belief that prescribed treatment is helping them or the lack of support from the patients environment (financial or employment situation, situation at household not allowing to comply with treatment, difficulties with transportation) (table 2).27 in order to increase patients adherence physicians should understand that their recommendations for patients have to include clear explanations of rationale for treatment, why adherence to prescribed regimen is crucial to therapeutic success, review of therapeutic assignments given during past visits (homework) including discussion of any difficulties patients may have encountered, attempt to address them and praise success (table 3).27 levy and feld suggested 10 recommendations addressing the reasons for nonadherence (table 4). they underline the crucial role of the proper physician-patient contact in establishing the pattern of patient s adherence. although levy and feld published their recommendations in 1999 when anti-tnf agents were emerging for treatment of cd their recommendations may be also applied to medications administered intravenously or subcutaneously in order to increase patient s adherence. for example, quality of the physician/patient relationship and patient s trust in physician s recommendations certainly would lead to increased adherence to any medication that is warranted. the major goal of treatment of cd is to induce and maintain disease in remission. it has been demonstrated that remission in patients with cd is associated with reduced hospitalizations and surgeries, increased employment and improved quality of life.29 this is why it is important to determine factors associated with adherence and factors associated with non-adherence to anti-tnf agents in order to improve adherence if patient does not adhere or to maintain adherence if patient adheres to prescribed regimen. until now and at the time of writing this manuscript, there have been no published studies on the adherence to czp in patients with cd. however, there are some data available on adherence to other anti-tnf agents in cd, namely infliximab and adalimumab. there have been only two studies that evaluated the adherence of infliximab30,31 and one study that assessed adherence to adalimumab32 in patients with cd. two studies assessed factors predictive of non-adherence for infliximab30 and adalimumab using multivariable models32 (table 5). in their first study kane et al collected data from outpatient databases that included 274 patients with cd who were scheduled to receive 1185 infusions with infliximab within 17 month period.32 the authors defined non-adherence as patient no-show without prior rescheduling of appointment by the patient.30 the observed non-adherence rate was 4% (48/1185) and a female sex and time from the initial infliximab infusion greater than 18 weeks were found to increase risk of non-adherence 2-fold.30 in an attempt to assess the impact of the adherence to infliximab on health care costs in patients with cd an analysis of the integrated health care information service national managed care benchmark database including medical histories of over than 25 million patients enrolled in managed care within the us was performed and identified 571 patients with cd who were receiving infliximab maintenance treatment over the 4 year time period (at least four consecutive infusions) within the first year after the initial infusion.31 non-adherence was defined as less than 7 infusions during the first year of treatment.31 its rate was found to be 34.3% and it was associated with nearly 3-fold increase in an all-cause hospitalizations (or=2.7; p<0.001) and 2.5-fold increase in cd-related hospitalizations (or=2.5, p<0.001).31 in addition, non-adherence to infliximab was also associated with increased by 73% adjusted total medical costs excluding infliximab cost (p<0.001), by 115% adjusted all-cause hospitalization cost (p<0.001) and by 29% adjusted all-cause outpatient cost excluding infliximab cost (p<0.001) when compared to adherence to infliximab.31 similarly, non-adherence to infliximab was associated with increased by 90% adjusted cd-related medical cost excluding infliximab cost, by 115% adjusted cd-related hospitalization cost and by 43% adjusted outpatient cost excluding infliximab cost when compared to adherence to infliximab (p<0.001).31 a group of french researchers performed a 21 month prospective observational multicenter study of adherence to adalimumab in patients with cd.32 non-adherence was defined as either delay or miss of at least one injection of adalimumab within 3 months prior to the study.32 among 108 patients with cd 49 (45.4%) of them were non-adherent to adalimumab injection with 16 patients (14.8%) missing at least one of injection and 33 patients (30.6%) delaying at least one injection.32 the reasons for non-adherence were forgetfulness (24.6%), infection (24.6%), travel (20%), intentional non-adherence (10.8%), pharmaceuticals supply problems (9.2%), side effects (7.7%), pregnancy (1.5%) and hospitalization due to cd (1.5%).32 overall, duration of disease greater than 93 months and adalimumab injection 80 mg every other week were negatively associated with injection delay or miss.32 the injection regimen of adalimumab at the dose of 40 mg every other week was associated with nearly 4-fold increase in injection delays whereas the presence of at least one relapse within last 12 months was a negative predictor of a delayed injection.32 duration of cd greater than 90 months was negatively associated with missed injection.32 current evidence strongly suggests that czp is an effective therapy for patients presenting with moderate to severe cd in anti-tnf nave patients as well as in patients with secondary loss of response or intolerance to infliximab. czp has expanded the spectrum of anti-tnf agents available for the treatment of patients with cd. patients with increased serum markers of inflammation (crp) belong to the subset of patients in whom czp is of particular benefit. studies have demonstrated czp to be an effective for induction and maintenance therapy in patients with cd and improving health-related quality of life for these patients.33,34 future studies on large number of patients are warranted to evaluate the efficacy of czp in the setting of clinical practice. there have been no published studies that assessed adherence to czp in patients with cd. there are limited data on adherence to other anti-tnf therapies, infliximab and adalimumab. treatment with czp is associated with improvement in quality of life and lessened work impairment in patients with cd. at this moment, health care professionals should be encouraged to follow ten adherence recommendations presented in table 4 to attempt to increase patient adherence to medications and regimens. patients should be encouraged to adhere to treatment with czp not only due to its efficacy in maintaining clinical remission in cd but also because of improvement in quality of life and reduction of work impairment. future studies should determine what factors are associated with non-adherence to czp and also other anti-tnf agents. this would allow us to make evidence-based steps necessary to increase patients compliance.

treatment with anti-tumor necrosis factor (anti-tnf) therapy has become a mainstay of therapy for patients with cd who are unresponsive to conventional medical management. currently there are three anti-tnf antibodies that have been approved by the us food and drug administration for the treatment of cd, namely infliximab, adalimumab and certolizumab pegol (czp). several double blind placebo controlled trials determined that czp is effective as induction and maintenance treatment in adult patients with cd regardless of their prior exposure to other anti-tnf antibodies. this review discusses the efficacy of czp and adherence to therapy with anti-tnf antibodies in patients with cd.

PMC3987757

pubmed-1208

use-dependent plasticity shapes neuronal networks within sensory systems during early life to optimally represent sensory stimuli. experience-dependent organization of eye-specific inputs is a major mechanism whereby refinement of synaptic connectivity is achieved in the developing visual system [24]. monocular deprivation during development leads to a loss of cortical connectivity of the deprived eye resulting in a shift of the ocular dominance in the visual cortex, which will become permanent if the md persists to adulthood [5, 6]. although neuronal plasticity of the developing brain gradually decreases with age, recent findings suggest that it can be reactivated in the adult visual cortex and other regions, such as the amygdala. a variety of experimental manipulations, including enzymatic treatments [10, 11], environmental enrichment [1215], food restriction, genetic manipulations [17, 18], and other manipulations, promote this kind of plasticity [1921]. although the mechanisms behind the adult induced plasticity are still unclear, we are beginning to understand the key factors involved. for example, the developmental maturation of neuronal inhibition, mainly through the parvalbumin containing interneurons [22, 23], is known to be involved in both the opening and the closure of the critical period. several extracellular matrix components, such as psa-ncam or the perineuronal nets, have been shown to play a role in the maturation of the inhibitory circuitries and experimental manipulations removing these extracellular matrix components, can trigger an early closure or a reopening of the critical period, respectively. similarly, a variety of other molecules, such as transcription factors or proteins involved in chromatin structure remodeling, are also key factors in regulating the closure and reopening of the critical period. the main pharmacological approaches to experimentally regulate critical period plasticity in the adulthood are those affecting the action of ascending projection systems, such as the serotoninergic or cholinergic systems [21, 2729]. in this line, we have investigated the plastic effects of antidepressants, such as fluoxetine, that modulate serotoninergic transmission, and we have shown that these drugs, in a long-term treatment, are able to trigger critical period plasticity in the adult brain, through an early epigenetic modification that regulates gene expression. here, we have used fluoxetine in combination with an experience-dependent paradigm of visual deprivation, to analyze the large-scale gene expression patterns, to understand the temporal-dependent changes that allow the reopening of the critical periods in the adult brain. a total amount of 32 long-evans hooded rats were used in this study, equally distributed in 4 experimental groups (n=8 animals per group), as explained later (figure 1(a)). animals were group-housed under standard conditions with food and water ad libitum in plexiglas cages (40 30 20 cm) and kept in a 12: 12 light/dark cycle. adult rats at the postnatal day 70 (p70) were systemically treated with fluoxetine (fluoxetine-hydrochloride, 0.2 mg/ml drinking water) for 23 days. three weeks after the beginning of the fluoxetine treatment, rats were anaesthetized with avertin (1 ml/100 g) and mounted on a stereotaxic apparatus to perform the eyelid suture for monocular deprivation (md). eyelids were inspected daily until complete cicatrisation; subjects with even minimal spontaneous reopening were excluded. great care was taken during the first days after md to prevent inflammation or infection of the deprived eye through topical application of antibiotic and cortisone. two days after md, the binocular region of the primary visual cortex was dissected. for all microarray experiments, total rna was purified using rna extraction kit (macherey nagel), and amino allyl crna labeling kit (ambion) was used to label crna according to manufacturer's standard protocols. agilent whole rat genome microarray kits (4 44 k) were hybridized following provided protocols. images from hybridized microarrays were segmented and the median intensity of each spot was estimated by the software genepix v.5.0 (axon). data was imported into the software (http://cran.r-project.org/) and preprocessed by the bioconductor package limma. the statistical analysis used was a linear model followed by t-test for finding the differentially expressed genes. in order to increase the reliability of the statistical analysis in addition, we also increase the reliability of the analysis through validation of the results using multiple rt-pcrs. lists of significant genes were screened by the david 6.7 annotation tools in order to find overrepresented biological themes. purified rna was treated with dnase (fermentas) and cdna was synthesised from 1 g of rna (invitrogen). real-time pcr was carried out to determine relative enrichment in the samples using the sybr green method according to the manufacturer instructions (sybr green i master, light cycler 480, roche diagnostics). the comparative ct method was used to determine the normalized changes of the target gene relative to a calibrator reference; in particular, values were normalized to gapdh levels. previous studies have shown that 7 days period of monocular deprivation in fluoxetine-treated adult rats is sufficient to bring about a change in the ocular dominance. to reveal early transcriptional changes that precede and underlie the functional change, we analysed gene expression, using dna microarrays, at two days after md. microarray analysis revealed only relatively few genes that were significantly regulated by either flx (n=197, see supplementary table s1 in the supplementary material available online at http://dx.doi.org/10.1155/2013/605079) or md alone (n=239, table s2), treatments that themselves do not produce any changes in the ocular dominance plasticity. however, the combination of flx and md, the treatment that promotes changes in ocular dominance, altered the expression of a significantly larger number of genes (n=1603, table s3, figure 1(b)). notably 1237 out of 1603 (77%) of the genes in the group receiving both md and flx were downregulated, whereas in the groups receiving either md or flx, 111 out of 239 and 88 out of 197 genes were downregulated, respectively, comprising of roughly 50% of all the regulated genes. hence, the combination of the treatments apparently has two major effects on gene expression; first, it increases the number of regulated genes when compared to the single treatments, and second, it has a striking effect on downregulating most of the genes, indicating that silencing of genes normally expressed during basal conditions is likely involved in the triggering of plasticity of the adult brain. the representation of biological themes was screened using fisher's exact test on the lists of differentially expressed genes in each comparison. chronic fluoxetine treatment induced a regulation of genes related to chromatin remodelling, nervous system development, and plasticity, as well as regulation of gene expression and transcription in the binocular visual cortex (table s4). md altered the expression of a significant number of genes related to cell differentiation, cell plasticity, and neurogenesis. several genes of the ion homeostasis and regulation of transcription were also found overexpressed (table s5). the combination of md and fluoxetine treatment downregulated the majority of the differentially expressed genes, altering the expression of genes represented in a variety of functional processes, including genes related to neuronal development, plasticity, and apoptosis. in addition, genes involved in the synaptic transmission, ion and intracellular calcium homeostasis, and vesicular secretion were found differentially expressed. blood circulation and lipid metabolism were among the most significantly overrepresented families (table s6). to provide validation of the microarray data, we next examined single patterns of gene expression by means of real-time pcr, in the same experimental groups and using the same experimental paradigm (figure 1(a)). in particular, we focused our attention on genes whose expression may alter molecular and cellular processes involved in the closure of the critical period for visual cortex plasticity, such as the balance of inhibitory and excitatory transmission [22, 31, 32], transcription factors regulating gene expression, extracellular matrix remodeling, myelination, and chromatin structure remodeling [26, 29], as well as genes involved in processes of synaptic plasticity, neuronal differentiation, and outgrowth (see table 1). we observed that fluoxetine produced a significant increase in the expression of genes involved in inhibitory neurotransmission when comparing both animals with binocular vision and animals with monocular deprivation with their respective controls (bv-sal versus bv-flx and md-sal versus md-flx; figure 2(a)). specifically, in animals with binocular vision, we found an increased expression of the vesicular gaba transporter (vgat; 60% increased expression; p=0.001), while in rats with monocular deprivation together with fluoxetine treatment, we observed an increase in the expression of gabra4 (30% increased expression; p=0.02). we did not observe many changes in the composition of nmda receptor subunits in either of the experimental groups (see figure 2(b) and table 1). the only significant change we found was a decrease in the expression of the nr2a subunit (nmda-2a; 20% decreased expression; p=0.04) in the animals with monocular deprivation treated with fluoxetine. we detected increases in the gene expression of transcription factors in the animals with binocular vision treated with fluoxetine (figure 2(c)). in particular, nfkb1 and dlx1 increased their expression (50% and 30% increased expression, resp. however, in those animals with monocular deprivation, fluoxetine treatment produced a decrease in the expression of transcription factors, such as egr-2 (p=0.04). the expression of reelin, transcript that encodes a glycoprotein that mediates synaptic plasticity at hippocampal level, was significantly increased in md animals treated with fluoxetine (figure 2(d); 40% increased expression; p=0.001). the expression of additional transcripts that encode proteins involved in neuronal differentiation and outgrowth processes as well as synaptic plasticity increased in both groups. in animals with binocular vision, fluoxetine promoted an increase in the expression of clcn3 (20% increased), kcnv1 (20% increased), and kcnq3 (30% increased), which encode ion channels that mediate chloride and potassium conductance (p<0.05), and in animals with monocular deprivation fluoxetine produced also an increase in the expression of clcn3 (50% increased expression; p=0.01). the expression of mmp2 and mmp9 was markedly changed between animals treated with fluoxetine and with binocular vision and those with monocular deprivation (figure 2(e)). mmp2 and mmp9 encode for proteolytic enzymes that degrade extracellular matrix components [3537] and play a key role in mediating synaptic plasticity at the level of the hippocampus [38, 39]. in particular, mmp2 gene expression was decreased in animals treated with fluoxetine alone (50% decrease; p=0.02), while animals with combined monocular deprivation and chronic fluoxetine treatment had an increased expression of both mmp2 (60% increased; p=0.01) and mmp9 (50% increased; p=0.01). changes in the expression of transcripts that encode an enzyme that regulate chromatin susceptibility to transcription were detected in animals with binocular vision after chronic fluoxetine treatment. in particular, we found that hdac3 expression was enhanced (figure 2(f); 30% increased p=0.02). on the other hand, the expression of mbp, which encodes a basic protein of myelin, a repressive factor for visual cortex plasticity, was significantly reduced by fluoxetine treatment in animals with both binocular vision (40% decreased; p=0.01) and monocular deprivation (40% decreased; p=0.001). this study provides a large-scale analysis of changes in patterns of gene expression associated with the reopening of the critical period of plasticity in the adult visual system induced by the combination of fluoxetine treatment and monocular deprivation. our findings suggest a scenario where an enhanced serotoninergic transmission induced by long-term fluoxetine treatment induces a shift of the inhibitory-excitatory balance [8, 29], which in turn promotes an alteration in the expression of genes involved in different biological themes that may underlie the functional modifications in the adult visual cortex related with the reopening of the critical period plasticity. our results reveal that the process of plasticity reactivation in adulthood involves both (i) a transient activation of neural mechanisms normally present during early stages of brain development and (ii) a removal of molecular factors that inhibit plasticity in adulthood. gene expression patterns involved in processes of synaptic plasticity, neuronal differentiation, and outgrowth were, indeed, differentially regulated by chronic fluoxetine treatment. reelin is an extracellular glycoprotein involved in the migration and correct development of the cerebral cortex [41, 42]. reelin is highly expressed by cajal-retzius neurons during development, but its expression is limited to a subpopulation of interneurons during the adulthood [43, 44]. although the function of reelin in adult neurons remains unclear, its overexpression has been shown to enhance plasticity and learning, affecting presynaptic transmission [34, 45]. our results demonstrate an upregulation of reelin after chronic fluoxetine treatment, suggesting that the overexpression of molecules involved in the juvenile plasticity plays an important role in the reopening of the critical periods during the adulthood. the proteolytic enzyme mmp2, on the other hand, may drive mechanisms of synaptic plasticity by degrading extracellular matrix components that are inhibitory for plasticity, as observed in the adult hippocampus. increase of mmp2 expression, indeed, was paralleled by a decrease of mbp: a basic component of myelin, which is a repressive factor for visual cortex plasticity. our analysis of gene expression points towards a downregulation of mbp following long-term antidepressant treatment, supporting the hypothesis that the removal of factors that are inhibitory for plasticity may provide a permissive environment for structural and functional changes of neuronal circuitries in the adult nervous system. chronic fluoxetine administration has been shown to promote structural changes in both excitatory [46, 47] and inhibitory circuits [4850]. although there is evidence that long-term fluoxetine administration promotes a reduction of gaba-mediated inhibition in adult visual cortical circuitries [8, 29], a compensatory mechanism might explain the increase in the expression of vgat or gabra4 that we observe in our experiment. these results are also in agreement with previous studies, in which fluoxetine treatment in combination with monocular deprivation produces an increase in the elongation of the tips of interneuronal dendrites, supporting the idea that inhibitory neurotransmission plays a key role in the reopening of the critical periods [20, 22, 23]. similarly, the change of nmda receptor subunit composition, evidenced by the decrease in nmda-2a gene expression following antidepressant treatment, is particularly interesting in this respect. the expression of the nr2a subunit has been correlated with a progressive decrease of nmda receptor currents during development [51, 52]. this raises the possibility that a decrement of the nr2a/b ratio may increase nmda receptors sensitivity thus causing the strengthening of synapses required for the potentiation of the nondeprived input. another highly significant notion that emerges from our data is that the changes promoted by the combination of fluoxetine with monocular deprivation, regarding the expression of transcription factors and proteins of the extracellular matrix, are opposed to those promoted by fluoxetine alone. this indicates that these molecules might be underlying the structural plasticity changes driven by monocular deprivation to produce the shift in the ocular dominance and its consolidation in the visual system. our findings support the hypothesis that the therapeutic effect of antidepressant drugs is dependent on changes in neuronal plasticity [55, 56]. importantly, these results open up new insights into the understanding of the mechanisms underlying the reopening of the critical period in the adult brain, by providing the basis of gene expression patterns for a visual deprivation paradigm that demonstrates the ability of the nervous system to translate environmental stimuli into structural and functional changes of neural circuitries.

the nervous system is highly sensitive to experience during early postnatal life, but this phase of heightened plasticity decreases with age. recent studies have demonstrated that developmental-like plasticity can be reactivated in the visual cortex of adult animals through environmental or pharmacological manipulations. these findings provide a unique opportunity to study the cellular and molecular mechanisms of adult plasticity. here we used the monocular deprivation paradigm to investigate large-scale gene expression patterns underlying the reinstatement of plasticity produced by fluoxetine in the adult rat visual cortex. we found changes, confirmed with rt-pcrs, in gene expression in different biological themes, such as chromatin structure remodelling, transcription factors, molecules involved in synaptic plasticity, extracellular matrix, and excitatory and inhibitory neurotransmission. our findings reveal a key role for several molecules such as the metalloproteases mmp2 and mmp9 or the glycoprotein reelin and open up new insights into the mechanisms underlying the reopening of the critical periods in the adult brain.

PMC3710606

pubmed-1209

procalcitonin (pct) is a 14-kd protein encoded by the calc-1 gene and synthesized physiologically by thyroid c-cells. under normal conditions, serum pct levels are negligible. after shock or tissue injury (i.e. burn, trauma, surgery) or infections and sepsis, pct mrna expression has been documented in human extra-thyroidal tissues. thus, systemic pct concentrations are considered as a component of the inflammatory response and as an acute-phase marker. in cardiac acute patients, data on pct some studies [3, 4] reported that pct levels were increased in acs patients on admission, whereas other investigations [5, 6] documented that plasma pct concentrations were in the normal range in patients with uncomplicated acute myocardial infarction. elevated concentrations of pct have been reported in patients with cardiogenic shock. in a more recent retrospective study, it was observed that cs patients showed high pct concentrations, especially in the presence of multiorgan failure (mof) and in absence of signs of infections (cultures and clinical findings). we recently observed that the degree of myocardial ischemia (clinically indicated by the whole spectrum of acs, from unstable angina to cardiogenic shock st-elevation following myocardial infarction) and the related inflammatory-induced response are better reflected by crp (which was positive in most acute cardiac care patients of all our subgroups) than by pct which seems more sensible to a higher extent of inflammatory activation, being positive only in all cs patients. in these patients, the clinical interpretation of absolute pct values (both in diagnostic and prognostic terms), represent a major challenge since they may be influenced by several factors, such as the degree of systemic inflammatory response, the coexistence of multiorgan dysfunction, the presence/absence of infections and finally by the time of measurements during hospital course (i.e. the dynamics of pct levels). no data are so far available on the dynamics of pct levels in patients with cardiogenic shock. the aim of this preliminary investigation was therefore to evaluate the serum evolution of pct during intensive cardiac care unit (iccu) staying in a group of patients with cardiogenic shock (cs) following st-elevation myocardial infarction (stemi) submitted to primary percutaneous intervention (pci) with no laboratory or clinical sign of infection. ten consecutive patients with cardiogenic shock following stemi were submitted to pci and then admitted to our 12-bed iccu in florence, a tertiary center, from 1 september 2008 to 31th march 2009. to be eligible for the present study, all patients had to be free of infection at the time of blood sampling, as evidenced by both clinical and microbiological examinations, including urinary cultures and microbiological examinations of tracheal aspirate in mechanical ventilated patients and blood cultures. a clinical diagnosis of cardiogenic shock was made if all the following criteria were present: 1. systolic blood pressure persistently less than 90 mmhg or vasopressors required to maintain a systolic blood pressure of more than 90 mmhg; 2. signs of hypoperfusion (e.g. urine output less than 30 ml/hour or cold/diaphoretic extremities or altered mental status); 3. clinical evidence of elevated left ventricular filling pressure (e.g. pulmonary congestion on physical examination or chest x-ray). pulmonary artery catheterization was not required when all clinical criteria and echocardiographic evidence of left ventricular dysfunction without mechanical complications were present. the day after iccu admission blood samples were obtained for cardiac biomarkers (tni<0.15 ng/ml), leucocytes count (4000-10000/l), crp (< 9 mg/dl), uric acid (< 6.5 mg/dl), nt-pro brain natriuretic peptide (nt-probnp, in males 0-50 yrs:<88 pg/ml,>50 yrs:<227 pg/ml; in females: 0 -50 yrs:<153 pg/ml;>50 yrs:<334 pg/ml) and procalcitonin measurements (normal values<0.5 ng/ml). transthoracic 2-dimensional echocardiography was performed on admission in order to evaluate left ventricular ejection fraction (lvef). apache ii (acute physiology and chronic health evaluation ii) score was also assessed. the study design was approved by the local ethic committee and inform written consent was obtained for each patient. statistical analysis was performed by means of spss 13.0 package (spss inc, chicago, il). data are reported as frequencies and percentages or means s.d. and analyzed with fisher s exact test and student s t test, respectively. since pct was determined once a day, its mean value for each day of hospital stay in either group of patients has been calculated and graphically plotted in order to obtain two linear regression lines, whose slopes have been subsequently compared by means of an f test. in all analyses, table 1 shows the clinical characteristics of cs patients following stemi included in the study after pci. clinical characteristics of patients included in the study. compared with survivors, dead patients exhibited higher values of apache ii score, as well as trend towards higher serum concentrations of troponin i, though it did not reach statistical significance. no significant differences were detectable in regard to age, sex, infarct location, left ventricular ejection fraction, and mean arterial pressure between the two subgroups. as depicted in table 2, higher values of glycemia, nt-probnp and crp were detectable in dead patients in respect to survivors. basal serum concentrations of procalcitonin were higher in (dead) survivors patients, though this difference did not reach statistical significance due to high values of standard deviation. as depicted in figure 1, the pattern of pct variations during iccu course was significantly different in cs patients who survived in respect to those who died (survivors: slope=-3.760.71 standard error; dead: slope=-0.810.35 standard error, p=0.004). the main finding of the present investigation, though preliminary and performed in a small subset of patients, is that the patterns of temporal pct variations throughout iccu course were heterogeneous in patients with cs and no clinical or laboratory signs of infection. a progressive reduction in pct values was observed in cs patients who survived, whereas the lack of changes in pct concentrations was documented in cs patients who died. conversely, basal absolute pct values were not significantly different between the two subgroups, since they exhibit a wide range of values in the overall population. sponholz et al. described the evolution of serum procalcitonin levels after uncomplicated cardiac surgery and observed a progressive return to normal levels within the first week. peak pct levels were reached within 24 hours postoperatively and this increase seemed to be dependent on the surgical procedure, being more invasive procedures associated with higher pct levels, and on intraoperative events, including aortic cross-clamping time, duration of cardiopulmonary bypass and duration of surgery. in infected patients, pct levels were elevated throughout the first week postoperatively [19, 20, 21], with a more pronounced trend in bacterial and fungal infections than in viral infections of sirs. [22, 23]. the authors concluded that the dynamics of pct levels, rather than absolute values, may be more important for identifying patients with infectious complications after cardiac surgery. more recently prat et al. confirmed a slight increase in pct values in the first postoperative day after cardiac surgery, in agreement with previous results [25, 26, 27] and with adamik et al., who showed that the development of postoperative complications after cardiac surgery with cardiopulmonary bypass was associated with increased postoperative neopterin and pct levels. similarly, after heart transplantation, serum pct levels display a rise in response to surgery, with a peak on day two, whereas high peak levels with delayed return to normal values should lead to a search for inflammatory processes, as they are often associated with increased morbidity and mortality. likewise, in patients with cardiogenic shock and no sign of infections we documented a reduction of pct levels only in survivors cs patients. this time course of procalcitonin can probably be explained, both in postsurgical and in cs patients, by normal pct kinetic. in healthy subjects the injection of endotoxin is followed by a rise in pct, reaching a maximum 24 hours thereafter. the return of pct levels to normality within a few days in surgical patients (after an uncomplicated postoperative course) and in cs survivors patients can be explained by half-life of pct (18 to 24 hours), in absence of a further insult that might induce pct production. our findings, along with those in cardiac surgery strongly support the contention that the dynamic approach may be more reliable that the static one (that is the absolute single pct value) especially in the challenging conditions characterized by a systemic inflammatory response, such as cardiac surgery and cardiogenic shock. indeed, in a cohort of unselected critically ill patients, jensen et al. observed that a pct increase was an independent predictor of 90-day survival and that the pct day-by-day changes was able to identify critically ill patients at a higher risk of icu mortality. on the other hand, the initial ct level did not predict mortality, even though many patients were admitted with a pct 1.0 ng/ml. this suggests that several pct measurements should be made consecutively to assess the critically ill patient s infection-related mortality risk (to monitor treatment of infection day-by-day). in our investigation we further confirmed that higher values of nt-probnp are associated with increased mortality in cs patients and that a more marked systemic inflammation (as inferred by higher values of crp) and higher severity score (as indicated by apache ii) were associated with an ominous prognosis. the main limitation of the present investigation is represented by the small number of patients. however, the population is homogeneous, comprising patients with cardiogenic shock following stemi all submitted to pci, with no clinical and laboratory sign of infections. it is interesting to note that, despite the small number of subjects, the behavior of pct was clearly detectable. according to our preliminary findings, patterns of temporal pct variations throughout iccu course were heterogeneous in patients with cs following stemi submitted to pci and no clinical or laboratory signs of infection. a progressive reduction in pct values was observed in cs patients who survived, whereas a lack of changes in pct concentrations was documented in cs patients who died. our findings strongly support the contention that the dynamic approach may be more reliable that the static one (that is the absolute single pct value) especially in the challenging conditions characterized by a systemic inflammatory response, such as cardiogenic shock. we further confirmed that higher values of nt-probnp are associated with increased mortality in cs patients and that a more marked systemic inflammation (as inferred by higher values of crp) and higher severity score (as indicated by apache ii) were associated with an ominous prognosis.

introductionprocalcitonin concentrations are considered as a component of the inflammatory response and as an acute-phase marker, after shock or tissue injury (i.e. burn, trauma, surgery) or infections and sepsis. no data are so far available on the dynamics of procalcitonin levels in patients with cardiogenic shock following st-elevation myocardial infarction, with no clinical or laboratory sign of infection. methodswe evaluated procalcitonin values every day during intensive cardiac care staying in ten cardiogenic shock patients admitted to our intensive cardiac care unit. nt-pro brain natriuretic peptide, c reactive protein and apache ii score were also assessed. resultssix patients survived, whereas 4 patients died. a progressive reduction in procalcitonin values was observed in cardiogenic shock patients who survived, whereas the lack of changes in procalcitonin concentrations was documented in cardiogenic shock patients who died (survivors: slope=-3.76; dead: slope=-0.81, p=0.004). furthermore, higher values of glycemia, nt-pro brain natriuretic peptide and c reactive protein (as well as higher apache ii scores) were detectable in dead patients in respect to survivors. conclusionsin our preliminary study we observed that in patients with cardiogenic shock and no sign of infections a reduction of procalcitonin levels was detectable only in survivors. moreover, higher values of nt- brain natriuretic peptide, a marked systemic inflammation (higher values of c reactive protein) and higher severity score (as depicted by apache ii) are associated with an ominous prognosis in cardiogenic shock patients.

PMC3484587

pubmed-1210

cell growth is tightly linked with the cell's perception of its nutritional environment. in particular, microorganisms, such as the yeast saccharomyces cerevisiae, that spend most of their time in the stationary phase in the wild, therefore, yeast is a good model organism for the study of nutrient detection and response. autophagy is one of several responses to nutrient starvation (klionsky and ohsumi 1999). a large number of cytoplasmic components are nonselectively enclosed within a double-membrane structure called an autophagosome, in which they are transported into the vacuole/lysosome to be degraded by resident hydrolases. such turnover of a large amount of cytoplasm mediated by autophagy is essential for survival under nutrient-depleted conditions. we have isolated several genes essential for autophagy (termed apg) and have been investigating the function of the gene products (tsukada and ohsumi 1993; funakoshi et al. 1997; matsuura et al. 1998). genetic and morphological analyses revealed that the degradative process of autophagy shares mechanistic components with the cytoplasm-to-vacuole targeting (cvt) pathway (harding et al.; baba et al. 1997), which is biosynthetic, delivering a resident hydrolase, aminopeptidase i (api), to the vacuole (klionsky and ohsumi 1999). on the other hand, autophagy and the cvt pathway are distinct in many aspects. the two pathways appear to be regulated separately; the cvt pathway is mainly observed under growing conditions, whereas autophagy is induced by starvation (baba et al. furthermore, cvt vesicles and autophagosomes, the vesicles formed in the cvt pathway and autophagy, respectively, are clearly different in size (baba et al. 1997). recently, the t-snare tlg2 and sec1-homologue vps45 were found to be required for the cvt pathway, but dispensable for autophagy, suggesting that these two pathways are mechanistically distinct (abeliovich et al. 1999). tor is a phosphatidylinositol kinase-related kinase that promotes cell cycle progression in response to nutrient availability (thomas and hall 1997). treatment with the immunosuppressant rapamycin, a specific inhibitor of tor, induced cell cycle arrest at g0. for example, accumulation of glycogen, repression of genes that are stimulated in growing cells, and stimulation of genes that are induced during starvation, all result from inhibition of tor (hardwick et al. our laboratory has found that rapamycin induces autophagy in yeast (noda and ohsumi 1998). however, the molecular mechanism by which tor negatively regulates autophagy remains to be determined. here, we present evidence that apg1, a protein kinase essential for autophagy, plays a pivotal role in induction of tor-regulated autophagy. an nh2-terminally truncated (8 amino acid) form of apg1 open reading frame, subcloned into pgbd-c2 vector, was used as bait to screen a yeast genomic library, and interacting proteins were identified by dna sequencing. a dna fragment including the entire apg17 gene was cloned from yeast genomic dna using pcr. the kinase-negative apg1 mutation was obtained using the quikchange site-directed mutagenesis kit (stratagene). antibody against apg13 protein was raised against a glutathione s transferase (gst)-apg13 fusion protein. specifically, a 1.9-kb bglii-xbai fragment of the apg13 gene coding for 396 amino acids of apg13p was subcloned into the vector pgex-2 t. the expressed fusion protein was purified with glutathione-sepharose 4b (amersham pharmacia biotech). expression and purification of the fusion protein was carried out according to the manufacturer's instructions. immunoprecipitation, kinase assay, and immunodetection of nh2-terminally hemagglutinin (ha)-tagged apg1 (apg1) were performed as described previously (kamada et al. apg1 was immunoprecipitated with anti-ha mab (16b12; babco), and an in vitro protein kinase assay was performed in the presence of [p]atp and myelin basic protein (mbp, substrate). yeast cells exponentially grown in yepd medium were treated with zymolyase 100 t (seikagaku kogyo) to generate spheroplasts. the resultant spheroplasts were treated with or without 0.2 g/ml of rapamycin and broken by resuspending in lysis buffer (pbs, ph 7.4, 1 mm edta, 1 mm egta, 2 mm na3vo4, 50 mm kf, 15 mm na2h2p2o7, 15 mm p-nitrophenylphosphate, 20 g/ml leupeptin, 20 g/ml benzamidine, 10 g/ml pepstatin a, 40 g/ml aprotinin, 1 mm pmsf, and 0.5% tween-20). cell lysate was cleared by 10-min centrifugation at 6,500 g and 30-min incubation with protein g apg1 in the cleared cell lysate was bound to anti-ha mab, and apg13 was detected with anti-apg13 antibody. the resultant immunoprecipitates were also analyzed by protein kinase assay and immunoblot with anti-ha. for in vivo labeling of apg13, cells (tfd13-w3) expressing apg13 were in vivo-labeled with 50 ci of s (trans, icn) for 10 min, or 50 ci of pi overnight in sd medium, and transferred to yepd or nitrogen-depleted medium sd(n) for 1 h. apg13 protein was immunoprecipitated following tca precipitation. immunoprecipitated apg13 was treated with 5 u of alkaline phosphatase for 1 h at 30c. progression of autophagy was estimated by the increase of alkaline phosphatase activity in the cells expressing a cytosolic proform of the phosphatase protein (pho860p; noda et al. in an effort to study the mechanism of autophagy induction, we focused on the apg1 gene, which encodes a protein kinase whose activity is essential for autophagy (matsuura et al. nh2-terminally ha-tagged apg1 (apg1) was immunoprecipitated with anti-ha ascite and the resultant immunocomplex was analyzed using an in vitro kinase assay. apg1 kinase activity was found to be highly elevated in cells grown under starvation conditions (fig. 1 a). after a 6-h incubation in nitrogen-depleted medium, sd(n), the amount of activated apg1 had apparently increased, and was accompanied by slower gel migration, presumably because of autophosphorylation (fig. the increase in apg1 kinase activity is not due to this apparent increase in the protein amount, because shorter treatments with rapamycin (for example, see fig. apg1 activity was also increased by rapamycin treatment, but the effect of rapamycin was abolished in a rapamycin resistant tor1 mutant (tor1-1; kunz et al. these results suggest that apg1 activation is required for the induction of autophagy and that it is mediated by tor proteins. a kinase-negative apg1 mutant (k54a; this indicates not only that the enhanced apg1 kinase activity is required for autophagy, but that basal apg1 activity in growing cells (fig. next, we performed a two-hybrid screening with apg1 as bait to identify apg1-associating proteins, which may regulate apg1 activity. the following three genes were obtained from the screen: apg13 (funakoshi et al. 1997) and two novel genes, which were subsequently found to be essential for either autophagy or the cvt pathway, or both. one gene, designated as apg17 (ylr423c), was essential for only autophagy and was not required for the cvt pathway (fig. 2 a). the other, cvt9 (harding et al. 1996; d.j. klionsky, personal communication), was required for the cvt pathway, but not for autophagy. among the 16 apg genes discovered so far, it is interesting to note that apg1 binds to proteins whose function is specific to either autophagy (apg17) or the cvt pathway (cvt9). overexpression of apg1 in an apg13 mutant partially rescues the autophagy defect (funakoshi et al. similarly, the apg17 mutant was also rescued by overexpression of apg1 (data not shown), indicating that these three genes interact functionally. in the apg13 mutant, attenuated apg1 activity was observed. in rapamycin-treated apg17 cells, apg1 activity also was found to be largely impaired (20% of the wild-type). on the other hand, deletion of cvt9, which is not needed for autophagy, resulted in rapamycin-induced activation of apg1 to 50% of wild-type. the effects of deleting apg13 and apg17 on apg1 activity are not the result of a general autophagy defect, because deletion of other apg genes, such as apg5 (mizushima et al. 1998), does not affect the activation of apg1 (data not shown). these results indicate that the activated state of apg1 is required for autophagy induction, and that apg13 and apg17 play a key role in the activation of apg1 in response to tor inhibition. the next question we addressed was how these apg1-associating proteins transmit the starvation signal from tor to apg1. overexpression of apg13 resulted in a smeared apg13 band on the immunoblot caused by retarded migration, indicating that it was modified in some way (fig. this modified form was observed only in growing cells, and after starvation or rapamycin treatment, the slower-migrating form disappeared. in particular, it was noted that the disappearance of the slower-migrating form occurred within five minutes after rapamycin treatment. in vivo labeling and in vitro phosphatase treatment revealed that the apg13 bandshift was due to phosphorylation (fig. these results suggest that apg13 is phosphorylated in a tor-dependent manner, which was confirmed by the observation that dephosphorylation of apg13 in response to rapamycin was not seen in tor1-1 cells (fig. the dephosphorylated, faster-migrating form of apg13 in starved cells was rapidly phosphorylated after readdition of yepd medium (fig. 3 d), suggesting that the phosphorylation state of apg13 is extremely sensitive to nutrient conditions. this excludes the possibility that the phosphorylated form of apg13 is degraded upon starvation and apg13 is de novo synthesized after medium addition. the faster-migrating form of apg13 (as well as the slower-migrating form) was found to be labeled with pi (fig. 3 b, lane 8), suggesting that apg13 remains partially phosphorylated under starvation conditions. to confirm that there is a physical association between apg1 and apg13 a vacuolar protease-deficient strain was used for these experiments because apg13 is quite labile in cell lysate. when apg1 and apg13 were expressed from a high-copy plasmid, only the faster-migrating form of apg13 3 e), indicating that the hyperphosphorylated form of apg13 has little affinity for apg1. next, we performed the experiment using a low-copy plasmid to more closely approximate physiological cellular conditions. the amount of apg13 bound to apg1 increased rapidly (as quickly as 10 min) after rapamycin treatment (fig. 3 f), which corresponds well to the time course of apg13 dephosphorylation. these results strongly indicate that tor negatively regulates apg1 kinase activity by means of (hyper)phosphorylation of apg13, which reduces the affinity of apg13 for apg1. from these results, we hypothesized that apg13 binding to apg1 is required for the induction of autophagy, but not for the cvt pathway. the two-hybrid assay was used to determine the apg1-binding site on apg13, using various apg13 open reading frame fragments as prey. a central 89-amino acid region (432520) we also isolated a cooh-terminal truncated form of apg13, apg13(1448), whose ser449 was mutated to a stop codon using in vitro mutagenesis (kaiser et al. this mutant has a mutation within the putative apg1-binding site, and is unable to associate with apg1, as confirmed by the two-hybrid assay and coimmunoprecipitation (fig. 4 a, and data not shown). this result indicates a role for a domain of apg13 around amino acid 448 in binding apg1. we tested several cooh-terminal truncated apg13 mutants including apg13(1448) for autophagy activity. 4 b, bottom), presumably due to the absence of the apg1-binding domain. another cooh-terminal truncated form, apg13 (1568), which contains the entire putative apg1-binding domain, displayed partial, but significant, autophagic activity, when compared with apg13(1448). apg13(1568) was less competent for autophagic import than full-length apg13, suggesting that additional sequences downstream of the apg1 binding site are important for maximal activity. on the other hand, apg13(1448) partially, but significantly, rescued vacuolar-targeting of precursor api (fig. 4 b, top), suggesting that this truncated protein is still functional for the cvt pathway. apg1 activity in the apg13 mutant was partially restored in the presence of either apg13(1448) or apg13(1568), and was fully recovered in the presence of the whole apg13 construct (fig. 4 c). the kinase activity of the apg13(1568) transformant was clearly higher than that of the apg13(1448) transformant. these results confirmed the hypothesis that the apg1apg13 association and subsequent activation of apg1 are required for autophagy induction in response to starvation. it is currently thought that tor signaling in yeast is bifurcated (thomas and hall 1997). one pathway involves the small gtpase rho1, which is responsible for actin organization and is not affected by rapamycin. the other involves tap42, a rapamycin-sensitive phosphatase-associating protein that is necessary for the initiation of protein translation and amino acid permease turnover (di como and arndt 1996). tap42 is known to be located directly downstream of tor, and is required for tor-mediated signaling, especially the rapamycin-sensitive branch (jiang and broach 1999). to investigate this issue further, we tested the ability of a tap42 mutant (tap42-11; di como and arndt 1996) to induce autophagy at a nonpermissive temperature. accumulation of vacuolar autophagic bodies in tap42 cells was found to be comparable to that in wild-type cells, confirming that induction of autophagy is not controlled by tap42 (fig. 5). furthermore, deletion of npr1, whose product is a protein kinase negatively regulated by tap42 (schmidt et al. 1998), did not affect the induction of autophagy (data not shown). therefore, we concluded that tap42 does not transmit a signal from tor in the autophagy induction pathway. the association between apg1 and apg13 is negatively regulated by tor signaling. in nutrient-rich conditions, dephosphorylated apg13 possesses a high affinity for apg1, which is activated upon apg13 binding, leading to induction of autophagy. this tight apg1apg13 association is required for autophagy, but not for the cvt pathway, an observation that supports a model in which the apg1apg13 complex plays an important role in switching from the cvt pathway to autophagy in response to nutrient conditions (fig. the function of apg17 is still unclear, but our preliminary results suggest that it may be involved in the apg1apg13 interaction. when both apg1 and apg13 were overexpressed, apg1apg13 binding was observed, even in cells grown in yepd (fig. 3 d), resulting in a small amount of apg1 activation, insufficient to induce autophagy (e.g., see fig protein sorting to the plasma membrane and vacuolar degradation of amino acid permeases, gap1 and tat2, are also regulated in response to nutrient conditions, and are dependent upon tor, npr1, and tap42 (but not on apg1; roberg et al. tap42 plays a key role in the rapamycin-sensitive tor pathway, suppressing npr1 activity (schmidt et al. our observation that tor regulation of apg1 activity and autophagy induction is rapamycin-sensitive, but tap42-independent, implies that the apg1apg13 interplay comprises a novel tor signaling pathway regulating autophagy induction. it is possible that tor directly phosphorylates apg13, but this remains to be investigated. recently, nuclear transport of transcription factor gln3 has been shown to be under the control of tor signaling, but the involvement of tap42 in this system is still controversial (beck and hall 1999; cardenas et al. 1999). deletion of gln3 did not affected autophagy (data not shown). in mammalian cells, two targets of mammalian tor (mtor) have been identified: 4e-bp1 and p70s6k (thomas and hall 1997). another recent study reported a relationship between mammalian tor and autophagy (shigemitsu et al. apg1 homologues of caenorhabditis elegans (ogura et al. 1994) and mammals (yan et al.

autophagy is a membrane trafficking to vacuole/lysosome induced by nutrient starvation. in saccharomyces cerevisiae, tor protein, a phosphatidylinositol kinase-related kinase, is involved in the repression of autophagy induction by a largely unknown mechanism. here, we show that the protein kinase activity of apg1 is enhanced by starvation or rapamycin treatment. in addition, we have also found that apg13, which binds to and activates apg1, is hyperphosphorylated in a tor-dependent manner, reducing its affinity to apg1. this apg1apg13 association is required for autophagy, but not for the cytoplasm-to-vacuole targeting (cvt) pathway, another vesicular transport mechanism in which factors essential for autophagy (apg proteins) are also employed under vegetative growth conditions. finally, other apg1-associating proteins, such as apg17 and cvt9, are shown to function specifically in autophagy or the cvt pathway, respectively, suggesting that the apg1 complex plays an important role in switching between two distinct vesicular transport systems in a nutrient-dependent manner.

PMC2150712

pubmed-1211

a change in the genetic code, also known as mutation, is the primary source of genetic variation which gives rise to diversity within a population. when accumulated over generations, these genetic variations may improve the adaptability; hence, the survival of organisms in different environmental conditions [1, 2]. this may in turn induce or preferentially select for further advantageous changes for better adaptation within the environment [35]. although mutations-conferring advantageous traits have been observed in animals such as lizards and fish, it is difficult to study these effects in a laboratory setting due to space and time constraints. for example, it took 36 years for the lizards to show distinct features. on the other hand, bacteria has a number of advantages-fast generation time, ability, to fossilize, and resurrection of historical generations. escherichia coli, a common intestinal bacterium, has been used in a long-term evolutionary experiment spanning more than 2 decades [3, 911]. a number of stress adaptation studies had demonstrated that the growth phases may impact e. coli adaptation. nair and finkel suggested that a nonspecific dna binding protein, dps, may confer multiple stress tolerance at stationary phase, which concur with jolivet-gougeon et al.. in addition, the genome of several strains of e. coli had been sequenced, representing a reliable source of genetic knowledge. in terms of the effects of chemical treatments, bacterial resistance and tolerance to antibiotics are well established and the mechanisms have been widely studied [1417]. in contrast, mechanisms of insusceptibility to nonantibiotic agents, such as food preservatives and antiseptics which might include tolerance or resistance, are less well understood. for example, citric acid inhibits the growth of proteolytic strains of clostridium botulinum, sodium chloride can inhibit the growth of many bacteria such as listeria monocytogenes, ochrobactrum anthropi, and lactobacillus plantarum by lowering the water activity, and fatty acid such as formic, propionic, and acetic acid [24, 25] are also capable of inhibiting bacterial growth. as an intestinal bacterium, e. coli comes into contact with the food and chemicals that we consume. a study which treated pigs with ampicillin, a common antibiotic, demonstrated a significant increase in the occurrence of ampicillin-resistant e. coli from 6% to more than 90% after a course of 7 days. it has been suggested that incomplete absorption in the large intestine led to the presence of subtherapeutic doses of antibiotics in the faeces, resulting in evolutionary pressure for intestinal bacteria such as e. coli towards antibiotic resistance [27, 28]. although the interactions between antibiotics and bacteria have been well studied [16, 26, 29, 30], the interactions between food additives and bacteria remain elusive. it had been demonstrated that benzoic acid and sodium chloride [32, 33] can affect e. coli physiology. firstly, we aim to examine the adaptability of e. coli atcc 8739 (a sequenced strain) in a long-term culture environment in the presence of benzoic acid, sodium chloride, and monosodium glutamate (msg, a common taste enhancer in asian cooking), singly and in combination. two concentrations of each additive were used to evaluate the effects of concentration in the adaptability of e. coli. generation time across passages is used as an estimation of adaptation where decreased generation time across passages in an additive demonstrated that the cells are growing faster compared to an earlier passage. thus, a decrease in generation time across passages indicates that the cells are adapting to the additive, whereas an increase in generation time suggests stress. secondly, we aim to estimate the genomic effects of these adaptations using nei and li distance to estimate the genetic distances between the samples after polymerase chain reaction (pcr)/restriction fragments length polymorphism (rflp). we hypothesized that, e. coli atcc 8739 is able to adapt to the food additives; thereby, demonstrating decrease in generation time across passages. generation time analysis demonstrated e. coli atcc 8739 is able to adapt to the additives over extended culture, and dna fingerprinting suggests that benzoic acid, sodium chloride, and monosodium glutamate are exerting evolutionary pressure on the bacterium. lysophilised escherichia coli atcc 8739 strain (reference passage 4 from atcc) was revived on nutrient agar plate and incubated at 37c before inoculating into 8 different treatments supplementation in 10 ml nutrient broth. these 8 treatments consist of 4 sets of additives with 2 different concentrations each as shown in table 1. subculturing was performed by transferring 1% (100 l) of the previous culture on every monday, wednesday, and friday to the next passage in order for adaptation to occur at the stationary phase of growth. optical density (od) readings were taken before the next subculture at 600 nm wavelength to estimate the number of generations within the current passage and to also determine the number of cells that are being inoculated into the new passage. glycerol stocks for each treatment were made from 1% of the culture for every 12th passage after culturing on macconkey agar. the swap experiment was done fortnightly (6-7 passages interval), involving the transfer of escherichia coli cells cultured in different treatments to other treatments for the measurement of generation time. four types of swaps were carried out, whereby the cells were inoculated into the new treatment in a 100 times dilution. the first set of swap involves the inoculation of basal medium- (l salt) treated cells into the six nonsalt treatments. an example would be inoculating cells grown in l salt into h msg treatment. for the second set, cells cultured in high and low concentrations of each treatment were swapped for all treatments. for example, cells growing in h msg were inoculated into the l msg media and vice versa. in the third set, cells of high concentration treatments (h msg, h ba, and h salt) the last set is similar to the previous set except that cells of the low concentration were swapped. cells from low concentration treatments (l msg, l ba, and l salt) were each inoculated into l comb media. od600 readings were recorded down at intervals, and generation times were calculated for each interval. treatment cultures from every 12th passage interval were used for genomic dna extraction using the phenol-chloroform method of dna extraction for gram-negative bacteria. the dna pellet was air-dried and dissolved to 100 ng/l in ph 8.0 tris/hcl buffer and stored at 20c. each reaction consisted of 50 l of mixture prepared using 200 ng of dna template in 10pmoles of dntps, 50 pmoles of primer, 1 unit of taq polymerase, and 1x standard buffer (with 1.5 mm of mgcl2) provided by the supplier (new england biolabs, inc.). primer 5, cgcgctggc; primer 6, gctggcggc; primer 7, caggcggcg were used separately. the pcr reaction was carried out (hybaid limited, pcr express) under the cycling condition of initial denaturation at 95c for 10 minutes; 35 cycles of amplification at 95c for 1 minute, 27c for 1 minute, 72c for 3 minutes, followed by a final extension at 72c for 10 minutes before gel electrophoresis in 2% (w/v) agarose gel with 1x gelred. the primers used were generated by a previously described method using the following rules: (1) the same primer must be suitable as forward and reverse primers, (2) each primer must be between 6 to 15 bases, (3) the predicted amplicon size must be between 300 to 3100 bases in order for resolution in 2% (w/v) agarose gel, and (4) each primer should be predicted to yield 3 or 4 amplicons. 11 l of pcr product was digested with 1 unit of restriction endonuclease (taqi, hinfi, or mspi), in a reaction mixture consisting of 1x restriction digestion buffer and 100 ng/l acetylated bsa made to a total volume of 20 l with distilled water. hinfi and mspi reaction mixtures were incubated at 37c, while the taqi reaction mixture was incubated at 65c. all reaction mixtures were incubated for 16 hours before analysis in 2% (w/v) agarose gel with 1x gelred. cell density was calculated from od600 readings using the correction suggested by sezonov et al.. briefly, the cell density is directly proportional to od600 readings when od600 reading is below or equal to 0.3, at which the cell density is equivalent to 5 10 cells per milliliter. if od600 reading is above 0.3, the cell density is estimated by the equation of cell density=52137400 in (od600 reading)+118718650. generation time for all experiments was calculated from difference in cell density at intervals between 120 and 300 minutes after the inoculation of cells into fresh media, and the geometric mean was calculated. changes in generation time across passages were tested using t-test for regression coefficient. the migration distance of the bands of pcr and rflp of different treatments within the same passage was tabulated and a nei-li dissimilarity index (di), where the maximum value of 1 is obtained when there are no common bands when comparing between the 2 treatments, while a minimum of 0 will be obtained when the 2 treatments have exactly the same bands. the correlation coefficient (cc) value between dis across passages statistically tested against the cc value of 0.95 (~1) using the z-test for two correlation coefficients where the p value of more than 0.05 would indicate that the null hypothesis (cc is equal to 0.95) is not rejected. analysis of the generation times showed that all eight treatments over the passages displayed different rates of decreasing generation times as shown in table 2. the steepest decline in generation time occurs in h comb treatment where approximately 2.02 minutes reduction in generation time per passage over 70 passages was observed, followed by l msg (1.87 minutes), l ba (1.39 minutes), l salt (1.24 minutes), l comb (1.22 minutes), h ba (1.15 minutes), h salt (1.12 minutes), and finally h msg (0.906 minutes). the regression intercept may be used to estimate the generation time of the cells in each treatment media for the first passage which is indicative of the level of initial stress on the cells. on this basis, the treatment exerting the highest level of stress on the cells would be h comb, followed by l msg, h ba, l ba, l comb, l salt, h salt, and h msg. the linear regression of the generation time across passages demonstrated that the gradients of the equations are not equal to zero which indicates that the generation times are not constant for the six swaps. although there are changes in the general trend of generation time across the passages, the p values calculated for the six swaps were more than 0.05 which is not significant: l salt cells to h msg media, 0.475509; l salt to l msg media is 0.421721; l salt cells to h ba media is 0.250415, l salt cells to l ba media is 0.4660235; l salt cells to h comb media is 0.484887; l salt cells to l comb is 0.443381. the generation times trend of the four swaps (msg, ba, salt, and combination) from low-treatment to high-treatment over 12 swaps change in a decreasing manner (table 3). with reference to the regression equations, the linear regression gradient of low treatment cells into high treatment media for combination treatment is the steepest followed by that for ba, msg, and salt treatments. at swap count between eight and nine, the generation time is almost the same for msg, salt, and combination, but the generation time for ba is still distantly higher. the generation time trends of the four swaps (msg, ba, salt, and combination), from high treatment to low treatment over 12 swaps, changed in a decreasing manner. with reference to the regression equations, the linear regression gradient of high-treatment cells into low-treatment media for msg treatment was the steepest followed by that for ba, combination, and salt treatments. the generation time of high-treatment cells into low-treatment media for msg treatment was almost the same as for salt treatment between swap count four and five, and as for combination treatment between swap count eight and nine. the generation time of swapping high-concentration treated cells into high-combination medium showed similar trends. at the 8th swap, after the 10th swap, generation time for all treatments remain, constant at 200 minutes till the 12th swap. the generation time of swapping low-concentration treated cells in to low-combination medium showed similar trends. at the 2nd swap, after the 4th swap, generation time for all treatment followed a similar trend till the 12th swap. electrophoresis agarose gels of the pcr and rflp products for the eight treatments were used to study the differences between the genome of the e. coli cells of the treatments across the passages. nei-li dissimilarity index (di), which had been shown to be suitable for rflp, was utilised to mathematically calculate the dissimilarity between pair-wise comparisons of the treatments. the dissimilarity index of the 28 comparisons showed a trend of convergence from pcr/rflp number 4 onwards (figure 2). this trend is further elaborated with the estimation of the maximum and minimum mean values (figure 3) which shows converging linear regression line across the 6 pcr/rflp. the similarity among the six resulting effects is that each type of effects had two originating comparisons. therefore by plotting the two comparisons against each other and testing for significance, we can deduce whether the genomic differences in each of the two comparisons are actually a consequent effect from the resulting effects. this suggests that the pcr/rflp-inferred genetic distance between h msg and h comb, and h ba and h salt varied independently (not correlated). in this paper, we present one of the first comprehensive investigations of the effect of e. coli cells ' adaptations to a variety of food additives using a long-term culture approach. our results suggest that cells grown under different stress condition are able to adapt to the environment which can be observed by decreased generation time and genetic variations. since e. coli cells were grown in nb with the various supplementations of treatments, it was important that any changes to the cells were a direct result of the treatments rather than from the nb. the generation time trend for e. coli cells from l salt (nb) inoculated into six different media (table 3) showed that nutrient broth did not appear to impact on adaptability as none of the regression gradients were statistically different from a gradient of zero suggesting that the general generation time trend remained almost the same; therefore, nutrient broth (l salt media) was unlikely the cause of any adaptations observed. the low-concentration treated cells were observed to be adapting to their environment as seen from the decreasing generation time. the cells are dividing at a faster rate with increasing passages suggesting lowered stress level in later passages comparing to early passages (table 3). however, as the concentration of additives is the same throughout the passages, decreased generation time suggests that the cells are adapting to the stress. low-treatment cells inoculated into high-treatment media also showed decrease in generation time across the swaps. growth rate has been used as a measure for adaptation to a stressed environment in previous studies where chen and shakhnovich had demonstrated increase in growth rate of 35 bacterial species upon adaptation to thermal stress. in addition, zhu and yang also demonstrated increase in growth rate; thus, decrease in generation time, when clostridium tyrobutyricum adapts to the presence of butyric acid. our results suggest that the low-treatment cells had gradually adapted to its own individual treatment before the swap, causing it to be less stressed when swapped into high-treatment media. the generation times of cells from low single treatments to l comb were observed to increase gradually across passages (table 3). this suggests that low msg, ba, or salt treatments were not stressful enough to induce significant adaptations such that when they were placed into the l comb treatment which now contains additional stress inducers, the cells could not cope. this may suggest that the cells may be gradually optimized to grow in a specific treatment; thus, increasingly specialized to their specific environment. similar cases had been reported in other evolutionary studies using e. coli [4244]. the effects of l comb did not increase the adaptability but instead decreases it as seen from the generation time analysis (table 2). it has been suggested that the presence of msg counteracts the effects of drop in ph caused by ba. this is achieved by increasing the resistance of e. coli cells against the lowered ph, which will otherwise kill the cells. this suggests that the effects of l msg and l ba cancel each other out, leaving only l salt which is further supported by the similarity between the adaptability of l salt (1.24 minutes per generation, table 2) and l comb (1.22 minutes per generation, table 2). e. coli cultured in h comb treatment had the greatest decline in the generation time over 70 passages. since higher stress level may force the cells to adapt quickly in order to survive [45, 46], suggesting that the e. coli cells in h comb treatment experienced the highest level of stress among the eight treatments (table 2) in contrast to h msg which induced the lowest decrease in generation time. this may suggest that the presence of glutamate in msg may be aiding the growth of cells as glutamate can serve as an additional source of nutrient for the cells. thus, h msg may cause the least amount of stress but instead led to better growth resulting in a lower rate of adaptation; hence, the lowest decrease in generation time. when high-concentration treated cells were swapped into low-treatment media containing the same type of stress, reduced generation time was observed. although both high and low concentrations appear to result in some adaptations as measured by generation time, the rate of adaptation differs. the general decline in generation times of the cells from low-treatment to high-treatment media is steeper than that of the reverse. high-concentration treated cells inoculated into low-concentration treatment media appeared more stressed. this is surprising as low-concentration treated cells inoculated into high-treatment media should experience more stress than high-concentration treated cells inoculated into low-treatment media, provided that the type of stress is similar. a possible explanation for this is that the type of stress may differ, even though low and high treatment contained the same type of additives and differing only in concentration. this may be explained by the catabolism of stress-induced molecules. in takatsu, the level of cardiolipin, a salt stress marker of staphylococcus aureus, took longer to return to basal level upon reculture in basal media after stressed in 5% nacl than in 10% nacl.. it may be plausible that high-concentration treated cells may induce stress-induced molecules which may add to the level of metabolic stress at a lower concentration. the swap from individual high-concentration treatments (h msg, h ba, and h salt) to h comb showed decreased generation time. this may suggest that pretreatment of cells in a stressed environment may condition them to adapt to another stress environment which has been demonstrated by other adaptation studies using e. coli [5053]. however, the swap from individual low-concentration treatments (l msg, l ba, and l salt) to l comb showed the opposite trend with increased generation time. this further corroborates that the adaptive nature of different concentrations may be different; thus, requires further studies. our results from pcr/rflp showed a converging trend in di indicating that the e. coli from all treatments are getting similar (figure 3) suggesting that they mutate in a similar manner. this suggests that they may evolve the same type of stress mechanism and dna repair. however, all the treatments originate from the same bacterial clone, suggesting that the initial stress adaptation may involve mutation as it has been suggested that hypermutation is a feature in initial stress adaptations. it is known that e. coli exposed to stresses would respond to counteract the effects. tucker et al. has shown that e. coli in nitric oxide (no) will reduce the no to nitrous oxide under anaerobic conditions which is harmless to the cell. in another study, while the cells from all treatments may have experienced different types and levels of stresses, it is likely that the cells might have adapted and activated similar stress-responsive mechanism by evolving similarly. this interpretation is supported by a number of studies suggesting the presence of global stress response in e. coli [5759]. in addition, cebrin et al. found that adaptation to ph stress may protect staphylococcus aureus against oxidative stress by hydrogen peroxide, suggesting that adaptation to particular stress may confer tolerance to other stresses. in this paper, the similar response and mutations to the e. coli of the treatments primers 5, 6, and 7 amplified a random sample amounting to 0.37% of the whole genome which is a limitation of this study. however, several studies had demonstrated that pcr-based dna fingerprinting using a small number of primers is suitable to examine genetic diversity in e. coli, candida dubliniensis, and mackerel. another limitation is that dna fingerprinting was performed on the entire population and not on isolated colonies. hence, this paper can only imply on areas of the genome that were amplified, and analysis was approaching genetic similarity at a population scale. this method had been used other studies examining metagenomics in environmental bacterial samples [63, 64] and human myopia. the genes responsible for stress-handling mechanism may also not be present in the amplified regions of the genome. it is unlikely that the genetic distance of e. coli in the eight treatments reaches zero as a population, suggesting that the declining trend is likely to taper off. on the other hand, previous studies in e. coli and herminiimonas arsenicoxydans demonstrated the presence of ecological specialization. our results showed that the generation time decreased over passages, suggesting the possibility of ecological specialization. this may indicate the presence of both global stress response and ecological specialization in e. coli. global stress response allows for adaptation to new stress environments, but extended stress may lead to ecological specialization. hence, it may be hypothesized in future studies that continued culture may lead to ecological specialization which may be seen as a divergence in the genetic distance. statistical analysis of the selected comparisons indicates that the effects of all the treatments were insignificant except 10ba+salt (table 4). statistical tests for msg and ba effect suggested that different gels provided constant results; thus, suggesting reliability in our study. statistical tests suggest that msg and ba, and msg and salt are likely to interact with each other (table 4). this suggests that high-combination media contains 10msg+10ba+salt and the interacting effects of msg and ba, and msg and salt. results from swap analysis indicated that low-salt cells when swapped to high-combination media showed an increase in generation time. this suggests that the high stress in high combination media results in difficulties for the cells from l salt to grow and caused an increase in doubling time. this might be due to the additional combined stress produced by msg interacting with ba and salt. the presence of the additional interacting stress of the combination treatment can also be observed from the analysis of generation time where the stress level of h comb is much higher than the three individual high-concentration treatments. ba kills bacteria by lowering the ph of the media, whereas msg has the effects of ph resistance on the cells. hence, the presence of msg may aid the growth of the e. coli living in low-ph environment caused by the presence of ba. on the other hand, combined effects from msg+s could be harmful to the cells as salt may increase the high sodium content provided by msg in media. this paper had demonstrated that e. coli is able to adapt to food additives over an extended period of time by observing the decreased in generation time over a period of 70 passages. this may have implications in using sublethal doses of bacteriocidal agents such as disinfectants and preservatives. hence, it may be hypothesized that increasing passages may demonstrate a shift towards ecological specialization.

escherichia coli is commonly found in intestine of human, and any changes in their adaptation or evolution may affect the human body. the relationship between e. coli and food additives is less studied as compared to antibiotics. e. coli within our human gut are consistently interacting with the food additives; thus, it is important to investigate this relationship. in this paper, we observed the evolution of e. coli cultured in different concentration of food additives (sodium chloride, benzoic acid, and monosodium glutamate), singly or in combination, over 70 passages. adaptability over time was estimated by generation time and cell density at stationary phase. polymerase chain reaction (pcr)/restriction fragments length polymorphism (rflp) using 3 primers and restriction endonucleases, each was used to characterize adaptation/evolution at genomic level. the amplification and digestion profiles were tabulated and analyzed by nei-li dissimilarity index. our results demonstrate that e. coli in every treatment had adapted over 465 generations. the types of stress were discovered to be different even though different concentrations of same additives were used. however, rflp shows a convergence of genetic distances, suggesting the presence of global stress response. in addition, monosodium glutamate may be a nutrient source and support acid resistance in e. coli.

PMC3658543

pubmed-1212

laparoscopic surgery has established itself as a durable alternative for both gynecologic and general surgical procedures. with increasing popularity and greater utility, the types and number of reported complications are increasing. we describe the case of a 70-year-old male undergoing routine laparoscopic cholecystectomy for gallstone pancreatitis who developed asystolic cardiac arrest intraoperatively. a review of the literature revealed 2 cases of asystolic cardiac arrest during laparoscopy: one was during laparoscopic cholecystectomy and one was during diagnostic laparoscopy for gynecologic evaluation. a 70-year-old male with a past medical history negative for ischemic heart disease with asymptomatic myocardial infarction was admitted to the hospital with a 24-hour history of severe, acute epigastric abdominal pain. his point of maximal impulse was nondisplaced, and he had no evidence of jugular venous distension. his abdomen was soft with no masses, hernias, or palpable evidence of organomegaly. he demonstrated mild to moderate tenderness to palpation of his epigastrium and right upper abdominal quadrant. initial investigations included the following tests: complete blood count: white blood cell count 14.6; hemoglobin/hematocrit 15.1/44.5, platelets 399 000; basic metabolic profile: na 138, k 4.1, cl 103, co2 26, urea 14, creatinine 0.9, glucose 138; liver function tests: total bilirubin 0.8, albumin 3.1, aspartate aminotransferase (ast) 576, alanine aminotransferase (alt) 417, alkaline phosphatase 352, ldh 1639; amylase 1173; lipase 5339. initial electrocardiography revealed regular rhythm with first-degree av block and no evidence of acute coronary ischemia. a chest xray showed mild cardiomegaly, hyperinflated lungs, and no evidence of infiltrate or congestion. ultrasonography of the gallbladder showed several gallstones with a common bile duct measurement to 8 mm, no wall thickness, and no evidence of pericholecystic fluid. he was not allowed to take anything by mouth and was started on empiric intravenous antibiotics for presumed persistent common bile duct obstruction. over ensuing days, the patient's laboratory evaluations showed a continuously decreasing amylase and lipase until normality was achieved on hospital day number 4. it was deemed that the patient would require endoscopic retrograde cholangiopancreatography (ercp) and cardiology clearance prior to undergoing laparoscopic cholecystectomy. because the patient's acute symptomology had entirely resolved and he was tolerating a regular diet without recurrence of pain, he was discharged home with plans to undergo ercp and cardiac evaluation as an outpatient. ercp was attempted by a well-experienced gastroenterologist; cannulation of the sphincter of oddi was unsuccessful secondary to a duodenal diverticulum, and the procedure was aborted. cardiac assessment consisting of a stress test was performed, which was negative for unstable coronary circulation. the patient returned to the hospital 6 weeks after the initial evaluation for laparoscopic cholecystectomy. in the interim, he had been doing well, tolerating a low-fat diet, and had stopped smoking 2 weeks prior to the scheduled surgery. preoperative medication comprised mefoxin and was discontinued approximately 30 minutes prior to the scheduled surgery. in the operating room, the patient was preoxygenated with 80% oxygen to achieve a peripheral pulse oxygen saturation of 100%. appropriate intubation was confirmed by positive end-tidal co2 and auscultation of administered breaths in bilateral lung fields. anesthesia was maintained with nitrous oxide and fentanyl. in the supine position, access into the peritoneal cavity was attained using the open hassan technique via an intraumbilical incision. pneumoperitoneum was initially achieved to an intraperitoneal pressure of 15 mm hg by first administering low-flow co2 followed by high-flow co2. the gallbladder was then grasped with a gallbladder grasper and retracted in the lateral/cephalad direction for approximately 15 seconds resulting in an asystolic episode for 30 seconds. the patient was administered atropine, with subsequent resumption of sinus cardiac rhythm. with resumption of stable vital signs, reinsufflation of the peritoneal cavity to 15 mm hg sinus rhythm was achieved within 5 seconds of releasing the gallbladder and relieving the pneumoperitoneum. a third attempt at gallbladder manipulation was made without insufflation but with direct visualization of the gallbladder. cardiac rhythm resumed approximately 5 seconds after the gallbladder was released. throughout all 3 episodes, adequacy of ventilation was confirmed bilaterally, oxygen saturation remained at 100%, capnometry readings of 35 to 40 mm hg were obtained, and the patient remained normothermic. no acute electrocardiographic changes were appreciated with resumption of cardiac rhythm in the operating room. the patient was extubated in the operating room, transferred to the postanesthesia recovery room, and maintained on telemetry. postoperative cardiology and electrophysiology evaluation did not reveal a primary cardiac event as the cause of the patient's asystolic episodes. additionally, the patient's cardiac isoenzyme profile over the 24-hour period was negative for acute myocardial injury. given these findings coupled with the patient's respiratory stability intraoperatively, it was deemed that a severe vagal reaction in response to gallbladder retraction was the source of the patient's asystolic episodes. therefore, the patient underwent insertion of a temporary pacemaker prior to a reattempt at a laparoscopic cholecystectomy. the pacer was set to a rate greater than the patient's resting heart rate. the second procedure was performed using a pneumoperitoneum of 15 mm hg and was accomplished without incident. the pacemaker was removed in the early postoperative period, and the patient was discharged home without further incident. the causes of cardiovascular collapse during laparoscopy include co2 pulmonary embolization, cardiac arrhythmias, vagal reactions secondary to peritoneal distention during insufflation or viscus manipulation, and diminished cardiac preload secondary to caval compression. asystolic cardiac arrest is a potential manifestation of these hemodynamically significant events. in a review of the literature, the american association of gynecology reports an incidence of one in 2500 cases of asystolic arrest during laparoscopy. we were able to identify only one previously described case of asystole during laparoscopic cholecystectomy. the clinical manifestation generally includes a diminished end-tidal co2, tachycardia, diminished breath sounds in a specific lung field on auscultation, and a classic cardiac murmur associated with gas embolization. the general mechanism is perceived to be infiltration of insufflated co2 into venous/lymphatic channels with subsequent pulmonary migration. it is unlikely that the asystolic arrest in our case is secondary to gas embolization because our patient failed to exhibit any of the above signs. an alternative cause of hemodynamically significant cardiovascular changes during laparoscopy is hypoxia or hypercapnia resulting in cardiac arrhythmias. it is believed that the combination of the trendelenburg positioning and elevated intraabdominal compartment pressures predispose a patient to aspiration, resulting in hypoxia and possibly hypercapnia. however, it is unlikely that clinically significant elevations in co2 levels on blood-gas measurements can be detected. elevated intraabdominal pressures can diminish venous return to the heart, preload, resulting in diminished cardiac output. with intraabdominal pressures ranging from 12 to 15 mm hg, however, with intraabdominal pressures in excess of 40 mm hg, a significant caval compression leads to a decreased preload and cardiac output. the first is an increase in central blood volume due to the forcing of blood out of the splanchnic circulation. the second is a diminished preload secondary to peripheral pooling of blood in the lower extremities in combination with the reversed trendelenburg position. the result is a temporary increase in circulating blood volume followed by a sustained decrease in central pressures. these typically occur in patients with primary pulmonary pathology, such as pulmonary/mediastinal blebs, which rupture under positive pressure. lehman et al delineate the possibility of tension pneumothorax during insufflation secondary to a congenital diaphragmatic defect. cases of pneumothorax/pneumomediastinum generally present with hypotension, tachycardia, diminished breath sounds in a lung field, and possibly subcutaneous emphysema. shifren et al1 describe a case of asystolic cardiac arrest during gynecologic laparoscopy that is attributed to rapid peritoneal distension during insufflation. under circumstances of elevated intraabdominal pressures, it is postulated that manipulation of certain pelvic structures/ organs may further elevate intraabdominal pressures. we do not believe this to be the cause in our case. even during gallbladder handling without pneumoperitoneum, asystole occurred.1 it is our opinion that the asystolic cardiac arrest in our case was secondary to a severe vagal reaction that was triggered by manipulation of the gallbladder. this was validated by the fact that recurrent asystole was documented for approximately 5 seconds upon grasping the gallbladder without elevated intraabdominal pressures. additionally, no reproducible hemodynamic sequelae occurred during the successful attempt at laparoscopic cholecystectomy, once the temporary pacemaker was in place. we could not identify any clinical criteria, including altered co2 levels, hypoxia, diminished breath sounds, or tachycardia to suggest any of the other proposed mechanisms of cardiovascular collapse during laparoscopy that are described above.

laparoscopic surgery has become a durable alternative for both gynecologic and general surgical procedures, but reported complications are increasing. we describe the case of a 70-year-old male undergoing routine laparoscopic cholecystectomy for gallstone pancreatitis who developed asystolic cardiac arrest intraoperatively. a review of the literature revealed 2 cases of asystolic cardiac arrest during laparoscopy: one was during laparoscopic cholecystectomy and one was during diagnostic laparoscopy for gynecologic evaluation.

PMC3015508

pubmed-1213

voltage-gated sodium channels (nav) are membrane-bound proteins that initiate action potentials in nerve and muscle cells and are critical elements of proper function in these tissues. the channels open when the voltage across the cell membrane is depolarized by a few millivolts above the normally negative resting membrane potential. channel activation allows sodium ions to enter the cell and further depolarize the membrane potential. the movement of sodium ions through the membrane comprises the rising phase of the action potential. this step, along with the activation of voltage-gated potassium channels, allows the membrane to repolarize and ends the action potential. action potentials act as electrical messages that travel along the axons of nerve cells and the surface of muscle fibers initiating the release of neurotransmitters by neurons and coordinating contractions in muscle. sodium channels are formed by a 260 kda -subunit that is associated with one subunit (1) in skeletal muscle cells and with two subunits (3 and 1 or 2) in the central nervous system. the subunit forms the pore and the protein contains components required for other aspects of channel function including voltage-dependent activation and fast inactivation (figure 1). although the kinetics and voltage dependence of channel gating are modified by the subunits [3, 4], all the elements of channel function can be reconstituted when the subunit is expressed alone in heterologous expression systems, e.g. xenopus laevis oocytes [57]. an early model of the two-dimensional folding pattern of the subunit predicted that the protein consists of four homologous domains (di-iv) composed of six -helical transmembrane segments (s1s6) and that the regions of the protein between segments s5 and s6 of all four domains reenter the membrane to form the outer pore of the channel through which sodium ions enter the cell. further work on sodium channels has supported the general features of this model including the identification of the s4 segments as the voltage sensor. a repeated motif of positively charged amino acids separated by two hydrophobic amino acids is found in the portions of the channel assigned to the s4 transmembrane segments. when the positively charged residues in the s4 transmembrane regions are replaced with uncharged amino acids, the voltage-dependence of channel gating is altered as would be expected in the region of the channel that acts as a voltage sensor [9, 10]. additionally, there is evidence that portions of the s4 segments move outward in response to the membrane depolarization. residues in the s4 segments become more accessible to reaction with extracellular reagents and less accessible to reaction with intracellular reagents when the membrane is repeatedly depolarized [11, 12]. after channels open in response to membrane depolarization, they rapidly inactivate which stops the flow of sodium ions into the cell. this form of channel gating occurs when the linker between the third and fourth domains of the channel physically occludes the intracellular mouth of the channel pore [9, 1316]. channel opening, ion selectivity and inactivation are each controlled by separate regions of the -subunit. voltage-gated sodium channels are encoded by a multigene family [17, 18]. different types of excitable tissue express different members of the sodium channel gene family and the tetrodotoxin (ttx) sensitivities of nerve or muscle cells are dependent on the type of channels expressed in the cell. there are ten members of the gene family in the three mammalian species where all members of the gene family have been identified. in the weakly electric fish sternopygus macrurus, six genes have been identified although there maybe as many as eight sodium channel isoforms in fish (personal communication by h. zakon;). the nine mammalian channel isoforms that have been identified and functionally expressed (nav1.1 nav1.9) have greater than 50% identical amino acid sequence within each of the four domains and the linker between domains iii and iv. the tenth sodium channel isoform nav has a more divergent sequence and may represent a distinct subfamily. this channel isoform has never been functionally expressed in heterologous cells, but evidence from nav expression patterns and nav knockout mice indicate that the channel may not function as a voltage-gated channel but rather be important for sensing and regulating extracellular salt in the hypothalamus and visceral organs [23, 24]. the genes that encode nav1.1 through nav1.9 in humans and mice are found on four chromosomes and the chromosome segments containing sodium channel genes are paralogous. a segment of chromosome 2 contains genes encoding nav1.1, nav1.2, nav1.3 and nav1.7 that have more than 90% amino acid sequence identity [18, 21]. the marine toxin ttx has long been recognized as a potent inhibitor of sodium currents in nerve and muscle. a more comprehensive review of ttx and its actions relative to other marine toxins is discussed elsewhere in this issue (al-sabi et al.). ttx sensitivities of nerve or muscle cells are dependent on the type of channels expressed in the cell. products from the cluster of genes located on chromosome 2 are all blocked by nanomolar concentrations of ttx and are expressed in neurons. a second cluster of genes containing nav1.5, nav1.8 and nav1.9 is located on chromosome 3. these isoforms have 75% amino acid sequence identity as genes in the chromosome 2 cluster but are blocked by micromolar concentrations of ttx. the genes have more limited expression patterns, with nav1.8 and nav1.9 primarily expressed in neurons of the dorsal root ganglion and nav1.5 primarily expressed in cardiac muscle cells. the two final sodium channel isoform genes, nav1.4 and nav1.6, are each located on separate chromosomes despite the fact that they have greater than 85% sequence identity with the genes clustered on chromosome 2 and are also blocked by nanomolar concentrations of ttx. one channel isoform, nav1.6, is expressed in many types of neurons but nav1.4 appears to be solely expressed in skeletal muscle fibers. subtle differences in channel function may explain differences in the expression pattern of channel isoforms, but an understanding of the correspondence between channel function and expression is still at an early stage. recent work has shed some light on the correspondence between channel isoform function and expression. both nav1.2 and nav1.6 are expressed in neurons of the central nervous system but their expression patterns differ in the cell types and even the region within the same neuron in which they are found. the nodes of ranvier are the regions along the length of axons without myelin and glial cell wrapping that allow for the saltatory conduction of action potentials. the predominant isoform at the nodes of ranvier in sensory and motor neurons of the adult central and peripheral nervous system is nav1.6. however, in developing retinal ganglion cells both nav1.2 and nav1.6 are clustered at immature nodes of ranvier, and only as myelination proceeds does nav1.6 replace nav1.2 [28, 29]. if nav1.6 is required for proper function of mature axons, this suggests that nav1.6 allows axons to transmit high frequency action potentials. indeed, nav1.2 and nav1.6 respond differently to a rapid series of depolarizations, currents through nav1.2 decrease and currents through nav1.6 increase [30, 31]. currents through nav1.6 increase because repeated stimulations of the channels cause a use-dependent potentiation of channel opening where channels activate faster after repeated depolarizations. currents may decrease through nav1.2 after repeated stimulation because channels more rapidly enter a slow inactivated state and fewer channels are available to open with repeated stimulations. the slow inactivated state develops after prolonged membrane depolarization or repeated stimulation and sodium ions can not pass through channels in this state [32, 33]. the expression of nav1.6 in mature nodes of ranvier may allow the rapid, repeated depolarization of the membrane and the transmission of high frequency action potentials along the length of the axon. cell maturation is accompanied by another change in sodium channel isoform expression in skeletal muscle tissue. however, by postnatal day 35, mrna encoding nav1.5 is undetectable and mrna encoding nav1.4 has increased 10-fold [34, 35]. intriguingly, after denervation nav1.5 is expressed again and nav1.4 transcript expression declines as if the muscle were reverting to an earlier developmental stage. cardiac muscle tissue where nav1.5 is primarily expressed has a very different pattern of activity than adult skeletal muscle where nav1.4 is primarily expressed. action potentials in the heart are repetitive and sustained with the membrane remaining depolarized for several hundred milliseconds. in contrast, during the action potential in skeletal muscle fibers membrane depolarization lasts a few milliseconds and can occur at high frequency [37, 38]. if subjected to the prolonged depolarizations that occur during the cardiac action potential nav1.4 channels enter the slow inactivated state and are no longer excitable unless the membrane is held at a negative potential for seconds. however, nav1.5 currents do not slow inactivate completely even after long depolarizations lasting seconds. these channels can function in the activity pattern found in heart muscle tissue and may also be able to function better in the altered activity pattern of immature and denervated muscle fibers that have depolarized resting membrane potentials and spontaneous action potentials. several mutations in nav1.4 that are linked to the skeletal muscle diseases hyperkalemic periodic paralysis and paramyotonia congenital impair the ability of the channels to enter the slow inactivated state and reduce the use-dependent inhibition of sodium current after rapid stimulation [41, 42]. this defect accentuates the muscle membrane depolarization and hyperkalemia sensitivity that lead to muscle paralysis. thus, an alteration of the sodium channel isoforms expressed in a tissue may impair the proper functioning of that tissue. although a change in sodium channel expression pattern might allow certain tissue to resist the effects of ttx, the expression of an isoform with different kinetic and voltage-dependent properties might alter the functional properties of the tissue. tetrodotoxin blocks sodium channel activity by binding to the outer pore of the channel that is formed by s5s6 linkers. the portion of the linkers that interacts with ttx forms the pore -helix, the selectivity filter and the outer vestibule of the pore [43, 44]. sodium ions entering the cell pass through the outer vestibule of the pore and the narrow selectivity filter before they can enter the inner pore of the channel (figure 2). two rings of mostly negatively charged amino acids line the outer vestibule (shown in pink in fig. 2), and the inner ring, composed of aspartate (di), glutamate (dii), lysine (diii), and alanine (div) (the deka filter shown as red, red, blue, and green space filling structures in fig. 2) forms the selectivity filter [45, 46]. structural support for the selectivity filter is provided by interactions of the amino acids between the negatively charged rings and those in the pore helices [43, 44]. the positively charged guandinium group and hydroxyls of ttx (figure 3) interact with the side chains of the amino acids that form the negatively charged rings [45, 47, 48]. changes in the amino acids of the s5s6 linker from the pore helix to the outer negatively charged ring effect ttx binding either by altering the electrostatic interaction between ttx and an amino acid side chain directly or by altering the shape of this narrow portion of the pore where ttx binds [4345, 4749]. the binding affinity of the channel for ttx is also altered by changes in membrane potential [50, 51]. the percentage of channels blocked by ttx increases with repeated stimulation of the channel. one model of use-dependent block interprets the increased block that accompanies repeated membrane depolarizations as indicative that the binding site for ttx becomes accessible and the probability of ttx binding increases when the channel activates in response to membrane depolarization [50, 51]. another model of ttx use-dependent block suggests that toxin binding in the pore can be affected by a cation (na or ca) bound in the closed pore [52, 53]. when the channel opens the ion passes into the interior of the cell and allows ttx to bind to the outer pore. the determination of which model correctly describes use-dependent block will probably best be made with data from the crystal structure of the pore in the open and closed state. these data should show whether the conformation of the outer pore is different when the channel is in the closed and open state. in addition to the block of ion permeation by ttx, external application of toxin appears to affect sodium channel gating. the steady state voltage dependence of gating charge immobilization the restriction of voltage sensor movement during inactivation is hyperpolarized by ttx in crayfish giant axons. the effect of ttx on charge immobilization is reduced, but not entirely eliminated, by internal perfusion of n-bromoacetamide (nba). this result suggests that ttx alters the voltage dependence of both fast and slow inactivation, or that nba alters channel structure in such a way that the effect of ttx on inactivation is reduced. although a number of amino acid changes have been identified that affect ttx binding to the channel through mutational analysis, naturally occurring differences in ttx sensitivity among members of the sodium channel gene family arise from differences at a single amino acid position in the outer pore (figure 1). tetrodotoxin-sensitive members of the gene family can be distinguished by the presence of an aromatic amino acid at the domain i position above the selectivity filter and ttx-resistant members have a cysteine or a serine at the same position [5660]. in tetrodotoxic animals, ttx resistance appears to be derived from the substitution of non-aromatic amino acids at this critical position in domain i in the ttx-sensitive members of the sodium channel gene family [61, 62]. a hydrophobic portion of ttx is thought to interact with the aromatic amino acid above the selectivity filter in domain i [43, 47]. the type of amino acid at this position also affects the ability of the channel to conduct sodium ions through the membrane. the ttx-resistant channel nav1.5 that is primarily expressed in heart muscle tissue has lower permeability to sodium ions compared to the ttx-sensitive channel nav1.4 expressed in skeletal muscle tissue. the replacement of a tyrosine with a cysteine at the critical position in domain i of nav1.4 reduces single channel conductance from 53 ps to the level measured for nav1.5, 43 ps [58, 63]. the relationship between channel permeability and the functional requirements of excitable tissues is not clear but it is unlikely that the differences reflect a requirement for ttx resistance in heart muscle. the effects of ttx on the human body differ among various excitable tissues based upon the sodium channel isoforms expressed in the cells of that tissue type. the dangers of ttx intoxication include effects on tissues that primarily express ttx-sensitive sodium channel isoforms such as skeletal muscle tissue and peripheral nerves. the diaphragm is composed of skeletal muscle fibers and may become paralyzed during ttx intoxication. heart muscle tissue that primarily expresses a ttx-resistant channel isoform can in most instances continue to function after ttx is ingested. other effects include paralysis of the limbs that can progress to generalized flaccid paralysis, dizziness that may be accompanied by a sensation of floating perhaps as proprioceptive input is lost, and numbness of the mouth that can progress to the tongue, face and periphery. the most severe cases of ttx poisoning can include symptoms of hypotension and bradycardia perhaps as the ttx-sensitive channels found in arterial smooth muscle cells and the sinoatrial node of the heart are blocked [6769]. ingesting newts of the genus taricha that have high concentrations of ttx in their skin is lethal to almost every one of their potential predators. one predator, the garter snake thamnophis sirtalis, has evolved resistance to ttx that allows the snake to eat newts. the effects of attacking a toxic newt can range from reduced locomotor performance to paralysis and death from respiratory failure depending on the level of resistance of the individual snake. variation in ttx resistance among snake populations is extreme and spans three orders of magnitude. thamnophis sirtalis is found across north america, but it is only in snake populations that are sympatric with toxic newts that extreme resistance to ttx has evolved suggesting that snakes have evolved resistance in a coevolutionary arms race with tetrotodoxic newts. phylogeographic evidence indicates that ttx resistance has evolved independently at least twice within t. sirtalis populations in western north america [71, 72]. studying the evolution of ttx resistance in garter snakes has provided an opportunity to determine what changes are possible in a conserved portion of the nav that still allow for proper channel function. elevated resistance to ttx is due, at least in part, to mutations in the outer pore of a nav expressed in the skeletal muscle fibers of resistant snakes [73, 74]. an analysis of the nucleotide sequence of the channel cloned from snake skeletal muscle demonstrates that the channel has the highest homology to other vertebrate skeletal muscle sodium channel genes (nav1.4). tetrodotoxin resistance in garter snakes has evolved through mutations in an important functional region of a ttx-sensitive sodium channel gene, tsnav1.4 and not through changes in gene expression where a ttx-resistant channel gene is expressed in skeletal muscle tissue. mutations observed in ttx-resistant snake sodium channels are at positions in the outer pore other than those previously identified, and may therefore have effects on channel function that differ from those imparted by changes in the domain i sequence [58, 63]. snakes from three ttx-resistant populations (warrenton, benton, and willow creek) have an aromatic amino acid in the critical position of the domain i sequence but novel mutations in the domain iv s5s6 linker of tsnav1.4 that increase the ttx concentrations required to block the channel. the domain iv outer pore sequence of tsnav1.4 from a non-resistant snake population (bear lake) matches that of rat and human nav1.4 and is blocked by low concentrations of ttx. changes in the amino acid sequence of channels from ttx-resistant snakes are in a highly conserved portion of the s5s6 linkers that forms the outer vestibule and selectivity filter of the channel. amino acid substitutions that alter the structure or charge in this portion of the pore can affect both ttx-binding and channel function [43, 45]. for example, ttx binding, single-channel conductance and ion selectivity are all affected by amino acid substitutions that replace the conserved domain iv aspartic acid in the outer ring of charged amino acids in the outer pore with an uncharged residue [45, 75, 76]. one of the amino acid substitutions in the willow creek channel sequence replaces the aspartic acid in the equivalent position of domain iv with an uncharged asparagine. it is reasonable to predict that this dramatic change at a critical residue may affect other aspects of channel function. additional substitutions in the channel sequence that affect ttx binding are in regions of the channel that affect the structure of the selectivity filter and outer vestibule. the population with the most extreme resistance values (willow creek) has the most changes in the outer pore sequence of tsnav1.4 and the four amino acid substitutions increase the ttx resistance of the channel by three orders of magnitude. this population also has significantly slower action potential (ap) rise rates than the three other populations suggesting that the changes in the structure of the outer pore may have altered channel function. two populations from another lineage with intermediate ttx resistance (benton and warrenton) have more conservative amino acid substitutions and the change in ttx resistance they impart is more modest. the ap rise rates from benton snakes are the same as those recorded from nonresistant snakes (bear lake population), however ap recordings from warrenton snakes have the surprising result that the ap rise rates are faster than those recorded from other snake populations. this result is not predicted by the single change in the pore sequence of the warrenton channel and suggests that another mechanism may also play a role in ttx resistance of this snake population. specifically, snake from this population may express an increased number of sodium channels in skeletal muscle fibers. an increase in the sodium current of skeletal muscle fibers would increase the ap rise rate and could increase the concentration of ttx required to block activity in the skeletal muscle fibers of this population. the proper functioning of electrically excitable cells depends on maintaining a balance between ion currents that alter the membrane potential. adaptations that allow common garter snakes to eat tetrodotoxic newts may have altered sodium channel function and this change could alter the magnitude of sodium currents in skeletal muscle cells. changing sodium current magnitude could alter the balance of currents that influence cell excitability and action potential propagation. there is a physiological trade-off between ttx resistance and locomotor performance that manifests as more resistant snakes having slower maximum crawl speeds. differences among populations in ap rise rates suggest that changes that alter the ttx resistance of the skeletal muscle may also affect cell physiology. the slowing of the ap rise rate in one population and its increase in another suggest that changes in channel function and possibly channel expression affect cell physiology. the evolution of ttx resistance in thamnophis sirtalis has occurred through a series of unique substitutions in a protein that is vital for nerve and muscle cell activity and in a region of the protein that is critical for its function. further work in this system has the potential to increase our understanding of how ion channel function influences cell physiology through an opportunity to measure the benefits and costs of changes in sodium channel structure on the proper functioning of nerve and skeletal muscle tissue.

tetrodotoxin (ttx) is a highly specific blocker of voltage-gated sodium channels. the dissociation constant of block varies with different channel isoforms. until recently, channel resistance was thought to be primarily imparted by amino acid substitutions at a single position in domain i. recent work reveals a novel site for tetrodotoxin resistance in the p-region of domain iv.

PMC3663409

pubmed-1214

viruses are the most abundant and diverse biological entities in the biosphere, and their global numbers have been estimated at 1.2 10 and 2.6 10 in the ocean and soil, respectively. thus, viruses are key elements that contribute to the life cycles of cellular organisms. however, the study of viral ecology in natural habitats has been limited due to the difficulties of viral culture. the lack of a ubiquitous marker gene, such as the 16s rrna gene shared by all bacteria and archaea, also hampered our understanding of the genetic diversity of viruses, prior to the introduction of metagenomics into the field of viral ecology [1, 4]. recently, viral metagenomics has enabled researchers to explore the community structure and diversity of viruses in various natural ecosystems. this methodology depends on a priori knowledge of the viral types that may be present [6, 7]. the first viral metagenome of uncultured marine viral communities was published in 2002, and there have been many subsequent advances in the methodologies (e.g., methods for amplifying the initial viral genomes) and tools used for bioinformatics analysis in viral metagenomics. these technical developments have facilitated explorations of the abundance and diversity of viruses from a wide range of natural habitats in korea. in korea, viral metagenomics was applied for the first time to unique samples, including fermented foods and atmospheric samples, as well as habitats where viruses are expected to have significant roles, such as rice paddy soil, seawater, and the human gut. this review describes recent advances in viral metagenomics and provides summaries of studies that have been conducted to characterize korean viral metagenomes. in addition, the advantages and disadvantages of the most widely used viral dna amplification methods are discussed, based on empirical knowledge. viral metagenomics is the study of viral metagenomes (known as viromes), which are obtained directly from environmental samples using viral particle purification and shotgun sequencing. viral metagenomic studies have increased gradually since the expansion of metagenomic approaches to viral ecology; i.e., over 200 (61 reviews) investigations of the viral communities in environmental samples have been published (fig. 1) [8-13]. in 2002, breitbart et al. used shotgun library sequencing and reported that the majority of the sequences in marine viral metagenomes shared no similarity with any genes in public databases, which suggested that most environmental viruses remained uncharacterized. subsequently, many studies have surveyed the viral diversity of unexplored habitats using viral metagenomics. cultivation in a host is usually necessary to obtain a virus from the environment that is being investigated. however, the application of metagenomics techniques based on genetic information can circumvent this obstacle. based on the physical characteristics of virions, viral particles can be isolated from environmental samples, which are enriched using a combination of size filtration (e.g.,<0.22 m) and density gradient centrifugation (e.g., 1.35-1.5 g/ml cesium chloride), so that the virome can be obtained from the purified viral particles. there is a lack of evolutionarily conserved genes, such as the prokaryotic 16s ribosomal rna gene, in viral genomes. therefore, the fragmented viral metagenomic sequences obtained by whole-genome shotgun dna sequencing are used to analyze the viral ecology instead. viral genomes are small (on average, they comprise a few to several dozen kilobases); so, valuable genome coverage can be achieved easily by dna sequencing. the advent of high-throughput sequencing techniques, such as 454 pyrosequencing and illumina sequencing, makes it easier to achieve suitable genome coverage at low cost. indeed, over 90 of either nearly complete or complete novel viral genomes were assembled using these methods in recent studies [9, 10, 15-20]. however, it is inevitable that an amplification step is necessary for small viral genomes prior to dna sequencing. linker amplified shotgun library (lasl) and multiple displacement amplification (mda) are the main methods used to amplify viromes in viral metagenomics. the application of adapter attachment is restricted to double-stranded dna sequences; so, only double-stranded dna (dsdna) viruses can be detected using the lasl method. thus, the dominance of dsdna tailed bacteriophages was reported initially in uncultured marine viral assemblages, as well as other environmental samples, such as kimchi, aquatic water, and feces. the mda technique amplifies dna isothermally using the phi29 polymerase and random hexaprimers and has been used before in microbiology. the high amplification efficiency of this method means that it is suitable for amplification during viral metagenomics applications. particularly, the phi29 polymerase of the mda technique selectively amplifies circular genomes (estimated at 100 times), and it has facilitated the discovery of abundant single-stranded dna and rna viruses in environmental samples [23, 24] (described in detail below). the analysis of viral metagenome data using bioinformatics is one of the most challenging aspects of viral metagenomics. one million to 1 billion reads of viral metagenomic sequences are typically generated by high-throughput sequencing platforms (with an average read length of 350-400 bp using 454 gs-flx titanium and 2 150 bp using illumina hiseq 2500). after removing any low-quality, redundant, and chimeric sequences, the viral sequences are compared to sequences in public databases (e.g., the genbank non-redundant nucleotide database, mg-rast, and camera) using blast or usearch. the identification of viral sequences based on significant amino acid similarity (e-value of<10) was first described by breitbart et al., and it has since been extended to the exploration of environmental viromes, although the e-score applied to viral metagenomic studies appears to be regarded as " a loose standard ". most of the environmental viromes detected by viral metagenomics are defined as orphan (unassigned) sequences. the majority of viral sequences shares no amino acid similarity with previously observed genes (average 40% to 50%, occasionally up to 90%, of sequences); so, they are characterized as " unknown ". comparisons of viral sequences with the data in public databases have demonstrated that little is known about environmental viruses. thus, the majority of the unassigned sequences in viral metagenomes is often regarded as " junk sequences " due to a lack of suitable bioinformatics tools and viral databases for their characterization [1, 28]. at present, the viral databases are biased toward animal and plant viruses, although viruses that infect prokaryotes (bacteriophages) are sparsely represented. most of the latter are restricted to phages that infect bacteria belonging to the phyla proteobacteria, firmicutes, and actinobacteria [29, 30]. moreover, even " known " viral sequences share low amino acid similarities (< 50%) with viral protein sequences [9, 11, 13, 31]; so, the majority of environmental viruses representing novel viral species and their viral diversity is much greater than considered previously. the observation of a high percentage of orfans (open reading frames with no homologs in known genes in the databases) in viral genomes also supports the novelty of environmental viromes. thus, researchers could discover novel viruses in orphan sequence pools that currently remain " untapped resources. " these findings indicate our current lack of knowledge about viral genetic information and emphasize the need for physiological studies of viruses to understand viral ecology based on genomic data. using viral metagenomic approaches, viral diversity and abundance have been investigated in various natural ecosystems in korea, including rice paddy soil, fermented foods, the human gut, seawater, and the near-surface atmosphere (table 1). 2). sipho- and podo-like tailed viruses were found in fermented foods (fig. 2a-2c), while non-tailed small viruses were detected in the near-surface atmosphere (fig. various types of tailed, non-tailed, circular, and long linear viruses were found in the human gut (fig. in general, the virions ranged in size from 30 to 60 nm. in agreement with the results of previous viral metagenomic studies [23, 31], over half of the sequences in the korean environmental viromes were described as orphan sequences, based on comparisons with viral proteins in public databases (table 1). most of the sequences identified were assigned to the siphoviridae, podoviridae, and myoviridae families of dsdna viruses and the circoviridae, germiniviridae, nanoviridae, and microviridae families of ssdna viruses (fig. the first viral metagenomic study in korea surveyed uncultured viral assemblages in rice paddy soil in 2008, where mda was used with phi29 dna poly merase and random hexaprimers to amplify viral dna and to construct clone libraries for metagenome sequencing. the soil was found to contain a rich pool of unknown ssdna viruses and dsdna viruses. this study also focused on the effect of mda amplification on different types of genomic dna and showed that mda preferentially amplified circular dna genomes. this was also demonstrated using an environmental sample from surface seawater, where dsdna viruses alone were retrieved in the lasl library, whereas ssdna viruses were overwhelmingly represented in the mda library. thus, the amplification methods used in viral metagenomics can affect the ratios of viral sequences greatly and lead to inaccurate estimates of viral diversity. next, park et al. investigated the abundance and diversity of uncultured viral assemblages in fermented shrimp, kimchi, and sauerkraut-fermented foods that have been consumed for a long time around the world. in contrast to the soil virome, dsdna bacteriophages from the families myoviridae, podoviridae, and siphoviridae dominated the fermented foods, and they contained a low complexity of viral assemblages compared with other environmental habitats, such as seawater, human feces, marine sediment, and soil. however, it is possible that the viral diversity of the viromes detected in fermented foods may have been constrained to dsdna viruses by the lasl method. a large number of unknown microbes, such as bacteria, archaea, microbial eukarya, and viruses, constitute up to 10 bacteria per gram of feces in the human gastrointestinal tract, and it is expected that gut viruses will affect the relationships among viruses, bacteria, and gut epithelial cells. kim et al. investigated the abundance and diversity of dna viruses in fecal samples from five healthy koreans, particularly ssdna viruses. using epifluorescence microscopy with sybr gold staining, the viral abundance ranged from 10 to 10 per gram of feces, which was 10-fold less than the bacterial abundance in many other environments that harbored 10-100-fold more viruses (e.g., aquatic environments). moreover, the diversity of gut viral assemblages was lower than that of gut bacteria. who found that viral-microbial interactions in the human intestine could not be described as a predator-prey relationship, and instead, it was referred to as " kill the winner, " which was driven by a lytic life cycle. airborne viruses are now regarded as major environmental risk factors for complex disease pathogenesis [36-38]. however, the atmosphere remains " one of the last frontiers of biological exploration on earth ". using viral metagenomics with an advanced airborne particle sampling system, whon et al. conducted the first study of the diversity and community composition of airborne viruses in the near-surface atmosphere. the viral abundance in the atmosphere exhibited seasonal changes (increasing from autumn to winter before decreasing until spring) in the range of 10 to 10 viruses per m, and the temporal variations in viral abundance were inversely correlated with seasonal changes in temperature and absolute humidity. plant-associated ssdna geminivirus-related viruses and animal-infecting circoviruses dominated the viral assemblages, with low numbers of nanoviruses and microphages in air viromes, which suggests that airborne viral assemblages are affected greatly by terrestrial plants and animal activities. thus, the compositions of the viral assemblages detected in fermented foods and marine samples are biased toward dsdna viruses, such as sipho-, podo-, and myophages, when the lasl method is used, whereas the viral assemblages detected in rice paddy soil, human gut, marine, and near-surface atmosphere samples contain high proportions of ssdna viral sequences, due to the use of mda, as shown in fig., the compositions of the viral assemblages characterized in korean environments tend to depend on their specific microbial features. the human gastrointestinal tract and fermented foods are exposed to massive numbers of gut bacteria and lactic acid bacteria, respectively [39-41]. by contrast, the atmosphere contains far less cellular metabolism and reproductive activity than other environments, such as the soil, seawater, fermented foods, and the human gut. on this basis, the lowest abundance of eukaryotic viruses was observed in the viral assemblages in the human gut and fermented foods, whereas a high abundance of eukaryotic viruses was detected in the near-surface atmosphere. high levels of prokaryote and eukaryote cells are present in rice paddy soil and seawater, and so, comparable amounts of bacteriophages and eukaryotic viruses were detected in their viromes. the development of viral metagenomics has facilitated the discovery of novel, previously undescribed viral species. an artifact of the mda method is that it selectively amplifies the circular genomes of ssdna viruses, so that a large number of ssdna viral sequences have been identified in environmental viromes. thus, there is great interest in the distribution and host range of ssdna viruses. in particular, microphages from the family microviridae have been identified in a wide range of environments [13, 45, 46]. in contrast to the ecology of marine dsdna phages, marine ssdna phages in the family microviridae have distinct spatial and temporal distributions. ssdna microphages were abundant in the healthy human gut and their genotypes were much more diverse than those reported previously. moreover, prophage-like elements in the genomes of gut microbes, such as bacteroides and prevotella spp., were characterized as a novel subgroup in the family microviridae [13, 47], while viral sequences from the human gut were clustered with prophage-like elements from bacteroides and prevotella spp.. eukaryotic ssdna viruses that infect plants and mammals have been identified in many environmental viromes. circoviruses that are known to infect birds and pigs have also been identified in the viromes of invertebrates and a fish [48, 49], while geminiviruses that cause plant diseases have been detected in whiteflies, which act as insect vectors of plant viruses. a recent study by whon et al. investigated airborne dna viral assemblages in near-surface atmosphere samples and showed that a high number of viruses (log 6 to 7 viruses per m) were present in the air, which were dominated by geminivirus-related viruses., sclerotinia sclerotiorum hypovirulence-associated dna virus 1-which indicates that the airborne viral assemblages in the near-surface atmosphere may have strong interactions with plants. these results highlight the extensive distribution of ssdna viruses in a wide range of environments, and their host ranges may be wider than previously recognized. thus, the discovery of novel genomes of ssdna viral families in metagenomic studies could revolutionize our knowledge of the ecology and evolution of ssdna viruses. in the last decade, viral ecologists have focused on community-level analyses of viruses to understand their abundance and genetic diversity in specific environments. the ecological effects of viruses, particularly bacteriophages, are known to control host populations via the " kill the winner system, " while they drive mortality and evolutionary change in microorganisms via lateral gene transfer by infecting their host bacteria, although the basic issue of " who infects whom " is poorly understood [2, 29, 51, 52]. in the ocean, for example, viruses regulate the microbial abundance, release dissolved organic matter, and affect global biogeochemical cycles by killing up to 40% of host bacteria per day [53, 54]. in contrast, symbiotic functions of viruses, such as host survival, competition, and protection from pathogenic infections, are beginning to be understood, and evidence for a beneficial interaction in phage-host interactions was found in the mammalian gut ecosystem [29, 56]. when host survival is threatened, a variety of environmental factors can trigger prophage induction, and the liberated prophages may become completely virulent. overall, these studies suggest that prophage induction may responsible for triggering dysbiosis and changes in the microbial population by altering host phenotypes, thereby leading to a new environmental niche. traditionally, host culture-dependent techniques, such as plaque assays, have been widely used for the identification of phage and host bacteria interactions. however, plaque assays require isolated host bacteria; so, they are low-throughput methods. this method is also difficult to apply to environmental samples where lysogenic infections are prevalent, because the method relies on observations of visible plaque formations, which are often absent from lysogenic infections [3, 58, 59]. recently, deng et al. demonstrated a new technique, known as " viral tagging, " for identifying the interactions between cultivated host bacteria and their phages, which used the nucleic acid stain sybr gold to generate fluorescently labeled phages, so that the host cells fluoresced with viral tagging, thereby allowing the sorting of virus-tagged cells by flow cytometry [52, 60]. this emerging technique is undoubtedly helpful for not only exploring virus-host interactions in their natural habitats when the method is combined with other experimental tools, such as single viral genomics and phagefish, but also identifying viral receptors in macro-organisms (e.g., the mammalian gut) if the method is combined with a fluorescently labeled receptor protein during histological examinations. the emergence of viral metagenomics has facilitated advances in virology and allowed us to understand novel aspects of viral ecology. at present, viral metagenomics is a powerful and sensitive technique for detecting viruses that can not be identified by traditional culture- and sequence-based approaches. most importantly, viral metagenomics suggests that novel viruses interact constantly with the human population. thus, viral metagenomics can facilitate the improved surveillance of viral pathogens in the fields of public health and food security. this technique can be used to understand viral ecology by exploring the environmental viromes that are generated by viral metagenomics.

the introduction of metagenomics into the field of virology has facilitated the exploration of viral communities in various natural habitats. understanding the viral ecology of a variety of sample types throughout the biosphere is important per se, but it also has potential applications in clinical and diagnostic virology. however, the procedures used by viral metagenomics may produce technical errors, such as amplification bias, while public viral databases are very limited, which may hamper the determination of the viral diversity in samples. this review considers the current state of viral metagenomics, based on examples from korean viral metagenomic studies-i.e., rice paddy soil, fermented foods, human gut, seawater, and the near-surface atmosphere. viral metagenomics has become widespread due to various methodological developments, and much attention has been focused on studies that consider the intrinsic role of viruses that interact with their hosts.

PMC3794084

pubmed-1215

postoperative cognitive dysfunction (pocd) is the deterioration of cognitive function, especially learning and memory, which may last for days, months, or even years [13]. pocd occurs after cardiac and noncardiac surgeries and increases first-year morbidity and mortality after surgery [46]. there is no doubt that aged individuals are more likely to develop pocd [79]. maze's group demonstrated that learning and memory were impaired after anesthesia and surgery in 3-4-month-old mice.. demonstrated that there were no signs of neuroinflammation or cognition impairment after surgery in adult mice (46-month-old). similarly, wuri et al. observed no learning or memory changes after partial hepatectomy in adult mice (4-month-old). according to the work of finlay and darlington, mice after two months accordingly, 36-month-old mice and 2-month-old mice are biologically equivalent to 3040-year-old human and college freshmen, respectively. while maze's group investigated the learning and memory after anesthesia and surgery in 3-4-month-old mice, no data in younger adult mice, that is, 2-month-old, are available. unfortunately, this is an age at which particular diseases will arise and may need surgeries, such as appendicitis, osteosarcoma, and leukemia. in the present study, we investigated whether the spatial reference memory of 2-month-old mice will be affected after anesthesia and surgery. this study was approved by the animal care and use committee of shanghai jiao tong university, school of medicine. all animal procedures were performed in accordance with the national institutes of health (nih) animal care guidelines. two-month-old male c57bl/6j mice were provided by the animal research center of shanghai jiaotong university, school of medicine. the animals were housed in standard cages under controlled laboratory conditions (temperature of 22 2c, 12-hour light/12-hour dark cycle) with free access to regular rodent pellets and water. all mice were allowed to adapt to their new environment for 7 days before beginning the experiments. mice were randomly divided into three groups: nave group, anesthesia group, and surgery group. splenectomy was performed with neuroleptic anesthesia (intraperitoneal injection of 200 g/kg fentanyl and 10 mg/kg droperidol, as reported previously [15, 16 ]) in the surgery group. for the splenectomy, a small incision was made in the left upper abdominal quadrant, and the spleen was mobilized, isolated, and removed. a single dose of butorphanol (0.4 mg/kg, s.c.) was administered for postoperative analgesia at the end of surgery. spatial reference memory was evaluated in the mwm using a computerized video tracking system (days 37 and days 6266 in figure 1, n=15). the reference memory test was performed on four days (days 36 and days 6265 in figure 1). for the reference memory, briefly, a hidden round platform was placed 1 cm below the water surface and located in the center of the northeast quadrant in a circular pool (110 cm in diameter and 30 cm in depth). the water was maintained at 2325c, and the pool was situated in a room with visual cues. in all the trials, each mouse was released into the water facing the pool wall from one of four separate quadrants and allowed to swim until it landed on the platform. once the mouse found the platform, the trial was terminated, and the mouse was allowed to stay on the platform for 15 s. if the mouse failed to find the platform within 60 s, it was gently guided to the platform and allowed to remain on the platform for 15 s. four trials were conducted per day, separated by a 5-minute intertrial interval, and the platform remained at the same location throughout the test. the amount of time spent finding and mounting the platform (escape latency) and the swimming speed were calculated from the recorded videos using mwm software (shanghai jiliang software technology co. ltd., china). the probe test was performed on the day after the reference memory test (day 7 and day 66 in figure 1). in this test, the platform was absent, and the animals were allowed to swim freely for 60 s, starting from the quadrant opposite the platform. seven days or 66 days after surgery (day 8 and day 67 in figure 1, n=15), the avoidance learning task was performed as previously described [17, 18] in a y-maze equipped with electric grids in the floor. the grids were controlled by a computer, and a camera on the top of the y-maze recorded and provided the position of the animals to the computer. the animals had to leave the start arm within 5 seconds and escape into the correct arm to avoid foot shocks. active avoidance errors were recorded if the animals did not leave the start arm within 5 seconds. if the mouse chose the foot shocks were administered for 7 seconds each until the animals chose the correct arm. the foot shock level was changed individually (maximum: 40 v) according to the performance of the mouse in the first trial or until the mouse suddenly lifted one or two paws from the grid at the bottom of the y-maze after the shock. one trial per minute was performed until the mouse reached the final criterion of correctly performing seven out of eight consecutive trials. in order to avoid odor confounding, the numbers of total trials, active avoidance errors, and discrimination errors were recorded. mice in each group were sacrificed 2 h, 6 h, 24 h, and 48 h after surgery. hippocampus dissections were performed on ice-cold frosted glass, and tissues were quickly frozen in liquid nitrogen. the mouse hippocampus was homogenized in sterile 0.1 m pbs containing a complete protease inhibitor cocktail (roche). the homogenates were centrifuged at 10,000 rpm for 15 min at 4c, and the supernatants were analyzed for il-1 and il-6 using elisa kit (r&d systems, minneapolis, mn). the protein concentrations of all samples were measured using a bca protein assay kit (pierce). for immunohistochemical analysis of microglia, all mice (n=4) were deeply anesthetized using equithesin [1% pentobarbital/4% (v/v) chloral hydrate; 3.5 ml/kg, i.p.] and perfused intracardially with saline followed by 4% paraformaldehyde in 0.1 m phosphate buffer (pb, ph 7.4). brains were then harvested, postfixed in the same fixative for 4 hours at 4c, and immersed in 1030% gradient sucrose in pb for 2448 hours at 4c for cryoprotection. brain tissue was freeze-mounted in oct embedding medium, and 16 m thick coronal sections of hippocampus were cut sequentially and mounted on superfrost plus slides. slices were permeabilized in 0.4% triton x-100, blocked with 5% bovine serum albumin in 0.1% triton x-100, and incubated overnight at 4c with mouse anti-cd11b (abcam, cambridge, uk, 1: 100). after rinsing in 0.1% triton x-100 in pbs, sections were incubated with secondary antibodies conjugated with alexa fluor 488 (1: 500; invitrogen; paisley, uk) for 1 hour in the dark. all the procedures for negative controls were processed in the same manner except omitting primary antibody. the statistical package for the social sciences (spss) v.20.0 was used for the statistical analyses. two-way anova with repeated measures was used to analyze the water maze escape latency and average speed. one-way anova was used for the probe quadrant trial data, avoidance learning task data, and the il-1 and il-6, followed by post hoc bonferroni correction. upregulation of these two cytokines was observed at 6 hours after surgery and decreased again by 24 hours postoperatively (figures 2(a) and 2(b)). anesthesia per se did not affect the level of il-1 or il-6 at any time point compared with the nave controls. cd11b immunostaining showed that very few microglia were activated in the hippocampus from mice of nave or anesthesia groups (figure 3). on the contrary, surgery profoundly induced microglia activation in the hippocampus, which was intensive at 6 and 24 hours after surgery but started to decline by 48 hours (figure 3). the results of the morris water maze performed two days and two months after surgery revealed no differences among the three groups in latency, swimming speed, or swimming time in the target quadrant during the probe test (figures 4 and 5). similarly, no significant differences were observed in the results of the avoidance learning task in the y-maze, regardless of the number of learning trials, avoidance errors, or discrimination errors or voltage (figures 4 and 5). we demonstrated in the present study that (a) anesthesia and surgery but not anesthesia only temporally increased hippocampal il-1 and il-6, as well as microglia activation in the hippocampus in 2-month-old young adult mice and that (b) anesthesia and surgery can not hurt the short-term and long-term reference memory of such aged mice. these findings indicate that central inflammation induced by surgery does not necessarily lead to reference memory impairment in young adult mice. our present results are consistent with previous studies demonstrating that neuroinflammation can be induced by surgery.. demonstrated that the concentration of il-6 in cerebrospinal fluid significantly increased 1 week after cardiac surgery. [11, 20, 21] demonstrated that serum tnf- induced by surgery disturbed the blood-brain barrier (bbb), which then stimulated macrophage migration into the hippocampus and promoted hippocampal neuroinflammation. we assume that the increases of il-1 and il-6 in hippocampus after surgery in the present study may arise through the same pathway, but more studies are needed to confirm the assumption in the young adult mice. other proinflammatory cytokines may also be involved in the development of pocd in aged animals. ma et al. demonstrated that tnf-, il-1, il-4, and il-6 in the hippocampus increased after surgery and the tnf- receptor antagonist attenuated the elevation of these cytokines. wang et al. found that the hippocampal il-1, tnf-, and ifn- were overexpressed after surgery in aged mice.. demonstrated that the hippocampal il-6, il-12, and il1 increased after surgery in aged rats. more studies should be done to investigate whether such proinflammatory cytokines changed after surgery in young adult animals. however, the neuroinflammation detected in the current study is insufficient to cause learning or memory impairment in such young adult mice, as we did not observe any short- or long-term changes in these aspects after surgery. we postulate that this is because the immune system in young adult mice is very strong so that the anti-inflammatory pathways, such as the vagus nerve pathway, are rapidly activated to reduce the inflammation. in many previous experiments reporting impaired learning or memory ability after surgery, at least one other contributing factor exists. for example, fidalgo et al. observed impaired learning and memory after surgery in adult mice when they were simultaneously infected with lps (50 ng/kg, a subclinical dose). the surgery-induced learning and memory impairment demonstrated by he et al., wan et al., and cao et al. together, results from current and previous findings all indicate that other factors, such as old age or subclinical infection, are required so that the neuroinflammation induced by surgery would be deleterious enough to alter spatial reference memory in mice. morris water maze and active avoidance test were used in the present study to measure the spatial reference memory changes after anesthesia and surgery. spatial reference memory is the ability to remember the relevance of spaces, which is a relative long-term memory compared with working memory. working memory is a limited capacity that is responsible for the transient holding, processing, and manipulation of information, which is a relative short-term memory. more studies should be done to investigate the changes of working memory after anesthesia and surgery. age is probably one of the key determinants of the responses to anesthesia and surgery. as most previous studies utilized 46-month-old mice [15, 20, 21, 30], the present study used for the first time two-month-old mice, which are biologically equivalent to 18-year-old human. differences in ages among our study and previous studies are very possibly the reason for the different behavior tests results. since the spleen is an immunoregulatory organ and splenectomy may deteriorate the immunological system and therefore lead to an exaggerated inflammatory status, we are unable to rule out the possibility that the absence of spleen also contributed to the development of neuroinflammation. other surgical procedures are needed to further determine the role of surgical trauma in the neuroinflammation and the following learning and memory changes. anesthesia and surgery lead to neuroinflammation. however, such neuroinflammation is insufficient to impair the spatial reference memory of young adult mice.

postoperative cognitive dysfunction (pocd) increases morbidity and mortality after surgery. but the underlying mechanism is not clear yet. while age is now accepted as the top one risk factor for pocd, results from studies investigating postoperative cognitive functions in adults have been controversial, and data about the very young adult individuals are lacking. the present study investigated the spatial reference memory, il-1, il-6, and microglia activation changes in the hippocampus in 2-month-old mice after anesthesia and surgery. we found that hippocampal il-1 and il-6 increased at 6 hours after surgery. microglia were profoundly activated in the hippocampus 6 to 24 hours after surgery. however, no significant behavior changes were found in these mice. these results indicate that although anesthesia and surgery led to neuroinflammation, the latter was insufficient to impair the spatial reference memory of young adult mice.

PMC5124473

pubmed-1216

the over enthusiastic approach in maintaining tight blood sugar control so as to reduce the long-term complications of diabetes have resulted in increase in frequency of this complication. this situation seems to get worsened with use of combination of anti-diabetic drugs. the american diabetes association defines the hypoglycemia as any abnormally low plasma glucose concentration that exposes the subject to potential harm, and proposes a threshold of<70 mg%. the spectrum of symptoms depends on duration and severity of hypoglycemia and varies from autonomic activation to behavioral changes to altered cognitive function to seizures or coma. the short and long-term complications include neurologic damage, trauma, cardiovascular events and death. there can be a six fold higher incidence of death, increased costs of medical care, and loss of productivity due to hypoglycemia. apart from patient-related factors like lifestyle and comorbid conditions of the patients, various other factors like choice, dose, timing and combination of anti-diabetic drugs together with simultaneous use of other interacting drugs can increase the risk of hypoglycemia in diabetics. some studies have been conducted to evaluate the knowledge and awareness about hypoglycemia in diabetics. but in this study we have tried to evaluate how successful can diabetic education prove to be in improving the awareness of hypoglycemia and the practices adopted by the diabetics for its prevention. this would be helpful in formulating certain strategies that can keep a check on this common complication of diabetes treatment. the diabetic patients attending out-door facility of the hospital and who were being treated with oral hypoglycemic drugs were included in the study after obtaining their written informed consent. some important demographic characteristics of the patients like age, gender, education level were studied. the questionnaire was given in hindi for easy comprehension of the patients and their attendants. they were then prior asked for ability to read and comprehend the questionnaire. in the case of illiterate patients and attendants, the questionnaire had 20 questions to assess the knowledge (7), attitude (7) and practices (6) (kap) of the diabetic patients toward prevention of hypoglycemia. the knowledge part of the questionnaire was to assess the knowledge of the possibility of hypoglycemic episodes in the diabetic patients and its common symptoms. the questions asked from the patients to judge his knowledge aspect are given in table 1. attitude part of the questionnaire was to evaluate the beliefs of the patient regarding simple preventive measures for avoiding hypoglycemia. the practice part of the questionnaire was to judge how the knowledge and attitudes of the patients are practically put into action. each correct response was given a score of one and each wrong answer or unsure response was given a score of zero. seven questions from stanford questionnaire were also included in the performa to check the incidence of hypoglycemic symptoms in the patients in the past 1-week. the symptoms enquired were morning headaches, nightmares, night sweats, light headedness, shakiness or weakness, intense hunger and passing out episodes. the maximum possible score for each of the observed parameter was seven except for practices parameter where the maximum score was 6. after filling the performa, the diabetic patients and their attendants were educated by the treating doctor regarding possibility of hypoglycemia as a complication of diabetes treatment, its consequences, common hypoglycemic symptoms and some simple precautions to be taken to avoid its occurrence. patients were also advised to adopt the practice of self-monitoring of blood glucose. the patients were followed up after a month and again given the same kap questionnaire to check for improvement in their kap toward hypoglycemia. the incidence of hypoglycemic episodes in the last 1-week was again checked with the help of stanford questionnaire. the baseline scores and the follow-up scores were compared by paired t-test to assess the effect of diabetes care education on prevention of hypoglycemic episodes and improvements in kap of diabetic patients toward hypoglycemia. out of 137 patients who were given questionnaire, only 109 patients were included for the final analysis. rest of the patients were lost to follow-up. out of these 109 patients, the mean age of the patients at the time of inclusion in the study is 53.8 1.1 years. demographic characteristics of the patients the mean baseline scores and postdiabetic education scores for each parameter like kap and hypoglycemic symptoms are summarized in table 3. it is evident that there is a significant (p<0.001) improvement in the kap of diabetics after diabetic education. mean baseline scores and scores after diabetic education the percentage of patients responding correctly to each individual question of each parameter is indicated in [figures 1-3]. frequency distribution of diabetics according to their knowledge of hypoglycemia frequency distribution of diabetics according to their attitude toward hypoglycemia prevention frequency distribution of diabetics according to their self-reported practices frequency distribution of diabetics according to their hypoglycemic symptoms there is a big lacuna in the existing knowledge and attitude regarding hypoglycemia in diabetics. although many patients give importance to timely intake of meals and medicines, but the attitude toward other parameters like self-monitoring of blood glucose, keeping toffees or candies for an emergency situation and avoiding excessive exercises is largely lacking. with regard to practices, the situation is even worse. many patients who had good knowledge and beliefs about hypoglycemia did not still put it into practice. apart from a lack of awareness, forgetfulness and busy job schedule of the patients were the most common reasons, which did not allow a large number of patients to be self-disciplined regarding timely intake of meals and medicines. among the patients who knew about the importance of self-monitoring of blood glucose levels, many were not able to implement it due to lack of resources and education. many patients suffered from hypoglycemic symptoms in the past 1-week, as judged by the stanford questionnaire. out of all the symptoms, weakness, shakiness, and intense hunger were most frequently complained of. with diabetic education, there is a significant improvement both in knowledge and attitude of the patients. a significant number of patients now know about the possibility of hypoglycemia in diabetics and the dangerous nature of hypoglycemic episodes. a lot of patients have started believing in the importance of knowing about the hypoglycemic symptoms so as to prevent them. although there is an improvement in the practices of the patient also but it was not equivalent to the improvement in knowledge and attitude of the patients. best-followed practices were regular and timely intake of meals and medicines. a large number of patients also started paying attention to warning episodes of hypoglycemia. but, unfortunately, the practice of self-monitoring of blood glucose and keeping toffees and candies as an emergency measure was least commonly followed. although there has been a significant decrease in the overall hypoglycemic symptom score (stanford score) after diabetic education, but the complaint of night sweats and light headedness did not decrease even after diabetic education. the decrease in overall hypoglycemic symptom score is due to less hypoglycemic episodes seen in the patients and is indicative of the good influence of diabetic education on the patients. thus, proper diabetic education provides us with a ray of hope of improving the knowledge and attitude of the patients and decreasing the hypoglycemic episodes in diabetics. but one of the major challenges in the way of diabetic education is busy time schedule of the doctors that does not allow adequate time for their interaction with the patients. low literacy level of the patients and their attendants leading onto incomplete interpretation of the instructions is another major problem. many of the patients tend to forget the advices given by the health care providers, but this problem may largely be overcome by repeated health education and motivation. the hypoglycemic symptoms as judged by stanford questionnaire were not backed up with biochemical confirmation. recall bias was another limitation of the study as the answers to the questionnaire were largely subjected to patient's memory. only one session of diabetic education was given to the patients before their next evaluation. proper diabetic education of diabetic patients can prove to be very valuable tool for prevention of hypoglycemia. but the important hurdles in its way are busy and hectic schedule of health care providers, low literacy level and forgetfulness of the patients and their attendants, busy jobs of some of the patients and their low socio-economic levels. in spite of these obstacles, repeated and regular education, motivation and encouragement of the patients can not only improve the knowledge of the patients but also reduce the gap between knowledge and practices.

aims: to assess the role of diabetic education in increasing awareness about hypoglycemia and decreasing hypoglycemic symptoms in diabetics. materials and methods: this is a longitudinal study involving the use of a structured questionnaire for obtaining baseline information related to knowledge, attitude and practices (kap) of diabetic patients regarding hypoglycemia. then the patients were given diabetic education by the treating doctor regarding hypoglycemia, its symptoms and prevention; the effect of which was assessed by repeating the same questionnaire after a month. the occurrence of hypoglycemic symptoms was also compared before and after diabetic education. results:there is a significant improvement in all parameters like kap with diabetic education. the hypoglycemic episodes also decrease significantly. conclusions:proper diabetic education is seen to improve the knowledge and attitude of the diabetic patients toward hypoglycemia. this leads to improved practices of such patients and decrease hypoglycemic episodes in them.

PMC4366778

pubmed-1217

during human gestation the placenta as a temporal villous organ fulfills a wide spread panel of pregnancy maintaining functions, including exchange of gases and metabolites, regulation of water balance, and secretion of endocrine factors. the vast majority of placenta derived endocrine factors are synthesized in the syncytiotrophoblast, which as a unique epithelium-like layer without lateral cell borders covers all placental villous trees as well as parts of the inner surfaces of chorionic and basal plates. thus, the syncytiotrophoblast lines the intervillous space and hence is exposed to maternal blood. beside analogues of virtually all known classical hypothalamic and pituitary hormones, the human syncytiotrophoblast also synthesizes steroid hormones, monoamines, adrenal-like peptides, cytokines, and chemokines [2, 3]. chemokines are classified into four subfamilies according to the number and spacing of the first two cysteine residues in a conserved cystein structural motif. these four subclasses are referred to as c, cc, cxc, and cx3c, where c is a cysteine and x any amino-acid residue. the cx3c subclass was discovered in the late 1990s and contains only one member, termed fractalkine, or cx3cl1. fractalkine is synthesized as a 373 amino-acid transmembrane molecule, comprising an extracellular n-terminal domain, a mucin-like stalk, a transmembrane -helix, and a short cytoplasmic tail [6, 7]. the extracellular domains, representing the chemokine domain and the mucin-like stalk, can be shed by metalloproteases into a soluble isoform [810]. thus, fractalkine exists as both, a membrane-bound and a soluble form a situation considered as unique amongst the group of chemokines. while the soluble form has chemoattractive activity for monocytes, natural killer cells, and t-cells, the membrane-bound form promotes flow resistant adhesion of leukocytes to endothelial cells via its corresponding g protein-coupled, 7-transmembrane receptor cx3cr1. based on that, fractalkine may be considered as inflammatory chemokine expressed in activated endothelial and epithelial cells, as well as in dendritic cells, lymphocytes, osteoblasts, neurons, and microglial cells [1214]. according to tissue distribution analysis fractalkine mrna is most abundantly expressed in brain, heart, kidney, lung, and pancreas but can also be detected in human placenta. however, current knowledge on placenta derived fractalkine and its implications on pregnancy is limited and based on a small number of studies. placental fractalkine expression was initially demonstrated in villous trophoblast and the amniotic epithelium, which was suggested as resource for substantial release of soluble fractalkine into amniotic fluid of human second and third trimester pregnancies. studies by hannan et al. showed fractalkine expression by semiquantitative rt-pcr in primary endometrial epithelial cells and the trophoblast cell lines jeg-3, ac1m-32, and ac1m-88. migration and adhesion studies by the same group suggested fractalkine to be involved in embryo implantation processes. recently, increased placental fractalkine expression was suggested to contribute to increased microvessel density in placental tissue from pregnancies complicated by diabetes mellitus. in the light of the broad panel of factors released from human placenta thus, we aimed to analyse the spatiotemporal expression of placental fractalkine and tested the hypothesis whether it can be shed and released into the intervillous space, that is, the maternal circulation. the study was approved by the ethical committee of the medical university of graz and informed consent was obtained from the women. first trimester placentas (mean gestational week: 9.4 1.7) were obtained from women (mean maternal age: 28.1 6.2 years; mean body mass index: 24.2 5.0) undergoing pregnancy terminations for psychosocial reasons. term placentas were obtained after delivery (mean gestational age: 39.4 0.9 weeks) from healthy women (mean maternal age: 34.8 3.7 years; mean body mass index: 23.4 4.4) with singleton pregnancies and no clinical evidence of infection. pregnancies complicated by hypertension, preeclampsia, metabolic disease, steroid treatment, aids, alcohol abuse, and/or drug abuse were excluded. formalin fixed and paraffin-embedded (ffpe) tissue sections (5 m) from ten first trimester and ten term placentas were mounted on superfrost plus slides (menzel/thermo fisher scientific). after standard deparaffination, tissue sections were subjected to antigen retrieval by boiling slides in epitope retrieval solution ph 9.0 (novocostra, leica) for 7 min at 120c in a decloaking chamber (biocare medical). sections were immunostained using a staining robot (autostainer 360, thermo fisher scientific) and the ultravision large volume detection system hrp polymer kit (thermo fisher scientific) according to manufacturer's instruction. in brief, washing steps with tbs including 0.05% tween 20 (tbs/t; merck) were followed by background blocking with ultra v block for 5 min. monoclonal anti-human cx3cl1/fractalkine antibody (clone 81513, r&d systems) was diluted 1: 1000 (0.5 g/ml working concentration) in antibody diluent (dako) and incubated on slides for 30 min at rt. after tbs/t washing steps, primary antibody enhancer was applied to slides for 10 min at rt. following another washing, detection was achieved by incubation with the anti-mouse/rabbit ultravision hrp-labelled polymer system (15 min) and 3-amino-9-ethylcarbacole (aec, dako), according to manufacturer's instructions. for negative controls, slides were incubated with negative control mouse igg1 (dako) at the same concentration as mentioned above. moreover, specificity of monoclonal anti-human cx3cl1/fractalkine antibody was evaluated by an antibody preadsorption approach. for this purpose monoclonal anti-human cx3cl1/fractalkine antibody (1: 1000, 0.5 g/ml working concentration) was mixed in antibody diluent with an excessive amount of recombinant human full length fractalkine (5 g/ml working concentration, rhcx3cl1/fractalkine, r&d systems) and incubated with gentle shaking 1 h at rt. a mixture containing solely monoclonal anti-human cx3cl1/fractalkine antibody was incubated in parallel and served as control. bewo cells were purchased from the european collection of cell cultures (ecacc) and cultured in dmem/f12 (1: 1, gibco), supplemented with 10% fcs, penicillin/streptomycin, amphotericin b, and l-glutamine, at 37c in a humidified atmosphere containing 5% co2 in air. differentiation of bewo cells was induced with forskolin, which was supplemented to the culture medium with a final concentration of 20 m (10 mm stock in dmso). for experiments testing different concentrations of the metalloprotease inhibitor batimastat (tocris bioscience), 1 10 bewo cells were seeded in 24-well culture dishes (nunc, thermo fisher scientific) and 1 ml/well of above described culture medium. for all other bewo cell experiments, cells were seeded in 12-well culture dishes (2 10 cells/well) and 2 ml/well culture medium. one day after seeding, cells were incubated with culture medium supplemented with or without forskolin (20 m) and batimastat (10 m). cells cultured in culture medium containing equal volumes of dmso served as solvent controls. at the end of incubation cells were washed with buffered saline and lysed with ripa buffer (sigma-aldrich) including protease inhibitor cocktail (roche diagnostics, indianapolis, ia, usa). the effect of batimastat on viability of bewo cells was analyzed by a methyl tetrazolium salt (mts) based cell viability assay (celltiter 96 aqueous one solution cell proliferation assay, promega), according to manufacturer's protocol. in brief, 2.5 10 bewo cells were seeded in 100 l culture medium per well in a 96-well dish. one day after seeding, cells were incubated in culture medium supplemented with batimastat (10 m) or solvent control dmso (0.1%) for 48 h. after incubation, 20 l mts solution reagent was added to each well and plates incubated for 1 h. thereafter absorbance was recorded at 492 nm using a plate reader and absorbance values for dmso control were set to 100%. primary trophoblasts were isolated from chorionic villi of four term placentas by enzymatic digestion and percoll density gradient centrifugation as described previously. cells were seeded in 6-well culture dishes (3 10/well) and 2 ml/well dmem (gibco) supplemented with 10% fcs and cultured in a hypoxic workstation (biospherix) under 8% oxygen at 37c. one day after seeding culture medium was exchanged with dmem/ebm (1: 1, gibco/lonza) supplemented with 7.5% fcs and cells incubated for another 48 h under 8% oxygen at 37c. villous tissues from human first trimester (n=7, between gestational week 7 and 12) and term placentas (n=3, between gestational week 38 and 40) were washed thoroughly in buffered saline and dissected into small pieces of approximately 5 mg moist mass. placental explants were cultured in dmem/f12 (1: 1, gibco) supplemented with 10% fcs, penicillin/streptomycin, amphotericin b, and l-glutamine with or without batimastat (10 m) in a hypoxic workstation (biospherix) under 2.5% oxygen (first trimester explants) and 8% oxygen (term explants) for 5 days at 37c. placental explants cultured in culture medium containing the same volume of dmso served as controls. after incubation, conditioned culture media were collected and placental explants homogenized in ripa buffer with protease inhibitor cocktail using a tissue homogenizer (ika ta10 basic, ultra-turrax). viability of placental explants was evaluated after culture by immunohistochemical staining of proliferation marker ki67 (clone mib-1, 1 g/ml, dako) and hcg (clone h-298-12, 1: 10, bioprime/biologo) as described in immunohistochemistry section. both batimastat treated and control explants showed proliferation of cytotrophoblasts and synthesis of hcg in the syncytiotrophoblast. moreover, effect of batimastat treatment was analyzed and compared with dmso control by measurement of released lactate dehydrogenase (ldh) activity in culture supernatants using ldh cytotoxicity detection kit (takara bio inc. obtained absorbance values were normalized to total protein of respective explant homogenates and dmso control set as one. total rna from trophoblasts and placental tissues was isolated using a column based rna isolation kit (sv total rna isolation system, promega) including an on column dnase treatment step. after quality check, total rna was subjected to quantitative gene expression analysis using a one-step rt-pcr kit (qiagen) and a predesigned expression assay for fractalkine (hs_cx3cl1_qf_1 quantifast probe assay, qiagen) according to manufacturer's instructions. in brief, 100 ng total rna of each sample was mixed with kit components in a total reaction volume of 20 l. samples were analyzed in triplicate in 96-well plates (roche diagnostics) and a bio-rad cxf96 real-time pcr system. cycle conditions included reverse transcription for 20 min at 50c, an initial pcr activation step for 5 min at 95c, and subsequent 2-step cycling with denaturing for 15 s at 95c and annealing/extension for 30 s at 60c for a total of 40 cycles. ct values were automatically generated by the cfx manager 2.0 software (bio-rad) and relative quantification of gene expression was calculated by standard ct method using the expression of beta-2-microglobulin (hs_b2m_qf_2 quantifast probe assay, qiagen) as reference. b2 m was validated by comparison with the expression of other reference genes, ribosomal protein l30 (hs_rpl30_qf_1 quantifast probe assay), hypoxanthine phosphoribosyltransferase 1 (hs_hprt1_qf_2 quantifast probe assay), and 18s rrna (hs_rn18s1_qf_2 quantifast probe assay) and showed no significant developmental or cell differentiation dependent changes. placental villous tissue was thoroughly washed with pbs and homogenized in ripa buffer including protease inhibitor cocktail using a tissue homogenizer. after determination of protein concentration according to lowry et al., 60 g total protein was applied and separated on precast 10% bis-tris gels (nupage, novex; invitrogen). 100 ng recombinant human full length fractalkine (rhcx3cl1/fractalkine, r&d systems) was applied as positive control. electrophoresis was followed by semidry blotting of proteins on 0.2 m nitrocellulose membranes (trans-blot, bio-rad laboratories). blotting efficiency was determined by staining membranes with ponceau s solution (sigma aldrich). immunodetection was conducted with a chemiluminescent immunodetection kit (western breeze; invitrogen) according to manufacturer's instructions. monoclonal anti-human cx3cl1/fractalkine antibody (clone 81513, r&d systems) was diluted in blocking solution 1: 500 (1 g/ml working concentration) and applied to the membrane overnight at 4c. for normalization membranes were incubated with monoclonal anti-beta actin antibody (1: 20.000; clone ac-15, abcam, cambridge, uk). images were acquired with fluorchem q system (alpha innotech, cell bioscienes, santa clara, ca, usa) and band densities were analyzed with alpha view sa software 3.4.0. results are presented as a ratio of relative fractalkine and beta-actin band densities, with first trimester samples set to one. fractalkine was measured in cell culture supernatants and cell lysates as well as tissue homogenates using a quantitative sandwich enzyme immunoassay (human cx3cl1/fractalkine quantikine elisa, r&d systems). cell culture supernatants were centrifuged at 1.500 g and 4c for 5 min. cell lysates and placental explant homogenates were centrifuged at 8.000 g and 4c for 10 min. after centrifugation 100 l of clear supernatants was subjected to immunoassays according to manufacturer's instruction. complete culture medium incubated without cells and ripa buffer served as blank for fractalkine measurement in conditioned supernatants and cell lysates, respectively. samples were measured in duplicate and obtained fractalkine concentrations normalized to total cell protein or total tissue protein, respectively, which was determined in lysates according to lowry method. data were subjected to normality test (shapiro-wilk test) and equal variance test. in case of normally distributed data differences between groups were tested using two-tailed t-test. otherwise mann-whitney rank sum test was applied. immunohistochemical staining of human first trimester placental sections localized fractalkine at the apical microvillous plasma membrane of the syncytiotrophoblast (figure 1(a)). the fetal endothelium, villous cytotrophoblasts, and extravillous trophoblasts in cell columns did not express fractalkine (figures 1(a) and 1(b)). in first trimester decidua fractalkine was detected at the apical plasma membrane of uterine glandular epithelial cells (figure 1(c)). neither spiral arteries nor uterine veins showed endothelial staining (figures 1(c) and 1(d)). in human term placenta fractalkine was detected at the apical plasma membrane of the syncytiotrophoblast (figure 1(e)). no staining was observed in the fetal vascular endothelium of terminal villi and stem villi (figures 1(e) and 1(f)). to get an idea of putative changes of placental fractalkine expression over gestation, placental tissues were analyzed at first trimester and term. quantitative gene expression analysis revealed a 15.1-fold (0.9) increase in placental fractalkine mrna expression at term, when compared to first trimester (figure 2(a)). on protein level, placental fractalkine was detected by immunoblotting of first trimester and term placenta tissue homogenates and corresponded with recombinant 90 kda full length fractalkine, which served as positive control (figure 2(b)). in contrast to quantitative gene expression analysis, semiquantitative band densitometry of immunoblots showed only a 1.7-fold (0.1) increase of placental fractalkine at term, when compared to first trimester (figure 2(c)). immunohistochemistry suggested the syncytiotrophoblast to be the main source of placental fractalkine expression. in order to substantiate this finding the trophoblast cell line bewo, a well accepted model for the villous trophoblast population, showing secretion of pregnancy-specific hormones as well as good syncytialization, that is, formation of trophoblast syncytia in vitro, was tested for its capacity to express and release the chemokine. while basal expression of fractalkine mrna was low but detectable in untreated bewo cells, incubation with forskolin, a reagent known to induce bewo cell differentiation and syncytialization, led to a time dependent increase over time with a 22.1-fold (1.6) upregulation compared to vehicle control after 48 h (figure 3(a)). forskolin induced fractalkine expression in bewo cells was accompanied by a 9.8-fold increase in the release of soluble fractalkine, which accounted for 2.91 (0.34) ng/mg cell protein after 48 h incubation (figure 3(b)). since trophoblast cell lines may differ in some aspects when compared to their primary counterpart, primary trophoblasts shown to spontaneously form syncytia in vitro were isolated from term placenta and tested for their capacity to release soluble fractalkine. analysis of supernatants from primary term trophoblasts showed continuous release of soluble fractalkine, which increased by 39.5% between 24 h and 48 h of incubation (figure 3(c)). data from trophoblast culture provided strong evidence that fractalkine is not only expressed but also released from villous trophoblast. since data on placental fractalkine release have virtually not been described so far, it was tempting to test if the previously described mechanism of metalloprotease mediated shedding of the transmembrane form also applies for human placenta, that is, human trophoblast. for this purpose the release of soluble fractalkine was first analyzed in forskolin treated bewo cells in presence and absence of the metalloprotease inhibitor batimastat, which has previously been shown to effectively block fractalkine shedding in other cell types such as smooth muscle cells and hepatic stellate cells [24, 25]. incubation with 5 m and 10 m batimastat decreased the release of soluble fractalkine by 67.1% and 91.6%, respectively, compared to cells treated without the inhibitor after 48 h (figure 4(a)). analyses of cell lysates from forskolin stimulated bewo cells incubated with or without batimastat revealed that inhibition of soluble fractalkine release was accompanied by a 3.1-fold increase of cell associated fractalkine compared to control after 48 h (figure 4(b)). to ensure that observed effects were not due to changes in cell viability, batimastat treated and control cells were analyzed using a mts based viability assay. accordingly, batimastat treatment slightly but not significantly decreased the relative number of viable bewo cells in proliferation by 4.3% compared to control after 48 h (figure 4(c)). the situation observed in bewo cells was at least in part reflected in explant culture of human first trimester and term placenta. in first trimester placental explants, batimastat decreased the release of soluble fractalkine by 17.8% after 5-day culture, which did not reach statistical significance. however, analyses of respective tissue homogenates revealed a significant 1.6-fold increase of tissue associated fractalkine in explants incubated with batimastat, when compared to controls after 5 days (figure 5(a)). in explants from human term placenta, incubation with batimastat showed a considerable decline of soluble fractalkine release by 56.3%, while at the same time the fraction of tissue associated fractalkine increased 1.5-fold when compared to controls (figure 5(b)). comparison of controls from first trimester and term explant experiments revealed a 5.0-fold increase in tissue associated as well as released fractalkine towards term. however, the ratio of released versus tissue associated fractalkine remained constant and was 2.1 in both first trimester and term placental explants, suggesting constitutive shedding of placental fractalkine. in order to determine any cytotoxic effects of batimastat on placental explants, the release of ldh into the culture medium was analyzed after culture and showed a slight but nonsignificant increase by 8.5% and 10.4% after batimastat treatment of first trimester and term explants, respectively, when compared to controls (figure 5(c)). the concept of the dual nature of fractalkine, acting both as soluble chemoattractive factor and transmembrane adhesion molecule, can be well applied for fractalkine expressed in human placenta. data from placental explant and trophoblast culture provide strong evidence that placental fractalkine is constitutively released from the syncytiotrophoblast into the maternal circulation via metalloprotease dependent shedding. when speculating about a putative role of shed placental fractalkine in the fetal-maternal cross-talk, important aspects of placental development should be considered. during early pregnancy perfusion of the intervillous space with maternal blood is not yet fully established, and thus shed placental fractalkine may not act locally on maternal cx3cr1 expressing cells, but rather in an endocrine way. in doing so, placenta derived fractalkine may contribute to the low grade systemic inflammatory responses described to occur in third trimester of pregnancy [2628]. this assumption is in good agreement with increasing expression and release of placental fractalkine towards term. mild inflammatory responses were suggested to contribute to maternal metabolic changes, resulting in insulin resistance and hyperlipidaemia, which accommodate increased energy demands of the growing fetus. contribution of placental fractalkine to the maternal pool of soluble fractalkine during gestation is hard to estimate and should include the fact that the entire surface of placental villi at term with approximately 1215 m represents only a very small area compared to approximately 40007000 m endothelium of maternal blood vessels [1, 29]. expression analysis of other proinflammatory cytokines, such as tnf-, il-6, il-1, and il-1, showed no difference between preeclamptic and normal placental explants, suggesting a rather marginal contribution of placenta derived cytokines to systemic inflammation. based on placental explant experiments, the ratio between shed to membrane-bound fractalkine seems to remain constant from first trimester until term but may be influenced by parameters such as gene expression, half-life of both variants, and metalloprotease dependent shedding. the phenomenon of increased membrane-bound fractalkine in cells treated with batimastat was explained as an accumulation of the membrane-bound form as a consequence of impaired shedding activity on the cell surface [24, 25]. since shedding is mediated by a disintegrin and metalloprotease (adam)10 and adam17 [8, 9], which both can be detected in the syncytiotrophoblast, it is tempting to speculate about an aberrant activity of these metalloproteases and its consequence on the release of placental fractalkine in pathological pregnancies. interestingly, expression of both metalloproteases has been shown to be increased in placentas from pregnancies complicated by preeclampsia [31, 32], suggesting increased shedding and release of placental fractalkine. this assumption is in line with a recent case-control study, showing elevated plasma concentrations of soluble fractalkine in women with preeclampsia. however, whether or not preeclampsia is accompanied with increased release of placental fractalkine remains open and requires further in-depth studies. detection of fractalkine expression in uterine glandular epithelial cells is in good agreement with previous studies showing fractalkine in apical regions of the glandular epithelium of actively secreting glands of nonpregnant endometrium as well as in early decidua. when speculating about a physiological function of fractalkine release by uterine glands it should be considered that early implantation processes, with the enlarging syncytiotrophoblast invading not only uterine capillaries but also uterine glands, give rise to connections between the latter and the intervillous space. this situation can be observed from approximately day 17 after conception throughout the first trimester, suggesting delivery of glandular secretion products, including nutrients, growth factors, and immunomodulatory cytokines into the intervillous space during first and early second trimester [35, 36]. recently, replacement of glandular epithelial cells by so-called endoglandular trophoblasts has been suggested as additional mechanism for opening and connection of the uterine glands towards the intervillous space. thus, uterine glands together with the growing syncytiotrophoblast may contribute to a continuous release of fetal fractalkine into the intervillous space, that is, maternal plasma. at this stage of pregnancy autocrine signalling by placental fractalkine however, with ongoing pregnancy autocrine effects of placenta derived fractalkine may be neglected, as cx3cr1 can only be detected in the fetal endothelium but not the villous trophoblast compartment at term [18, 38]. while placental fractalkine may contribute as soluble factor to low grade systemic inflammatory responses in the mother, its role as membrane-bound chemokine located on the surface of placental villi is rather unclear. adhesion of maternal leukocytes to the syncytiotrophoblast may be considered if at all as very rare event in normal pregnancy. however, mechanisms preventing cx3cr1 expressing maternal leukocytes from binding to the syncytiotrophoblast remain speculative. specific glycans, like sialyl lewis x and lewis a on glycosylated proteins, such as hcg, have recently been suggested to play a role in prevention of maternal leukocyte adhesion to trophoblast. nevertheless, under pathological conditions membrane-bound fractalkine could facilitate adhesion and transmigration of maternal leukocytes through the villous trophoblast layer giving rise to accumulation of maternal immune cells within inflamed villi, as has been described for infectious villitis and villitis of unknown etiology [4042]. the human placenta is a source for the chemokine fractalkine, which is expressed in the syncytiotrophoblast and released into the maternal circulation by metalloprotease dependent shedding. increased expression and release of placental fractalkine may contribute to low grade systemic inflammatory responses observed in third trimester of normal pregnancy. aberrant placental metalloprotease activity may not only affect the release of placenta derived fractalkine but may at the same time affect the abundance of the membrane-bound form of the chemokine.

the chemokine fractalkine is considered as unique since it exists both as membrane-bound adhesion molecule and as shed soluble chemoattractant. here the hypothesis was tested whether placental fractalkine can be shed and released into the maternal circulation. immunohistochemical staining of human first trimester and term placenta sections localized fractalkine at the apical microvillous plasma membrane of the syncytiotrophoblast. gene expression analysis revealed abundant upregulation in placental fractalkine at term, compared to first trimester. fractalkine expression and release were detected in the trophoblast cell line bewo, in primary term trophoblasts and placental explants. incubation of bewo cells and placental explants with metalloprotease inhibitor batimastat inhibited the release of soluble fractalkine and at the same time increased the membrane-bound form. these results demonstrate that human placenta is a source for fractalkine, which is expressed in the syncytiotrophoblast and can be released into the maternal circulation by constitutive metalloprotease dependent shedding. increased expression and release of placental fractalkine may contribute to low grade systemic inflammatory responses in third trimester of normal pregnancy. aberrant placental metalloprotease activity may not only affect the release of placenta derived fractalkine but may at the same time affect the abundance of the membrane-bound form of the chemokine.

PMC3976874

pubmed-1218

drug-induced dermatological reactions are common with the antiepiletic drugs such as carbamazepine (cbz), phenytoin, lamotrigine, ethosuximide, and phenobarbital. these reactions may occur in the mild form as benign rash or may be severe and life-threatening as erythema multiforme major or toxic epidermal necrolysis (ten). erythema multiforme major, also known as stevens-johnson syndrome, is usually caused by reactions to medications, rather than infections. cbz was originally introduced in therapeutic armamentarium as an anticonvulsant, and is known to produce such adverse drug reaction (adr), but the reports are rare. cbz is still used as a first-line agent along with lithium and valproic acid in the treatment of bipolar disorder. although there are case reports of stevens johnson syndrome (sjs) occurring in schizophrenia, bipolar affective disorder, and during a manic episode when treated with cbz, we were unable to find reports of sjs in patients having epilepsy with bipolar affective disorder. sjs is a blistering disorder, characterized by mucosal erosions at two or more sites with small blisters and purpuric macules. a 17-year-old woman was brought to psychiatry opd with complaints of episodes of decreased sleep, irritability, and occasional aggressive and violent behavior. she was already on sodium valproate 600 mg/day for generalized tonic-clonic seizures, since the last 2 years from a primary health centre of uttarakhand state. she had a history of 510 convulsions per day for 23 days and then seizure free period of 1015 days. she accepted noncompliance to the regular treatment, thrice for 1520 days duration each, during the last 2 years, and admitted that a further increase in seizure frequency during such breaks compelled her to resume the treatment subsequently. on examination, she had history of frequent mood swings of mania and depression associated with episodic behavioral changes which was undoubtedly suggestive of bipolar affective disorder. she was diagnosed as a case of bipolar affective disorder, currently in remission, with comorbid epilepsy. the patient was initiated on higher dose (800 mg) of sodium valproate which was further increased gradually to 1600 mg/day. at this stage, a further increase in dose caused significant sedation and was intolerable. at this stage, she was referred to a neurologist which she refused to comply with. cbz 200 mg once daily was added to her regimen of valproate 1600 mg/day in consultation with the hospital physician, which was increased to twice daily after 5 days and then three times daily after 10 days of initiation. she tolerated the treatment well until 19 day when she returned with high-grade fever, redness of eyes, swelling all over body, eruptions on lips, face in butterfly pattern [figure 1, 2] and was admitted in the dermatology ward. on examination her axillary temperature was 39c, blood pressure was 90/60 mmhg, and the pulse rate was 70 per min and regular. she had multiple bullae formation all over the body in the symmetrical pattern which gradually increased to involve>6065% of the body surface area. she had severe pain during deglutition, generalized body edema, and bleeding per vagina. examination by a gynecologist revealed that she was in the menstruating phase and vaginal mucosa was found ulcerated. lesions on patient's face in the butterfly pattern lesions on patient's trunk her lab investigations showed that heamogloblin was 13 g/ dl, total leukocyte count 6000/mm, platelets 1.5 lakhs/mm, creatinine 0.9 mg/dl, urea 49 mg/dl, sodium 139 meq/l, and potassium 4.8 meq/l. examination of urine revealed albumin (+) and full field rbcs, which correlated with her menstruation phase. biopsy was not done as her clinical presentation was compatible with diagnosis of erythema multiforme major which was confirmed with an opinion from the pharmacologist. she was treated with methylprednisolone, chlorpheniramine, and ceftriaxone parenterally, and clobetasol gentamicin combination topically, along with iv fluids and other supportive measures. after about 2 weeks of intensive indoor management, her conditions started improving and during third week she was again put on oral valproate 200 mg with an incremental increase of 200 mg per day till it reached 1600 mg/day on eighth day of initiation. the patient again started feeling drowsy, which may have had a psychological component as the patient was already anticipating the effect due to past experience. she refused any other addition to her treatment for fear of similar reaction and agreed to bear the possibility of few convulsions per month. two months after discharge, the patient returned for review and the dose of sodium valproate was increased since the sedation was tolerable. her dose was increased to 1800 mg and then to 2000 mg in the next month. with this dose she started having about 34 months of seizure free period and maintained on the same dose since the last 6 months. cutaneous lesions mostly occur on the palms, soles, dorsum of the hands, and extensor surfaces. mucosal involvement may include erythema, edema, sloughing, blistering, ulceration, and necrosis. this patient had been taking valproate for 2 years, and her investigations were not suggestive of any other etiology for causation of these adrs. cutaneous reactions started within 3 weeks of administration of cbz, which is the usual risk period for this adr. the causality assessment by naranjo's algorithm was done which revealed a score 7 suggesting a probable adr to cbz. in one such a report, a 6-year-old boy developed sjs five weeks after cbz was added to valproic acid, which he had been taking as sole antiepileptic therapy for several weeks. another study showed the incidence of skin rashes with the same dose of cbz in psychiatric patients (1215%) was nearly three times more than that in neurological patients (5%). however, no study showing incidence in comorbid neurological and psychiatric patients was found during our search, hence we lack any comparison with our case report. our patient also received sodium valproate which is also known to cause hypersensitivity reactions, but the patient had been taking valproate since the last 2 years without any adrs. it was only after the administration of cbz along with a higher dose of valproic acid that this patient developed severe adrs. immunological reactions due to hormonal changes during menstruation trigger various hypersensitivity reactions such as mucosal ulcerations, skin rashes, asthma, etc., so it may be an important factor in drug reactions which is yet an unreported factor and should be considered in further studies. approximately three persons per million per week may experience life-threatening dermatological syndromes with the use of cbz. the incidence of adrs may increase when cbz is given along with higher doses of valproic acid because of increased plasma concentrations of cbz. this was a rare case of life-threatening erythema multiforme major/stevens johnson's syndrome with the use of cbz when given along with higher doses of valproic acid. it was perceived that menstruation, and comorbidity of neurological and psychiatric illnesses might have had predisposing roles. there is a need of continued adr monitoring and reporting of adrs, more so with the use of cbz.

carbamazepine (cbz) is frequently used for epilepsy and various psychiatric illnesses. it is known for its dermatological side effects which may range from mild rash to life-threatening reactions as stevens johnson syndrome or toxic epidermal necrolysis. we hereby report a rare case of 17-year-old woman suffering from generalized tonic clonic epilepsy with comorbid bipolar affective disorder, who was initially treated with sodium valproate with partial improvement. after 19 days of addition of cbz to the therapy, the patient developed erythema multiforme major with>60% skin involvement and oral, conjunctival, intestinal, and vaginal mucosal involvement.

PMC3356969

pubmed-1219

rasmussen's aneurysm is a less frequently noticed entity usually associated with pulmonary tuberculosis presenting with massive hemoptysis due to involvement of small peripheral pulmonary or bronchial artery by the tubercular cavity or lesion. involvement of a large central pulmonary artery by metastatic mediastinal nodal lesions is unreported so far. despite the large size, a 65-year-old male of primary squamous cell carcinoma left buccal mucosa treated by left composite resection, and modified radical neck dissection 8 months back. postsurgery local radiation with 25 fractions of 2 grey (gy) each completed just 4 months before, presented with generalized weakness and pain right hip region. there was no history of fall, fever, loss of weight or bleeding from any site. clinical examination revealed no evidence of disease at the local site and no palpable masses anywhere in the body and there was no neurological deficit. hematological, bio chemical and metabolic parameters were within normal limits, except mild hypo chromic normocytic anemia. initial imaging with magnetic resonance imaging of lumbo sacral spine revealed t1 hypointense and t2 hyperintense signal intensities in right sacral ala and iliac bone lesions suggestive of metastatic lesions. patient was subjected to fluorine 18-fluoro deoxy glucose positron emission tomography/computerized tomography (f18-fdg) for restaging and further evaluation. the primary site of left buccal region showed postoperative status with no morphological or metabolic abnormality. there was a metabolically active enhancing nodular mass lesion on the dorsum of the tongue measuring 30 19 mm with a standardized uptake value maximum (suv max) of 5.77 [figure 1a], similar nodular deposit in the cervical nuchal muscle measuring 22 21 mm with an suv max of 4.82 [figure 1b]. there were multiple bilateral metabolically active necrotic, nodular, sub pleural and parenchymal pulmonary metastasis with associated mild degree pneumothorax [figure 2]. mediastinum showed multiple metabolically active enlarged lymphadenopathy encompassing bilateral hilar, sub aortic, and para aortic lymphadenopathy which also showed conglomeration and central necrosis. interestingly there was a large contrast filled cavitary area measuring 66.6 54.8 72.6 mm seen amidst the necrotic lymph nodal mass, devoid of any fdg avidity [figure 3]. on close scrutiny the radiographic contrast collection was traceable up to one of the adjoining first order branch of left pulmonary artery [figure 4]. there were multiple metabolically active mixed lytic sclerotic disseminated skeletal metastasis as well [figure 5]. due to his poor physical condition and the extensive disease load. small asymptomatic unilateral pneumothorax requiring no intervention and the large extravasated contrast being restricted within the confines of the cavitating nodal metastasis explaining the stark absence of hemoptysis, patient was managed conservatively without any active intervention shifting from a curative to palliative intent. (a) metabolically active enhancing nodular mass lesion on the dorsum of the tongue (dotted arrow). (b) metabolically active nodular deposit in the cervical nuchal muscle (thick arrow) axial positron emission tomography/computerized tomography images of chest showing multiple bilateral metabolically active sub pleural and parenchymal pulmonary metastasis (arrows) with associated left sided pneumothorax (dotted arrow) axial positron emission tomography/computed tomography images of chest showing multiple metabolically active enlarged mediastinal lymphadenopathy with conglomeration and central necrosis (arrows) and a contrast filled cavitary area amidst the necrotic lymph nodal mass devoid of any fluoro deoxy glucose avidity (dotted arrow) high resolution axial and coronal computerized tomography chest showing the radiographic contrast collection traceable up to one of the adjoining first order branch of left pulmonary artery (arrow) maximum intensity projection and coronal positron emission tomography/ computerized tomography images revealing multiple metabolically active mixed lytic sclerotic disseminated skeletal metastasis (arrows) erosive pseudo aneurysm of small caliber pulmonary arterial branch caused by an adjoining infiltrating pulmonary lesion is termed as rasmussen aneurysm. fritz waldemar rasmussen a 19-century danish physician first described the occurrence of dilation of the pulmonary artery in a tuberculous cavity, rupture of which causes hemorrhage and hemoptysis, often massive and life threatening. other documented causes are atherosclerosis, bronchiectasis, sarcoidosis, trauma, postcardiac catheterization and postnecrotizing pneumonias. pathogenesis implicated is the progressive weakening of pulmonary arterial wall adventitia and media by granulation tissue, resulting in thinning of the arterial wall and formation of pseudoaneurysm. the distribution is usually peripheral and beyond the branches of main pulmonary arteries. in the present era of selective catheter angiography, the entity of rasmussen's aneurysm is a retrospective detection encountered while addressing suspected bronchial artery erosion being the cause of massive, intractable and life-threatening hemoptysis. angiographic intervention is warranted to unearth the source of bleeding and simultaneously attempt to embolize the bleeding source from rasmussen's transformed culprit artery. our patient had extensive metastasis to skeleton, lungs, muscles, tongue and mediastinal adenopathy with significant necrosis. one of the aggressive and necrotizing lymphnodal mass has eroded the adjoining pulmonary artery branch, in this case a major branch vessel unlike smaller vessels usually seen in tuberculosis cavities. the leaked out blood was contained within the necrotic lymph nodal mass which explains the absence of any revealed hemoptysis and a possible emergency bleeding situation despite the large sized extravasation. conventional sites of aneurysm are peripheral and beyond the branches of main pulmonary arteries, however it was central in this case and involved a larger central pulmonary arterial branch. reported cases of rasmussen's aneurysm are the result of infective and chronic tuberculous cavity eroding the adjoining small caliber bronchial/pulmonary artery and none by an aggressive metastatic necrotic nodal mass. the case also highlights the aggressive, extensive and erosive nature of squamous cell carcinoma metastasis. rasmussen's aneurysm is a rare sequel of pulmonary tuberculosis presenting with massive hemoptysis usually involving a small peripheral pulmonary or bronchial artery. involvement of a large central pulmonary artery by an aggressive necrotizing mediastinal lymph nodal mass is unreported so far. our case is one such entity of this rarity with walled off collection of a large magnitude contained within the necrotic mass and nonhemoptytic manifestation being an additional associated rarity.

the authors report an extremely rare occurrence of a massive aneurysm of a major pulmonary artery branch vessel caused by adjacent necrotizing aggressive squamous cell carcinoma metastatic mediastinal nodes. despite the huge size, there was no hemoptysis due to the walling off effect by the necrotic nodes.

PMC4379684

pubmed-1220

during the course of hiv-1 infection, multifactorial t-lymphocyte (t-cell)-mediated mechanisms contribute to the progressive loss of host immune function [15]. in infected individuals, immune dysregulation occurs early and is characterized by a decrease in cd4+cell count, a concurrent rise in cd8+cells, a progressive decline in the cd4+/cd8+ratio, and defective thymocyte proliferation. during late-stage disease, loss of t-cell homeostasis also occurs [7, 8]. t cells are chronically activated throughout the course of hiv infection, as indicated by an increase in the expression of the antigens ki67, cd38, and human leukocyte antigen (hla)-dr, with cd38 recognized as the most reliable marker of immune activation [13, 5, 9]. immune activation provides the virus with a steady pool of target cells and has been linked with increased polyclonal t-cell proliferation and turnover, as well as increases in the apoptotic marker cd95 [1013] and activation-induced cell death [12, 1416]. concomitant with the decline of cd4 cells in the peripheral blood, the frequency of the cd4+cd28 null subset increases with disease progression and eventual progression to aids. the presence of cd28 on t cells is critically important for the generation of t-cell responses. interaction of this costimulatory molecule with its ligands increases the expression of antiapoptotic proteins and improves interleukin (il)-2 production. the increase in circulation of t cells with a cd4+cd28 null phenotype is consistent with a process known as replicative senescence [11, 13, 1719]. t cells that lack cd28 surface expression are nonanergic, oligoclonally expanded, and terminally differentiated, with limited replicative capacity and increased sensitivity to apoptosis [20, 21]. these alterations in phenotype are accompanied by cytokine changes consistent with a chronic proinflammatory state. the failure to produce adequate amounts of il-2 leads to a marked functional impairment of cellular and humoral immunity. reduced il-2 expression has been associated with a shift from th1 cytokine responses to th2 cytokine responses. the dysregulation of cytokine secretion during the course of hiv infection has been examined using a wide range of methods, including enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, t-cell cloning, and, more recently, flow cytometric intracellular cytokine detection. of these methods, only flow cytometry allows quantitative and qualitative determination of cytokine expression patterns in individual t cells. successful antiretroviral (arv) therapy results in improvements in circulating cd4+t-cell levels with decreases in cd8+counts and declines in immune activation and cd95 expression [15, 7, 9, 2226]. limited data on replicative senescence suggest a lack of significant improvement in the short term, despite adequate hiv suppression. some studies have suggested that protease inhibitor- (pi-) based regimens may have a greater effect on cd4+count recovery than nonnucleoside reverse transcriptase inhibitor-based regimens [27, 28]. some of the previous studies on immune recovery with arv therapy have been cross-sectional in design, conducted primarily in white males, and focused on absolute cd4+count or cd4+% recovery. furthermore, most of these studies did not comprehensively assess the functional aspects of immune recovery. of the studies that did evaluate the influence of arv therapy on functional immune parameters, most were drug class specific [3032]. grace (gender, race, and clinical experience) is the largest arv trial to focus on women with hiv-1 in north america and was designed to assess sex-based and race-based differences in efficacy, safety, and tolerability of the pi darunavir with low-dose ritonavir (drv/r) plus an optimized background regimen over 48 weeks in a diverse, treatment-experienced patient population. the aim of this prospective substudy was to quantitatively and qualitatively evaluate the recovery of functional immunity (t-cell function) with a drv/r-based regimen in a subset of patients from the grace study. grace was a multicenter, open-label, single-arm, phase iiib study conducted at 65 sites across usa, puerto rico, and canada that enrolled 429 treatment-experienced patients (viral load 1000 hiv-1 rna copies/ml) aged at least 18 years with documented hiv-1 infection. patients received drv/r 600/100 mg twice daily plus an investigator-selected background regimen that could include etravirine. patients at participating grace study sites were eligible for the prospective immunology substudy, which aimed to enroll up to 100 subjects. all subjects were required to sign a separate, independent ethics committee/institutional review board (irb) informed consent form specific to this substudy. the irbs were quorom review inc., seattle, wa, office of human research ethics unc irb, chapel hill, nc, and aids research consortium of atlanta, atlanta, ga. each irb approved of this study. the study was conducted in accordance with the principles of the declaration of helsinki and followed good clinical practice guidelines. viral suppression in this analysis was defined as achieving hiv-1 rna less than 50 copies/ml at week 48. immune function and phenotype were determined by flow cytometry at baseline and weeks 12 and 48 in virologically suppressed and nonsuppressed patients. changes in immune phenotype were determined from subsets of cd4+and cd8+t cells, with immune activation defined as increased expression of t-cell cd38 and hla-dr surface markers, and immune replicative senescence by increased frequency of circulating cd28 null t cells. changes in immune function were assessed by lymphocyte proliferation in response to candida and tetanus (recall antigens), phytohemagglutinin (pha) and pokeweed (mitogenic plant lectins), and cd3+/cd28+and by intracellular cytokine expression of il-2, interferon-gamma (ifn-), and tumor necrosis factor-alpha (tnf-) in response to staphylococcal enterotoxin b. a whole blood lysing technique was used and subpopulations were assessed according to a standard protocol for 3-color and 4-color immunofluorescence flow cytometry using fluorochrome conjugated monoclonal antibodies and a fluorescence activated cell sorter. a panel of monoclonal antibodies was used to delineate total cd3 +, total cd4+and cd8+t cells, cd4+/cd8+ratio, activated t cells (cd38+/hla-dr+), immune replicative senescence (cd4+/cd28+or cd8+/cd28 +), and apoptotic cells (cd95 +). the vybrant cfda se cell tracer kit (molecular probes, inc.) lymphocytes were labeled with carboxyfluorescein diacetate and succinimidyl ester cfda se and then incubated at 37c with 5% carbon dioxide for 7 days. the label is inherited by daughter cells after division. labeled lymphocytes with carboxyfluorescein diacetate and succinimidyl ester were detected by flow cytometry using cd45 markers for gating strategy. the reagents used were candida, tetanus, cd3+/cd28 +, pha, and pokeweed. flow cytometric intracellular cytokine detection of th1 cytokines including ifn-, il-2, and tnf- was assessed using the bd cytofix/cytoperm plus fixation/permeabilization (golgiplug protein transport inhibitor; bd biosciences). peripheral blood mononuclear cells were stimulated with staphylococcal enterotoxin b (sigma) and a cef control peptide pool (cmv, ebv, influenza virus; nih reagent 9808). then, surface and intracellular staining antibodies were added in a single staining step (anti-hu-ifn-/cd69/cd4/cd3, anti-hu-il2/cd69/cd4/cd3, anti-hu-tnf-/cd69/cd4/cd3; bd biosciences). a total of 32 patients with hiv-1 were enrolled in the substudy; 25 (78%) patients had week 48 data and 19 (59%) were virologically suppressed at week 48. the normal comparator group consisted of 34 healthy, hiv-seronegative individuals, 17 (50%) of whom were women and 25 (74%) of whom were white. patient demographics and baseline characteristics for total and virologically suppressed patients are shown in table 1. the median (range) cd4+count at baseline for the total patient population was 191 (2, 463) and the median (range) cd4+count in virologically suppressed patients at baseline was 222 (2, 398) cells/mm (table 1). at week 48, the median (range) cd4+count for the total population and virologically suppressed patients was 337 (98, 812) and 398 (119, 812) cells/mm, respectively. in virologically suppressed patients, the cd4+% increased significantly from baseline to week 48 (p<.03; figure 1). the median (range) cd8+count at baseline for the total patient population was 912.5 (288, 3131) cells/mm, while the median (range) cd8+count in virologically suppressed patients at baseline was 1037 (288, 3131) cells/mm. the cd8+% decreased significantly from baseline to week 48 (p<.01) in virologically suppressed patients (figure 1). the median (range) cd4+/cd8+ratio at baseline was 0.22 (0.01, 0.70) in all patients and 0.22 (0.01, 0.70) in virologically suppressed patients. the cd4+/cd8+ratio at weeks 12 and 48 is displayed in figure 1 and table 2. the cd4+/cd8+ratio increased significantly from baseline to week 48 (p<.01) in suppressed patients. the percentage of cd4+and cd8+cells at weeks 12 and 48 in suppressed patients is shown over 48 weeks in figure 1. the percentage of cd4+cells significantly increased (p<.01) and cd8+cells significantly decreased (p=.03) from baseline to week 48. improvements in immune activation, as measured by decreases in cd38 and hla-dr expression on cd4+and cd8+cells over the course of the study, were observed in both the total patient population (table 2) and in virologically suppressed patients (table 2; figure 2). the percentage of apoptotic (cd95 +) cd4+cells in suppressed patients significantly increased from baseline to week 48 (p=.0142; table 2; figure 2). the percentage of apoptotic cd8+cells, on the other hand, significantly decreased from baseline to week 48 in suppressed patients (p=.0025; table 2; figure 2). changes in immune replicative senescence were measured by changes in the frequency of cd4+/cd28 or cd8+/cd28 cells (table 2). there was little change in the expression of costimulatory marker cd28+on cd4+cells from baseline to week 48 in the total patient population or the virologically suppressed group (table 2). there was a small decrease in the expression of cd28+on cd8+cells in the total patient population and the virologically suppressed population from baseline to week 48 (table 2). the ability of cd4+lymphocytes to respond to mitogens and recall antigens improved in grace patients over the course of the study. proliferation in response to cd3+/cd28+and pha was at, or near, normal levels by week 12 in virologically suppressed patients, and proliferation in response to pokeweed and candida was at normal levels by week 48 (figure 3). tumor necrosis factor-alpha and il-2 significantly increased in staphylococcal enterotoxin b-stimulated cd4+cells of virologically suppressed patients by week 48; there was no significant change in ifn- in the stimulated cd4+cells (figure 4). few published studies within clinical trials have prospectively assessed in vitro changes in immune function as measured by lymphocyte proliferation [34, 35], and none of these assessed intracellular cytokine production in response to arv therapy. this substudy from the grace trial evaluated t-cell function in a racially diverse, treatment-experienced population comprised of more than 30% women. as expected, based on results from previous studies [24, 27, 28], drv/r-based therapy resulted in increases in cd4+cell counts and decreases in cd8+counts in virologically suppressed patients, with an improved cd4+/cd8+ratio. in addition, we found that drv/r-based arv therapy was associated with progressive functional immune recovery over 48 weeks in virologically suppressed patients, as demonstrated by improved lymphocyte response to mitogens and recall antigens. this suggests that not only do the cd4+cell counts of hiv-1-infected patients improve during drv/r-based therapy, but their ability to respond in vitro to immune stimuli may improve as well. results from this report are consistent with a recent study that demonstrated significant decreases in immune activation and apoptosis in cd4+and cd8+t cells and a decrease in immune cd8+senescence following arv therapy. while there have been studies evaluating the influence of arv therapy on immune parameters, data typically represent class-specific rather than regimen-specific treatment approaches. for example, therapy with integrase inhibitors has been shown to result in larger cd4+cell count increases compared with other arv classes [29, 37, 38]. the work shown here represents a regimen-specific study, with combination drv/r treatment in addition to an optimized background regimen that could differ across patients. unfortunately, we can not assess differences between regimens, as our study was not comparative. the reduction in t-cell activation seen here has previously been shown to correlate with the reduction in viral load, as well as an improved response to recall antigens. the observed hiv-1-induced increase in apoptotic cd95+/cd4+cells has been suggested to be independent of arv activity; the pi saquinavir has been shown to decrease cd95 expression in cd4+cells from healthy donors whose cells were previously briefly incubated with hiv-1 virus. although anti-cd95-induced apoptosis declines with therapy, this change is only occasionally associated with a reduction in expression of cd95 on t lymphocytes. it is thus not surprising that in our study cd95 expression on t cells did not decline. in the grace study, no improvement in the cd4+/cd28 or cd8+/cd28 phenotype was seen, consistent with other studies that have suggested that cd28 expression is not normalized in hiv-1infected patients who have undergone up to 3 years of arv therapy [40, 41]. one possible limitation to this study is the small sample size, which may limit data interpretation. the study aimed to enroll 50 women and 50 men, but, due to a delayed start date, after initiation of the grace study, the patient pool for this substudy was limited and the enrollment goal was not reached. nonetheless, the data obtained are similar to those of other trials. both immune phenotype and function of cd4+and cd8+cells were significantly improved in treatment-experienced patients receiving drv/r-based therapy, as evidenced by positive changes in the capacity to proliferate and the expression of intracellular cytokines by cd4+cells. the functional recovery observed in virologically suppressed patients, as assessed by proliferative response and intracellular cytokine expression, was also seen in nonsuppressed patients, although to a lesser degree. antiretroviral therapy has been available for 25 years; however, there has been limited research on immune recovery after long-term treatment. furthermore, the focus of immune recovery has historically been an assessment of cd4+cell counts. in the current study, in addition to the evaluation of cd4+cell counts, cd4+cell activation (cd38+/hla-dr+, cd95 +), function, and senescence (cd28) were measured as well as cd8+cell counts, activation, function, and senescence. it should be noted that, as observed in this trial, despite the improvements in immune phenotype and function seen with arv therapy, complete normalization of cd4+and cd8+parameters is rarely achieved in hiv-1-infected individuals [24, 42]. however, we hypothesize that there may be some clinical benefit even in patients who do not experience measurable or significant increases in cd4+cell counts. it is feasible that, in patients with higher baseline cd4 levels, measurable increases in cd4+cell counts may not be observed. previous studies have shown that despite cd4+cell count normalization, the pretreatment nadir cd4+count and level of cd28 cd4+coexpressing lymphocytes determine ongoing immune competence, including responses to immunization. despite the successful hiv therapy noted in the grace substudy this possible treatment-resistant expansion and persistence of cells with a senescence phenotype may have potential for long-term cardiovascular, metabolic, and other aging-associated consequences. this grace substudy demonstrates that drv/r-based therapy improved cd4+cell recovery and was associated with progressive functional immune recovery over the 48-week study period. thus, initiation of arv therapy may be required not only to restore immune function, but also to diminish the effects of chronic inflammation .

objective. during the course of hiv infection, progressive immune deficiency occurs. the aim of this prospective substudy was to evaluate the recovery of functional immunity in a subset of patients from the grace (gender, race, and clinical experience) study treated with a drv/r-based regimen. methods. the recovery of functional immunity with a darunavir/ritonavir-based regimen was assessed in a subset of treatment-experienced, hiv-1 infected patients from the grace study. results. 19/32 patients (59%) enrolled in the substudy were virologically suppressed (< 50 copies/ml). in these patients, median (range) cd4+cell count increased from 222 (2, 398) cells/mm3 at baseline to 398 (119, 812) cells/mm3 at week 48. cd8+% decreased significantly from baseline to week 48 (p=.03). proliferation of cd4+lymphocytes in response to cd3+/cd28 +, phytohemagglutinin, and pokeweed was significantly increased (p<.01) by week 12. proliferation in response to candida and tetanus was significantly increased by week 48 (p<.01 and p=.014, resp.). staphylococcal enterotoxin b-stimulated tumor necrosis factor-alpha and interleukin-2 in cd4+cells was significantly increased by week 12 (p=.046) and week 48 (p<.01), respectively. conclusions. darunavir/ritonavir-based therapy demonstrated improvements in cd4+cell recovery and association with progressive functional immune recovery over 48 weeks. this trial is registered with nct00381303 .

PMC3874356

pubmed-1221

chronic diseases are a major health concern and major cost in australia and in the most developed and developing countries. since 1999, the australian government, through its national health insurance scheme known as medicare, has sequentially introduced a range of incentives including subsidised computers, payments to gps via the medicare schedule for chronic disease management (cdm) including general practice management plans (gpmps, item 721), team care arrangements (tcas, item 723), reviews of gpmps and/or tcas, home medicines reviews (hmr, item 900), and items for work undertaken by general practice nurses on behalf of a gp including administration of these care plans [35]. as in november 2012, according to the medicare schedule fees (for payment to gps) reimbursement for these items was: item 721 $141.40, item 723: $112.05, item 732: $70.65, and item 900: $151.75. several key factors have been identified for effective chronic disease management including: the availability and accessibility of information technology (it), accessibility of patients ' clinical information, patient/consumer participation in decision making, good linkages with community resources and services, longer consultations for patients with gps, gps delegating roles and responsibilities to other health professionals, and using models of care [69]. while medicare data suggests that less than 14% of patients with a chronic disease have a gpmp and/or a tca, only one in five of these plans is regularly followed up and reviewed at the recommended frequency. effective continuing professional development is also of importance for quality improvement in clinical practice [11, 12]. the breakthrough series methodology which includes a model for improvement (plan-do-study-act) has been described as a strategy for improving quality outcomes in general practice in australia and has been used to upskilled gps in chronic disease management [14, 15]. this mixed methods study tested a broadband-based service known as cdmnet, which creates web-based gpmps using disease specific templates by extracting and auto-populating data from gps ' computer records into the gpmp and sharing this with designated member of the care team [1417]. cdmnet was developed and is managed by an australian-based external provider, precedence health care (phc). comprising workshops and interviews, this study aimed to promote best practice in gp management of patients diagnosed with a chronic disease, in particular, using medicare item numbers 721, 723, 732, and 900 and cdmnet. while this study was conducted in australia, the impact of the introduction of information technology is of interest globally, where information technology is being introduced for chronic disease management. the breakthrough series methodology including the plan-do-study-act model [1315], comprising of four learning workshops and a followup workshop over a period of 8 months;semistructured interviews with gps who participated in the learning workshops [18, 19]. the breakthrough series methodology including the plan-do-study-act model [1315], comprising of four learning workshops and a followup workshop over a period of 8 months; semistructured interviews with gps who participated in the learning workshops [18, 19]. regarding learning workshops, a convenience sample of gps was recruited through the monash university, department of general practice networks. the letter of invitation was circulated to 508 gps, of those, 57 gps expressed interest; 24 gps, 2 practice staff and 6 research staff, attended the first learning workshop. regarding interviews, of the 24 gps who commenced workshop series, 15 agreed to participate. gps were invited to participate in the interviews during learning workshop 2; a followup telephone call was made to (a) confirm agreement to participate and (b) to arrange a time to conduct the interview. data were collected using three tools specifically developed for this study: a predisposing activity completed two weeks before each learning workshop, an evaluation completed during each learning workshop, a reinforcing activity completed two weeks after each learning workshop. a predisposing activity completed two weeks before each learning workshop, an evaluation completed during each learning workshop, a reinforcing activity completed two weeks after each learning workshop. interviews were conducted during august-september 2011 at the participating gps ' practices. qualitative data were analysed using content analysis [18, 19]; quantitative data were analysed using spss v.19. ethics for the project were submitted and approved by monash university human research ethics committee (muhrec). throughout the intervention period (from march to november 2011) and the period up until the followup workshop (november 2011), gps were asked to report the number of gpmps (721), tcas (723), reviews (732), and hmrs (900) created. during this time, the number of items created and completed initially increased, reduced, and then increased again (table 1). gps felt this peak and plateau similarly reflected that they were managing their usual throughput after managing their backlog. gps were also asked to report the number of item 721 that they had created for four chronic diseases: diabetes, osteoarthritis, chd/cvd, and coad/copd during the intervention period (table 2). a similar pattern emerged with the number of item 721s developed; increasing then plateauing after workshop 2-3. discussion during workshops indicated that this may have occurred because gps felt they had identified most of their patients who they had not previously considered for a gpmp, and then after the backlog had been addressed, the numbers reported at later workshops were more reflective of their usual throughput. in addition to the four chronic diseases (table 2), gps created gpmps and tcas conducted reviews and ordered hmrs for other chronic diseases including depression, asthma, chronic low back pain, chronic kidney disease, stroke, and type 1 diabetes (table 1). regarding the use of cdmnet, gps were asked to estimate any shift from not using to using cdmnet during the intervention period (figure 1). for gpmps (item 721), while there was an initial increase then a reduction of using cdmnet, there was a small but noticeable reduction in the number of gpmps developed not using cdmnet. a similar pattern emerged for tcas (item 723) using cdmnet, but the number developed not using it was suggested that this occurred because gps were developing tcas for gpmps that they had previously developed not using cdmnet. regarding reviews (item 732) and hmrs (item 900) that the increase and plateau using cdmnet and the reduction and increase not using cdmnet, it was felt that these patterns reflected the followup patterns for gpmps and tcas developed prior to the introduction of cdmnet. thus, a pattern was emerging suggesting an increase in the use of cdmnet for gpmps and tcas, but not for reviews and hmrs during the intervention period; however, it was felt that the pattern for reviews and hmrs would follow the pattern for gpmps and tcas in the future. this also suggests that an increase in the number of care planning items developed by gps using cdmnet may contribute to increase gp's income. gps also spoke about changes in their practice nurses ' and practice managers ' roles. practice nurses were becoming more involved in generating gpmps using cdmnet, identifying eligible patients, spending more time completing health assessments and preparing gpmps and reviews. eligible patients lists, assisting in coordination, entering data on the computer, and identifying patients for recalls and reviews. throughout the intervention period, gps evaluated the learning workshops as being of benefit. benefits and the value of the workshops reported by gps included the following: learning from other gps that they are also experiencing similar challenges, in particular, the low interest of specialists in management plans whether they are internet generated or not, resolving how to add lists of allied health providers to cdmnet, identifying the difference between lack of details generally recorded in paper-based care planning documents (gpmps and tcas) compared with the detail recorded in a cdmnet-generated plan, the ease at which cdmnet works compared to the initial perception that cdmnet is complex, the (reduced) time required to modify documents to meet individuals ' needs, when using cdmnet, an awareness that cdmnet is still an evolving process that requires it infrastructure and information sharing across the health system. learning from other gps that they are also experiencing similar challenges, in particular, the low interest of specialists in management plans whether they are internet generated or not, resolving how to add lists of allied health providers to cdmnet, identifying the difference between lack of details generally recorded in paper-based care planning documents (gpmps and tcas) compared with the detail recorded in a cdmnet-generated plan, the ease at which cdmnet works compared to the initial perception that cdmnet is complex, the (reduced) time required to modify documents to meet individuals ' needs, when using cdmnet, an awareness that cdmnet is still an evolving process that requires it infrastructure and information sharing across the health system. having computers available at the workshop for gps to practice was valued, as was the medico-legal session. other benefits included learning from each other about how to use cdmnet, which among other things, contributed to gps being more aware of patients ' eligibility for gpmps, tcas, reviews, and hmrs: practice makes perfect-by, using the information obtained from the education activity workshops and using the knowledge straight away (gp1). for some gps, implementation of cdmnet was delayed for reasons beyond the project team's control; thus, gps were at varying stages of learning the information presented, and subsequently discussion at workshops did not always reflect the stages for all gps: we were a bit late getting it loaded on because we had some software issues and other things we started doing a few things ourselves so we were getting familiar with the format and process and that sort of thing, it all made a bit of sense and at the education meetings they were trying to troubleshoot and ease the concerns and help us feel comfortable and answer questions about use and that sort of thing, so that was all fine being time poor, gps reported that their lack of time meant they did not have the opportunity or the time to create gpmps for patients: yeah, the more complex it is, when you're under pressure already running late, you think, no i can not do this today (gp 15). uploading allied health providers ' and specialists ' information onto the address book was an issue. although phc staff provided advice and assisted gps with this process, there were significant challenges in completing this task: one of the issues is having huge numbers of names to scroll through to find the ones that we use [in the address book] the majority of gps indicated they had some patients who were concerned with privacy issues and some patients who can not afford the cost of private ahp/specialists. generally, the community centres that the gps in this cohort referred to did not have cdmnet installed in their it systems: i had one patient with multiple chronic conditions and i wanted to do a gpmp, but i had to do the old paper system because she is very concerned about privacy and she said she does not have internet, she is one of those persons that is very concerned (gp2) most of the allied health people are at the local community centre and i suppose it's kind of made me realise in some ways this whole system does not work very well for my patients because most of them do not use private allied health people (6) it issues. identified information technology (it) issues variously impacted on the gps. some expressed concern that the information developed using cdmnet was not automatically recorded back into their medical software; others queried how cdmnet would affect the practice medical software systems and others queried compatibility: cdmnet can not be installed for all gps because cdmnet is not compatible with some medical software (gp7). overall, gps felt that using cdmnet raised their awareness of the number of patients with a diagnosis of chronic disease, resulting in more recognition of patients ' eligibility and subsequent increase in developing gpmps. generally, gps felt they had become more proactive in developing gpmps, tcas, and reviews for patients with chronic disease(s), rather than being reactive as they previously had been. the thing is, as you get more and more familiar; if i get really good on it and i could do them five or six, it would be fantastic (gp2). all agreed that the internet is seen to be the way of the future. with this in mind, the future use of cdmnet may depend on several factors such as the efficiency of the system, cost benefits for gps and practices, and better health outcomes for patients. the use of the breakthrough series approach provided significant insight into gps ' management of patients with a chronic disease, in particular, when using medicare items 721, 723, 732, and 900 and the broadband-based service, cdmnet. at each of the workshops, gps provided feedback about cdmnet which was of value to the developers to inform updating and further development of the broadband-based service. despite an initial lack of satisfaction with cdmnet, those who completed the gp education workshops generally reported satisfaction with many aspects of cdmnet. the eight gps who withdrew during the intervention period cited a range of reasons including time commitments, incompatibility with their practice medical it systems, and challenges with cdmnet. overall changes made by participating gps regarding their use of cdmnet varied from no change/having developed no gpmps using cdmnet to change/developing many gpmps using cdmnet. while the variation is significant, this may be due to many reasons, not the least, the timing of when cdmnet was installed for participating gps during the intervention period. feedback from the interviews with the gps reflected the comments made during the gp education workshops: time to learn the process was required, the uploading of the address book was a challenge for many because of allied health professionals, and specialists could not be included in gpmps and/or tcas if their information was not available in the address book. other it issues were also discussed for exploration by phc, the for example, the incompatibility of cdmnet with some medical it systems. nonetheless, gps generally agreed that using cdmnet raised their awareness about their patients, that gp education workshops prompted them to think about chronic disease management as a process that included a team, and that the internet is seen to be the way of the future. the gps who ceased using cdmnet either during or after completing the gp education workshops were influenced by several issues including time, cost and/or staff (including gps) unwilling to use the tool. while some felt the concept was good, they did not feel it was of benefit in their practice. the strengths of this study include the fact that the education workshops were undertaken in as close to real life as possible. gps indicated that they benefited from, among other things, collegiality, sharing of ideas, and identifying challenges. the data collection tools were developed to ensure that the gps had a record of the changes they had made throughout the intervention. interest in the study was strong, and significantly two-thirds completed the gp education workshops. most participating gps participated in face-to-face interviews and assisted with circulating questionnaires to patients who had gpmps developed during the intervention period. gps indicated that they felt that cdmnet could be sustained but would be enhanced if broadened to include other templates such as disability care planning and pain management. whilst the technology was seen as a potential challenge for some gps and patients, others felt that cdmnet could be sustained and enhanced by including a wider range of templates for diseases other than chronic disease. not all participating gps had cdmnet installed at the same time. with a difference of several months between the first and final installation, no comparison could be made between gps ' progress regarding use of cdmnet and the medicare item numbers. in addition, not all gps who commenced in the study completed all workshops; thus, the data is reduced. generalisability may be challenged by current and future technology regarding, for example, compatibility of cdmnet with medical software and/or adding providers into the address book, what happens in the general practice (general practice routine), and/or staff in the practice who are able/willing to be involved with the technology. similarly, transferability may depend on what happens in health professionals ' practices (gps, allied health professionals, and specialists), particularly whether health professionals choose to use cdmnet or not. during the intervention period, all gps developed gpmps and most developed tcas, with output for some increasing in the earlier part of the intervention period then levelling towards the end of the intervention period. the breakthrough series methodology facilitated upskilling gps ' management of patients diagnosed with a chronic disease, in particular, the use of medicare item numbers 721, 723, 732, and 900 and the application of broadband-based service cdmnet as an enabler to achieve this. future work may include the study being replicated with a larger study sample, randomization of participants, and conducting quantitative analysis of the outcomes. the work could include collecting demographics and professional characteristics for those who choose not to participate or commence participation then withdraw.

background. key factors for the effective chronic disease management (cdm) include the availability of practical and effective computer tools and continuing professional development/education. this study tested the effectiveness of a computer assisted chronic disease management tool, a broadband-based service known as cdmnet in increasing the development of care plans for patients with chronic disease in general practice. methodology. mixed methods are the breakthrough series methodology (workshops and plan-do-study-act cycles) and semistructured interviews. results. throughout the intervention period a pattern emerged suggesting gps use of cdmnet initially increased, then plateaued practice nurses ' and practice managers ' roles expanded as they became more involved in using cdmnet. seven main messages emerged from the gp interviews. discussion. the overall use of cdmnet by participating gps varied from no change to significant change and developing many the gpmps (general practice management plans) using cdmnet. the variation may be due to several factors, not the least, allowing gps adequate time to familiarise themselves with the software and recognising the benefit of the team approach. conclusion. the breakthrough series methodology facilitated upskilling gps ' management of patients diagnosed with a chronic disease and learning how to use the broadband-based service cdmnet.

PMC4041255

pubmed-1222

peptidyl prolyl cistrans isomerases regulate many biological processes by interacting with molecular switches whose conformations are modulated via cistrans isomerization of the prolyl peptide () bond.(1) many molecular switches are involved in cell signaling pathways, and deregulation of these pathways could trigger cellular transformation, oncogenesis, and other diseases.(2) there are three structurally unrelated classes of ppiases that are known to date: the cyclophilins that bind cyclosporine, the fk506 binding proteins (fkbp), and the parvulins, of which pin1 is a member. undoubtedly, cistrans isomerization of the peptide bond is one of the slowest conformational transitions found in proteins. the detailed atomistic understanding of the mechanism of ppiases is still lacking, and the bits and pieces that are known do not always form a coherent story. the local changes of the isomeric state of the prolyl peptide bond act as a switching mechanism in altering the overall conformation of proteins. protein signaling processes utilize the additional conformational variability that arises due to the resulting cis and trans isomers of peptide bonds. several molecular switches that are regulated by cistrans isomerization have been discovered over the years.(2) the role of cistrans isomerization of the prolyl peptide bond in interleukin tyrosine kinase (itk) sh2 domain that is regulated by cyclophilin, and ligand-gated 5ht3 ion channel,(5) are two recognizable examples. the binding site of the sh2 domain of itk discriminates between two different ligands, depending on the isomeric state of a distal prolyl peptide bond. similarly, the conformation of five prolyl peptide bonds, one in each subunit, was shown to determine the state of the ligand-gated 5ht3 ion channel. when the prolyl peptide bonds adopt the trans conformation, the channel is closed; when they are in the cis isomeric state, the channel is open, allowing ions to flow through. importantly, the hiv virus has also been shown to use the human cyclophilin during its final stages of viral replication, which has rekindled interest in this enzyme especially for drug design purposes. human cyclophilin catalyzes cistrans isomerization of a prolyl peptide bond of the hiv capsid in order to trigger a conformational change necessary for viral packaging. cistrans isomerization of prolyl peptide bonds is characterized by a very high activation energy barrier of around 1622 kcal/mol, and the rate is in the order of tens to hundreds of seconds. therefore, cistrans isomerization is involved in slow conformational changes, including the rate-limiting step in protein folding. nature has provided the ppiase enzymes to circumvent this very slow kinetics by catalyzing the cistrans isomerization and decreasing the time scale from seconds to the more biologically relevant millisecond time scale. the mechanism of the ppiases is still not well understood and is controversial, and has been the subject of many experimental and computational studies. for example, it was earlier thought that the remarkable speedup is achieved by a nucleophilic attack to the carbonyl carbon atom of the preceding residue that would result in the loss of the pseudo-double-bond character of the peptide bond. this possible loss in pseudo-double-bond character could then result in a lower activation energy barrier and therefore lead to a faster rate of isomerization. however, this mechanism was shown to be implausible due to the retention of catalytic activity of cyclophilin after mutagenesis studies that were carried out on all the residues that have the ability to act as the nucleophile.(16) therefore, ppiases are one of the rare enzymes in biology that carry out their function in the absence of any actual bond formation and cleavage. how do the ppiases then achieve this remarkable speedup of more than 5 orders of magnitude? several hypotheses have been proposed over the years that include the effect of substrate desolvation and the idea of preferential transition-state binding in the active site.(1) it was shown that the effect of removing the substrate from aqueous solution to the hydrophobic pocket of the ppiases, as shown in figure 1, could result in a speedup of cistrans isomerization. this effect is partly due to the weakening of the pseudo-double-bond character of cn in nonaqueous environment, resulting in a small reduction of the transition barrier height by about 1.3 kcal/mol.(17) similarly, a speedup of up to about 20-fold of the rate of cistrans isomerization was later observed in micelles that also resulted in a small decrease in the barrier height by about 1.8 kcal/mol, assuming that the speedup is purely due to barrier reduction.(18) likewise, we have previously observed a speedup in the rate of cistrans isomerization using molecular dynamics simulations in the absence of explicit water molecules around the prolyl peptide bond due to a reduction in the effective roughness on the energy landscape that results in a change in the kinetic prefactor.(19) the kinetic prefactor depends on the diffusion coefficient on the landscape, which in turn depends on the effective roughness of the landscape. also, the speedup can simply be a consequence of the change in the frictional drag experienced by the substrate in moving from an aqueous environment to the dry hydrophobic cavity of the binding site of the ppiases. however, these prefactor effects and slight reduction in barrier height due to the lack of aqueous medium could not account for the more than 5 orders of magnitude increase in the observed rate of cistrans isomerization due to ppiases. also, it has previously been shown that an increase in the rate of cistrans isomerization of the angle can be achieved by constraining the peptide bond in a loop conformation, but the extent of the role of this phenomenon in the catalysis of cistrans isomerization of the peptide bond by ppiases is not known. cyclophilin catalyzes cistrans isomerization of a -gly-pro- motif on the exposed loop structure of the hiv capsid (left). the binding site of cyclophilin (right) has a very hydrophobic pocket with the nonpolar residues shown as white, an arginine residue at the entrance of the pocket shown as blue surface, a histidine shown as cyan surface, and two asparagines shown as green. the side-chain ring of the proline residue fits very nicely into the hydrophobic pocket of the binding site. in order to fully understand the catalytic mechanism at the atomistic detail, one has to be able to observe the cistrans isomerization of the peptide bond. in this regard, all-atom molecular dynamics simulation has proven invaluable as a complementary technique to existing experimental results in fully understanding protein function.(22) however, normal molecular dynamics simulation has not been able to provide a complete picture of the catalytic mechanism of the ppiases because of the time scale limitation, and therefore the cistrans isomerization can not be simulated directly. the time scale of cistrans isomerization and even the time scale of the catalyzed process are beyond the submicrosecond time scale of normal molecular dynamics (md). therefore, earlier computational studies of this system have used umbrella sampling and restrained molecular dynamics in order to traverse the isomerization path. a limitation of these types of techniques is the reliance on a priori decisions about the transition path that could potentially bias the outcome. previously, we developed an accelerated md method(26) that was used to simulate for the first time the cistrans isomerization of the prolyl peptide bond,(27) and we were able to calculate the free energy barrier and rate constants in both implicit and explicit solvent. this method speeds up the transition over energetic barriers with little or no prior knowledge of the energy landscape. in this work, we have used the accelerated md method to fully study the catalytic mechanism of the cyclophilin a enzyme by simulating the cistrans isomerization of the free substrate taken from the hiv capsid and that of the enzymesubstrate complex, both in explicit solvent. therefore, we have used classical molecular mechanics to study the catalytic mechanism of this enzyme. we observed cistrans isomerization of the -gly-pro- bond of the free substrate ace-his-ala-gly-pro-ile-ala-nme from the accelerated molecular dynamics simulations in explicit water as shown in figure 2a. the substrate is derived from the loop region of the hiv capsid (figure 1) that is regulated by cyclophilin a. in addition to the crystal structures of cyclophilin complexed with the whole hiv capsid, cyclophilin has also been cocrystallized with the short piece taken from the full-length capsid. the free energy profile along the bond was estimated as shown in figure 2c, after reweighting the distribution of the peptide angle of -gly-pro-. the free energy barriers are similar regardless of the direction of rotation, with a barrier height of about 16.5 1.2 kcal/mol going from the trans to cis isomer and about 12.8 1.5 kcal/mol going from the cis to trans isomer. the similarity of the free energy profile in both directions can be attributed to the lack of the side chain in the preceding glycine residue, thus allowing for almost equal probability of undergoing clockwise and anticlockwise rotations. this result contrasts with our previous study of the -ser-pro- motif that has an asymmetric free energy profile, which could be attributed to the side chain of serine hindering the trans-to-cis clockwise rotation.(27) cistrans isomerization of the bond of the -gly-pro- motif of the free substrate (a) and enzymesubstrate bound complex (b) and the corresponding free energy profile after reweighting of the distribution (c) for the free substrate (black) and enzymesubstrate complex (red). after simulating the cyclophilinsubstrate complex with accelerated md simulations in explicit water, we also observed cistrans isomerization of the bond of -gly-pro- in the catalytic pocket of the enzyme that allowed us to monitor and study the catalytic mechanism of cyclophilin (figure 2b). an immediate observation of the time series of the angle is the directionality of the cistrans isomerization. the transition from trans to cis, and vice versa, is mainly unidirectional, and the directionality of the transitions is also obvious from the estimated free energy profile along the bond, also shown in figure 2c. the barrier height of the transition from the trans to cis isomer is lower for the anticlockwise direction, and that for the cis to trans transition is lower along the clockwise direction. also, the trajectory of cistrans isomerization of the enzyme-bound substrate is noticeably different from that of the free substrate as can be seen in figure 2, a and b. an ensemble of conformations around 90 of the angle of -gly-pro- is stabilized as compare to the free substrate. therefore, after reweighting the distributions, two other main observations from the free energy profile are the stabilization of the cis isomer against the trans isomer as compared to the free substrate and the lowering of the barrier height as we go from the trans isomer to the cis isomer (anticlockwise). the cis and trans isomers of the substrate in the binding site now have almost equal probability of occurrence, with the transition state at a higher free energy. the barrier height from trans to cis even though the barrier height of the transition state is lowered in the enzymesubstrate complex as compared to that of the free substrate, the barrier reduction is not enough to make the transition-state complex more stable than the complex with the cis or trans isomer. consequently, crystal structures of the enzymesubstrate complexes are expected to be found either in the cis or trans conformation, as is the case. the barrier height from the trans to cis isomer in the enzymesubstrate complex is now about 10.2 kcal/mol. the reduction in the barrier height is therefore estimated to be around 6.3 kcal/mol. as a result, the magnitude of the reduction of the barrier height coupled with a possible order of magnitude speedup due to the kinetic prefactor effect is enough to increase the rate of the isomerization by more than a factor of 10. since the time scale of the cistrans isomerization of the free substrate occurs in 11000 s, this speedup is enough to put the catalyzed process in the biologically relevant millisecond time scale. we can see from figure 2 that the transition state in the complex has lower free energy than that in the free substrate, relative to the trans isomer. the lower barrier height of the transition state is therefore partly responsible for the speedup of the rate of cistrans isomerization. also, the free energy of the cis isomer is similar to that of the trans isomer, thermodynamically increasing the population of the cis isomer relative to that in the free substrate. we analyzed the ensemble of structures of the transition state, and we observed that the transition state is formed when the carbonyl oxygen of gly forms a hydrogen bond with a backbone nh group of asn 102, as shown in figure 3. this favorable hydrogen-bonding interaction between the carbonyl oxygen and the backbone hydrogen of asn 102 is formed when the n-terminal of the peptide bond rotates clockwise (looking from the n-terminal to the c-terminal along the bond). the c-terminal of the peptide bond, which comprises the proline ring, never rotates for this particular substrate and stays snuggled in the hydrophobic pocket, as is depicted in figures 1 and 3. the rotation of the n-terminal of the peptide bond during catalysis has also been suggested from crystal structure analyses,(32) contradicting previously reported c-terminal rotation of the peptide bond.(33) however, the rotating end could be dependent on the sequence of the substrate or the family of ppiase. the enzyme-bound substrate is stabilized by three main interactions: the nonpolar interaction proline makes with the hydrophobic pocket, the hydrogen-bonding interaction between the guanidinium moiety of the conserved arginine (top) and the carbonyl oxygen of proline, and the hydrogen bond between the carbonyl oxygen of glycine and the backbone hydrogen (below) of asparagine. therefore, in order to further probe the extent of transition-state stabilization, we have constructed a thermodynamic cycle as shown in figure 4. the thermodynamic cycle links the free energies of binding between the trans, transition state, and cis isomers of the substrate and cyclophilin to the free energies of cistrans isomerization. it is clear from this analysis that the transition state binds more strongly to cyclophilin followed by the cis isomer, with the enzyme having the least affinity for the trans isomer. this result agrees with previous nmr experiments(34) which showed that the cis isomer binds 4 times stronger than the trans isomer to cyclophilin, which correlates with the fact that many of the cyclophilinsubstrate structures adopt the cis form. our suggestion that the transition state interacts more favorably with cyclophilin does not contradict the fact that all of the structures of the cyclophilinsubstrate complexes are either in the cis or trans conformation. despite the fact that the transition state interacts more favorably with the enzyme, the total free energy of the enzymetransition-state complex is higher than that of the ground state cis and trans isomers as can be seen in figure 2c, due mainly to the high penalty of activation. t, ts, and c represent the trans, transition state, and cis conformations, respectively. furthermore, stabilization of the transition state is due not only to the favorable interaction made by the carbonyl oxygen of the gly with the backbone hydrogen of asn and by the favorable nonpolar interaction of the proline residue with the hydrophibic pocket but also to a favorable interaction of the carbonyl oxygen of pro with the guanidinium moiety of the highly conserved arg 55, also shown in figure 3. arginine 55 is shown to interact quite differently with the trans, transition state, and cis isomers, and therefore partly responsible for the differences in binding affinity. in this regard, a catalytic antibody, abzyme, optimized to recognize and bind a transition-state mimic, accelerates the rate of cis/trans isomerization of the peptide bond, but to a much lesser extent than cyclophilin.(35) why would the optimized abzyme have a much smaller increase in the rate of cistrans isomerization of the peptide bond than cyclophilin? the answer, we believe, lies in the nature of the peptide bond mimic, an -ketoamide, that looks like a distorted peptide bond. the chemistry of the hapten (the distorted peptide bond mimic) on the n-terminal side of the proline residue is rather different from that of typical substrates. the peptide bond connecting the preceding residue and the proline is replaced by a dicarbonyl moiety in the hapten. this moiety allows the angle of the amide bond to be around 90, similar to that of the transition state of the peptide bond. therefore, this small change from the real substrate could compromise the activity of the abzyme on the real substrate, since the specificity of action of the abzyme was optimized for a substrate with a slightly different chemistry. the effect of arg 55 is visually evident from the crystal structure of the cyclophilinsubstrate complexes in which the guanidinium moiety forms hydrogen bonds with the carbonyl oxygen of proline, similar to the interaction shown in figure 3. arg 55 is highly conserved, and its replacement has been shown to decrease the catalytic activity of cyclophilin.(36) the catalytic efficiency (kcat/km) of the wild-type cyclophilin was estimated to be 16 m s, and that of the arg55ala mutant was estimated to be 0.016 m s, about 0.1% of the catalytic efficiency of the wild-type. therefore, these results suggest that arg 55 might be important for recognition, since some catalytic activity is retained for the arg55ala mutant. the binding affinity of cyclophilin for its substrates is very low, about 2040 m,(34) and therefore, this single arginine residue could potentially be critical for both recognition and stability. it was previously shown that arg 55 was stable during one nanosecond of md simulation, and elimination of its overall charge destabilized the transition state and the cis isomer complexes.(24) we therefore decided to explore the role of arg 55 in the catalytic process over a much longer time scale and the effect this residue might have on the stability of the transition-state complex. we carried out three normal md simulations, 50 ns each, on the enzymesubstrate complex with the substrate -gly-pro- bond in the trans, transition state, and cis state, bound separately to the enzyme. the angle of only the transition state was held at 90 by applying a 1000 kcal/mol/rad angle restraint on only that degree of freedom. the distance between the carbon of the guanidinium moiety of the arginine residue to the carbonyl oxygen of the substrate proline residue was monitored for the three simulations as summarized in figure 5 (black lines). it can be seen in figure 5 that this hydrogen-bond contact is not stable in the complex with the trans isomer. also, the hydrogen-bond formation is reproducibly correlated with the stability of the complex. at around 35 ns in this simulation of the enzymesubstrate complex of the trans isomer, the arginine residue disengages the carbonyl oxygen of the proline residue one more time, and this event is followed by the diffusion of the substrate out of the binding site (figure 5; blue lines). the blue line is the distance between the c- of the proline residue of the substrate and the c- of the phenylalanine residue in the hydrophobic pocket. hydrogen-bonding interactions between the binding site of cyclophilin and the substrate in the trans, transition state, and cis conformations. the black line represents the distance between the carbon atom of the guanidinium moiety of the conserved arg 55 residue in the binding site of cyclophilin and the carbonyl oxygen of the proline of the substrate. the gray line depicts the distance between the backbone nh group of asn 102 in the binding site of cyclophilin and the carbonyl oxygen of gly of the substrate. the blue line monitors the substrate in the binding site of cyclophilin and represents the distance between the c- atom of proline of the substrate and the c- atom of phenylalanine, one of the residues in the hydrophobic pocket of the binding site. similarly, but to a lesser extent, the distance between the enzymesubstrate complex with the substrate in the cis isomer is not stable and also correlated with the stability of the complex. the substrate of the cis isomer diffuses out of the binding site immediately as the arginine residue disengages with carbonyl oxygen of the proline residue. however, the hydrogen-bond contact that is formed between the arginine residue and the carbonyl oxygen of proline of the cis isomer is more stable than that of the trans isomer (figure 5). in contrast to the enzymesubstrate complexes of the trans and cis isomers, the hydrogen bond between the guanidinium moiety of the arginine residue and the carbonyl oxygen of proline of the transition-state complex is very stable and stays in the binding site during the course of the 50 ns simulation, as also shown in figure 5. arginine 55 therefore acts as an anchor for the substrate in the binding site by preferentially stabilizing the transition state over the trans and the cis isomers. consequently, mutation of arg 55 to ala would result in reduction of the catalytic efficiency, as was previously shown.(36) the transition state also makes an additional favorable contact (figure 3) due to the long-lasting hydrogen-bond interaction between the carbonyl oxygen of the gly residue and the backbone hydrogen of asn 102, as also shown in figure 5 (gray line). the gly of the trans isomer in the complex never forms a hydrogen bond with the backbone nh group of asn 102 as also shown in figure 5. the carbonyl oxygen of gly of the cis isomer in the complex does form a hydrogen bond with the backbone nh group of asn 102 (figure 5) as in the transition-state complex, but to a much lesser extent. therefore, the stabilizing role of arg 55 and the asn 102 qualitatively agrees with the predicted trend in binding energies; that is, the transition state binds stronger than the cis isomer, which in turn binds stronger than the trans isomer. cistrans isomerization of peptide bonds of proteins is a very important switching mechanism in biology that is involved in many cell signaling pathways. even with the help of peptidyl prolyl isomerases, such as cyclophilin, the resulting time scale is beyond that of normal molecular dynamics. it is clear from previous experiments that there are no bond formation and cleavage events occurring during catalysis; therefore, we have used classical molecular mechanics coupled with the accelerated molecular dynamics methodology to shed some light on this very important catalytic mechanism. using the accelerated molecular dynamics method that allows us to overcome the submicrosecond time scale limitation of normal molecular dynamics simulations, we have studied the catalytic mechanism of cyclophilin in full-atomistic detail in explicit water. aside from a possible small electronic contribution that has not been captured by the classical mechanics empirical force field, we are able to fully describe the catalytic mechanism of cyclophilin and provide quantitative estimates of the free energies associated with the process. the catalysis is shown to occur mainly through the stabilization of the transition state in the binding site due to a combination of favorable hydrophobic and very long-lasting hydrogen bonding interactions. cyclophilin decreases the barrier height of the trans to cis transition by 6.3 kcal/mol, which when coupled with other factors, such as a possible change in the kinetic prefactor, could speed up the isomerization process by as much as 10. a possible effect that the classical force field could not be able to fully capture is some of the small reduction in the barrier height of 1.3 kcal/mol(17) due to a small electronic effect of desolvation of the substrate. if we added this small correction to the calculated barrier height, the overall barrier reduction would be around 7.6 kcal/mol, which would further increase the estimated rate of isomerization. also, since the guanidinium moiety of the conserved arginine residue stabilizes the transition state through interactions with the carbonyl oxygen of proline, this also puts it somewhat close to the nitrogen of the proline. the closeness of the guanidinium moiety to the proline nitrogen could weaken the delocalization of the electron cloud along the pseudo-double peptide bond. however, as we can see, majority of the speedup has been captured using classical accelerated molecular dynamics.

peptidyl prolyl cistrans isomerases (ppiases) are ubiquitous enzymes in biology that catalyze the cistrans isomerization of the proline imide peptide bond in many cell signaling pathways. the local change of the isomeric state of the prolyl peptide bond acts as a switching mechanism in altering the conformation of proteins. a complete understanding of the mechanism of ppiases is still lacking, and current experimental techniques have not been able to provide a detailed atomistic picture. here we have carried out several accelerated molecular dynamics simulations with explicit solvent, and we have provided a detailed description of cistrans isomerization of the free and cyclophilin a-catalyzed process. we show that the catalytic mechanism of cyclophilin is due mainly to the stabilization and preferential binding of the transition state that is achieved by a favorable hydrogen bond interaction with a backbone nh group. we also show that the substrate in the transition state interacts more favorably with the enzyme than the cis isomer, which in turn interacts more favorably than the trans isomer. the stability of the enzymesubstrate complex is directly correlated with the interaction the substrate makes with a highly conserved arginine residue. finally, we show that catalysis is achieved through the rotation of the carbonyl oxygen on the n-terminal of the prolyl peptide bond in a predominately unidirectional fashion.

PMC2651649

pubmed-1223

preeclampsia/eclampsia (pe) syndrome, defined as new onset and persistent hypertension after 20 weeks of gestation in association with significant proteinuria, is a major cause of maternal-fetal morbidity and mortality worldwide. the pathophysiology of pe remains incompletely understood, and anticipation and appropriate management of this disorder are frequently insufficient. the prevalent pathogenic theory of pe includes the manifestation of two characteristic and sequential processes considered to be of paramount importance. the first corresponds to an insufficient placentation, which drives an increase in the resistance of the uteroplacental circulation, and the second involves the maternal reaction through the activation of an inappropriate inflammatory response with a (proposed) globally impaired endothelial function (ef). structural and functional alterations in large arteries have also been reported accompanying pe syndrome [4, 5]. impaired ef and arterial damage could occur for a certain time before significant proteinuria and clinical manifestations of pe become apparent [3, 6]. thus, the possibility of identifying early subclinical endothelial dysfunction, as well as structural and/or functional arterial alterations during pregnancy, could be of value in recognizing and classifying the different hypertensive disorders of pregnancy. hopefully, this will have a positive impact on the understanding of this syndrome, as well as on the appropriate and early management of these patients. celermajer et al. 's technique, commonly known as flow-mediated dilation (fmd), utilizes the vascular (or vaso-) reactivity test (vrt) and has become the most popular method to assess ef. the vrt consists of positioning a pneumatic cuff around the upper arm and provoking an arterial occlusion for five minutes (transient ischemia, ti). this maneuver elicits an increase in blood flow in the brachial artery once the cuff is deflated (i.e., reactive hyperemia, rh), which subsequently stimulates endothelium to release nitric oxide (no). finally, locally produced no results in a dilation of the brachial artery (assessed by b-mode ultrasound) and a reduction of arterial stiffness (changes in pulse wave velocity (pwv) assessed by mechanotransducers). the magnitude of the arterial dilation is used as an indicator of ef, and healthy pregnant women show an enhanced vascular response evaluated by this method compared with healthy nonpregnant women [9, 10]. whereas fmd provides information about the recruitability of ef (i.e., its responsiveness to a specific stimulus), it does not provide information concerning basal/tonic ef (i.e., release of endothelial autacoids before fmd measures are initiated). in this context, gori et al. described a novel index for assessing the response of the artery to low flow, which utilizes data obtained from the cuff occlusion period of an fmd test. synonymous to fmd, the vasoconstriction observed under conditions of reduced flow has been named low-flow-mediated vasoconstriction (l-fmc). inclusion of l-fmc data to traditional measurement of fmd could provide additional and/or complementary information, which, they propose, may improve the detection of patients with cardiovascular disease and profile the vascular response to exercise among healthy volunteers. whether the integration of l-fmc into traditional fmd studies will provide additional/complementary information among patients with hypertensive disorders in pregnancy is unknown. in addition, changes in arterial stiffness assessed by means of carotid-to-radial pulse wave velocity (pwvcr) due to the same test (vrt) have been proposed as an alternative tool for the evaluation of ef [8, 14]. pwv, in particular carotid-to-femoral pwv, is recognized as the gold standard parameter for the evaluation of regional aortic stiffness having a wide biomedical application. a reduction in pwvcr values (i.e., upper limb region) in response to vrt has been evidenced in healthy young adults, whereas a blunted reduction has been reported in pathophysiological circumstances such as hypertension and congestive heart failure. however, the impaired ef (which could follow hypertensive disorders of pregnancy) can be assessed by using pwvcr changes and if it provides additional or complementary information to those of brachial diameter assessment has not been studied yet. in this context, the aims of this work were as follows: firstly, to determine noninvasive central and peripheral arterial parameters in a group of healthy and hypertensive pregnant women, through the use of validated techniques and parameters; secondly, to determine and analyze basal and recruitable ef through the measurement of fmd, l-fmc, and pwvcr changes. the normotensive subjects (healthy pregnant women, hp; n=10) were recruited from the routine antenatal clinic. women with preeclampsia (pe; n=8) and with gestational hypertension (gh; n=8) were recruited from the antenatal hospital ward, where they were admitted due to mild hypertension (140/90 to 149/109 mmhg). the definitions used followed the classification of the gestational hypertensive disorders, as recommended by the report of the national collaborating centre for women's and children's health, hypertension in pregnancy, of the national institute for health and clinical excellence. under this classification, pe was defined as bp greater than 140/90 mmhg on two consecutive occasions more than 4 h apart, in combination with significant proteinuria (> 300 mg total protein in a 24 h urine collection) developing after 20 weeks of gestation in previously normotensive women. all pe included in the study were mild in terms of the severity of the syndrome. participants were asked to abstain from physical activity and vitamin supplementation for at least 4 hours prior to the examination. baseline demographic data were obtained by an obstetrician during a clinical interview and laboratory samples were extracted prior to the examination. the study protocol was approved by the ethics research committee of the school of medicine (republic university, uruguay) and all participants gave written informed consent. after recompilation of clinical and laboratory data, subjects were instructed to lie in a left lateral position (to avoid vena cava compression by the uterus) in a temperature-controlled (2123c) room, for at least 15 minutes, in order to establish stable hemodynamic conditions. heart rate (hr) and right brachial (peripheral) systolic and diastolic blood pressure (psbp and pdbp, resp.) were measured using an oscillometric device (omron hem-433int oscillometric system; omron healthcare inc., mean blood pressure (mbp) was derived from the standard equation usually employed at the peripheral level: mbp=pdbp+1/3(psbp pdbp). the carotid-to-femoral pulse wave velocity (pwvcf) was measured to analyze aortic regional stiffness. to this end, carotid and femoral artery waveforms were consecutively obtained with a high-fidelity applanation tonometer from the carotid and femoral regions simultaneously with continuous ecg monitoring (sphygmocor 7.01, atcor medical, sydney, australia) (figure 1). then, carotid-femoral propagation time (t3) was determined by subtracting the time delay between the peak of r wave of the ecg recording to femoral foot of the pressure waveform (t2) of the corresponding cardiac cycle and the time delay between the peaks of r wave to carotid foot of the pressure waveform (t1). the algorithm utilized to detect the so-called foot of the wave was the intersecting tangents. straight distance between the recording sites (carotid-to-femoral distance (c-f x)) was then carefully measured using tape on the body surface to reduce the influence of altered body contour in pregnancy. finally, pwvcf was automatically calculated as the quotient between c-f x and t3 (figure 1). the reported value of pwvcf for a subject was always the average of at least eight consecutive beats. pulse wave analysis (pwa) was used to assess central hemodynamics as well as systemic arterial stiffness and wave reflections. for this purpose, mean radial artery waveform was obtained (through the acquisition of many cycles) with the applanation tonometer from the wrist, and a corresponding mean ascending aortic pressure waveform was generated with a validated generalized transfer function using the same mentioned customized software (sphygmocor 7.01, atcor medical, sydney, australia). the radial pulse waveform was then calibrated using the diastolic and mean arterial pressure obtained at the brachial artery. central systolic, diastolic, and pulse blood pressure (csbp, cdbp, and cpp, resp.), heart rate (hr) corrected central augmentation index (ap/cpp 100[%] heart rate adjusted to a hr of 75 bpm; aix@75), and amplification ratio (ppp/cpp) were determined with the integrated software. ultrasound assessment of carotid arteries was based on the techniques and recommendations described in international consensus. high-resolution b-mode ultrasound images of both (right and left) common carotid arteries (ccas) were obtained using a 10 mhz linear-array transducer connected to a portable ultrasound system (sonosite, micromaxx, sonosite inc., 21919 30th drive se, bothell, wa 98021, usa). measurements (still images and video clips/cine loops) were digitally stored for off-line analysis (figure 1). near and far walls were analyzed and images were obtained from anterior, lateral, and posterior angles. at first, a carotid plaque screening was performed, for which the definition used was a focal structure that encroaches into the arterial lumen of at least 0.5 mm or 50% of the surrounding intima-media thickness or demonstrated a thickness of greater than or equal to 1.5 mm. then, longitudinal views of the ccas were acquired and a video (cine-loop) of at least 10 seconds was recorded and stored. the cimt and beat-to-beat diameter waveforms were obtained and analyzed off-line using a step-by-step border detection algorithm (based on changes in acoustic impedance (z)), applied to each digitized image (hemodyn-4 m software, buenos aires, argentina). a region of 1.0 cm proximal to the carotid bulb was identified, and the far wall cimt was determined as the distance between the lumen-intima and the media-adventitia interfaces (figure 1). the software performs multiple automated or semiautomated measurements along the centimeter and averages them, increasing the accuracy of the measures. the instantaneous diameter (from the leading edge of the near wall intima-media interface to the intima-media interface of the far wall) waveform then, complementary biomechanical parameters such as peterson's elastic modulus (ep) and beta stiffness-index () were calculated relating these measures with central blood pressure as follows:(1)ep=csbpcdbpsddd/dd,=lncsbp/cdbpsddd/dd, where csbp, cdbp, sd, and dd are central systolic and diastolic blood pressure and carotid systolic and diastolic diameter, respectively (figure 1). ep measures the ability of the arteries to change their dimensions in response to the pulse pressure caused by cardiac pulsatile ejection (pressure change required for (theoretic) 100% increase in diameter), whereas is considered to be relatively independent of blood pressure levels. once baseline noninvasive arterial evaluation was carried out, we utilized the theoretical basis, general protocol, and methodological aspects of the vrt recommended by the guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery [7, 18]. for this purpose, participants were submitted to five minutes of ischemia by occluding left radial and cubital arteries using a pneumatic cuff placed around the left forearm (just below the elbow to at least 50 mmhg above psbp) and several parameters of vascular reactivity were measured before, during, and after ischemia (figure 1). accepted methodology for the evaluation of ef (recruitability) and simultaneously for pwvcr measurement (see later), left brachial artery was visualized longitudinally above the antecubital crease using same high-resolution b-mode ultrasound device mentioned earlier (sonosite; micromaxx; usa) (figure 1). similarly, video sequences were recorded at rest, during forearm occlusion and after cuff deflation. subsequently and similarly to the processing of carotid images, recordings were analyzed off-line using same automated step-by-step algorithm applied to each digitalized image that allows the brachial diameter waveform obtainment and fmd and l-fmc calculation. brachial local stiffness (ep and) was also determined by relating brachial arterial pressure and brachial diameters, as was explained earlier for carotid measurements. fmd was quantified as the percentage of change in brachial dd, considering the basal levels and those measured one minute after cuff deflation:(2)fmd%=ddafter cuff deflationddbaselineddbaseline100. in addition, doppler signals were performed to acquire blood flow velocity in baseline conditions and at specific moments during the reactive hyperemia period. doppler signals were used to obtain the brachial shear rate (and its percentage of change), relating mean blood flow velocity (vm (cm/s)) to brachial mean diameter (dm) according to the following equations:(3)sr=vmdm, sr%=sr after cuff deflationsr baselinesr baseline100. sr is an estimate of shear stress without accounting for blood viscosity and was obtained for the characterization of the endothelial stimulus. noninvasive, carotid, and radial pressure waveforms were simultaneously obtained using strain gauge mechanotransducers (motorola mpx 2050, motorola inc., corporate 1303 e. algonquin road, schaumburg, illinois 60196, usa) by placing them on the skin over the carotid and radial sites (left hemibody). pwvcr was determined taking into account the given distance between these arterial sites (c-r x) and the time delay (t) between the carotid and radial waveforms onset (figure 1). the algorithm used for the detection of the foot waves was described and explained in previous work. although a four-minute recording after cuff release was obtained, one minute after ischemia was the specific moment where the analysis was especially taken, according to previous reports [8, 16] (figure 2). pwvcr levels corresponding to baseline and to postischemia period were determined by averaging eight consecutive beats. after that, percent of change of pwvcr (with respect to basal levels) was quantified as follows:(4)pwvcr%=pwvcrafter cuff deflationpwvcrbaselinepwvcrbaseline100.all structural and function arterial evaluations were done by the same trained operator. the statistical analyses were performed using the statistical package for social sciences (version 22.0). normality of the distribution of the data was examined using the shapiro-wilk test and q-q plot. all data are presented as mean value (mv) standard deviation (sd). two-way analysis of variance (anova) was employed for the evaluation of differences in variables within and between hypertensive and control pregnant women. differences in percentage of change of variables determined before and after the vrt (arterial diameter, pwv, and shear rate) were evaluated using two-tailed paired student's t-test. recordings were successfully obtained from all women and all studies were included in the analysis. the mean duration of the studies was 1 hour approximately and they were all well tolerated (without symptoms and/or complications). the mean gestational age at examination of all the pregnant women was 35 3 weeks. significant proteinuria in the daily urine collection could divide the group of hypertensive pregnant women in those with preeclampsia (with significant proteinuria>300 mg/24 hours, pe) and those with gestational hypertension (without or with only traces of proteinuria, gh). maternal age, gestational age, and number of previous gestations were similar between study groups. body weight and body mass index (bmi) were significantly higher in pe compared with hp and gh (p<0.05). uric acid levels were within normal values in hp and gh, while in pe they were abnormally increased. baseline peripheral sbp, dbp, and map levels were significantly higher in pe and gh in comparison with hp (p<0.001). no peripheral bp differences were found among groups with hypertension (gh versus pe). in addition, pe showed higher values of csbp compared with gh (p=0.004), without differences in cpp and cdbp. when compared with hp women, levels of csbp and cdbp in women with pe and gh were higher. aix@75 and the amplification ratio, two composite measures of systemic arterial stiffness and wave reflection amplitude, were analyzed and are presented in table 2. aix@75 was significantly higher in pe with respect to gh and hp (24.3 5.7% versus 11.8 7.6 and 12.2 12.4%, resp. no significant differences were found in this parameter between gh and hp. on the other hand, amplification ratio (cpp/ppp) was only statistically different between pe and gh, with pe having the lowest values. when analyzing muscular peripheral arteries (i.e., brachial artery) by local (ep and) and regional arterial stiffness parameters (pwvcr), no differences were found among groups. however, cca and aorta (i.e., elastic arteries) showed meaningful differences in stiffness. for example, right cca ep was significantly increased (duplicating approximately its values) in pe with respect to hp and gh (p<0.001 and p=0.004, resp.). similar tendencies were noticed in from the right cca but not reaching statistical differences, indicating that changes in carotid artery stiffness in pe and gh are pressure-dependent. on the left side, differences were observed in ep comparing pe and hp, and similar tendencies were maintained for. finally, hypertensive pregnant women showed higher values of pwvcf (regional aortic stiffness) compared with hp women. however, no differences were found between the groups with hypertension, although women with pe had a tendency to show higher values (p=0.14). none of the groups (hp, gh, or pe) presented atherosclerotic plaques. right, but not left, cimt was significantly elevated in pe with respect to hp women (p=0.010). taking into account the vrt (vascular reactivity test), all groups evoked endothelial stimulus (reactive hyperemia) evaluated by changes in shear rate before and after cuff deflation (p<0.001). in addition, peak sr and sr% were the same among groups (p=0.86 and p=0.39, resp.) (table 3). no significant changes were found in heart rate or blood pressure intra- and intergroup before and after cuff deflation, ensuring stable hemodynamic conditions during the maneuver (data not shown). regarding the fmd, all of them showed a dilatation of the brachial artery with respect to the basal state but without statistical significance in women with pe. as was expected, hp women showed quantitatively the highest fmd response (9.4 3.0%; p<0.001), while women with gh and pe reached the lowest values (3.6 3.3%; p=0.021; 2.2 2.9%; p=0.081, resp.). fmd mean values of gh and pe compared to hp were significantly different (p<0.001). as was mentioned above one minute after the cuff deflation, pwvcr decreased only in hp (7.0 1.6 to 5.9 0.8 m/s, p<0.01). gh showed a blunted hyperemic pwvcr response (7.1 0.9 to 7.0 0.8 m/s; p=0.627), while pe showed a tendency to increase arterial stiffness (6.0 1.1 to 6.4 1.3 m/s; p=0.06). pwvcr percentage changes [pwvcr (%)] differed comparing hp women with women with gh (13.9% versus 0.9%; p<0.01) and with pe (13.9% versus+7.0%; p<0.01). l-fmc of the brachial artery was different according to the pregnancy status (p<0.001). maximal vasoconstriction (negative values) was observed in hp women (7.8 3.7%, p<0.001) followed by women with gh (4.5 2.1%, p<0.001), while women with pe did not reach significant arterial constriction during the cuff inflation (0.7 3.5; p=0.576) (table 3). demographic, anthropometric, and laboratory variables shown in table 1 did not significantly correlate with any of the arterial parameters. in addition, there was no significant correlation between parameters of ef (i.e., fmd, l-fmc, and pwvcr%) and aix@75 or amplification ratio (data not shown). however, a low but statistically significant correlation was found between baseline pwvcf and l-fmc (r=0.45, p=0.04), without reaching statistical significance with other ef parameters. a significant correlation between fmd, l-fmc, and pwvcr% was seen among these parameters in the whole study population (figure 3). the present study is, to our knowledge, the first one to determine and assess simultaneously, in a group of healthy and hypertensive pregnant women, the vascular reactivity or ef by using three different but complementary methods in conjunction with the determination of central and peripheral arterial structural and functional parameters. the main results of this work were as follows: (1) central aortic blood pressure and wave reflections as well as elastic (aortic and carotid) arteries stiffness are increased in pe, with respect to peripheral blood pressure-matched gh and hp, and (2) pe showed both resting (l-fmc) and recruitable (fmd and pwvcr%) endothelial dysfunction. among the methods that allow measurement of vascular reactivity or ef in the clinical setting, fmd has rapidly gained popularity because of its simplicity, reproducibility, and noninvasiveness [7, 18]. however, as was mentioned earlier, one important limitation of fmd is that it only provides information about the recruitability of ef (i.e., its responsiveness to a specific stimulus) and not about concerning resting ef (i.e., release of endothelial autacoids before fmd measures are initiated). we here analyze in hypertensive pregnant women both types of functional aspects of ef: endothelial recruitability through fmd and pwvcr changes and resting endothelial tone through l-fmc. the magnitude of fmd observed in hp in response to vrt was similar to that described in previous reports [9, 10]. as it was expected, hypertensive pregnant women showed a reduction in fmd with respect to hp, in coherence with greater degrees of endothelial dysfunction [22, 23]. it is noteworthy that only pe did not reach statistical significance in the dilation of the brachial artery, obtaining a more complete blunted response. although the fmd of pe was numerically lower than those from gh, this difference did not reach statistical significance. this could be attributed or not attributed to the magnitude of the standards deviation of the mean due to the low sample size. therefore, the vascular profile from pregnant women with gh who might develop pe could be quite similar to those women with pe. however, there is a lack of information that compares fmd between groups with pe and gh and only few studies directly analyze this issue. according to quinton et al., the fmd at one minute of the cuff deflation was not different between the gh and pe in women who were not receiving any medication, while there were statistical differences between these groups when women were receiving medical treatment. nevertheless, in a prospective study conducted by filho et al., they did not find differences in fmd of the brachial artery in patients with two different forms of hypertensive disorders of pregnancy. when analyzing changes in arterial stiffness due to vrt, hp showed the major reduction in pwvcr values. on the other hand, women with hypertension showed not only a blunted response in pwvcr changes but also, in pe, a tendency to increase arterial stiffness one minute after the cuff deflation was evidenced. indeed, by means of this method, changes of pwvcr in pe tended to be higher in comparison to gh, indicating probably greater degree of impairment of ef. it is noteworthy that all participants showed the same increase in blood flow velocity with respect to basal conditions after cuff deflation (endothelial stimulus), and variables such as baseline levels of pwvcr, basal brachial diameter, blood pressure, and gestational age were similar among the groups. taking into account resting endothelial tone, our results show that, during cuff inflation, brachial artery responses varied between the studied groups. l-fmc of the brachial artery was significant only in hp and gh, without any constriction in pe, suggesting that pe develop also basal endothelial dysfunction. although l-fmc was firstly described and assessed at the radial artery, spiro et al. evidenced later that this phenomenon also occurs in healthy subjects at the brachial artery and it can be measured reliably. studies agree that radial artery vasoconstriction occurs during cuff inflation in nonpregnant women [12, 28, 29], whereas recent studies examining the brachial diameter during occlusion demonstrate conflicting results [27, 28, 3032]. differences in cardiovascular profile, methodological issues, and interobserver variability could explain the widely variable results. l-fmc of the brachial artery in a regimen of low but not zero blood flow (as it occurs in the radial artery) in a level that is upstream of the occlusion site. therefore, the magnitude of reduced blood flow in the brachial artery and its relationship with the basal levels (endothelial negative stimulus for vasoconstriction) should surely yield different brachial responses. as it was previously reported, we found that women with pe showed marked structural and functional alterations in peripheral and central hemodynamics [4, 3335]. pe had a strong tendency to present higher values in practically all studied parameters related to central hemodynamics. for instance, central sbp, aix@75, cca ep, and pwvcf were significantly higher in pe with respect to hp. we also found differences in central hemodynamics between women with hypertension, but this was not the rule as it was for pe versus hp. only csbp, aix@75, and right cca ep were markedly augmented in comparison to gh. these findings were not due to differences in peripheral blood pressure, which was elevated to a similar degree in both types of hypertensive states. these pieces of information analyzed together indicate that women with hypertensive disorders in pregnancy (mainly pe) have increased central bp overload, central arterial stiffness, and amount of wave reflections, probably related to a vasoconstriction state due to endothelial dysfunction. altered central hemodynamics in pe may signify an inadequately increased left ventricle afterload and myocardial oxygen demand in the mother circulation, as well as hemodynamic disturbances transmitted to the fetal circulation. blunted fmd, l-fmc, and pwvcr changes evidenced in pe are in consonance with the plasma uric acid levels that were found elevated only in this group. in previous reports, hyperuricemia was associated with an increase of plasma xanthine oxidase activity and/or a reduction in antioxidant systems related to increased formation of reactive oxygen species and endothelial dysfunction. could reflect an associated overweight/obesity state, differences in na and body fluid retention by the hemodynamic overload due to the hypertensive condition, or a combination of both. our results indicate that brachial artery responses to inflation and deflation of the cuff related to endothelial dynamics could share some vascular mechanism. however, there are confusing results around the fmd and l-fmc correlation, with variable results depending on the analyzed artery (brachial versus radial) and type of physiological or pathophysiological circumstance [1113, 27]. although both l-fmc and fmd are an expression of the vascular reactivity in response to changes in blood flow, their relationship is neither conceptually simple nor mathematically linear. on the other hand, when analyzing the relationship between fmd and pwvcr the analysis can also be a little more complex. according to moens and korteweg equation, pwv is determined by arterial diameter and also by the elastic modulus. if post-vrt changes in pwvcr in pe and gh would have followed only the changes in brachial diameter (fmd), the obtained changes in pwvcr would have shown an equal behavior to the geometrical change (change in diameter). however, in accordance with the obtained values in the groups with hypertension, a dissociation among these variables was evidenced, with an increase of arterial diameter (which would reduce the levels of pwvcr) without significant changes in pwvcr levels (or even a trend to increase in pe) after the cuff deflation period. this discordance between parameters behavior in response to the vrt indicates an increase in the elastic modulus in parallel with changes in the arterial diameter. thus, at least in pe, we evidenced a reciprocal and simultaneous change in the vascular wall intrinsic properties and the brachial diameter. an impaired response to changes in blood flow in a concrete vascular ledge (e.g., brachial artery), without simultaneous adequate change both in brachial diameter and in arterial stiffness, could have important hemodynamic consequences. at first, a reduction in the vasodilator reserve related to endothelial dysfunction as it was seen in other pathophysiological circumstances could implicate an incapacity of the arterial system to determine an appropriate vascular adjustment against hemodynamic changes in the long (fetal growth) and even in the short term (exercise, change of position, etc.). second, an impaired capability of response to hemodynamic changes due to endothelial dysfunction could yield other functional cardiovascular alterations that was seen in pe, like increased left ventricle afterload and diastolic dysfunction. this point is in consonance with altered values of central parameters found in pe mentioned above. the important additional information brought by introduction changes in pwvcr and l-fmc, together with the information of central and peripheral hemodynamics, is that these variables provide information concerning a different aspect of vascular reactivity and ef, therefore complementing (and not overlapping with) the information provided by fmd. this vascular approach may provide a more comprehensive assessment of vascular state and endothelial function in hypertensive disease of pregnancy. however, our findings were statistically significant and, by definition, this indicates that the study was adequately statistically powered. our technical approaches including the use of both multiple automated and semiautomated edge-detection/point software in ultrasound image and pressure wave assessment are largely operator-independent and also empower our findings. given the means of the different variables and sds observed in previous works and in the present sample, twenty-five subjects (n=25) of the total sample size (the sum of the sizes of comparison groups) would be required to detect a statistically significant effect of the pregnancy status with at least 80% of power. there is no isolated technique which satisfies completely this purpose with enough accuracy. at the present time, different combinations of clinical risk factors, biochemical markers, and doppler ultrasound of the uterine arteries are recommended. the detection rate of pe using only one clinical model of screening that includes risk factors (e.g., nulliparity, maternal age, family history of pe, etc.) is 45.3%, while only with doppler ultrasound of uterine arteries at the second trimester it is 63.1% and with a combined approach it reaches 67.5%, with a 25% of false positive rate. the clinical importance of improving detection of pe can also be stressed when confidential enquiries are analyzed, showing that in a substantial proportion of cases of fetal death due to preeclampsia a different management might have altered the outcome. moreover, the evidence demonstrates that administration of antiplatelet agents (primarily low dose of aspirin in different trials) to well-selected women leads to a significant reduction in the risk of developing preeclampsia and its serious consequences. for these reasons, an accurate prediction of preeclampsia or early diagnosis may, therefore, allow more efficient allocation of resources for monitoring and improving maternal and perinatal outcomes [1, 2]. on the other hand, the extensive and growing information that links endothelial dysfunction/arterial damage with pathophysiology of pe motivates researchers and clinicians to evaluate arterial parameters (including endothelial function) in this clinical setting. additionally, there is a need to count with a more comprehensive assessing ef in a patient in concrete. in that sense, the inclusion of validated arterial parameters and a more complete ef evaluation in the contemporary assessment of preeclampsia into multiparametric models could improve prediction of pe. in this small study, which addresses the feasibility of measuring these parameters simultaneously, simply, and noninvasively, we found encouraging results that we believe warrant further investigation in order to contribute to the early recognition of preeclampsia. this is the first study that measures and analyzes, in the same pregnant women, central and peripheral hemodynamics and ef by using different parameters that offer additional and complementary information. resting and recruitable ef from pregnant women can be assessed by using pwvcr changes and l-fmc, respectively. central aortic pressure and wave reflections as well as stiffness of elastic arteries are improperly increased in pe. future studies will have to determine if incorporation of these pieces of information together, assessing basal state and functional reserve or capability of response of the vascular system into multiparametric models that include clinical, obstetric, and laboratory variables and doppler ultrasound of uterine arteries, will be able to improve contemporary prediction of preeclampsia (from healthy pregnancy and from gestational hypertension). hopefully, this could change the clinical management and prognosis of the pregnant women with pe.

introduction. an altered endothelial function (ef) could be associated with preeclampsia (pe). however, more specific and complementary analyses are required to confirm this topic. flow-mediated dilation (fmd), low-flow-mediated constriction (l-fmc), and hyperemic-related changes in carotid-radial pulse wave velocity (pwvcr) offer complementary information about recruitability of ef. objectives. to evaluate, in healthy and hypertensive pregnant women (with and without pe), central arterial parameters in conjunction with basal and recruitable ef. methods. nonhypertensive (hp) and hypertensive pregnant women (gestational hypertension, gh; preeclampsia, pe) were included. aortic blood pressure (bp), wave reflection parameters (aix@75), aortic pulse wave velocity (pwvcf) and pwvcr, and brachial and common carotid stiffness and intima-media thickness were measured. brachial fmd and l-fmc and hyperemic-related change in pwvcr were measured. results. aortic bp and aix@75 were elevated in pe. pe showed stiffer elastic but not muscular arteries. after cuff deflation, pwvcr decreased in hp, while gh showed a blunted pwvcr response and pe showed a tendency to increase. maximal fmd and l-fmc were observed in hp followed by gh; pe did not reach significant arterial constriction. conclusion. aortic bp and wave reflections as well as elastic arteries stiffness are increased in pe. pe showed both resting and recruitable endothelial dysfunctions.

PMC4550743

pubmed-1224

the analysis is based on food consumption data collected in nine european countries: denmark, finland, germany, ireland, italy, poland, spain, the netherlands, and united kingdom. the data were collected by an international life science institute (ilsi) europe expert group (9). the selected micronutrients are vitamin a, retinol, vitamin d, vitamin e, niacin, vitamin b6, folate, calcium, magnesium, iron, zinc, phosphorus, iodine, selenium, and copper. data reflect intakes from natural sources plus mandatory fortification (practiced in denmark, ireland, and poland) other fortification and supplements are not included. the survey methods comprise seven-day records or diaries with estimated or weighed amounts of foods (denmark, ireland, italy, and uk) and 24 (poland) or 48-hour recall (finland, the netherlands, and spain). germany used a modified diet history and the dutch and spanish recalls were supplied with semiquantitative food frequency questionnaires. the data from spain and the netherlands have been adjusted to correct for short registration period (9). national dietary surveys with information on year, methodology, survey size, and age range intake of nutrients was calculated using national food composition databases. the intake data provide data from four groups: children 410 years, children 1117 years, adult women, and adult men. the analytical process included steps 2 and 3 of the following four steps for each of the selected vitamins and minerals and for each selected gender and age group: calculation of mean intakes from each of the dietary surveys (an estimate of mean european intake).calculation of the ratio between the 95th percentile and mean intake in each survey.calculation of the average of the ratios.calculation of the common estimate for high intake as the product between steps 1 and 3 (an estimate of european 95th percentile intake). calculation of mean intakes from each of the dietary surveys (an estimate of mean european intake). calculation of the common estimate for high intake as the product between steps 1 and 3 (an estimate of european 95th percentile intake). average ratios for each country and the overall average arranged in ascending order are shown in table 2. the results show that for each nutrient the ratio has approximately the same value from one survey to another, implying that the ratio between high and average intake of micronutrients follows a regular pattern across countries and survey methods. the polish ratios are systematically higher than those of the other countries due to the short registration period, which causes a wider intake distribution. the spanish and to some extent also the dutch ratios are lower than the others probably because of adjustment of primary data. in the present analysis average of ratios (p95:mean intakes) from national dietary surveys in nine european countries intake is not reported. note: the overall average is based on ratios from all gender and age groups, i.e. normally four ratios from each country. overall, the ratios can be divided into three categories: one category with overall ratios of the 95th percentile and mean intake between 1.48 and 1.58; this range in ratios is close to the energy ratio (1.45) and includes the nutrients: magnesium, phosphorus, zinc, iron, vitamin b6, niacin, and folate.another category has overall ratios in the range between 1.67 and 1.79 and includes the nutrients: calcium, selenium, vitamin e, iodine, and copper.a third category has overall ratios in the range of 2.072.32 and comprises the nutrients: vitamin a, vitamin d, and retinol.in table 3, ratios of energy and micronutrients for young and older children, and adult females and males are presented. ratios for the group of young children tend to be slightly lower than the ratios for adults. the intake distributions of children aged 1117 years are wider and thus the ratios higher because of a wider range in energy requirements in this group. an exception from this tendency is the ratios of vitamin a, vitamin d, and retinol, which are lower than other group values. one category with overall ratios of the 95th percentile and mean intake between 1.48 and 1.58; this range in ratios is close to the energy ratio (1.45) and includes the nutrients: magnesium, phosphorus, zinc, iron, vitamin b6, niacin, and folate. another category has overall ratios in the range between 1.67 and 1.79 and includes the nutrients: calcium, selenium, vitamin e, iodine, and copper. a third category has overall ratios in the range of 2.072.32 and comprises the nutrients: vitamin a, vitamin d, and retinol. average of ratios (p95:mean intakes) from all counties divided into age and gender groups note: figures in parentheses are the number of countries contributing to the average. the results of the present study show that the ratio between high and mean intake for each micronutrient is fairly stable from one survey to another. the similarity occurs despite differences in dietary habits across countries and despite several differences of methodological nature (type of dietary survey, food composition data base, sampling procedure, etc.). this similarity probably reflects the biological nature of eating and eating a variety of foods. we divided the ratios into three rough categories in an attempt to explain the findings. since most nutrients are widely distributed in rather low concentrations in many foods, their ratios are close to the ratio between the 95th percentile and mean energy intake (category 1). in category 2, intakes still follow energy intake but the intake distribution may be influenced by a few foods, which are important nutrient sources and has a skewed intake distribution, e.g. milk, which is the major source of calcium. the third category includes highly skewed intake distributions due to only a few significant food sources of the micronutrient in question. this effect may explain why the ratios for vitamin a, retinol, and vitamin d are lower among the children aged 1117 years than the three other groups, probably reflecting that most teenagers dislike fish, liver, and carrots foods very rich in these nutrients. the only identifiable factors clearly affecting the ratios are the length of registration period and modification of primary data. because of the pragmatic purpose of this study and because excluding, i.e. polish data does not change the overall ratios much, we decided to accept all survey results. the reason for this was the assumption that most underreporters are individuals found in the lower end of the intake distribution (i.e. below mean intake) and that the pattern of underreporting is the same in all populations. if the intakes of underreporters were increased to true levels, the intake distribution would be narrowed probably without any significant increase in levels of high intakes. this means that the mean values will increase and the ratio p95/mean will be reduced. the final product between the overall higher mean and lower ratio will be approximately unchanged. we are fully aware of the limitations of the applied procedure where we used aggregated micronutrient data. with access to the individual data from each country but even if the calculations are not mathematically (statistically) correct they will still create an estimate of high intake, which is sufficiently valid for the purpose of the present study. the uncertainty is hardly any larger than that of results from any of the single surveys. one could argue that simply calculating the average of 95th percentiles would be a more simple and direct way to a common estimate of high intake. this is true and the results would probably be close to the results calculated via the ratios. another argument against the present approach is the use of mean instead of median values. but using the ratio between 95th percentile and median did not change the final results (results not shown) and since it is important to find figures that will satisfy all member states in the eu, we suggest the use of mean values instead of median values. using the present method makes it possible to include mean intake estimates from dietary surveys that are not able to describe the intake distribution. this will allow us to use data from all or at least most european countries and thus increase the acceptability of the results across all eu member states. flynn et al. (4) used a different approach in their model for calculating safe additions to foods. they expressed in each survey the high intakes (9097.5th percentiles) as multiples of recommended intakes and used the mean of these multiples as a representative value. however, this method does not allow use of results from surveys giving only mean intakes. unfortunately the ilsi study does not provide data for all nutrients but we assume that the remaining vitamins and minerals will fit into a similar pattern. it still has to be shown based on dietary intake data whether this assumption is true, before the proposed model for calculating common european estimates for high intakes of all vitamins and mineral can be implemented in administrative practice. the average ratio of the 95th percentile and the mean intakes of energy and micronutrients from foods show a remarkable similarity across countries and age groups. for each nutrient it is possible based on dietary surveys from different countries to calculate a representative european average. the average ratio of the 95th percentile and the mean intakes is a simple and suitable measure for calculation of an estimate of high intake from an estimated average intake by the four steps described in the methods section. it is possible to include mean intake estimates from dietary surveys, which are not able to describe the intake distribution. the authors declare that they have no conflict of interest and have received no funding for the present paper.

background: a central element in establishing maximum amount of micronutrients in fortified foods and supplements is to reach to an agreement on how to estimate high intakes of vitamins and minerals from the european diet. objective:to examine whether ratios between the 95th percentile and mean intakes of vitamins and minerals show similarities across different countries independent of dietary habits and survey methods and if so, to suggest a simple and pragmatic way to calculate common estimates of high micronutrient intakes from foods.design:intake data of selected vitamins and minerals from nine european countries were examined for adult females and males and for children aged 410 and 1117 years. the ratios between the 95th percentile and mean intakes were calculated for each micronutrient, country, and age group. results:the ratios for each micronutrient follow a fairly regular pattern across countries and survey methods with differences between age groups.the nutrients fall into three categories: nutrients with ratios between 1.45 and 1.58 energy, magnesium, phosphorus, zinc, iron, vitamin b6, niacin, and folate; nutrients with ratios between 1.67 and 1.79 calcium, selenium, vitamin e, iodine, and copper; nutrients with ratios between 2.08 and 2.32 vitamin a, vitamin d, and retinol. conclusion:sufficiently precise estimates of high micronutrient intakes across european countries can be reached by multiplying the overall average of ratios (p95/mean intakes) for each micronutrient with the corresponding mean intakes from all available dietary surveys in europe. this approach is a simple and pragmatic way to create common european estimates of high micronutrient intakes from foods.

PMC2767238

pubmed-1225

keratoconus is a progressive noninflammatory thinning disorder of the cornea leading to a decrease in visual acuity as a result of myopia and irregular astigmatism [1, 2]. corneal collagen cross-linking (cxl) can effectively halt the progression of the disease, but visual acuity following cxl remains poor. in patients intolerant to rigid gas permeable contact lenses after corneal cxl, additional interventions are often necessary to improve their vision [1, 2]. many visual rehabilitation options are available to manage keratoconus including intracorneal ring segment implantation (icrs), phakic intraocular lenses (piol), and photorefractive keratectomy (prk) and all can be combined with cxl [36]. in patients with poor best-corrected visual acuity icrs implantation the prk is used to correct mild refraction error [3, 4], while the piol's are used to correct moderate to severe ametropia in patient with good best-corrected visual acuity [5, 6]. our study group recently published the 6-month data on the safety and efficacy of cxl followed by insertion of a phakic toric implantable collamer lens (icl) (visian toric v4 icl; staar surgical, monrovia, ca) in the posterior chamber for correction of myopia and astigmatism in patients with keratoconus. in this paper, we report the long-term safety and efficacy of sequential cxl, then icl implantation, separated by 6 months, in a larger cohort of patients with moderate to severe keratoconus with moderate to severe myopia and astigmatism, and good best-corrected visual acuity. this was a retrospective study of patients with keratoconus who underwent sequential cxl-icl procedure between december 2010 and march 2012 at the beirut eye specialist hospital (besh), beirut, lebanon. this study was approved by the institutional review board at besh and complied with the declaration of helsinki. all patients signed an informed consent prior to treatment and all surgical procedures were performed by one surgeon (e.j). jarade's protocol were included if they had a preoperative best-correct visual acuity better than 20/40, were hard contact lens intolerant (defined as a comfortable wearing time of less than 8 hours per day), had an endothelial count>2,200 cells/mm (noncon robo, konan medical), had history of progressive keratoconus in one or both eyes (defined as an increase in maximum keratometry of 1.00 diopter (d) or more in 1 year and/or the need for new contact lens fitting more than once in the previous 2 years), and did not have any corneal surgery (including prk and icrs) before or after the cxl and icl implantation. patients were considered eligible for icl implantation after cxl only if the keratoconus was considered stable (defined as subjective refractions [5, 9, 10] within 0.50 d of spherical equivalent at 4 and 6 months postoperatively and was most of the time equivalent to the refraction prior to cxl). the exclusion criteria for enrollment in this study (those who could not undergo the cxl and phakic iol procedures consecutively) were central corneal thickness of less than 450 m (measured by optical pachymetry (pentacam; oculus optikgerate gmbh, wetzlar, germany)), mean k reading>56.00, endothelial cell count of less than 2,000 cells/mm measured on the central part of the cornea by specular microscopy, anterior chamber depth of<2.8 mm from endothelium to anterior capsule measured by pentacam (oculus optikgerate gmbh, wetzlar, germany), corneal opacification or scars, history of keratitis (any form), peripheral marginal degeneration, previous corneal and/or intraocular surgeries, and autoimmune and/or connective tissue disease. the central corneal thickness limit of 450 m would account for around 400 m of remaining stromal thickness after removal of the epithelium, which is considered as the safety thickness for the residual stroma to avoid endothelial cell damage during the cxl procedure. the criteria for diagnosing keratoconus were based on a combination of computed slit-scanning videokeratography of the anterior and posterior corneal surfaces, keratometric readings, and corneal pachymetry [1316]. keratoconus was classified, according to the amsler-krumeich criteria, into four stages based on corneal power, thickness, transparency, and astigmatism. contact lens use was discontinued for at least 3 weeks for rigid lenses and 1 week for soft lenses prior to any ophthalmic examination, investigation, and treatment. it included uncorrected distance visual acuity (udva), corrected distance visual acuity (cdva), manifest and cycloplegic refractions, anterior and posterior segments evaluation with dilated fundus examination, and keratometric evaluation. since the autorefractometer results of refraction are not always accurate in keratoconus and after both cxl procedures, all refractions were based on refined refraction using trial lenses, and the axis of astigmatism was chosen according to the best visual acuity obtained while rotating the astigmatism trial axis. follow-up examinations were scheduled at baseline and at 1, 3, 6, and 12 months and every 6 months thereafter. the eye to be treated was anesthetized by applying proparacaine hydrochloride 0.5% drops on three occasions at 5-minute intervals. after positioning the patient under the operating microscope, an eyelid speculum was inserted and the central 9 mm corneal epithelium was removed with a blunt spatula. a mixed riboflavin 0.1%20% dextran solution was instilled every 5 minutes until the riboflavin penetrated the cornea (i.e., approximately 30 minutes). the ultraviolet lamp (uv-x illumination system, version 1000; iroc ag, zurich, switzerland) was then focused on the apex of the cornea at a distance of 5 cm for a total of 30 minutes, providing a radiant energy of 3.0 0.3 mw/cm. the required irradiance of 3.0 mw/cm was calibrated prior to each treatment using an ultraviolet a meter (lasermate-q; laser 2000, wessling, germany). during ultraviolet a administration, the cross-linking procedure adopted in our study is in accordance with the standard dresden protocol, which has been shown to result in absorption limited to the anterior two-thirds (200400 m) of the stroma as demonstrated by stress-strain measurements, thermomechanical measurements, and swelling studies. thinnest and central corneal thickness were continuously monitored (sonogage pachymeter; sonogage, inc., cleveland, oh) to ensure that neither of the two parameters dropped below 400 m. after treatment, the eye surface was washed with balanced salt solution and two drops of gatifloxacin 0.3% were instilled, followed by placement of a bandage soft contact lens. postoperatively, patients received acetaminophen 500 mg twice daily for 3 days, one drop of gatifloxacin 0.3% six times daily for 7 days with one drop of tobramycin-dexamethasone 0.1% four times daily for 10 days, and one drop of loteprednol 0.5% five times daily, slowly tapered over 5 weeks. the bandage soft contact lens was removed on postoperative day 4 and the eye examined by slit-lamp microscopy to confirm complete corneal epithelialization. complete assessment was performed 1 and 6 months postoperatively and included udva, cdva, refraction, and anterior/posterior topography. no further progression of keratoconus was noted in any eyes throughout the 6 months of follow-up period. the appropriate icl size was determined based on the horizontal white-to-white distance measured manually with a caliper, and the anterior chamber depth was measured with the pentacam. a minor clinical adjustment of anterior chamber depth was performed by subtracting no more than 0.2 mm whenever corneal anterior bulging was advanced. regarding the inaccuracy of the autorefractometer in predicting the k-reading in many keratoconus cases and to obtain accurate icl choice using the online icl calculator software, adjustment of extreme values of k readings obtained by autorefractometer was performed by attenuating the k-reading values to reflect the magnitude of astigmatism obtained by manifest refraction and the chosen axis of astigmatism was always the axis obtained by manifest refraction. the pupil was dilated with cyclopentolate and phenylephrine drops, instilled 30 minutes prior to surgery, and the horizontal axis was marked by the surgeon with the patient upright to control for cyclotorsion. a 3.2 mm clear corneal tunnel incision was performed in the horizontal temporal meridian (regardless of the astigmatism axis). the icl was inserted in the posterior chamber through the incision using the injector cartridge supplied by the manufacturer. after the icl was gently positioned in the sulcus with the axis properly aligned, the remaining viscoelastic material was completely washed out of the anterior chamber with balanced salt solution and a miotic agent was instilled. tobramycin-dexamethasone 0.1% eye drops were used four times a day for 10 days and then slowly tapered over 3 weeks. descriptive statistics were reported as mean and standard deviation for continuous variables. repeated-measures analysis with the bonferroni test for post hoc analysis and the wilcoxon signed rank test were computed. the study included 30 eyes of 19 patients, among those 13 males and 6 females. mean age was 30.44 8.14 years (range: 20 to 45 years). mean follow-up was 16 5.75 months; all patients (100%) had complete follow-up from baseline up to 12 months after icl implantation; only 10 (33%) eyes of 10 patients had 24 months of follow-up. the visian toric icl was implanted in all eyes; 11 patients underwent bilateral implantation while the remaining 8 patients had unilateral icl implantation. preoperative mean spherical power was 8.37 3.89 d (range: 20.5 to 4 d) and mean cylindrical power 2.95 1.40 d (range: 1 to 5.25 d). according to the amsler-krumeich classification, 6 eyes had stage i, 14 eyes had stage ii, and 10 eyes had stage iii keratoconus at baseline. among the eyes that completed the 24 months of follow-up after icl implantation, 6/10 had stage i, 2/10 had stage ii, and 3/10 had stage iii keratoconus at baseline. the preoperative values were compared to values starting 6 months after cxl, because visual acuity and corneal keratometry vary significantly in the first few months after cxl. according to table 1, both udva and cdva values at 6 months after cxl did not differ from baseline (p=1.000 and 0.231, resp.). at 6 months after icl implantation, there was significant improvement in mean udva from 1.57 logmar to 0.17 logmar (p<0.001) and mild improvement in cdva from 0.17 logmar to 0.11 logmar (p<0.001). both cdva and udva remained stable thereafter up to 24 months (tables 1 and 2). no eye lost 2 or more lines in cdva in the study (figure 1). at 12 months, 43% (13 of 30) of eyes gained 1 line in cdva, and in the smaller subset of eyes with 24 months follow-up 60% of eyes gained 1 line in cdva. overall, 60% (18 of 30) and 50% (5 of 10) of eyes had udva of 20/30 or better 12 months and 24 months after icl implantation, respectively. at 6 months after cxl, the small changes in se and the spherical component of refraction were not significant from baseline (p=0.611 and 1.000, resp.), unlike the mean change of 0.21 d in cylindrical component (p=0.012) (table 1). however, the changes in se, sphere power and cylindrical power at 6 months after icl implantation were all clinically and statistically significant from baseline and their values remained relatively stable up to 12 months (table 1). however, in the smaller subset of 10 eyes with 24 months of follow-up (table 2), small hyperopic shifts of 0.25 d in se (p=0.012) and 0.20 d in spherical power (p=0.005) were noted after 6 months after icl visit. overall, 63.3% and 40% of eyes were within 1.0 d se at 12 and 24 months after icl implantation, respectively (figure 2). all keratometric values showed a gradual decrease after cxl, up to the 24 months of follow-up. according to table 1, the decrease in mean k (flat) from baseline became statistically significant 6 months after icl implantation, while the decreases in mean k (steep) and mean k (max) from baseline were statistically significant starting 6 months after cxl. overall, the safety index=[mean postoperative cdva (logmar)/mean preoperative cdva (logmar)] at 12 months and 24 months after icl implantation was 0.73 0.29 and 0.72 0.25, respectively. the efficacy index=[mean postoperative udva (logmar)/mean preoperative cdva (logmar)] at 12 months and 24 months after icl implantation was 1.03 0.26 and 1.04 0.26, respectively (figure 3). all epithelial defects healed within 4 days after cxl. in this study, none of the patients had infectious keratitis, lens rotation, vaulting problem, cataract formation, pigment dispersion, or pupillary block. also, none had development of clinically significant haze at any of the follow-up periods. there was, however, a transient increase of intraocular pressure that was observed in most patients during the first week after icl implantation that was controlled with topical drops. providing optimal refractive and vision results to patients with progressive keratoconus remains challenging to the refractive surgeon. while corneal collagen cross-linking (cxl) can halt progressive disease, patients with high refractive error and poor vision at baseline would remain so, after cxl, even without keratoconus progression [10, 18, 19]. therefore, cxl is used to set the stage for other interventions to be performed. management after cxl is tailored according to the patient's best-corrected visual acuity and refractive status. in patients with good-best corrected visual acuity and high residual refractive error after cxl, piol implantation provides adequate correction of ametropia. several types of toric piol were reported to be effective and safe in eyes with keratoconus, but only a handful of studies have evaluated their use following a cxl procedure [6, 9, 10, 18, 2024]. the visian toric icl has demonstrated good efficacy and safety profiles for the correction of high ametropia in patients without keratoconus [2532]. in our previous study, toric icl implantation 6 months after cxl was proven to be an effective and safe method of improving visual acuity and refraction in selected eyes with moderate to severe keratoconus. in this paper, we assess the long-term (up to 24 months) safety and efficacy of that same procedure in 30 eyes with mild-to-moderate progressive keratoconus. stability of keratoconus following cxl in preparation for icl implantation has been previously defined using stability of refraction data [5, 9, 10]. as such, icl implantation was performed 6 months after cxl, since most patients had a stable visual acuity and manifest refraction by 4 months. this flattening was not significant enough to alter the mean se manifest refraction at the time of icl implantation, or the outcome of the icl procedure at 12 months. in the small subset of 10 eyes with 24 months of follow-up, the small hyperopic shift in se might have resulted from the continuous flattening in k readings; however, the change did not affect vision. the continuous flattening in k readings and its effect on se is most likely due to the effect of cxl. it is unlikely that the 3.2 mm clear corneal incision at the time of icl implantation (surgically induced astigmatism) would have contributed to the change in se; the incisions were placed at the temporal site according to the surgeon's preference, regardless of the axis of manifest astigmatism. another possible yet unlikely culprit of the change in se is the rotation of the toric visian icl with loss in the refractive corrective effect. although possible, the effect of a rotation on refraction and visual acuity would have been uncovered earlier, and all our patients were happy with the end-result. after stabilization of keratoconus with cxl and icl implantation, 60% (18 of 30) and 50% (5 of 10) of eyes had udva of 20/30 or better at 12 months and 24 months, respectively. results of our study compare favorably to other reports in terms of gain in udva and cdva [9, 10, 21], as reflected by the safety and efficacy indices. in our study, the slight myopic se refraction at post-icl implantation was related to 2 factors. first, there is no way to customize the icl to exactly fit the patient's refraction, and in most cases we had to use what was available (undershoot the target refraction of plano). second, one patient had a high refractive power that exceeds the capacity of the icl (which is limited to 18.0 d of manifest refraction at the eyeglasses plane). only 2 other studies have evaluated the safety and efficacy of visian toric icl following cxl [6, 10]. both kymionis et al. and shafik shaheen et al. in a case report published encouraging results of this procedure; at 3 months, udva improved from counting fingers to 20/40 and cdva improved from 20/100 to 20/30. shafik shaheen et al., in a case series of 16 eyes with early-stage (undefined) keratoconus, showed a favorable outcome in terms of visual acuity and se at 3 years of follow-up; mean cdva improved from 20/35 to 20/22, mean udva improving to 20/23 and mean se improving from 8.5 d to 0.25 d. in our previous study on mild to severe keratoconus, the 6-month results revealed that mean cdva improved from 0.15 logmar to 0.12 logmar, mean udva decreased from 1.67 logmar to 0.15 logmar, and mean se decreased to 0.89 d with no complication. izquierdo jr et al. employed the iris-fixated artiflex phakic iol (ophtec, usa) in 11 eyes with progressive keratoconus. results were favorable in terms of visual acuity, sphere, and cylinder at 12 months. employed the toric artiflex/artisan phakic iol in 17 keratoconic eyes; at 24 months, 14 eyes were within 0.50 d of the attempted se correction and 13 eyes were within 1.00 d of the attempted cylinder correction. icl implantation after cxl depends on the stability of keratoconus (both refraction and keratometry) since progression would lead to refractive changes and drop of visual acuity. a continuous flattening in k readings after cxl occurred in our study with no significant effect on se, udva, nor cdva at 1 year; in the smaller subset of 10 eyes a statistically but nonclinically significant change in se was observed following cxl at the 2-year follow-up, but both udva and cdva were not affected. although we do believe that a longer time interval would possibly show a greater change in keratometry, we are still uncertain whether an equivalent amount of change in refractive error would accompany this flattening, possibly related to the altered biomechanics of cross-linked corneas. as demonstrated in our results, the change in keratometry did not significantly alter the se and more importantly did not alter the udva and cdva. the small hyperopic shift observed in our study deserves further investigation with long-term studies to assess its long-term impact on vision, but it does not warrant delaying icl implantation. the continuous flattening effect of cxl with the accompanying risk for a hyperopic shift can last more than 2 years [35, 36]; therefore, targeting mild undercorrection rather than delaying the icl implantation for 12 months would improve predictability and may be a solution. moreover, implanting an icl at 6 months as opposed to 12 months offers the patient the benefit of earlier functional visual recovery. in conclusion, the results of toric icl implantation 6 months after cxl at 1 year and at 2 years compare to the outcomes at 6 months in a previous study; it is an effective and safe method of improving visual acuity and refraction in keratoconus eyes with high myopia and astigmatism and good best corrected visual acuity.

purpose. to evaluate the long-term safety and clinical outcome of phakic visian toric implantable collamer lens (icl) insertion after corneal collagen cross-linking (cxl) in progressive keratoconus. methods. this was a retrospective study of 30 eyes (19 patients), with progressive keratoconus, who underwent sequential cxl followed by visian toric icl implantation after 6 months. results. at baseline, 6 eyes had stage i, 14 eyes stage ii, and 10 eyes stage iii keratoconus graded by amsler-krumeich classification. at 6 months after cxl, only k (steep) and k (max) decreased significantly from baseline, with no change in visual acuity or refraction. flattening in keratometric readings was stable thereafter. there was significant improvement in mean uncorrected distance visual acuity (1.57 0.56 to 0.17 0.06 logmar, p<0.001) and mean corrected distance visual acuity (0.17 0.08 to 0.11 0.05 logmar, p<0.001) at 12 months after icl implantation that was maintained at the 2-year follow-up. mean cylinder power and mean spherical equivalent (se) also decreased significantly after icl implantation. a small hyperopic shift in se (+ 0.25 d) was observed at 2 years that did not alter visual outcomes. conclusions. visian toric icl implantation following cxl is an effective option for improving visual acuity in patients with keratoconus up to 2 years.

PMC4383407

pubmed-1226

ternary boron carbonitride (b c n) nanotubes have recently attracted much attention because of their excellent mechanical properties, electrical properties, and anti-oxidant capacities. in addition, theoretical studies have revealed that the band gaps of b c n nanotubes can be tailored over a wide range by simply varying the chemical composition rather than by geometrical structure [3-7], which is superior to their carbon and boron nitride (bn) counterparts. this gives b c n nanotubes potential for use in electronics, electrical conductors, high temperature lubricants, and novel composites. compared with the very extensive study about carbon and bn nanotubes, however, very little work was reported about b n nanotubes in 1994, several methods have been devoted to the synthesis of b c n nanotubes, such as arc-discharge, laser ablation, chemical vapor deposition (cvd), template route, and pyrolysis techniques. particularly, single-walled b n nanotubes have been recently synthesized by wang et al. via a bias-assisted hot-filament method. however, most of them usually used risky reagents such as diborane (b2h2)/ammonia (nh3), or produced nanotubes with low purity and high-cost and encountered the phase separation problem of bn and c. thus, it is of great significance to explore novel and simple routes to prepare b the current work reports a relatively safe and effective approach for growing high-purity b c n nanotubes directly on commercial stainless steel foil, by using simple raw materials of boron, zinc oxide (zno), and ethanol absolute. the reaction of boron and zno at high temperature produces boron oxide vapor that is the source of b, while ethanol absolute and nitrogen provide the source of c and n, respectively. it is interesting that the stainless steel foil is not only the support substrate but also the catalyst for the growth of the nanotubes. the obtained nanotubes have an average diameter of about 90 nm and the b, c, and n elements are found to be homogeneously distributed in the nanotubes. the growth mechanism of the nanotubes is also investigated in this study. to the best of our knowledge, it is the first time to report the synthesis of ternary b the growth of nanotubes was carried out in a conventional tube furnace. an alumina boat loaded with about 1.0 g mixture of hexagonal zno and amorphous b powder (with a zno: b molar ratio of 1.5:1) commercial stainless steel-304 foil with a thickness of 0.05 mm was inserted into the quartz tube as the substrate. prior to heating, the chamber was flushed with high-purity n2flow to eliminate the residual air. then the furnace was heated to 1150 c under a mixture gas flow of n2(60 ml min) and h2(40 ml min). ethanol absolute (ar grade) was introduced into the chamber when the furnace temperature reached at 1150 c, which was carried by another n2flow with a rate of 20 ml min. finally, the furnace was cooled naturally to ambient temperature under the protection of n2flow. after taken from the furnace, the stainless steel substrate was found to be covered with white gray deposit in the temperature range of 10001100 c. the product was characterized by field-emission scanning electron microscopy (fe-sem, hitachi s5500), high-resolution transmission microscopy (hrtem, jem-2010f), x-ray energy dispersive spectrometer (eds), and electron energy loss spectroscopy (eels), respectively. figure 1shows the sem images of the product grown on the surface of stainless steel substrate. 1a) indicates a high production of one-dimensional (1d) nanostructures was synthesized. 1a is a high-magnification cross-sectional image of some 1d nanostructures, illustrating the hollow structure of the product. figure 1b clearly reveals that the nanotubes are shaped in bamboo consisting of a number of compartments. the surfaces of the nanotubes are very clean and no impurities can be observed, which indicates the high purity of the nanotubes. the diameters of the nanotubes are approximately 60120 nm, with an average value of about 90 nm. furthermore, it can also be found that nanoparticles are attached at the ends of nanotubes, which could be regarded as a typical symbol of vapor liquid solid (vls) growth model. figure 1c and d are the secondary electron and back scattering electron (bse) images of the same area of the product. it can be seen that the attached particles (bright particles) are distinguished clearly in the bse image, which further confirms the vls growth mechanism of the nanotubes. alow-magnification sem image of the product, showing the large quantity of the 1d product grown on the stainless steel substrate. the inset is the high-magnification cross-sectional image of some nanotubes, illustrating the hollow structure.bhigh-magnification image ofa, indicating the bamboo-like structure and high purity of the nanotubes.canddthe secondary electron and back scattering electronic (bse) sem images of the same area of the product the tem images of the product are shown in fig. the bamboo structure of the nanotubes with clean surface and uniform diameter along the nanotube length can be clearly seen (fig. 2a is the edx result, which shows the dominating peaks of b, c, and n with a low level of o, cu, and si. the existence of cu peak should be caused by the copper tem grid, while the si peak might come from the stainless steel substrate. furthermore, quantitative analysis gives the b: c: n atomic ratio of about 0.45:0.31:0.24. n nanotubes. in figure 2b, a particle attached at the end of the nanotube the edx spectrum indicates that the particle is mainly composed of fe with a small amount of ni, cr, cu, and c (inset in fig., the cu and c peaks should come from the carbon film-coated copper grid. while the existence of fe, ni, and cr in the particle should originate from the stainless steel substrate. hence, we believe that the stainless steel substrate play a catalyst role during the vls growth of the nanotubes. atem image of a nanotube. the inset is the edx spectrum of the nanotube.btem image shows a catalyst particle attached at the end of a nanotube. the inset is the edx spectrum of the particle figure 3a shows the hrtem image of the edge part of the nanotube wall. it can be seen that the lattice fringes are well-defined, which suggests that the nanotube wall has a high degree of crystalline perfection. the interlayer spacing is approximately 0.348 nm, corresponding to the (002) plane of hexagonal system of b c n crystal (jcpds no. 35-1292). the spots in the fast fourier-transformed ed (fft ed) pattern can be indexed as the (002) basal planes of the b figure 3b is the hrtem image of the joint, showing the tight connection between nanotube wall and compartment. the compartment is also well-crystallized with the identical lattice spacing of about 0.343 nm, which also corresponds to the (002) plane of b c n crystal. the fft ed pattern (inset in fig. 3b) is also indexed as the (002) planes of b c n crystal. figure 3c shows a representative eels spectrum taken from a segment of the nanotube wall, which demonstrates that the distinct absorption peaks of b, c, and n characteristic k-edges at 188, 201, and 401 ev, respectively. the k-edge signals show a discernible *peak, as well as an *band, indicating that the b, c, and n atoms are in the sp-hybridized state. the eels spectrum of the compartment shown in fig. 3d also indicates the distinct absorption features of k-edges of b, c, and n atoms. furthermore, the elemental maps reveal that b, c, and n elements are homogeneously distributed in the nanotube (fig. therefore, it can be concluded that the product is composed of ternary compound b ahrtem image of nanotube wall and the corresponding fft ed pattern (inset).bhrtem image of a joint between the wall and compartment and the corresponding fft ed pattern (inset).canddeels spectra taken from the nanotube wall and the compartment, respectively, revealing the dominating composition of b, c, and n elements in the wall and compartment elemental maps of a nanotube, implying the uniform distribution of the b, c, and n species in the nanotube as described above, a simple approach to synthesize b c n nanotubes was proposed by involving an additional role of catalyst of the stainless steel substrate. and based on the above results, vls model is believed to be responsible for the growth of the current nanotubes. similar to the literatures reported about bn nanotubes and nanowires [17-19], fig. firstly, the reaction of b and zno generates zn and boron oxide (b2o2) vapor at high temperature (1150 c). meanwhile, the surface of the stainless steel may partially melt at this temperature assisted by the erosion of n2 and h2. and thus liquid alloy droplets with main composition of fe cr ni are formed on the surface, as are verified by the edx result. then the liquid droplets adsorb the growth species from the surrounding vapors of b2o2, c2h5oh, n2, and h2 (fig. 5a). the involving species of b and c could lead to the further decrease of the melting temperature of stainless steel and promotes the formation of liquid droplets. the reactions among these vapors produce b/c/n atoms, which diffuse through fe cr ni alloy droplets. when the concentrations of species are greater than the saturation threshold, b n crystals begin to precipitate and initially form the cap on the liquid droplets, as is shown in fig., the cap will be lift from the droplet due to the stress under the curvature and then the hollow tip forms. at the same time, due to the diffusion of b/c/n atoms through the surface and bulk, the b when the nanotube wall grows, the b/c/n atoms also precipitate inside the nanotube and result in the formation of compartment layer. the compartment layers connect with the wall and grow together for a period, and finally depart from the droplet due to the stress accumulated under the curved compartment layers (fig. moreover, the flowing character of b2o2, c2h5oh, n2, and h2 vapors could lower their partial pressures in the chamber, which is also favorable for the formation of 1d nanotubes. it should be noted that the produced zn vapor is transported by the carrier gas to a much lower temperature zone (below the melting point of zn), where it deposits on the substrate in the form of zn products. in addition, it is found that the hydrogen in the mixture gas is essential for the growth of b c n nanotubes. if only pure n2 flow is introduced, no b n nanotubes can be obtained, adding weight to the proposed mechanism and suggesting that hydrogen plays an important role during the growth of the b n nanotubes directly onto commercial stainless steel foil is demonstrated by using raw materials of boron powder, zinc oxide powder, and ethanol absolute. the nanotubes are pure with bamboo-like morphology and an average diameter of about 90 nm. during the formation process, the stainless steel foil plays a catalyst role additionally besides the substrate role for the b the authors acknowledge financial support from the national natural science foundation of china (nsfc, grant no. 208106), tsinghua university state key laboratory of new ceramics&fine processing and guangxi university (grant no.

in this study, a novel and facile approach for the synthesis of ternary boron carbonitride (b c n) nanotubes was reported. growth occurred by heating simple starting materials of boron powder, zinc oxide powder, and ethanol absolute at 1150 c under a mixture gas flow of nitrogen and hydrogen. as substrate, commercial stainless steel foil with a typical thickness of 0.05 mm played an additional role of catalyst during the growth of nanotubes. the nanotubes were characterized by sem, tem, edx, and eels. the results indicate that the synthesized b c n nanotubes exhibit a bamboo-like morphology and b, c, and n elements are homogeneously distributed in the nanotubes. a catalyzed vapor liquid solid (vls) mechanism was proposed for the growth of the nanotubes.

PMC2894111

pubmed-1227

the strength of the knee extensor and flexor muscles has been extensively studied and is a relevant clinical indicator of health status and functional capacity in coronary artery disease8, neuromuscular diseases9, the elderly10, renal disease11, and fibromyalgia12. in sports medicine, knee extensor and flexor muscle strength is often monitored with the aim of preventing or treating orthopedic as well as muscular injuries13,14,15,16,17,18,19. therefore, practitioners need to have reliable tools for easy and reproducible assessment of muscle strength. the isokinetic concept of exercise20 is currently considered the best method for the assessment of knee extensor and flexor strength because of its well-established validity and reproducibility21,22,23,24,25. however, the acquisition and maintenance costs, the time required to perform an isokinetic assessment, as well as the absence of portability of isokinetic dynamometers can limit their use. conversely, hand-held dynamometers (hhd) are characterized by specific features such as low cost, portable design, and rapid data acquisition. an hhd provides reliable and reproducible results when used for the evaluation of muscle groups that produce little or moderate amounts of force. however, the results obtained for strong muscles, such as the knee extensors, are less convincing26,27,28. a systematic review comparing muscle strength assessment by hhd and isokinetic testing29 showed a positive correlation between these methods. however, the heterogeneity of the protocols and devices evaluated made the interpretation of the results difficult. therefore, the aim of this study was to: 1) assess the validity of hhd vs. isokinetic dynamometry in the evaluation of knee extensor and flexor muscle strength, 2) establish the reproducibility of hhd measurements of knee extensor and flexor muscle strength, and 3) compare the flexor/extensor ratios obtained with the 2 methods. the study was carried out at the muscle evaluation unit at the clemenceau rehabilitation university institute (strasbourg, france) and involved 30 healthy volunteers (table 1table 1.characteristics of the population (n=30)mean sdrangeage (years)32.8 11.52059height (cm)176.9 10.3160197weight (kg)74.5 15.852109gendermales (n=20)females (n=10)lateralityright (n=27)left (n=3)test sequence iso then hhd (n=16)hhd then iso (n=14)iso: isokinetic dynamometer; hhd: hand-held dynamometer). iso: isokinetic dynamometer; hhd: hand-held dynamometer subjects were recruited by means of advertisement in the university network from january 2015 to november 2015. inclusion criteria were as follows: age older than 18 years, and without cardiovascular disorders, osteoarticular diseases of the knees, or previous knee osteoligamentous or thigh muscle injuries. all participants were fully informed about the risks and procedures associated with the experiment and provided written informed consent to participate in the study. each participant completed a 10-min warm-up on a cycle ergometer (power: 1 w/kg body weight; pedaling rate: 7080 rpm). the testing method sequence (hhd vs. isokinetic testing) was established according to a randomization table prepared by the biostatistics department of the strasbourg university hospitals using r software version 3.2.2 (r core team (2015). the randomization of the subjects was generated by a draw using a bernoulli distribution with a parameter of 0.5 to maintain a mean balance in the test order. a recovery period of 20 min was implemented between testing methods. for both methods, muscle strength was first measured in the dominant leg, and after a 10-min rest, in the non-dominant leg. to avoid inter-examiner variations, all measurements were carried out by a single investigator, specialized in physical medicine and rehabilitation (male, 31 years old, 180 cm, and 90 kg). the strength of the extensor and flexor muscles of both knees was assessed using an isokinetic dynamometer (con-trex mj; cmv ag, dbendorf, switzerland). each participant was positioned on an adjustable chair with the back set at 90 of posterior inclination. the knee range of motion was set at 90 (from 100 to 10 of flexion; 0 corresponding to the complete extension of the knee) after having aligned the leg segment to anatomical zero. finally, a measurement of gravity for the leg segment to be tested was carried out over the entire range of motion. the protocol of isokinetic evaluation included 6 series of contractions on the dominant and non-dominant sides including concentric, isometric, and eccentric evaluations of the knee extensor and flexor muscles (table 2table 2.protocol for muscle strength measurement with the isokinetic dynamometerseriesspeedmusclesmoderepetitionsrom/positionwarm-up180/secext/flexconcentric1090warm-up120/secext/flexconcentric1090series 160/secext/flexconcentric390series 2180/secext/flexconcentric690series 30/secextisometric190 of flexionseries 40/secflexisometric190 of flexionseries 560/secexteccentric390series 660/secflexeccentric390ext: knee extensors; flex: knee flexors; rom: range of motion). a recovery period of 60 s was observed between each maximal series. before each, participants received standardized oral information on the experimental procedure and were verbally encouraged in a standardized manner by the operator during the test. following 10 min of rest after the evaluation of the dominant leg, muscle testing was performed in exactly the same way for the non-dominant side. ext: knee extensors; flex: knee flexors; rom: range of motion the strength of the extensor and flexor muscles of both knees was assessed with hand-held dynamometry (microfet2, hogan health industries, inc. this dynamometer is a portable digital instrument than can be held in the palm of the hand giving muscle strength measurements in kilogram-force (kgf). as with manual muscle testing, this dynamometer is placed between the leg segment to be evaluated and the examiner s hand. participants were asked to sit with their legs dangling over the end of a standard, adjustable examination table, with hips and knees flexed to 90, in order to have a distance of 12 cm between the popliteal fossae and the table end. the height of the examination table was adjusted to have a distance of about 10 cm between the participant s feet and the floor. participants had to hold the side-edges of the table with their hands and carry out a maximal isometric voluntary contraction for 5 s. the examiner positioned a knee on the floor, with the arm fully extended in front of the lower limb to be tested. to evaluate the knee extensor muscles, the dynamometer was placed on the anterior part of the lower leg, above the talotibial joint line (fig. 1.position of an individual subject for the evaluation of knee-extensor (panel a) and knee-flexor (panel b) muscles using the hand-held dynamometer). the examiner placed one of his feet against the wall to better resist muscle contraction. to evaluate the knee flexor muscles, the dynamometer was placed on the posterior part of the leg, 12 cm above the lateral malleolus (fig. in both cases, the examiner produced a resistance force in the horizontal direction to counter the force developed by the participant and maintain an isometric contraction of the knee extensor and flexor muscles. the evaluation started with the extensor muscles of the dominant leg, followed by the flexors of the dominant side. after a recovery period of 10 min, the knee extensor and flexor muscles of the non-dominant leg were assessed using the same experimental design. for each muscle group, participants carried out 3 isometric maximal voluntary contractions of 5 s. a 60-s recovery period was observed between each muscle contraction. the position of the leg to be tested was verified initially with the help of a standard goniometer. before the evaluation, participants received standardized oral information on the test procedure and were encouraged orally during the test. position of an individual subject for the evaluation of knee-extensor (panel a) and knee-flexor (panel b) muscles using the hand-held dynamometer for each isokinetic series, the measurement criterion was the peak torque (pt) obtained during each series, expressed in newton-meters (nm). the maximal force developed during each contraction, expressed in kilogram-force (kgf), was recorded. for each muscle group, the maximal force was averaged over the 3 isometric contractions and used as the measurement criterion. thus, the 2 obtained variables were the peak torque (nm) and the maximal force (kgf). by assuming a correlation of 0.6 between the 2 measurements and a total magnitude of the confidence interval of 0.45 around the estimate, a sample size of 28 subjects was needed. the reproducibility of the hhd measurement was established using the coefficient of variation over 3 consecutive measures. according to stokes, reproducibility was considered acceptable when the coefficient of variation was 15% or below30. the statistical analysis was performed independently by the biostatistics department of the strasbourg university hospitals. all analyses were carried out using r version 3.2.2, with all the required packages, in the most updated version available at the time of the data analysis. the gaussian character of the variables was assessed with the shapiro-wilk test. when 2 variables followed a normal distribution, the pearson correlation coefficient was used; if not, the spearman correlation coefficient was employed. concerning the correlations, the test sequence and the laterality effects were evaluated by ensuring that the confidence intervals (ci) at 95% for each variable were overlapping in the 2 modalities. the results obtained with the 2 evaluation modes on the whole population are summarized in table 3table 3. results of the muscle strength measurementsmean sd ecart-typerangemedianisokinetic (nm)concentric 60/secext d179.1 52.2*$#100.3275.8180.7ext nd165.4 52.0*90.5260.3155.3flex d91.9 28.5$51.1164.793.8flex nd85.1 25.551.0143.878.1concentric 180/secext d132.3 39.4*$75.4207.8136.8ext nd130.4 39.6*71.5204.2134.1flex d72.2 22.9$32.1115.372.8flex nd71.8 24.1$32.0141.669.2isometric 0/secext d177.2 57.3*89.5291.8181.2ext nd167.4 56.6*88.0281.0162.2flex d84.0 25.248.0153.784.7flex nd79.3 25.945.0144.273.1eccentric 60/secext d201.9 61.9*110.9357.5191.5ext nd195.5 69.8*111.8357.0169.9flex d123.1 44.169.5252.3111.6flex nd116.7 36.865.9210.3103.8hhd (kgf)ext d52.9 16.7*22.787.457.6ext nd51.1 17.2*22.284. 351.8flex d26.7 6.017.440.927.2flex nd26.9 5.318.238.927.1ext: knee extensors; flex: knee flexors; d: dominant side; nd: non-dominant side; sd: standard deviation. $p<0.05 vs. eccentric 60/s in similar muscles. #p<0.05 concentric 60/s vs. concentric 180/s in similar muscles. the strengths measured for the knee extensor muscles were greater than the corresponding values for the knee flexor muscles, whatever the mode and velocity of the muscle actions (p<0.05). ext: knee extensors; flex: knee flexors; d: dominant side; nd: non-dominant side; sd: standard deviation. #p<0.05 concentric 60/s vs. concentric 180/s in similar muscles the knee flexor/extensor strength ratios did not change according to the evaluation mode, with no difference between the dominant and non-dominant sides (table 4table 4.flexor/extensor ratios in isokinetic and hand-held dynamometrydominant sidenon dominant sideconcentric 60/sec52 7.0%53 9.7%concentric 180/sec55 7.0%55 8.4%isometric 0/sec49 8.7%49 11.0%eccentric 60/sec61 8.1%61 10.9%hhd53 12.9%57 hhd: hand-held dynamometer the knee extensor muscle strength was significantly greater than for the knee flexor muscles, both on the dominant and non-dominant side (p<0.05). there was no difference between the dominant and non-dominant side in a given muscle group (table 3). the ratios of knee flexor/extensor muscle strength were similar in dominant and non-dominant sides and were not significantly different compared to isokinetic ratios (table 4). the coefficient of variation of the 3 isometric muscle strength measurements using the microfet 2 hhd ranged from 3.2 to 4.2% according to the muscle group evaluated. mean difference (absolute and relative values) as well as limits of agreement were similar between muscle groups (table 5table 5.reproducibility of muscle strength evaluation with microfet 2 hand-held dynamometercoefficient of variationmean differencelimits of agreement(%)(kgf)(%)(kgf)(%)dominant knee extensors3.20.1 3.70.1 7.07.37.613.814.0non-dominant knee extensors3.30.0 3.70.4 7.47.57.515.114.4dominant knee flexors4.20.4 2.51.4 8.94.65.416.419.2non-dominant knee flexors3.70.3 2.10.9 7.73.94.614.416.3). the correlation coefficient between the isokinetic and hhd muscle strength values ranged from r=0.87 (0.750.94) to r=0.72 (0.480.86) (p<0.01), indicating that the correlation between testing methods was generally good (table 6table 6.correlation coefficients and 95% confidence intervals for knee muscle strength assessments obtained by hand-held (hhd) and isokinetic dynamometers (con-trex)con-trexeccentricisometricconcentricconcentric60/sec0/sec60/sec180/sechhdextensors dr=0.87r=0.85r=0.87r=0.85(0.750.94)(0.70 0.92)(0.730.93)(0.710.93)extensors ndr=0.84*r=0.87r=0.86r=0.87(0.690.92)(0.740.94)(0.720.93)(0.740.94)flexors dr=0.80*r=0.75r=0.81r=0.85(0.620.90)(0.530.87)(0.630.91)(0.700.93)flexors ndr=0.75*r=0.72*r=0.83*r=0.82(0.530.87)(0.480.86)(0.670.92)(0.660.91)*spearman correlation coefficient; d: dominant side; nd: non-dominant side.*spearman correlation coefficient; d: dominant side; nd: non-dominant side. all coefficients of correlation are significant (p<0.01). the flexor/extensor ratios were significantly correlated between the hhd and isokinetic dynamometer in concentric 180/sec and isometric 0/sec modes on the dominant and non-dominant sides (p<0.05). the flexor/extensor ratio measured in concentric 60/sec mode was correlated to the hhd only for the non-dominant side. no correlation was observed for the flexor/extensor ratios between hhd and isokinetic eccentric 60/sec mode (table 7table 7.coefficients of correlation for knee flexor/extensor muscle strength ratios obtained with hand-held or isokinetic dynamometrydominantnon dominantconcentric 60/sec0.230.46*concentric 180/sec0.39*0.45*isometric 0/sec0.37*0.46*eccentric 60/sec0.040.13*p<0.05). the results of the present study show: 1) good reproducibility of the hhd for knee extensor/flexor muscle strength assessment, 2) clinically acceptable agreement between the values obtained with the hhd versus the isokinetic dynamometer, and 3) the need for caution when interpreting the flexor/extensor ratios using hhd compared to isokinetic dynamometry. the muscle strength values using isokinetic dynamometry recorded in this study are in good agreement with those found in the literature31, particularly in the study by maffiuleti et al. in 200723. for a similar population of healthy individuals (age: 30 5 vs. 33 11 years old; height: 175 8 vs. 17710 cm; weight: 70 13 vs. 74 16 kg), the strength measurements using the con-trex isokinetic module were very close, with a peak torque for the knee extensors on the dominant side of 178 46 vs. 179 52 nm (concentric at 60/s); 132 38 vs. 132 39 nm (concentric 180/s), and 202 68 vs. 202 62 nm (eccentric 60/s) when compared with our values. similarly, the muscle strength values recorded with the hhd are in agreement, albeit with greater absolute data, with literature data32, 33 and with the recent study by douma et al., who established reference values for knee extensor and flexor muscle strength in the active dutch population by using the microfet 2 hhd34. the magnitude of difference between our values and previously published data using hhd is likely related to the characteristics of the participants, who were younger and fitter in our study compared to previous studies. greater limits of agreement were previously observed for reproducibility of muscle strength using hhd in strong muscle groups (i.e., knee extensors) compared to weaker muscle groups (i.e., knee or elbow flexors)34. consequently, hhd was not considered as suitable methodology for muscle strength assessment in knee extensors35. conversely, our results are at odds with this view as we report that the reproducibility of muscle strength assessment is not altered by the level of muscle strength developed by the subjects. indeed, the variation coefficients were lower for the 3 measures of knee extensors compared to knee flexors, despite higher levels of absolute strength in the former. similarly, we did not observe greater limits of agreement (loa) in the knee extensors compared to the knee flexors. of note, absolute values of loa in the present study are somewhat lower than those previously published34. we suggest that if hhd devices are manipulated by experienced operators, hhd can be used for the evaluation of muscle strength, even in strong muscles such as the knee extensors. it should also be kept in mind that the strength of the operator might be key in stabilizing the hhd device during the measurement procedure, and therefore the operator s own strength is likely to be of great importance for the accuracy of the measurements36. when compared with previous results reporting the coefficient of variation (cv) of isokinetic measurements using a contrex device (1.93.4% for knee extensors and 2.73.6% for knee flexors), the cvs reported in the present study are very similar for hhd (3.23.3% for knee extensors and 3.74.2% for knee flexors). this result suggests that the reproducibility of the strength measurements using hhd is in close agreement with that observed with isokinetic testing23. in a previous literature review comparing hhd with isokinetic dynamometry, stark et al. showed that correlation coefficients ranged between 0.43 and 0.99 for the knee extensor and flexor muscles29. however, the heterogeneity of the protocols as well as the different models of dynamometers make the results difficult to interpret. the present study is the first to analyze the correlation between these specific dynamometers (contrex isokinetic vs microfet 2), and we report a good correlations between the 2 methods (r=0.720.87). of note, the present data were gathered using a simple protocol, easily reproducible without any additional equipment, in order to be established in routine clinical practice and to take the greatest advantage of the practical aspects of hhd. when looking at subjects engaged in regular sports practice, only one study previously explored the correlation between hhd and isokinetic measurements37. the study reported lower correlation coefficients (r=0.334 0.110.617 0.7) compared to our results, but it should be emphasized that the number of subjects, the type of dynamometers, and the protocol employed were different. lastly, the present study is also the first to provide a correlation analysis between hhd and isokinetic testing for the evaluation of knee flexor/extensor muscle ratios, an index used routinely in the field of sports medicine. our results show correlation coefficients ranging from 0.37 to 0.46 (all p<0.05). although statistically significant, these results indicate a moderate level of correlation between the 2 methodologies, and even no correlation at all when comparing flexor/extensor ratios obtained using hhd with that from isokinetic testing in eccentric mode. therefore, flexor/extensor muscle strength ratios measured using hhd should be interpreted with caution until further investigation is performed in this area. a limitation of our study is the fact that our population consisted of healthy, relatively young volunteers without osteoarticular or neuromuscular pathologies. compared muscle strength measurements by using hand-held and isokinetic dynamometers in a population of individuals with various unilateral orthopedic pathologies of the knee5. they found a significant correlation between both methods (r ranging from 0.57 to 0.80). however, the evaluation by hhd did not highlight a statistically significant difference between sides, whereas the isokinetic evaluation found a force deficit on the injured side. the pain that the patient may experience during the evaluation could be a confusing factor and must be taken into account when interpreting the strength results in the presence of pathologies. another limit of this work is that hhd and isokinetic devices do not provide strength assessment with similar units of measurement. this makes direct comparison of absolute strength values difficult and limits the present comparison to correlation analysis. to obtain strength values expressed in the same units between the 2 devices (newtons or kgf) would have required an evaluation of the lever arm, which is likely to have introduced more variability in the data. each evaluation method has its specific advantages and limitations. for a practitioner looking for rapid and regular muscle strength testing knee extensor and flexor muscle strength recorded with an hhd is reproducible and significantly correlated with the isokinetic values, indicating that this method may in some cases be a useful replacement for isokinetic strength measurement, even for the assessment of strong muscles. however, for strength ratio assessment, and when judged against the isokinetic standard, hhd is not a valid option.

[ purpose] to compare measurements of knee extensor and flexor muscle strength performed using a hand-held dynamometer and an isokinetic dynamometer in apparently healthy subjects. [subjects and methods] thirty adult volunteers underwent knee muscle strength evaluation using an isokinetic or a hand-held dynamometer. [results] strong positive correlations were found between the 2 methods, with correlation coefficients r ranging from 0.72 (95% confidence interval [ci], 0.480.86) to 0.87 (95% ci, 0.750.94), depending on the muscle group and the isokinetic evaluation mode. the reproducibility of the hand-held dynamometer findings was good, judged by a coefficient of variation of 3.24.2%. however, the correlation between the 2 methods for the assessment of flexor/extensor ratios ranged from 0.04 to 0.46. [conclusion] knee extensor and flexor muscle strength recorded with a hand-held dynamometer is reproducible and significantly correlated with the isokinetic values, indicating that this method may in some cases be a useful replacement for isokinetic strength measurement. however, for strength ratio assessment, and when judged against the isokinetic standard, a hand-held dynamometer is not a valid option.

PMC5080149

pubmed-1228

genome-wide biases in nucleotide content have been extensively studied in a wide variety of organisms. during the past decade, there has been accumulating evidence that these variations at the dna level can result in parallel changes in the frequencies of amino acids in the encoded proteins. for example, the gc content of bacterial genomes has been shown to influence the amino acid composition of the proteome. in addition to the variations in gc content, however, bacterial genomes can also show significant compositional asymmetry between the two dna strands. this strand asymmetry is usually measured as gc or at skew (see methods) and it can be due to such factors as different substitutional patterns between the leading and lagging strands during replication. by comparing the amino acid compositions of proteins encoded on the leading strand with those encoded on the lagging strand, it has been shown that these nucleotide skews can also affect the amino acid composition of bacterial proteins. among eukaryotes, the correlations between nucleotide content and amino acid compositions have been studied primarily in animal mitochondrial genomes. it has been shown that the variations in gc content affect the amino acid composition of the encoded proteins but the effects of variations in nucleotide skew between genomes have not been studied. the recent availability of a very large number of completely sequenced mitochondrial genomes allows us to fill this gap. mammalian mitochondria, including those of humans, are characterized by negative gc skews and positive at skews, i.e. the major coding strand of mammalian mitochondria is relatively rich in the nucleotides c and a, and correspondingly poor in g and t. for instance, as noted by perna and kocher (1995), although the human mitochondrial genome contains more than 40% gc pairs, the frequency of g on the coding strand is only 5%. the strength of the skew varies between species, and some species of invertebrate show opposite skews to those found in mammals, i.e. the coding strand is rich in g and poor in c. in recent years, there has been an explosion of data on mitochondrial whole genome sequences. since our goal was to measure the effect of strand asymmetry on amino acid composition, we chose a group of species, the platyhelminthes (flatworms), in which the gc and at skews are opposite to those seen in mammals. among the flatworms, the coding sequences are rich in g and t and correspondingly poor in c and a, i.e. a complete contrast to the patterns seen in mammals for example, the main mitochondrial coding strand in schistosoma mansoni (a flatworm) contains only 6.7% c, but it contains 25% g. despite the contrasting gc and at skew patterns, however, both mammals and flatworms contain essentially the same set of homologous mitochondrial genes and they span similar ranges of gc content. thus a comparison between these two groups provides us with an opportunity to assess the potential effects of dna strand bias on the amino acid composition of a well-characterized set of orthologous proteins. we downloaded all of the publicly available complete mitochondrial (mt) genome (mtdna) sequences from both mammals and platyhelminthes from the ncbi refseq organelle genome database (http://www.ncbi.nlm.nih.gov/genomes/organelles/mztax_short.html) (released in july, 2006). there were a total of 170 mt genomes from mammals and 13 mt genomes from platyhelminthes (supplementary table s1).+-strand (the major coding strand used by ncbi for annotation) for each species. the gc and at asymmetry is measured in terms of gc- and at-skews according to the following formulae given in perna and kocher: gc-skew=(gc)/(g+c); at-skew=(a t)/(a+t), where c, g, a, and t are the occurrences of the four nucleotides. we predicted the amino acid compositions based on the mitochondrial genetic code (table 1) by partitioning the mitochondrial codons into ca-rich codons, gt-rich codons, and other codons. the frequency of synonymous codon usage was measured by the nucleotide content of g+t or c+a at the third codon positions of fourfold degenerate codon families: ggn (glycine), gtn (valine), cgn (arginine), acn (threonine), gcn (alanine), and ccn (proline) (supplementary fig. s1). partition of the vertebrate mitochondrial genetic code into gt-rich, ca-rich and other codons gt-rich codons (italic) include gt, tg, gg, tt codons at the first two codon positions. ca-rich codons (bold) include ca, ac, cc, aa codons at the first two codon positions. different codon assignments in mammals and platyhelminthes are underlined. in platyhelminthes, aga and agg code for ser, ata for ile, and aaa for asn. the numbers following each codon are codon usage per thousand codons in the 11 conserved proteins of mammals (the first number) and platyhelminthes (the second number). the statistical significance of the average differences in amino acid composition between the two groups of species was scored using a student's t-test. we first confirmed the published reports (see introduction) of contrasting gc and at skews in the mitochondria of mammals and flatworms. as can be seen from fig. 1, there is a negative gc skew, and a positive at skew, in the major coding strand among the mammalian species, whereas the opposite is true among the flatworms. despite the variations between species within both groups, there is a large average difference between them and this difference between the groups is statistically highly significant (p<0.0001). as previously noted by perna and kocher and le et al., the major coding sequence of mammalian mtdna is relatively rich in c and a, whereas g and t are much more common in the flatworm coding sequences. contrasting patterns of dna strand asymmetry in the mitochondrial coding sequences of mammals and platyhelminthes. (a) the gc skew, (g c)/(g+c), is negative for all mammalian species (shown in blue), and positive for all platyhelminthe species (shown in red). (b) the at skew, (a t)/(a+t), is positive for mammals (shown in blue) and negative for platyhelminthes (shown in red). despite their differences in strand asymmetry, the major coding strands of the two groups encode essentially the same set of 11 conserved mitochondrial proteins including cytochrome b, three subunits of cytochrome c oxidase (subunit 1, 2, and 3), six subunits of nadh dehydrogenase (subunit 1, 2, 3, 4, 4l, and 5), and atp synthase f0 subunit 6 (atp6). the exception is that nadh dehydrogenase subunit 6 (nd6) is encoded on the major coding strand in flatworms, but is encoded on the opposite strand in mammals. given the contrasting patterns of mitochondrial dna strand asymmetry between these two groups of animals, we wished to investigate if there was a corresponding difference between the two groups in the frequencies of encoded amino acids. specifically, because of their negative gc skews and positive at skews (which reduce the frequencies of g and t nucleotides on the coding strand), we expected the mammalian coding strands to encode proteins that are relatively low in the proportions of cysteine (c), valine (v), phenylalanine (f), glycine (g), and tryptophan (w), all of which are encoded by gt-rich codons (table 1). the combined proportions of these five amino acids among the mammals are approximately half the value observed in the flatworm orthologs. since these gc and at skews result in a corresponding enrichment of c and a nucleotides on the mammalian coding strand, we expected the mammalian proteins to show a corresponding relative increase in the proportions of glutamine (g), threonine (t), proline (p), histidine (h), asparagine (n), and lysine (k), all of which are encoded by ca-rich codons. again, this prediction is borne out (fig. 2b) and again the average difference between the two groups of species is approximately twofold. not only are the average differences in the predicted direction, but they are statistically highly significant (p<0.0001) and they are consistent over all species within each group (see details in supplementary table s1). in addition to the consistency over species, there is also a consistency over all amino acids within the two codon groups. these differences are surprisingly large, given that we are dealing with a set of conserved orthologous proteins. although taken as a group, the amino acid frequencies show approximately a twofold difference between the two groups (fig. 2), at the level of individual amino acids, some of these differences are threefold or greater (fig. 3). again, the individual amino acid differences shown in fig. 3 are highly statistically significant (p<0.0001). thus, we can conclude that strand asymmetries at the level of dna have had a major influence on the composition of these mitochondrial protein sequences. (a) the proportions of cysteine (c), valine (v), phenylalanine (f), glycine (g), and tryptophan (w) are relatively low in mammalian proteins (shown in blue) and relatively high in flatworms (shown in red). (b) the proportions glutamine (q), threonine (t), proline (p), histidine (h), asparagine (n), and lysine (k) are, in contrast, relatively high in mammals (shown in blue) and relatively low in flatworms (shown in red). the proportions of individual amino acids that were most affected by dna strand asymmetry in mammals and platyhelminthes. (a) the proportions of cysteine and valine are low in mammalian proteins (shown in blue) and high in flatworm proteins (shown in red). (b) the proportions of glutamine and threonine are high in mammals (shown in blue) and low in flatworms (shown in red). these results show that differences in the patterns of strand asymmetry between the coding and template strands of a mitochondrial gene can produce very significant changes in the amino acid composition of the encoded proteins. the magnitude of these changes is comparable to those noted previously by foster et al. for the effects of differences in mitochondrial nucleotide composition, it should be noted, however, that the strand asymmetries described in this study do not affect the same subsets of the amino acids as those affected by changes gc content. as an illustrative example, we can compare a single pair of mitochondrial genomes, one mammal and one flatworm, those of the red deer (cervus elaphus) and the liver fluke (fasciola hepatica). in both species, the overall nucleotide content of the mitochondrial coding sequences is virtually identical at 38% gc and proportions of gc-rich (garp) and at-rich (fymink) amino acids (see foster et al. for details) are also very similar between these two species. in other words, their similarity in nucleotide composition is reflected in a similarity in the proportions of gc-rich and at-rich amino acids. but when we compare the same two species for the levels of ca-rich (qtphnk) and gt-rich (cvfgw) amino acids, we see large differences reflecting the differences in strand asymmetry between the mammals and the flatworms. for example, the liver fluke proteins contain more than twice as many valine residues and more than five times as many cysteine residues as do their orthologs in the red deer. on the other hand, the liver flukes have approximately a third as many glutamines and threonines as are found in the red deer. these differences are also statistically highly significant (p<0.001) and they are entirely consistent with what we see for the average differences between mammals and flatworms (fig. 3 and supplementary table s1). a possible alternative explanation for these results is that the amino acid differences are the cause, rather than the consequence, of the strand asymmetries. first, there is an even larger strand asymmetry at the synonymous codon sites (supplementary fig. s1) suggesting that the nucleotide skew is counterbalanced, to some extent, by functional constraint at the protein level. in other words, protein function does have an effect, but as a constraint rather than a cause. another way to illustrate this point is to calculate the gc skew at each codon position separately. the results (supplementary table s2) show that the greatest differences in gc skew occur at the third codon position and the least skew occurs at the second position. this is consistent with the fact that many changes at the third codon position alter the codon usage but do not affect the protein sequence. secondly, in mammalian mitochondria, one gene (nd6) is encoded on the opposite strand from the other 12 genes and, in accordance with our prediction, the amino acid composition of this mammalian protein displays a pattern that is similar to that of the flatworm proteins rather than the other 12 mammalian proteins (supplementary fig. both of these observations indicate that the primary effect is at the level nucleotide asymmetry between the two strands of the mitochondrial genome and that this dna bias causes a secondary effect at the level of protein composition. we performed a number of further tests to explore the interplay between functional constraint at the protein level and nucleotide skew at the dna level. since we limited our comparison to orthologous mitochondrial genes, we have eliminated the effect of different types of proteins in the two groups of species. moreover, since mitochondrial function is highly conserved in metazoan animals, we expect that the orthologous proteins are performing essentially the same functions in the two species groups. there is still, however, the remote possibility that there are differences in physiological conditions between the mitochondria of mammals and those of flatworms and that these differences could contribute to the differences in protein composition which we observe between the two groups. to control for this possibility, we examined the sequences of three mitochondrial ribosomal protein genes: l11, l15, and l20 (supplementary table s2). although these proteins function in the mitochondria, they are encoded by genes in the nuclear genome. the results show that the gene sequences do not show the characteristic mitochondrial gene skews and there is no significant difference between mammals and platyhelminthes, either at the dna level or at the protein level. this indicates that the key factor underlying the species differences is not the functional environment of the proteins but rather the location of the genes encoding those proteins. since our results point to a mutational force at the dna level that is counteracted by functional constraint at the protein level, we asked what would happen if we confined our analysis to a region of the protein where there was reason to believe that the functional constraint would be especially strong. the transmembrane domain portions of mitochondrial proteins provide an opportunity to do such an analysis. specifically, we looked at the patterns of gc skew and amino acid composition in the transmembrane domain regions of the coxi gene. the results (supplementary table s2) show that the increased functional constraint on amino acid composition does indeed lead to a decreased difference in gc skew between the two species groups. for example, the difference in gc skew between mammals and flatworms for the complete dataset is 0.84, whereas it is reduced to 0.57 for the transmembrane domains, but the same pattern remains both at the dna and protein levels. for instance, even within the transmembrane domains, the mammalian sequences have less cysteine and valine, and more glutamine and threonine than do their flatworm orthologs, reflecting the same patterns as shown in figure 3. in other words, increased functional constraint decreases the effect of nucleotide skew, but it does not completely eliminate it. overall, our results show that the dna strand asymmetry of animal mitochondrial genomes affects the amino acid composition of encoded proteins. a similar effect has been noted previously in bacterial genomes but it has not been reported in animal or plant genomes. since we infer that the dna strand bias is the cause rather than the consequence of the protein differences, this raises the question of what causes the dna strand bias in the first place. in bacterial genomes, there is good evidence that it is related to dna replication, based primarily on the fact that the direction of the bias switches at the origin of replication. recent work indicates that although dna strand biases are widespread in prokaryotes, eukaryotes, and viruses, the magnitude and the direction of the bias is variable, suggesting that the underlying causes are multifactorial. in animal mitochondrial genomes, the strand bias appears to be caused by varying durations of time that the heavy strand spends in the mutagenic single-strand state during replication. in addition to the replication-associated effects, there is evidence that transcription can also generate dna strand asymmetry in eukaryotes due to transcription-coupled mutations. a recent study has shown that strand bias in mitochondrial sequences can lead to artefactual results in phylogenetic reconstructions. this misleading result can be minimized by a recoding scheme that excludes transitions at rapidly evolving neutral sites. our results, however, show that mitochondrial strand biases can also have significant effects at non-neutral sites that change the amino acid sequence. this means that some degree of bias remains even after the correction has been implemented. as stated by jones et al. (2007) in the case of mitochondrial genes, strand-bias should be of particular concern and the previous use of mitochondrial genomes in resolving deep phylogenies requires critical re-evaluation. it remains to be seen if dna strand asymmetry can also affect the composition of proteins encoded by eukaryotic nuclear genes, as has been shown for biases in the gc content of nuclear genes. it is already known that dna strand asymmetries exist in nuclear genes but their effects on the composition of nuclear-encoded proteins have not been studied. this work was supported by a grant to dah from the natural science and engineering research council of canada.

abstractvariations in gc content between genomes have been extensively documented. genomes with comparable gc contents can, however, still differ in the apportionment of the g and c nucleotides between the two dna strands. this asymmetric strand bias is known as gc skew. here, we have investigated the impact of differences in nucleotide skew on the amino acid composition of the encoded proteins. we compared orthologous genes between animal mitochondrial genomes that show large differences in gc and at skews. specifically, we compared the mitochondrial genomes of mammals, which are characterized by a negative gc skew and a positive at skew, to those of flatworms, which show the opposite skews for both gc and at base pairs. we found that the mammalian proteins are highly enriched in amino acids encoded by ca-rich codons (as predicted by their negative gc and positive at skews), whereas their flatworm orthologs were enriched in amino acids encoded by gt-rich codons (also as predicted from their skews). we found that these differences in mitochondrial strand asymmetry (measured as gc and at skews) can have very large, predictable effects on the composition of the encoded proteins.

PMC2779903

pubmed-1229

calcific aortic valve stenosis has become the most common acquired valve disorder with the highest prevalence in the 8th or 9th decade of life. comorbidities such as diabetes mellitus, stroke, coronary heart disease, peripheral artery disease, pulmonary disease, and renal impairment appear to markedly increase the risk of conventional valve replacement in the elderly and may limit the benefit of surgery in these patients. nevertheless, the outcome of untreated aortic stenosis is dismal once congestive heart failure, angina pectoris, or syncope occur. therefore alternative, less invasive treatment options are needed. since the first-in-man transcatheter aortic valve implantation (tavi) by cribier et al. in 2002, more than 50000 procedures were performed worldwide and this intervention has become an accepted assumingly less invasive treatment alternative for high risk surgical patients. however, due the comorbidities in these patients, even tavi is associated with a number of complications that may lead to impaired outcome. their closer evaluation and the definition of risk factors as well as measures to reduce their occurrence are essential and require further research. acute kidney injury (aki) is a well-known complication of angiography with the use of iodinated contrast media that accounts for a significantly prolonged hospital stay and worse in-hospital outcome. furthermore aki has been shown to be an independent predictor of mortality [58]. the most important risk factor for aki in patients undergoing standard heart catheterization is preexisting chronic kidney disease [9, 10]. other risk factors include volume depletion, hemodynamic instability, and the use of nephrotoxic drugs. tavi requires the administration of contrast media and preexisting kidney disease is frequent in the currently treated patient population. however, the incidence of aki, predictors of this complication, and its impact on outcome in patients undergoing tavi have so far been poorly defined. therefore, we sought to assess the incidence of aki, search for its predictors, and analyze its impact on 30-day as well as midterm outcome in a sizeable group of consecutive patients undergoing tavi. a total of 150 consecutive patients with symptomatic aortic stenosis who underwent tavi in our institution because they were either not suitable for conventional surgical valve replacement or were considered at high operative risk by a multidisciplinary team including cardiologists and cardiac surgeons were included in this study. ten out of 150 patients had been enrolled in a chronic dialysis program and were therefore excluded from analysis concerning aki. before intervention, all patients received left and right heart catheterization. according to the institution policy, written informed consent blood samples for hematology and serum chemistry were drawn one day prior to intervention and daily up to 72 hours after treatment. patients with a previously impaired kidney function (estimated glomerular filtration rate, egfr<60 ml/min/1,73 m) received an intravenous prehydration protocol consisting of saline 0.9% with 1200 mg of n-acetyl-cysteine both 12 hours before and after the procedure. the vascular access site was evaluated by color-coded doppler sonography and ct-angiography. valve replacement was performed under general anaesthesia, except in one case which was done under local anaesthesia. all procedures were performed in the catheter laboratory using fluoroscopic guidance and nonionic isoosmolar contrast media iopromide (ultravist 370 (tm), schering ag, berlin, germany) and transoesophageal echocardiography. the patients received either a 23 or 26 mm edwards-sapien valve prothesis. renal function at baseline and after 48 hours was determined from serum creatinine determined by the method of jaff. since creatinine is known to be an insufficient marker of renal function but estimated glomerular filtration rate (egfr) is considered most suitable we used the modification of diet in renal disease (mdrd) formula for calculation: (1)egfr (ml/min/1,73 m2) =186(creatinine, mg/dl)1,154(age, years)0,203 (0,742 in women). acute kidney injury is divided into three stages by the acute kidney injury network: stage 1 is defined as a rise in serum creatinine 26.5 mol/l compared to baseline values or an increase in serum creatinine of more than or equal to 50% or a reduction in urine output as documented oliguria of less than 0.5 ml/kg per hour. in this study aki was defined as a 26.5 mol/l rise in serum creatinine 48 hours after procedure compared to baseline data drawn 24 hours before intervention. logistic euroscore was calculated by the web-based system (http://www.euroscore.org/) in advance and results taken for clinical decision making. sts score was evaluated retrospectively for further analysis including the predicted risk of renal failure (http://209.220.160.181/stswebriskcalc261/de.aspx). renal failure by sts score is defined as an increase of serum creatinine>176.8 mol/l, a 50% or greater increase in serum creatinine over baseline preoperative value, or new requirement for dialysis. differences in basic clinical characteristics between groups were tested by chi-square test for categorical and the anova f-test for continuous variables. uni- and multivariate predictors of aki were assessed by logistic regression analysis and odds-ratios (or) are reported. univariate predictors of mortality during followup were analyzed by cox regression and calculation of hazard rate ratios (hr) with 95% confidence intervals (95% ci). multivariate analysis of mortality was performed by cox regression analyses with potential covariates (adjusted hr). as covariates for adjustment, those parameters were chosen which were found to have a p value lower than 0.05 in univariate analyses of death. the mean age in the patient group was 81 7 years. in total, 96 patients received valve replacement via transfemoral (tf) and 54 patients via transapical (ta) approach. patients with transapical approach were more frequently male and had significantly more underlying comorbidities such as hypertension, pad, chd, previous cabg, previous stroke, and impaired kidney function based on baseline serum creatinine measurement (see table 1). ten patients (7%) had already been enrolled in a chronic dialysis program before intervention (5% of tf versus and 9% of ta patients, p=0.006) and were therefore excluded from analysis concerning aki. the average amount of contrast media used in all patients was 147 58 ml. patients with transfemoral approach received with 160 57 ml of contrast media a significantly greater amount than patients with transapical access who received 125 53 ml (p<0.0001). the rate of aki after pre-tavi diagnostic right and left heart catheterization in our patient population was 9.2% (n=13). only 4 patients (2.8%) developed aki after both diagnostic coronary angiography and tavi procedure (median 15 days between diagnostic catheterization and valve procedure). after exclusion of the ten patients who had already been enrolled in a chronic dialysis program before tavi, 140 patients were left for the analysis concerning the occurrence of acute kidney injury. two patients without aki needed short-term dialysis (one patient was hemofiltrated due to low cardiac output and consecutive renal impairment, another patient acquired septic shock with renal failure). there was no significant difference regarding weight, height, baseline creatinine, and hemoglobin values in pts. who developed aki after intervention compared to those who did not (see table 2). patients with aki were significantly younger (79 9 yrs versus 82 6 yrs, p=0.008) and had more frequently comorbidities such as hypertension and previous cabg whereas differences in peripheral arterial disease, cerebrovascular disease, chd, and hypercholesterolemia did not reach statistical significance. this difference did not also reach statistical significance which could have been due to the sample size. the amount of contrast media used during the procedure was also very similar between groups (147 71 ml versus 148 56 ml, p=0.93). with aki than in those without aki (20 12 days versus 15 10 days, p=0.03). both, 30-day-mortality (29% versus 7%, p<0.0001) and cumulative mortality after a median followup of 309 days were significantly higher in aki patients (43% versus 18%, p<0.0001). aki was associated with significantly worse survival (hrr 2.7, ci 1.345.41, p=0.006, figure 1(a)). mortality in aki pts. was even higher (hrr 3.8, ci 1.3710.37, p=0.01, figure 1(b)) after adjusting for risk factors (age, diabetes, pad, hypertension, previous myocardial infarction and cabg, left ventricular dysfunction, amount of contrast dye, baseline creatinine, and hemoglobin). predictors of aki occurrence in univariate and multivariate regression analysis are shown in tables 3 and 4. of all included variables (age, diabetes, hypertension, pad, previous cabg, myocardial infarction, left ventricular function, baseline creatinine, and hemoglobin and amount of contrast dye) only age was found to be significantly associated with aki in univariate analysis and was detected as an independent predictor of aki in multivariate analysis (or 0.93, ci 0.870.99, p=0.03). including vascular access site in the model, transapical approach was also a significant predictor of aki (or 1.8, ci 1.8518.4, p=0.003). neither euroscore (27 19% versus 23 13, p=0.18) nor sts score (6.0 3.5% versus 6.0 3.4%, p=0.97) predicted the marked difference in mortality rates between aki and non-aki pts (figure 2). when applying the sts score renal failure definition to our patient population, 19 pts. however, the predicted rate of renal failure by sts score was only 7.3% (p=0.023) for all pts. the predicted risk for renal failure based on the sts score did not significantly differ between the ta and tf treatment groups (6.8 3.5% versus 8.3 5.1%, p=0.054). the observed rate of aki was however significantly higher in the ta group (31% versus 11%, p=0.001) and exceeded thereby markedly the predicted rate of renal failure 1.6-fold in the tf and 3.7-fold in the ta pts. acute kidney injury after the use of iodinated contrast media in angiography is known to account for a number of adverse effects such as prolonged hospital stay and to be an independent risk factor of mortality [710]. several investigators have shown that aki is a relatively frequent complication after tavi and that it is associated with an increased mortality [1417]. however it remains unclear which of the underlying comorbidities contribute most to the adverse outcome following aki. moreover, reliable predictors of aki in this patient population still need to be defined. in particular, the value of risk scores developed for patients who undergo open heart surgery remains so far unknown. in the present study ,. found very similar rates with 19% for aki and 2% for temporary dialysis. while bagur et al. reported a lower rate of aki (11%) and need of dialysis after tavi (1.4%), aregger et al. and kong et al. found markedly higher rates of 28% and 7.4%, and 28.8% and 6%, respectively. one explanation for this wide variation of aki rates could be that the average amount of contrast media used in these studies differed markedly, too. while it was 148 ml in the present study, bagur et al. with the lowest rate of aki reported<100 ml. in contrast, aregger et al. with the higher rate of aki used 242 ml on average. the amount of contrast media used during angiography is indeed considered one major risk factor for the development of aki. nevertheless, in the present study no significant difference in contrast media use could be found between aki und non-aki patients. the observation that aki was not related to the amount of contrast used has also been reported by other groups [15, 16] suggesting that other factors may be more important for the development of renal impairment in the population currently undergoing tavi. prehydration in addition to intravenous n-acetyl-cysteine application prior to contrast media exposure is a well-known measure to reduce aki rates in patients with renal impairment. in our study patients with an egfr<60 ml/min/1,73 m were treated with 1000 ml of saline 0.9% and 1200 mg of n-acetyl-cysteine which may have prevented more patients from experiencing aki than without these protective measures. nevertheless, this prevention strategy in general and even more in this specific patient population is not effective enough to thoroughly avoid occurrence of aki. therefore hydration therapy may have contributed to the nonsignificant association between baseline creatinine and acute kidney injury risk. comparing aki rates and patients after diagnostic and tavi catheterization no correlation between aki after diagnostic and valve procedure could be seen implying the lack of a patient-inherent predisposition for aki occurrence after exposure to contrast media. this finding complies with the results of van linden et al. who stated that early contrast media exposure (17 days) by cardiac catheter or ct-scan did not increase the risk of aki or rrt. in this context it should be kept in mind that after cardiac surgery without any use of contrast media, the rate of aki can also reach up to 30% with 1% requiring dialysis treatment [21, 22]. bagur et al. reported a 25% incidence of aki after surgical aortic valve replacement in patients with preprocedural chronic kidney disease compared to 12% in patients undergoing tavi. the markedly adverse effect of the occurrence of aki on the outcome of tavi underlines the importance of identifying predictors of this complication as well as appropriate measures for its prevention. in the present study, 30-day mortality and midterm mortality were as high as 29% and 43% in patients with aki compared to only 7% and 18% in those who did not develop this complication. the difference in survival was even more pronounced when adjusting for differences in baseline characteristics. similar findings have been reported by other investigators [1417, 23]. despite these rather consistent findings with regard to incidence of aki after tavi and its adverse impact on outcome, the data with regard to risk prediction and options for prevention of aki remain controversial. it appears obvious that preprocedural chronic kidney disease should be a major risk factor for the development of postprocedural aki.. indeed showed impaired renal function with moderately elevated serum creatinine values before intervention and aki occurrence unrelated to the amount of contrast media to be the strongest predictors of 1-year mortality among tavi patients. although baseline creatinine in the present study was slightly higher in the aki group (126.4 59.2 this may be due to small sample size and thus lack of statistical power. as a matter of fact, preinterventional serum creatinine was found to be a significant predictor of aki only in the study by elhmidi et al. whereas several other studies could not confirm this observation [14, 15, 17, 19]. paradoxically, younger age turned out to be the only independent preprocedural risk factor for the development of aki in the present study. this observation must be seen with caution. to qualify for tavi instead of conventional surgery, younger patients must assumingly have been markedly sicker than older patients. hypertension was also found to be a predictor of aki in other studies [14, 19]. without consistency, peripheral artery disease [15, 16], previous myocardial infarction, chronic obstructive pulmonary disease, systemic inflammatory response [16, 17], residual aortic regurgitation, and periprocedural red blood cell transfusion [14, 15, 19] have been reported to predict aki after tavi. in accordance with kong et al., transapical tavi was found to be associated with a higher risk of aki in the present study. although this could be partially due to the worse baseline characteristics of these patients, transapical access remained a significant predictor after consideration of such differences. whether the more invasive nature of this approach, higher bleeding rates and requirement for blood cell transfusion account for this difference remains to be shown. in addition, similar to previous reports, the observed 30-day mortality was markedly lower than predicted by the logistic euroscore (11% versus 24%) whereas the sts score was indeed lower (6%). this is in agreement with the observation of piazza et al. who found lower estimates of operative mortality by the sts score stating that this scoring system has suboptimal discriminatory power and calibration for tavi patients. sts score as a surgical risk algorithm obviously omits several risk factors in the tavi population leading to different patient selection and thus mortality rates. the present study also demonstrates that sts score for prediction of renal failure has little value for prediction of renal failure after tavi. this underlines once more the importance of developing appropriate scores for the risk of death as well as of the risk of renal failure and other complications in patient populations currently treated with tavi. in addition to preexisting factors, hemodynamic instability with consecutive extreme hypotension caused by rapid pacing, balloon valvuloplasty, and prosthesis deployment during tavi may account for a significantly higher risk of aki in patients undergoing tavi compared to simple angiography or pci. this must be considered when developing measures to reduce the occurrence of aki after tavi. although the data were collected prospectively in consecutive patients undergoing tavi, the analysis with regard to incidence and predictors of aki was performed retrospectively. potentially relevant factors such as red blood cell transfusion, after procedure thrombocytopenia and hemoglobin drop, procedure time, hemodynamic complication or the use of angiotensin converting enzyme inhibitors, and/or angiotensin receptor blockers could not be evaluated. although the study comprised a sizeable number of tavi patients, it reflects a single-center experience only and a much larger population is required to perform extensive multivariate analyses in order to better identify risk factors for the development of aki with relevant impact on the decision making in clinical practice. its occurrence does not appear to be primarily related to the amount of contrast dye used. the occurrence of aki markedly increases hospital stay as well as 30-day and midterm mortality even after consideration of the baseline risk profile. thus, improvements in predicting the risk of aki after tavi as well as effective measures to reduce the rate of this complication would be essential.

background. transcatheter aortic valve implantation (tavi) is widely used in high risk patients (pts) with aortic stenosis. underlying chronic kidney disease implicates a high risk of postprocedural acute kidney injury (aki). we analyzed its occurrence, impact on hospital stay, and mortality. methods. 150 consecutive pts underwent tavi in our institution (mean age 81 7 years; logistic euroscore 24 15%). aki definition was a creatinine rise of 26.5 mol/l or more within 48 hours postprocedural. ten patients on chronic hemodialysis were excluded. results. aki occurred in 28 pts (20%). baseline creatinine was higher in aki pts (126.4 59.2 mol/l versus 108.7 45.1 mol/l, p=0.09). contrast media use was distributed evenly. both, 30-day mortality (29% versus 7%, p<0.0001) and long-term mortality (43% versus 18%, p<0.0001) were higher; hospital stay was longer in aki pts (20 12 versus 15 10 days, p=0.03). predicted renal failure calculated sts score was similar (8.0 5.0% [aki] versus 7.1 4.0% [non-aki], p=0.32) and estimated lower renal failure rates than observed. conclusion. aki remains a frequent complication with increased mortality in tavi pts. careful identification of risk factors and development of more suitable risk scores are essential.

PMC3541560

pubmed-1230

study population-in total, 49 patients with chronic viral hepatitis, consisting of 28 hbv-infected patients and 21 hcv-infected patients, and 33 healthy, non-infected controls were included in the study. the patients were recruited from the clinic of infectious diseases of julio muller hospital (federal university of mato grosso, cuiab, state of mato grosso, central-west brazil). chronic hbv infection was confirmed by persistent hbv surface antigenemia lasting more than six months. chronic hcv infection was confirmed by the presence of hcv rna in blood tests. cirrhosis was diagnosed by liver biopsy or based on clinical observations, laboratory tests or ultrasonographic evidence. non-infected subjects were recruited from a group of healthy blood donors at the public blood bank of mato grosso state. information about alcohol consumption, tobacco use, ethnicity and age was obtained from medical records and from an interviewer-administered questionnaire, which also included questions about exposure to mutagens and any history of cancer in the individual. cytokinesis-block mns (cbmn) assay-the cbmn assay was performed as described by fenech and morley (1985), with minor modifications. in total, 5 l of venous blood was collected in heparin-vacutainer tubes (becton&dickinson, franklin lakes, nj, usa) and lymphocyte cultures (2 per subject) were established using 0.3 ml of whole blood added to rpmi-1640 medium (sigma-aldrich, st. louis, mo, usa) supplemented with 20% foetal calf serum (cultilab, campinas, sp, brazil), 0.001% penicillin (vetec, duque de caxias, rj, brazil), 0.0005% streptomycin (sigma-aldrich) and 2% phytohemagglutinin (cultilab). after the cultures were incubated for 44 h at 37c in a bod incubator (eletrolab, so paulo, sp, brazil), cytochalasin b (sigma-aldrich) was added to the cultures (6 g/ml). the cells were harvested by centrifugation at 72 h after the culture was initiated. the lymphocytes were treated with a hypotonic solution (1% sodium citrate w/v) and fixed in a solution of methanol: acetic acid (3:1 v/v); in both cases, the solutions were ice cold and freshly prepared. the cell suspension was dropped onto a pre-cooled microscope slide and air dried before being stained for 5-7 min with 5% giemsa in sorensen phosphate buffer (0.06 m na 2 hpo 4 and 0.06 m kh 2 po 4, ph 6.8). microscopic analysis was performed with a light microscope (nikon, melville, ny, usa) at 400x magnification. for each individual, 2,000 binucleated cells were analysed for the presence of mn, npbs and nbuds in accordance with previously established criteria (fenech et al. we calculated the frequency of each biomarker (number in 1,000,) using the following formula: of x =(number of x/2,000) x 1,000, where x is mn, npbs or nbuds (montero et al. mutagen sensitivity evaluation-to determine mutagen sensitivity, the well-established mutagen dxr was used to treat lymphocytes from patients and non-infected subjects at 44 h after the culture was initiated. the cells were treated with 0.15 g/ml dxr (bergamo, taboo da serra, sp, brazil) diluted in sterile distilled water for 28 h, which brought the total culture time to 72 h. the experimental conditions for the dxr treatment were previously established in preliminary experiments. the conditions for cell harvesting and slide preparation were described in the previous section. sensitivity to dxr was expressed as induced dna damage: [(mean mn after dxr)-(basal mean mn)]. statistical analysis-an age comparison between groups was performed using one-way anova and the bonferroni post-hoc test. when the data exhibited unequal variance, the median numbers of mn, npbs and nbuds were compared between the groups using the non-parametric kruskal-wallis test followed by dunn s post-hoc test. similarly, the median numbers of mn obtained in the mutagen sensitivity test were compared between the groups using the mann-whitney u test. the mean measurements of the induced dna damage were compared using the student s t test. the g-test or the test was used to compare ethnicity, sex, alcohol intake and tobacco intake between the groups. a linear regression model was constructed using the stata 8.2 software programme (statacorp, college station, tx, usa) to verify the independence of the frequencies of mn, npbs and nbuds from the virus type, sex, age, alcohol intake, tobacco intake and the use of antiviral drugs. the statistical analyses were performed using the statistical software programme bioestat 5.0 (ayres et al. ethics-this study was approved by the ethical research board of julio muller hospital (protocol 439/cep-hujm/07) and informed consent to voluntarily participate was given by all of the subjects. of the 49 patients included in the study, 28 were chronically infected with hbv (21 males and 7 females) and had a mean age of 36.1 years (ranging from 21-56 years), while 21 were chronically infected with hcv (13 males and 8 females) and had a mean age of 44.1 years (ranging from 27-55 years). the non-infected control group consisted of 23 males and 10 females with a mean age of 37.4 years (ranging from 20-52 years). no differences were observed between the non-infected group and the hbv or hcv-infected patients with regard to their sex (p=0.97) and smoking habits (p=0.16). the mean age was similar between the infected patients and the non-infected control subjects; however, the mean age of the hcv-infected patients was significantly higher than that of either the chronically hbv-infected group or the controls (p<0.05). alcohol consumption among the study subjects ranged from 0-8 g/day and was more frequently reported by the non-infected control subjects (45.45%) than by the hbv-infected patients (17.86%) or the hcv-infected patients (14.28%) (p=0.003). cirrhosis was present in eight hbv-infected patients (28.6%) and three hcv-infected patients (14.29%) (p=0.31) (table i). seven patients (6 hbv-infected and 2 hcv-infected patients) were undergoing antiviral therapy at the time of the study (1 lamivudine and tenofovir, 2 ribavirin and -interferon and 3 tenofovir). table idemographic and clinical characteristics of patients chronically infected with hepatitis b virus (hbv) or hepatitis c virus (hcv) and of control subjectsdemographic and clinical parameterscontrols (n=33) n (%) total patients (n=49) n (%) hbv (n=28) n (%) hcv (n=21) n (%) males23 (69.7)36 (69.4)21 (75)13 (61.9)females10 (30.3)15 (30.6)7 (25)8 (38.1)age (years) range37.4 10.4 (20-52)37.4 10.3 (20-55)36.1 10.36 (27-55)37.41 7.7 (20-52)smoking habit1 (3.03)8 (16.3)4 (14.3)4 (19.04)alcohol consumption15 (45.4) 8 (16.3)5 (17.9)3 (14.3)cirrhosis-11 (22.4)8 (28.6)3 (14.3) a: anova and bonferroni post-hoc test, p<0.05; b: g test, p=0.004. a: anova and bonferroni post-hoc test, p<0.05; b: g test, p=0.004. the numbers of mn, npbs and nbuds observed in the patient group were 7.01 3.23, 2.76 2.08 and 4.57 2.98, respectively. the frequencies of mn and npbs, but not nbuds, were significantly increased (p<0.0001) compared with the frequencies in the controls (4.41 2.15 and 1.15 0.97 for mn and npbs, respectively, in the controls). considering the hbv-infected patients and the hcv-infected patients separately, the numbers of mn in the hbv-infected patients (7.18 3.57) and hcv-infected patients (6.78 2.80) were significantly higher than in the non-infected group (4.41 2.15) (p< 0.0001). the numbers of npbs and nbuds in the hbv-infected patients (3.27 2.40 and 4.71 2.79, respectively) and in patients infected with hcv (2.09 1.33 and 4.38 3.28, respectively) were increased relative to those of the control group (1.15 0.97 and 2.98 1.31, respectively). however, only the difference between the hbv-infected group and the control group was statistically significant (p=0.03) (table ii). the results were independent of age, sex, alcohol consumption, tobacco intake and the presence of cirrhosis for mn (r=0.22, p=0.02), npbs (r=0.23, p<0.001) and nbuds (r=0.15, p=0.03). the antiviral therapy did not significantly influence any of the analysed parameters. the frequency of mn was significantly influenced by sex only for males aged<40 years in the control group, who showed a significantly lower frequency of mn (3.46 1.12) than did females (5.63 1.60) of the same age (p=0.01). the frequency of mn and npbs was significantly higher in males, among all patients and within the hbv-infected group in comparison with the controls (p<0.01) (table iii). the frequency of npbs was significantly higher in females aged>40 years in comparison with the controls (p<0.01). the frequency of mn, npbs and nbuds was not significantly higher in male patients with cirrhosis in comparison with male patients without cirrhosis (table iv). table iifrequency of micronucleus (mn), nucleoplasmatic bridges (npb) and nuclear buds (nbuds) in hepatitis b virus (hbv) or hepatitis c virus (hcv)-infected patients and controlsgroupsmn (mean sd)npb (mean sd)nbuds (mean sd) controls (n=33)4.41 2.151.15 0.972.98 1.31total patients (n=49)7.01 3.23 2.76 2.08 4.57 2.98hbv (n=28)7.18 3.57 3.27 2.40 4.71 2.79 hcv (n=21)6.78 2.80 2.09 1.334.38 3.28 a, b: statistically significant in comparison to the controls (a: p<0.0001; b: p=0.03; kruskal-wallis test with dunn s post-hoc test); sd: standard deviation .. a, b: statistically significant in comparison to the controls (a: p<0.0001; b: p=0.03; kruskal-wallis test with dunn s post-hoc test); sd: standard deviation .. table iiithe effect of age and sex on frequency of micronucleus (mn), nucleoplasmatic bridges (npb) and nuclear buds (nbuds) n hepatitis b virus (hbv) or hepatitis c virus (hcv)-infected patients and controlsgroupsmn (mean sd)npb (mean sd)nbuds (mean sd) controls (n=33) males<40 years (n=14)3.46 1.12 1.32 1.312.86 2.67 males 40 years (n=9)4.06 1.590.83 0.52.67 1.17 females<40 years (n=4)5.63 1.601.50 0.824.13 0.85 females 40 years (n=6)6.33 3.541.0 0.633.0 1.26total patients (n=49) males<40 years (n=14)6.79 4.49 3.5 2.97 4.03 2.45 males 40 years (n=20)7.25 2.92 3.08 1.45 5.10 3.72 females<40 years (n=6)6.92 2.853.0 2.344.0 2.61 females 40 years (n=9)6.89 2.103.50 1.44 4.61 2.25hbv (n=28) males<40 years (n=12)6.71 4.82 3.27 3.14 4.42 2.45 males 40 years (n=9)7.33 2.03 3.27 1.51 5.89 3.46 females<40 years (n=4)7.0 3.633.25 2.963.25 2.90 females 40 years (n=3) 8.83 1.533.12 0.484.33 0.76hcv (n=21) males<40 years (n=2)7.25 2.475.0 01.75 0.35 males 40 years (n=11)7.18 3.59 2.67 1.37 4.45 3.97 females<40 years (n=2)6.75 1.062.50 0.715.50 1.41 females 40 years (n=6)5.92 1.663.80 1.92 4.75 2.79 a: statistically significant in comparison to females of the same age-class (p=0.01, mann-whitney u test); b: statistically significant in relationship to controls of the same age class (p<0.01, anova, post-test student t or kruskal-wallis, post test student-newman-keuls); c: groups with n<4 were not included in statistical analysis; sd: standard deviation. a: statistically significant in comparison to females of the same age-class (p=0.01, mann-whitney u test); b: statistically significant in relationship to controls of the same age class (p<0.01, anova, post-test student t or kruskal-wallis, post test student-newman-keuls); c: groups with n<4 were not included in statistical analysis; sd: standard deviation. the effect of cirrhosis on frequency of micronucleus (mn), nucleoplasmatic bridges (npb) and nuclear buds (nbuds) in males hepatitis b virus (hbv) or hepatitis c virus (hcv)-infected patientsgroupscirrhosis (n)mn (mean sd)npb (mean sd)nbuds (mean sd) total patientsno (23)6.82 4.102.98 2.533.67 1.75yes (11)7.54 2.272.68 1.585.00 2.59hbvno (15)6.56 4.363.26 2.914.13 1.72yes (8)8.25 2.233.06 1.685.25 2.39hcvno (9)7.31 3.802.43 1.632.81 1.53yes (3)7.00 2.901.66 cells from 15 chronically infected patients (9 hbv-infected and 6 hcv-infected patients) and 14 non-infected individuals were tested for dxr sensitivity. the numbers of mn were not significantly different between the hcv-infected and the hbv-infected patients; therefore, the patients were grouped together for comparison with the non-infected individuals to improve the statistical analysis. the patients exhibited significantly higher numbers of mn in both untreated (6.76 3.03, p=0.03) and treated (15.03 4.94, p=0.001) cells compared with the cells of non-infected individuals (4.25 1.45 and 9.39 3.39 in untreated and treated cells, respectively). dxr treatment significantly increased the number of mn in both the control group (p=0.0002) and the patient group (p<0.0001). the mean measurements of the induced dna damage were higher in the patient group (8.30 5.40) than in the non-infected control group (5.18 3.45). however, this difference was not found to be statistically significant, which was possibly due to a type 2 error (p=0.06). chronic hepatotropic virus (hbv and hcv) infections are characterised by potentially mutagenic cellular events, such as an increase in oxidative stress and viral integration into the dna of the host cell (farinati et al., we demonstrated that the lymphocytes of hbv-infected patients and hcv-infected patients exhibited an increased frequency of mn compared with lymphocytes from healthy, non-infected individuals. however, these authors reported a higher frequency of chromosome breaks, leading to the formation of acentric chromosome/chromatid fragments that ultimately contributed to the formation of mn. furthermore, the mn may have resulted from an inability of whole chromosomes to travel to the spindle poles during mitosis (fenech et al.. a higher frequency of chromosome gaps, aneuploidy and polyploidy in the peripheral blood mononuclear cells (pbmcs) of hcv-infected patients was reported previously (machida et al. the present results suggest that chronic infection with hbv or hcv accounts for chromosomal instability in lymphocytes and this phenomenon is characterised by the formation of acentric fragments and/or aneuploidy. npbs may occur when dicentric chromosomes originating from chromosome breaks or telomere-to-telomere end fusions are pulled to opposite poles of the cell during mitosis. nbuds are primarily considered to be formed from amplified dna that is being eliminated from chromosomes (fenech et al. although gene amplifications (myc and erbb2) were demonstrated in hcc tissue samples that were infected with hbv or hcv (al-qahtani et al. 2010), no reports have described this genetic alteration in pbmcs from individuals with viral hepatitis. dicentric y chromosomes have also been reported in the pbmcs of hcv-infected patients (machida et al. 2010). together with our results, these data suggest that the chromosomal instability in lymphocytes that results from hbv infection (and likely also from chronic hcv infection) is characterised by the presence of dicentric chromosomes and gene amplification. in this study, females and subjects aged 40 years exhibited more mn than did males and subjects<40 years, respectively, among both controls and hbv-infected patients. although these differences were not statistically significant, the results are in accordance with the well-known influence of sex and age on mn frequencies, as reported before (fenech 1998, bonassi et al. reports on the effects of sex and age on npbs and nbuds are conflicting in the literature (donmez-altuntas& bitgen 2012, nefic&handzic et al., we found statistically significantly higher frequencies of mn in males than in females; however, females did not present cirrhosis in our sample. it is known that dna damage levels in the leukocytes of hbv-infected patients and hcv-infected patients significantly correlate with the presence of liver lesions (farinati et al. in fact, in the current study, the frequencies of mn (in hbv-infected patients) and nbuds (in hbv-infected patients and hcv-infected patients) were increased in cirrhotic males, but this difference was not significant. whether the extension of liver lesions in male patients is correlated with the frequency of mn found in lymphocytes is not completely clear in the sample investigated here. even considering that the statistical analysis may have been influenced by the small size of our sample, these results should be considered with caution, especially because chromosome damage detected by the mn assay is an important biomarker for cancer prediction (bonassi et al. hbv and hcv may contribute to increased chromosomal aberrations in infected cells by direct and indirect pathways. regarding the indirect pathway, it has been shown that the presence of reactive oxygen species (ros) resulting from cytokine activity during chronic inflammation has a potent mutagenic effect (yan et al. a relationship between infection with hbv or hcv and increased production of ros, chromosomal aberrations and other dna damage has been reported previously (hagen et al. 1994, machida et al. 2010). furthermore, leukocytes from patients who are chronically infected with hbv or hcv exhibit higher levels of 8-ohdg, which is the most frequent ros-induced base lesion (farinati et al. the integration of hbv into the human genome affects the expression of genes located near the site of insertion and also causes more widespread alterations of chromosomal stability (saigo et al. 2012). because viral genome integration into the host dna also frequently occurs in the pbmcs of chronically hbv-infected patients (murakami et al. 2004), this process may contribute to genomic instability in these cells. recently, it was demonstrated that in chronic hcv infection, the presence of double strand breaks occurs concomitantly with shortened telomeres in t lymphocytes. this phenomenon is associated with the level of fibrosis and may influence the response to treatment (hoare et al. 2013). the frequency of mutations is directly influenced by the efficiency of the dna repair mechanisms because failure to remove a lesion can facilitate mutational fixation. extensive evidence has demonstrated that proteins produced by hbv and hcv interact with the proteins of the dna repair machinery and inhibit their functions in host cells (chen et al. therefore, it is possible that the cells of infected patients exhibit less efficient dna repair mechanisms due to the effects of the viral proteins, which may contribute to the elevated frequency of dna damage detected in this study. au et al. (2010) reported that when the cells of exposed populations (in this case, hcv-infected patients or hbv-infected patients) are challenged with a dna-damaging agent in vitro, the in vivo exposure-induced repair deficiency is dramatically amplified. additionally, the deficiency will be detectable in a challenge assay as an increase in the number of chromosomal aberrations, mn or unrepaired dna strand breaks. it has been shown that b lymphocytes infected with hcv in vitro exhibit increased sensitivity to bleomycin due to the action of the ns3 protein and the core viral protein (machida et al. furthermore, it was demonstrated that lymphocytes from hbv or hcv-infected hcc patients exhibit an increased sensitivity to bleomycin and benzo(a)pyrene-diol-epoxide, which is associated with an increased risk of cancer development (wu et al. the results obtained in the present study showed that the lymphocytes of hbv-infected patients and hcv-infected patients are not more sensitive to dxr than the lymphocytes of non-infected subjects. however, a challenge assay with other substances, such as bleomycin, needs to be performed to allow more definite conclusions about mutagen sensitivity in these patients. in summary, the present study demonstrated that the lymphocytes of patients who are chronically infected with hbv or hcv exhibit greater chromosomal instability, characterised by the presence of mn, npbs and nbuds. although we did not observe a statistically significant result, a possible influence of cirrhosis on these parameters should be considered for further investigation .

in this study, we analysed the frequency of micronuclei (mn), nucleoplasmic bridges (npbs) and nuclear buds (nbuds) and evaluated mutagen-induced sensitivity in the lymphocytes of patients chronically infected with hepatitis b virus (hbv) or hepatitis c virus (hcv). in total, 49 patients with chronic viral hepatitis (28 hbv-infected and 21 hcv-infected patients) and 33 healthy, non-infected blood donor controls were investigated. the frequencies () of mn, npbs and nbuds in the controls were 4.41 2.15, 1.15 0.97 and 2.98 1.31, respectively. the frequencies of mn and npbs were significantly increased (p<0.0001) in the patient group (7.01 3.23 and 2.76 2.08, respectively) compared with the control group. when considered separately, the hbv-infected patients (7.18 3.57) and hcv-infected patients (3.27 2.40) each had greater numbers of mn than did the controls (p<0.0001). the hcv-infected patients displayed high numbers of npbs (2.09 1.33) and nbuds (4.38 3.28), but only the hbv-infected patients exhibited a significant difference (npbs= 3.27 2.40, p<0.0001 and nbuds=4.71 2.79, p=0.03) in comparison with the controls. similar results were obtained for males, but not for females, when all patients or the hbv-infected group was compared with the controls. the lymphocytes of the infected patients did not exhibit sensitivity to mutagen in comparison with the lymphocytes of the controls (p=0.06). these results showed that the lymphocytes of patients who were chronically infected with hbv or hcv presented greater chromosomal instability.

PMC4005534

pubmed-1231

development of the central nervous system (cns) is a complex process, which requires integration of many cellular processes including neural stem cell proliferation, migration, and neuronal differentiation. the major neuronal migration occurs in humans between the 12th and the 24th weeks of gestation and results in the formation of the cortical plate. however, the cortex continues to develop, and late migrations from the germinal matrix into the cerebral cortex continue until five months postnatally. classification of neuronal migration disorders is based on morphological criteria and includes schizenccphaly, porencephaly, lissencephaly, argyria, macrogyria, pachygyria, microgyria, and micropolygyria. pathogenesis of these malformations is multifactorial and includes genetic factors and environmental agents. in recent years few epidemiological studies have addressed the impact of prenatal exposures, although several maternal factors such as use of ethanol or drugs, viral infections, maternal diabetes, and untreated phenylketonuria can potentially influence the neuronal migration. the aim of this population-based study was to describe perinatal characteristics in infants diagnosed with neuronal migration disorders aiming at identifying potential risk factors. the infants included in the study were identified as those hospitalized with a diagnosis related to a neuronal migration abnormality and confirmed by a computer tomography (ct) or magnetic resonance imaging (mri). all swedish residents are assigned a unique 12-digit national registration number, which is used for official population-based registers. this number makes it possible to identify individuals and collect certain information within registers and also to link information between different registers. the inpatient register is held by the national board of health and welfare, and it records information concerning hospitalizations, including date of admission and discharge and primary and secondary diagnoses together with the national registration number. from 1987 and the swedish medical birth register was established in 1973 and includes data on more than 99% of all births in sweden. a standardized set of medical records is used by all antenatal care clinics and delivery units and at the examination of the newborn infant. selected information from the records is computerized and forwarded to the register, which is held by the national board of health and welfare. for all births, medical information on maternal demographic data, the reproductive history, maternal smoking habits, the registration starts at the first visit to the antenatal clinic and is completed when the mother and newborn infant are discharged from the hospital. the records, which include copies of referrals for radiologic examinations, were used to identify the children who had been examined with ct or mri and to validate the diagnosis of a neuronal migration disorder. information concerning factors that might influence neuronal migration disorders was obtained through the swedish medical birth register. for the mothers we obtained information about age, body mass index at first antenatal visit (bmi, calculated as maternal weight in kilograms at first visit to the antenatal clinic/maternal height times height), chronic diseases prior to index pregnancy, reproductive history (parity, years of infertility), and smoking in early pregnancy. bmi was classified according to who definition as underweight<18.5 kg/m, normal weight 18.5 kg/m and<25 kg/m, overweight 25 kg/m and<30 kg/m, and obesity 30 kg/m. smoking was classified as no smoking, smoking 19 cigarettes/day, and>10 cigarettes per day. we also obtained information on maternal diseases during pregnancy and mode of delivery (noninstrumental and instrumental vaginal delivery and emergency and elective cesarean section). as chronic diseases we included kidney failure, celiac disease, epilepsy, ulcerative colitis, diabetes mellitus, asthma, and systemic lupus erythematosus. the information was retrieved by the international classification of diseases as icd-9 or icd-10 codes, and all icd-9 codes were manually transformed into icd-10 codes. for the infants we obtained information on sex, gestational age at birth, birth weight and birth length, and the apgar scores at one and five minutes. small for gestational age (sga) was defined as birth weight more than 2 standard deviations (sds) below the mean birth weight for gestational age and sex according to a swedish birth weight curve. appropriate for gestational age (aga) was defined as a birth weight between 2 sd and+2 sd and large for gestational age (lga) as a birth weight more than 2 sd above the mean. all infants born alive from 1980 through 1999 and diagnosed with a neuronal migration disorder according to mri or ct scan sometime during this period were eligible for inclusion in the study. a total of 820 infants had been hospitalized with a diagnosis such as congenital myopathies and malformations of the cns that could be related to a neuronal migration disorder (icd 9: 359.1-359.24, 742.1-24, 759.23, and icd 10: q 040.0-89.9, g 71.2). we requested information on performed ct or mri scans by a letter sent to all paediatric and radiological clinics in the country, and about half of the 47 clinics responded to our request. in 120 of the patients, the diagnosis of neuronal migration was confirmed by the local radiologist in 17 subjects, 9 boys and 8 girls, all of whom were included in the study. as the study was purely descriptive, no formal testing was possible. for certain characteristics (maternal age, bmi, gestational age at birth, and birth weight), the mother's median age was 29 years, and most women were born in sweden (table 1). one of the mothers was underweight with a bmi below 19 at the start of pregnancy, and none was obese. the mean bmi of the mothers at first antenatal visit was 21.3 (range 17.628.5). one woman suffered from chronic multifocal osteomyelitis and necrosis and another was involved in a motorbike accident. maternal care for (suspected) damage to the fetus by radiation was reported in one case. nine male infants and eight female infants were born after mean 39.3 gestational weeks and with a mean birth weight of 3327 grams. all children were born as singletons and were aga, and all had a full apgar score. most infants were classified to have an undefined migrational disorder, a disturbed migration of the immature brain or a migrational disorder of unknown type. congenital malformations of the spleen or the heart, potter's syndrome, and facial asymmetry were reported, including malformation of the eye and lens, microphthalmus, and cleft palate. two infants had skin problems at birth, and one infant suffered from obstruction of the intestine by gallstones or meconium (table 2). all infants were born at term and of ordinary birth weight to healthy mothers with normal or subnormal bmi and after normal pregnancies. most of the infants had a concomitant malformation, and two infants were diagnosed with congenital rubella infections. the use of modern techniques of brain imaging, especially mri, has dramatically improved the ability to detect these neurological disorders while the child is still alive. over 25 syndromes related to disturbances of neuronal migration. the incidence of disturbances of the neuronal migration is not known, but is increasing in the pediatric population probably due to the use of mri techniques for brain imaging. epidemiological studies and experimental data have demonstrated the importance of nutritional factors in fetal brain development and the deleterious effects of nutritional deficits [911]. bmi was recorded for 12 of the women in the study, and more than half of them had subnormal or near subnormal bmi at start of pregnancy. since 1992 maternal bmi has been recorded in sweden at the first antenatal visit, and an increase from 23 to 24 in mean bmi was noted between 1992 and 2002. one can speculate whether the generally lower bmi among these 12 mothers of infants with neuronal migration disorder, as compared to the general pregnant population might have influenced the fetal brain development. however, it was not possible to calculate the adjusted association between maternal bmi and risk of neuronal migrations disorder in this small descriptive study. the impact of maternal bmi at start of pregnancy and/or weight gain during pregnancy on the fetal brain development and neuronal migration needs to be evaluated in a larger study. it is well known that maternal diseases during pregnancy, such as diabetes and nontreated phenylketonuria, and conditions where the placental blood flow is reduced, such as preeclampsia, could interfere with the fetal brain development [1, 9, 13, 14]. even a less severe maternal hypothyroidism during the first half of gestation might affect the fetal neurodevelopment and neuronal migration. few studies have addressed the impact on maternal health on specific migration disorders. in our study however, conditions related to an abnormal pregnancy and conditions which all can cause suboptimal growth conditions for the fetus were reported in four women. neuronal migration disorders have been described in humans and/or in animal models following in utero exposures to infections. some viruses, such as herpes simples, cytomegalovirus (cmv), or hiv, are potentially capable to persist in a latent form within the central nervous system. several studies have described the teratogenic effect of cmv infection and the relation to neuronal migration disorders [1618]. adverse impacts of maternal infections due to rubella, group b streptococcus, cytomegalovirus, toxoplasmosis, and chorioamnionitis in the fetal brain are known. however, two children were reported with rubella syndrome at birth, which is more than expected. the percentage of susceptible pregnant women with rubella was gradually reduced from 12% in 1975 to just below 2% in 1994. rubella syndrome is very uncommon, and between 1975 and 1985, only a mean of two cases per year were recorded in sweden. since 1985, no child with the rubella syndrome has been registered. it can be speculated that rubella had an impact on the neuronal migration in two cases in this study. it is well known that maternal smoking increases the risk of intrauterine growth restriction and affects the fetal brain by inducing intrauterine hypoxia or by acting directly on the developing brain [2123]. smoking habits among women have changed over time in sweden. during the 1960s as many as 25% of the women smoked, this was reduced to 14% in 2004. in this study 23% of the women smoked, which was more than in the general pregnant population during this time period. the detection of cases was difficult mainly because of our strict criteria for eligibility, which included a ct or mri scan confirming the diagnosis. in 120 of all hospitalized children, a ct or mri scan of the brain was preformed, and only 17 of them were confirmed with a neuronal migration disorder. despite several reminders, only 50% of all hospitals in sweden responded to our request to send in ct and mri scans. none of the hospitals actively rejected participation in the study. the cause of this dropout is not known but was evenly distributed between level i, ii, and iii hospitals over the country. it can be speculated that journals from the study period were not easily found, and the time for searching these journals was regarded as too expensive for the hospitals. the study refers to a time of great medical advances, and the use of ct and mri scan is more widely used today. the cases were identified through the national patient register, and the responding hospitals were evenly spread over the country. considering the increasing use of ct and mri scans, a study performed in more recent years might detect more children with neuronal migration disorder. this descriptive study indicates that there might be an impact of low or subnormal maternal bmi before and during pregnancy, maternal infection, such as rubella, and maternal smoking on fetal brain development, including neuronal migration. the roles of maternal bmi and congenital infections should be tested in future analytical studies.

the development of the central nervous system is complex and includes dorsal and ventral induction, neuronal proliferation, and neuronal migration, organization, and myelination. migration occurs in humans in early fetal life. pathogenesis of malformations of the central nervous system includes both genetic and environmental factors. few epidemiological studies have addressed the impact of prenatal exposures. all infants born alive and included in the swedish medical birth register 19801999 were included in the study. by linkage to the patient register, 820 children with a diagnosis related to a neuronal migration abnormality were identified. through copies of referrals for computer tomography or magnetic resonance imaging of the brain, the diagnosis was confirmed in 17 children. median age of the mothers was 29 years. at the start of pregnancy, four out of 17 women smoked. almost half of the women had a body mass index that is low or in the lower range of average. all infants were born at term with normal birth weights. thirteen infants had one or more concomitant diseases or malformations. two infants were born with rubella syndrome. the impact of low maternal body mass index and congenital infections on neuronal migration disorders in infants should be addressed in future studies.

PMC3324140

pubmed-1232

more than 25 years after the identification of the causative agent of aids, hiv/aids is still a major challenge to society. the latest who/unaids report (2010) states that the number of people living with hiv has risen to 33.3 million, with more than 2.6 million new cases annually and almost 5000 aids-related deaths per day. with the introduction of the first hiv-1 protease inhibitor (pi) (saquinavir) in 1995 and the development of highly active antiviral therapy (haart) the clinical outcome of hiv/aids changed from a lethal to a manageable, but chronic, disease in the developed world. the early pis suffered from poor pharmacokinetic profiles and caused severe side effects such as hepatic toxicity and lipodystrophy. for these reasons and with a frequent daily dosing regimen, they were not the first-hand choice in haart. the most common combinations in early haart were instead two nucleoside reverse transcriptase inhibitors (nrtis) together with a non-nucleoside reverse transcriptase inhibitor (nnrti). the development of nnrti- and/or nrti-resistant hiv strains and the introduction of new pis, with a once-daily dose regime and improved effect profiles, have made the combination of a pi together with two nrtis a more frequent choice for first line treatment in haart. although saquinavir has, to date, been followed by eight other pis (ritonavir, indinavir, fosamprenavir, nelfinavir, lopinavir, atazanavir, tipranavir, and darunavir), improving pharmacokinetic properties and reducing adverse effects are still issues that need to be addressed. further, the rapid replication and the high mutation rate of the hiv-1 virus, together with the mutation pressure induced by today s pharmacotherapies, will lead to an increase in the problems associated with resistant virus strains. thus, we can not expect the good results currently seen with haart to continue if new drugs are not developed and introduced onto the market. we have been engaged in the development of novel hiv-1 pis since 1997. in our most recent program we developed novel classes of potent hiv-1 pis incorporating a shielded tertiary alcohol as part of the transition state mimic. inspired by the structure of the potent inhibitor atazanavir (atz) (figure 1), we used a similar hydrazide moiety in the prime side of our new tert-hydroxy-containing pis. by altering the length of the central backbone, using a one-, two-, or three-carbon spacer (figure 1, series a, b, and c, respectively), we focused on optimizing the interaction with the catalytically active aspartic acid residues of the enzyme. class b, with the two-carbon spacer, yielded the best results, with values of ki and ec50 as low as 1 and 3 nm, respectively. in all three series (a c), inhibitors with high membrane permeability were identified, as well as inhibitors with good metabolic stability, providing pharmacokinetic properties well in the range of hiv pis already on the market, e.g., atz. c cocrystallized with the enzyme revealed binding modes that were not completely successful in establishing strong symmetric hydrogen bonds (< 3.0) with both the catalytic residues asp25 and or asp125, originating from each monomer of the hiv-1 protease. therefore, we decided to further elaborate the central transition-state mimic by relocating the hydroxyl group one position away from the backbone. this strategy was implemented by making use of a -hydroxy -lactam moiety equipped with a secondary alcohol. it was hypothesized that the -hydroxy -lactam would provide a better hydrogen bond arrangement for the catalytic asp residues and at the same time reduce the flexibility, providing a more rigid inhibitor. spacers are indicated in red: a, one-carbon spacer (ki=5.5 nm);b, two-carbon spacer (ki=2.3 nm);c, three-carbon spacer (ki=2.8 nm);d, novel lactam-based inhibitors with two-carbon spacer (ki=0.8 nm) and altered stereocenters indicated by asterisks; e, three-carbon spacer (ki=4.2 nm). atz is included for comparison (ki=2.7 nm). modeling studies supported the hypothesis that a hydroxyl group in the 4-position of the -lactam might provide a new conformationally constrained transition-state-mimicking scaffold for the development of novel hiv-1 pis. since both the (3r,4s) and the (3r,4r) stereoisomers provided good docking poses, we decided to synthesize and evaluate all four stereoisomers of the -lactam (figure 1, d). in addition, two different lengths of the central tether (two or three carbons) were investigated (figure 1, d and e). the prime-side hydrazide moiety, inspired by atz, has been successfully used in inhibitors in series a c and was therefore retained in the new series of lactam-based inhibitors. here we present the synthetic protocols and the inhibitory potency on enzyme level, as well as the activity in a cell-based assay, of the new inhibitors (d and e). also included are stability and permeability studies of selected compounds, together with x-ray analyses of three of the inhibitors cocrystallized with the hiv-1 protease. starting from (s)-4-hydroxydihydrofuran-2(3h)-one (1a) or (r)-4-hydroxydihydrofuran-2(3h)-one (1b), four hiv-1 pr inhibitors with a two-carbon spacer and with varied stereochemistry in the lactam ring were synthesized (scheme 1). encouraged by previously reported alkylations,1a and 1b were chosen as starting substrates for the two-step alkylation process. upon treatment with dmpu, lda, and the first alkylating agent (allyl bromide or benzyl bromide) at 50 c followed by a second portion of lda and the addition of the second alkylating agent (benzyl bromide or allyl bromide) at 40 c, the dialkylated -hydroxy -lactams 2a d were synthesized in isolated yields of 249% (scheme 1, paths a and b). path a: (a) dmpu, lda, allyl bromide, dry thf, added at 50 c, stirred at 50 c for 1 h; (b) lda, benzyl bromide added at 40 c, stirred at 30 c for 1 h, giving 2a and 2c in 49% and 33% isolated yield, respectively. path b: (c) dmpu, lda, benzyl bromide, dry thf, added at 50 c, stirred at 40 c for 1 h; (d) lda, allyl bromide, added at 40 c, stirred at 30 c for 1 h, giving 2b and 2d in 2% and 5% isolated yield, respectively. in the first alkylation, the allyl group in 2a and 2c (or the benzyl group in 2b and 2d) was introduced trans to the controlling 4-hydroxyl group as expected, showing facial selectivity, as previously reported by meyers et al. and others. in the second alkylation, the benzyl group (or the allyl group in 2b and 2d) was introduced trans to the 4-hydroxyl functionality. consequently, the second alkylation changed the stereochemistry of the first inserted group, forcing it to end up cis to the 4-hydroxyl group. to be able to collect enough material of 2b and 2d, with their low-yielding synthetic pathway, a method was developed to alter the stereochemistry at the hydroxyl group in 2a and 2c. martin reagent to the corresponding ketones was followed by reduction using nabh4, affording 2d and 2b following paths a and b, respectively (scheme 2), with ratios 2d/2a of 5.7:1 and 2b/2c of 5.9:1. martin, dcm, rt, 1 h; (b) nabh4, 1% methanol in thf, rt 2 h, 2d+2a (5.9:1) 92%, 2b+2c (5.7:1) 85%. lactamization of the lactones 2a d with tbs-protected indanolamine (3) was performed by adopting the methodology developed by orrling et al. lactams 4a d were isolated in good yields using the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([ bmim]bf4) under microwave irradiation at 180 c for 35 min, followed by protection of the alcohol moiety with tbsotf under basic conditions. although the mixture was heated to 180 c, these lactamization conditions are relatively mild compared to those previously reported. the use of highly polar [bmim]bf4 allowed lactamization to proceed smoothly without the need of brnstedt acid. reagents and conditions: (i) [bmim]bf4, 180 c, 35 min; (ii) triethylamine, tbsotf, dcm, 025 c, overnight, giving isolated yields of 4a 64%, 4b 53%, 4c 72%, and 4d 50%. to synthesize the prime-side moiety 5a, hydrazone 7 was prepared in almost quantitative yield starting from the boc-protected hydrazine 6, as previously reported in the literature (scheme 4). benzylation of 7 using koh and 4-bromobenzyl bromide in anhydrous toluene afforded 8 in good yield. catalytic quantities of the phase-transfer catalyst tetrabutylammonium hydrogen sulfate (tbahs) were used to improve solubility and increase the rate of the reaction. after the initial workup of the alkylation reaction only compound 8 was generated, but after flash chromatography purification, compound 9 was also formed (owing to hydrolysis of the hydrazone). however, purification in this step was necessary to remove excess quantities of 4-bromobenzyl bromide, which was foreseen to cause problems in the later steps. the mixture of 8 and 9 was deprotected with 4 m hcl in thf to yield the pure hydrochloride salt of 10. owing to the photosensitivity of the free nitrogen in the p-bromobenzylhydrazine 10, the coupling of 10 with 11, synthesized as previously reported, was performed in a reaction vessel wrapped in aluminum foil. moreover, 10, 11, and hobt were added under a nitrogen atmosphere at 0 c, and the mixture was stirred for 30 min. subsequently, 4-methylmorpholine (nmm) and n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride (edc) were added and the reaction mixture was gradually heated to 25 c and stirred under a nitrogen atmosphere for 15 h, giving 5a in good isolated yield (77%, 61% overall isolated yield starting from 38 mmol of 6). reaction conditions: (a) acetone, mgso4, acoh (cat.), reflux, 1 h, 98%; (b) (i) koh, anhydrous toluene, tbahs, 50 c, 20 min; (ii) 3, 100 c, 2 h, 81%; (c) hcl, thf, reflux, 3 h, quantitative yield; (d) edci, hobt, nmm, dcm, 025 c, 15 h, 77% (61% isolated yield over four steps.). steps a and c required no purification. next the allylic double bonds in lactams 4a d were oxidatively cleaved to give the corresponding aldehydes v viii using osmium tetraoxide and sodium periodate in thf/water (3:1) at room temperature (scheme 5). note that the nomenclature for the absolute configuration for the lactam carbon in position 3 changes when comparing the lactams 4a d, the intermediates v viii, and 12 and 13 because of changes in the assigned priority according to the sequence rule. reagents and conditions: (a) 4a d, oso4, naio4, thf/h2o, rt, overnight; (b) 5a, acetic acid, na(oac)3bh, dry thf, rt, overnight, provided 12a in 35% and 12d in 54% isolated yield from 4a and 4d, respectively; (c) tbaf, thf, rt, overnight, provided 13a in 38%, 13b in 60%, 13c in 46%, 13d in 34% isolated yield from 4a d, respectively. reductive amination between the crude aldehydes and the prime side (5a) was performed in dry thf using acetic acid, followed by treatment with na(oac)3bh, to afford the crude tbs-protected products. the tbs protecting groups were removed using tbaf, and the inhibitors 13a d, carrying a two-carbon tether, were isolated in good yields (scheme 5). the tbs-protected inhibitors 12a and 12d (but not 12b and 12c) were isolated, purified, and fully characterized before the final deprotection. to evaluate the effect of different p1 side chains, a small series of p1 p-phenyl- and p-pyridyl-substituted inhibitors was produced. the known problem of rapid protodeboronation of 2-pyridylboronic acid prevented us from conducting functionalization of 12a and 12d directly via suzuki thus, to introduce the 2-pyridyl as a para-substituent in p1, the 2-pyridine-substituted hydrazide 5b (scheme 6) was synthesized starting from the 4-(2-pyridinyl)benzaldehyde, as previously described. the alcohols 14a and 14d were isolated as side products in reductive amination reactions to produce 12a and 12d, respectively. martin reagent was used to oxidize 14a and 14d to the corresponding aldehyde intermediates (scheme 5, v and viii, respectively), followed by reductive amination with 5b using acetic acid and na(oac)3bh in dry thf and subsequent tbaf-mediated deprotection to give useful yields of the inhibitors 13e and 13f (scheme 6). martin reagent, dry dcm, rt, 1 h; (b) acetic acid, na(oac)3bh, dry thf, rt, overnight; (c) tbaf, thf, rt, overnight, provided isolated yields of 63% 13e and 38% 13h from 14a and 14d, respectively. the tbs-protected inhibitors 12a and 12d were decorated using the corresponding phenyl- or pyridylboronic acids in suzuki miyaura cross-coupling in which herrmann s palladacycle (0.1 equiv) was used as a palladium precatalyst together with k2co3 (3.3 equiv) and [hp(t-bu)3]bf4 (0.2 equiv) in dme/water. the reaction mixtures were heated to 140 c for 20 min under focused microwave irradiation in sealed reaction vessels. cross-coupling was followed by deprotection of the hydroxyl groups using tbaf in thf at room temperature, giving inhibitors 13g j in good isolated yields (scheme 7 and table 2). reagents and conditions: (a) (i) 12a or 12d, herrmann s palladacycle, k2co3, 3- or 4-pyridylboronic acid, [hp(t-bu)3]bf4, dme, water, microwave 140 c, 20 min; (ii) tbaf, thf, rt, overnight, providing isolated yields of 63% 13 g, 59% 13h, 74% 13i, and 66% 13j. to be able to incorporate the new lactam scaffold into inhibitors with the three-carbon spacer, corresponding to the previously published c series (figure 1), the allylic compound 4a was refluxed in thf at 80 c with 9-bbn for 6 h. after addition of naoh, h2o2, and ethanol at room temperature and another 2 h stirring, the primary alcohol 16 was isolated in good yield (scheme 8). the alcohol 16 was oxidized to the corresponding aldehyde (17) using 50% so3py in dmso together with triethylamine in dcm at 025 c. the aldehyde was thereafter used in a reductive amination reaction with 5a using na(oac)3bh as reducing agent at 35 c to give 18 in moderate isolated yield (scheme 8). the nomenclature for the absolute configuration of the lactam carbon in position 4 changes when comparing the 13a j and 19a e series because of changes in the assigned priority according to the sequence rule in iupac s guidelines. reagents and conditions: (a) (i) 9-bbn, dry thf, 80 c, 6 h; (ii) 2 m naoh, 30% h2o2 in h2o, ethanol, rt, 2 h, 78%; (b) et3n, 50% so3py in dmso, dry dcm, 020 c, 3 h; (c) 5a, acetic acid, na(oac)3bh, dry thf, 35 c, 3 h, 35%; (d) tbaf, thf, rt, overnight, 19a 61%; (e) (i) herrmann s palladacycle, k2co3, arylboronic acid, [hp(t-bu)3]bf4, 105 c, 1.5 h; (ii) tbaf, thf, rt, overnight, 19b 45%, 19c 35%, and 19d 30%; (f) (i) 2-(tributylstannyl)pyridine, pd(pph3)2cl2, cuo, dmf, 105 c, 2 h; (ii) tbaf, thf, rt, overnight, 19e 16%. deprotection of 18 using tbaf in thf gave inhibitor 19a in a good yield. inhibitor 18 was also used as starting material in suzuki miyaura cross-coupling with phenyl- and pyridylboronic acids together with herrmann s palladacycle, k2co3, and [hp(tbu3)]bf4, heated by microwave irradiation to 105 c for 1.5 h. deprotection of the tbs groups using tbaf in thf gave 19b d in 3540% isolated yields. by use of 2-(tributylstannyl)pyridine, compound 18 was subjected to stille type coupling in dmf under microwave irradiation (105 c, 2 h) using cuo and with pd(pph3)2cl2 as precatalyst. the stille coupling was followed by tbaf-mediated deprotection giving inhibitor 19e in moderate isolated yield. since the preliminary docking studies suggested that two of the stereoisomers in the lactam moiety (( 3r,4s) and (3r,4r)), in the two-carbon-tethered inhibitors would fit well in the enzyme, all four stereoisomers were synthesized and evaluated regarding binding and in a cell-based assay, giving the results summarized in table 1. comparisons with previous series of tertiary-alcohol-based hiv-1 pis (a c) could easily be conducted by using the indanolamide in the p2 position and the p-bromophenyl as the p1 side chain. in accordance with the initial docking studies, inhibitors 13a (3r,4s) and 13d (3r,4r) exhibited good activity in the enzyme assay (ki of 2.1 and 6.4 nm, respectively) as well as in the cell-based evaluation (ec50 of 0.64 and 0.35 m, respectively). the stereoisomers 13b (3s,4s) and 13c (3s,4r) did not show any activity and, as expected, neither did the tbs-protected inhibitors 12a and 12d. the metabolic stability and permeability of the two active inhibitors were investigated. because of its low solubility, inhibitor 13a was not tested in the permeability assay. compound 13b exhibited slight cell toxic properties, with a cc50 of 15 m. the lactam scaffold inhibitors 13a and 13d (( 3r,4s) and (3r,4r), respectively) yielded the most potent inhibitors and were therefore selected for further optimization. when the p1 position is optimized by replacing the p-bromo substituent of the p1 phenyl group in 13a and 13d with heteroaromatic moieties, the inhibitors showed improved protease inhibitor potency and, most importantly, increased antiviral activity (table 2, 13e isolated yields in the final reductive amination (12a, 12d) or reductive amination atz papp(caco-2)=5.3 10 cm/s. for preparation of inhibitors, see schemes 6 and 7. isolated yields of 13e f from 14a or 14d (scheme 6) or in the coupling deprotection step of 13g j (scheme 7). atz clint=90 l min mg [140 l min mg]. atz papp(caco-2)=5.3 10 cm/s. the best inhibitors, having (3r,4s) configuration and 3- or 4-pyridylbenzyl as the p1 moiety (13 g and 13i), exhibited 10 times higher potency than 13a in the cell-based antiviral activity assay, the best ec50 values being 40 nm (table 2). the 2-pyridyl-substituted inhibitor (13e) showed lower activity than the 3- and 4-pyridyl-substituted analogues (13 g and 13j, respectively). the improved ec50 upon decorations with 2-, 3-, and 4-pyridyls has previously been demonstrated showing the same trend. the (3r,4r) compounds showed less improvements, but all inhibitors decorated with pyridine functionalized in p1 were observed to have higher potency than the precursor bromo compound 13d. the position of the nitrogen in the heteroaromatic p1 group showed the same general trend as in the (3r,4s) inhibitors, with the meta- and para-positions providing the best potency (table 2). heteroaromatic functionalization of p1 provided inhibitors with increased stability compared to 13a and 13d. compound 13h gave the best result (clint of 120 l min mg). both 13 g and 13h were observed to possess moderate permeability in the caco-2 studies, with papp of 3.8 10 and 5.1 10 cm/s, respectively. when the backbone spacer was elongated from two to three carbons, as in 19a e, inhibitors with lower potency than the 13 series were obtained (table 3). this is in accordance with results previously reported for the linear series of tertiary alcohol inhibitors, e.g., comparing the b(26) and c(27) series (figure 1). however, with the p-phenyl or p-4-pyridyl groups in the p1 position, submicromolar values of ec50 were observed in the antiviral cell based assay (19b and 19d). as mentioned above, permeability (caco-2) and stability (clint) studies were performed on some of the inhibitors prepared (13a, 13d, 13 g, 13h, 19a, 19c, and 19e). compound 19a showed high permeability (> 20 10 cm/s), while all other inhibitors investigated showed moderate permeability (( 320) 10 cm/s). the value of clint varied from 120 to>300 l min mg (tables 13). these results are in the same range as those previously reported for atz (papp=5.3 10 cm/s, clint=90 l min mg [140 l min mg]). there was no major difference between the 13 and the 19 series with respect to clint and papp, and the rigidification of the backbone seemed to be well tolerated compared to the linear inhibitors. the metabolic stability was improved when the bromo group in 13a and 19a was substituted by the heteroaromatic pyridyls, although the permeability was unfortunately reduced at the same time. for conditions, isolated yields of 19a for the deprotection step, and isolated yields of 19b e for the coupling deprotection step. atz clint=90 l min mg [140 l min mg]. a drug-resistant strain of the hiv-1 protease (leu63pro, val82thr, ile84val) was cocrystallized with the active pis 13i, 19b, and 19d for x-ray crystallographic studies of the complexes. data were obtained for all complexes, and the structures were refined to high resolution (for refinement statistics, see supporting information). the resulting electron density maps allowed unambiguous modeling of the inhibitors within the binding site. previously published structures of hiv-1 pis 20,21, and atz are included for comparison (figure 3). comparison of the overall x-ray conformations and binding patterns of compounds 13i (top left, pdb code 2uxz), 19b (bottom left, pdb code 4a6c), and 19d (bottom right, pdb code 4a6b) in the active site of hiv-1 protease. compound 13i forms five direct hydrogen bonds to the protease and five more via water molecules. the corresponding binding interactions for 19b and 19d are four direct bonds and six more through water bridges. in all three complexes, two of the interactions via water are due to the structural water coordinating ile50 and ile150 in the protein flaps. a complicating factor for the comparisons of the inhibitor complexes was the fact that compounds 20 and atz were rotated 180 compared to compounds 13i, 19b, 19d, and 21. the overall binding configurations for 13i, 19b, and 19d to the protease are, as expected, in good accordance with those of previously published linear inhibitors 20,21, as well as with atz, despite the novel -hydroxy -lactam moiety. previously published pis for comparison: atz (pdb code 3el9), 20 (pdb code 2uxz), and 21 (pdb code 2xye). on the basis of the modeling studies, it was postulated that the -hydroxyl group of the lactam moieties forms hydrogen bonds with the catalytic aspartic acids (asp25 and asp125). in the two-carbon linker compound 13i this -hydroxy group forms hydrogen bond interactions to the two catalytic aspartic acids with 2.7 and 3.0. the -hydroxy group in the three-carbon inhibitors 19b and 19d only form hydrogen bonds to asp25, with 2.7 and 2.6, respectively (figure 2). this loss of a hydrogen bond for the 19 series compounds is due to the different spatial conformation of the lactam ring, apparently as a result of the longer central backbone (figure 4). as the only difference between the structures of 13i and 19d is the length of the backbone tether, this is a likely explanation of the lower antiviral potency of compound 19d compared with 13i. the position of the -hydroxy group of the lactame ring, involved in hydrogen binding to both asp25 and asp125 in 13i (gold), is different in 19d (purple) and 19b (not shown) exhibiting the three-carbon linker. this leads to a loss of a hydrogen bond to one of the catalytic aspartates. the position of the -hydroxy group involved in hydrogen binding in 19d is 2.1 from the position observed in 13i. none of the cocrystallized pis in these novel series formed a symmetrical binding pattern with the catalytic aspartic acids (asp25 and asp125) such as that seen in atz. together with the hydrazide carbonyl oxygen, the carbonyl oxygen in the lactam ring in both 13i and 19d creates hydrogen bonds to the structural water bridging the inhibitors and the ile50 and ile150 in the flap region with hydrogen bond lengths of 2.73.3 (figure 2). the position of the p2p3 indanolamide in 13i, 19b, and 19d is not markedly affected by the introduction of the -hydroxy -lactam, absent in 20. while in 20 the indanolhydroxyl group was close enough to form a hydrogen bond to arg108 and for the arg108 to make an edge-on cation interaction with the p1 phenyl group, the distance to the indanol group in 13i seems to prevent this bond from forming (figure 5). in accordance with the previously observed results for 21, the p1 outer phenyl group in both 13i and 19d interacts through a hydrophobic interaction with pro81 (3.33.8) and an edge face interaction to phe153 (3.73.8). the differences in length of the central motif as well as in the length of the extension of 13i, 19d, 20, 21, and atz in the p1 site are nicely accommodated through corresponding shifts in the positions of phe153 and pro81 (figure 5). these interactions are likely to improve the binding constant and is the most likely explanation of the better binding of compound 13i than 13d, differing only in the length of the extension in the p1 site. in a previously examined complex with compound 20, the interaction with phe153 was not possible, as the corresponding moiety only reached far enough for a van der waal interaction with pro81. neither is the interaction with phe153 observed in the complex with atz. since the binding modes of 19b and 19d are very similar (figure 2), only 19d was included in the analysis, as the structure of the complex could be interpreted at higher resolution. comparison of the positioning of the cocrystallized inhibitors in the s2s3 pocket and interaction with pro81 and phe153 in the s1 pocket. the effect on the s2s3 site is visualized at residues asp29, asp30, arg108, and pro181. (a) superimposition of 13i (gold) and 19d (purple). as a result of an additional ch2 group in 19d, the lactam group present in the new series of compounds as in 19d mimics the conformation of 20, also exhibiting the three-carbon linker, very well. with the lactam ring present, the position of the indanol ring, and therefore also asp30/130, is more similar to the situation in 13i comprising the two-carbon linker. (c) superimposition of 13i and atz (black). despite the differences of functional elements between 13i and atz in the s2s3 site, the common ribbon of the compounds overlap well. in the p1 site 13i and 19d compound 19d induced side chain and main chain atom displacements in phe153 and pro81 up to 2.5 and 1.7, respectively, compared to atz complex positions. none of the new compounds induced a shift in the position of arg108, as was seen in compound 20. the introduction of the -hydroxy -lactams as new scaffolds was intended to provide more rigid pis and to relocate the hydroxyl group from the backbone to enable more symmetric binding to the catalytically active asp25 and asp125 of the hiv-1 protease. the outcome of the dialkylation reactions performed to obtain 2a d was in accordance with the results described by amat et al. in 2007, although they observed a larger substrate-dependent variability. when introducing the benzyl moiety in the first alkylation, as in the cases of 2b and 2d, the yields were lower (2% and 5%, respectively) than when the allyl group was introduced before the benzyl moiety (as in 2a and 2c, with yields of 49% and 33%, respectively). the same trend has been reported by johnson et al. with 4-substitued lactams but was not observed in the 5-substitued examples presented by meyers et al., in which the order of addition did not affect the yields. probable reasons for the lower yields observed by johnson et al. were steric and/or electrostatic interactions between the 4-hydroxy group and the bulkier 3-benzyl moiety present after the first alkylation, compared to the smaller allyl group. these findings followed the reasoning presented by huang et al., who proposed stereoelectronic factors to be the major explanation in this class of stereoselective two-step alkylation reactions. in the present work, the diastereoselectivity controlled by the stereochemistry of the 4-hydroxy group was strong enough to allow highly enantiomerically enriched isomers to be obtained in all cases. there was an urgent need for a robust method for the synthesis of the prime side hydrazide moiety (5a). the procedures used previously were cumbersome and low yielding because of the use of toxic and environmentally hazardous hydrazine hydrate and/or tedious purification protocols. previously used synthetic procedures were not satisfactory, since the quantities of prime side were not sufficient to support our lead optimization program throughout. the synthetic route to the prime side hydrazide moiety 5a presented here provided an efficient way of producing sufficient amounts and constitutes an improvement in yield as well as a reduction in work compared to previous methods. with this convenient method, there was no need to use hazardous hydrazine hydrate, and the purification protocol resulted in a good yield. the biological results obtained from the novel lactam-containing inhibitors are summarized in tables 13. evaluation of the four stereoisomers (13a d) gave two active and two nonactive pis (table 1). the (3r,4r) and (3r,4s) stereoisomers in the lactam ring showed the best results, with 13a and 13d being the most potent compounds (ki<10 nm and ec50<1 m). the most important structure activity feature appears to be the direction of the benzyl in the p1 position. with r-stereochemistry at the -carbon (13a and 13d), the direction of the -hydroxy substituent (position 4) appears to be of less importance for inhibition with 13a and 13d being almost equipotent. with s-stereochemistry at the -carbon, 13b and 13c showed almost no inhibiting effect on the enzyme or in the cell-based antiviral activity assay (table 1) and, as expected, the tbs-protected inhibitors 12a and 12d did not show any inhibitory potency. when the p1 side chain was decorated with heteroaromatic moieties (table 2), at best a 10-fold improvement in inhibitory potency was observed (13 g and 13i, ec50=0.04 m). compared to the 2-pyridyl inhibitor 13e (ec50=0.190 m), the 3- and 4-pyridyl-substituted inhibitors (13 g and 13i, respectively) with (3r,4s) stereochemistry afforded 5 times higher potency, with ec50 of 40 nm. despite the fact that atz contains a 2-pyridinyl in position p1, our previous series with one- or three-carbon spacers showed better potency for the 3- and 4-pyridinyl-substituted inhibitors. with the linear two-carbon spacer the 2-, 3-, and 4-pyridinyls gave equipotent inhibitors. this result was also obtained with the lactam-containing inhibitors with the three-carbon extended pis in the 19 series. comparing p-bromide functionalized inhibitors 13a and 19a, a 5-fold loss of potency within measured ki and ec50 values were observed. however, the same trend is present in both series (13 and 19, table 3) as seen with the shorter inhibitors. the pyridyls (19c e) showed slightly better inhibition compared to the p-bromo compound 19a. the p-phenyl substituted 19b was among the most potent inhibitors, concurring with recent reports. we have successfully introduced -hydroxy -lactams providing a rigid backbone moiety and replaced the previously used tert-hydroxy group with a sec-hydroxy group. in addition, the length of the central spacer was varied (two or three carbons). functionalization of the two most potent stereoisomers (3r,4s) (13a) and (3r,4r) (13d) with heteroaromatic moieties in the p-benzyl p1 position improved the potency, rendering ki values down to 0.7 nm and ec50 values down to 0.04 m. three inhibitors were cocrystallized with the hiv-1 protease enzyme providing information about the binding of the hydroxy lactams to the enzyme. the change in binding pattern between the inhibitors with two- and three-carbon spacers was in good agreement with the observed variation in enzyme binding activity.

in an effort to identify a new class of druglike hiv-1 protease inhibitors, four different stereopure -hydroxy -lactam-containing inhibitors have been synthesized, biologically evaluated, and cocrystallized. the impact of the tether length of the central spacer (two or three carbons) was also investigated. a compound with a shorter tether and (3r,4s) absolute configuration exhibited high activity with a ki of 2.1 nm and an ec50 of 0.64 m. further optimization by decoration of the p1 side chain furnished an even more potent hiv-1 protease inhibitor (ki=0.8 nm, ec50=0.04 m). according to x-ray analysis, the new class of inhibitors did not fully succeed in forming two symmetric hydrogen bonds to the catalytic aspartates. the crystal structures of the complexes further explain the difference in potency between the shorter inhibitors (two-carbon spacer) and the longer inhibitors (three-carbon spacer).

PMC3310203

pubmed-1233

haploid saccharomyces cerevisiae yeast cells use a prototypic, g-protein coupled-receptor/map kinase cascade signaling system, the pheromone response system 1, to sense and transmit information about the concentration of mating pheromone secreted by cells of the opposite mating type (fig. 1). the more information about pheromone concentration the system can transmit, the better a cell can distinguish between different pheromone concentrations, an essential ability for proper partner choice and mating. for example, a yeast cell ringed by potential mating partners strongly prefers to mate with partners producing the most pheromone 2. first, a cell grows up the pheromone concentration gradient 3, a process that likely depends on measurement of precise differences in pheromone concentration at different points on the cell surface. second, after contacting its partner and forming a prezygote, a cell preferentially completes fusion and forms a diploid with a partner that produces high amounts of pheromone 4. these experiments indicate it is important for cells to distinguish among different pheromone concentrations at multiple steps during the mating process. prior work suggested that optimal transmission of information about pheromone concentration depends on both distinguishable receptor occupancies and distinguishable downstream system responses. differences in receptor occupancy are clearly important for mating partner choice and discrimination; for example, in the presence of exogenous pheromone at a concentration that saturates the receptor, cells lose the ability to discriminate high pheromone-secreting partners from low pheromone-secreting partners 2. however, distinguishable receptor occupancies are not sufficient for partner discrimination, since hypersensitive cells, in the presence of exogenous pheromone at a concentration that does not saturate the receptor but does saturate downstream responses, also lose the ability to discriminate between partners secreting different levels of pheromone 2. in complementary studies of orientation of mating projections in spatial gradients of pheromone, segall 3 showed that hypersensitive cells did not orient their mating projections as precisely as wild-type cells and suggested that this might result from saturation of downstream responses at most points in the gradient. however, after reducing the gradient pheromone concentrations 100 fold to concentrations at which downstream responses are not predicted to be saturated, hypersensitive cells oriented their mating projections less precisely than wild-type cells orient in gradients of higher pheromone concentrations 3. these observations suggest that hypersensitive cells are inherently less able to respond distinguishably to different pheromone concentrations (i.e., transmit less information about pheromone concentration), even when they are responding to pheromone concentrations that saturate neither receptor nor downstream responses. one characteristic of wild-type cells that we 5 and others 6 have previously found is that, despite the large number of intermediate signaling events in the system, the dose-response curve of receptor occupancy closely aligns with dose-responses curves of downstream system responses. (here called dora) between receptor occupancy and the amount of pheromone-activated ste12 (pathway subsystem output p, which is reporter gene expression corrected for inherent cell-to-cell differences in the ability to express proteins 5) (fig. interestingly, dose-response alignment is commonly observed in many mammalian cell signaling systems, including the insulin 7, acetylcholine 8, thyroid stimulating hormone 9, angiotensin ii 10, and epidermal growth factor 11,12 response systems. researchers in the past have often regarded alignment of curves for ligand binding by a candidate receptor and downstream response as evidence that the putative receptor was in fact the molecule that bound ligand and caused the cellular responses1315. however, to our knowledge, researchers have investigated neither the implications of dose-response alignment for yeast pheromone response nor its general consequences for the function of cell signaling systems. first, dora describes a linear relationship between receptor occupancy and downstream response; consequently, the entire range of receptor occupancies evenly corresponds to the entire range of possible responses (fig. by contrast, even a modest dose-response misalignment, such as a 20-fold shift in the ec50 of downstream response (fig. 2c), compresses the downstream responses corresponding to a wide range of receptor occupancies into a narrow range (fig. previous analysis of noise propagation in a synthetic gene circuit revealed analogous amplification of upstream noise in a system with misaligned dose-responses16. this reasoning suggested to us that cell signaling systems with misaligned dose-responses inherently transmit information with lower fidelity, even if downstream responses are not saturated, an idea consistent segall s observations that hypersensitive cells oriented mating projections less precisely in gradients than wild-type cells even at concentrations that did not saturate downstream responses 3. we hypothesized that dose-response alignment might indicate a system that can transmit large amounts of information, and therefore we sought to better understand the underlying molecular mechanisms required for dora and the linear relationship between upstream and downstream response that it defines. proportional negative feedback in electrical circuits, where a constant fraction of the output is subtracted from the input, can bring about a linear input-output voltage relationship17. biologists have also shown that negative feedback can make input-output relationships more linear in biological systems; for example, bhalla et al. showed that, in a mapk/pkc-mediated signaling system, increasing the amount of a mapk-activated phosphatase that inactivates the mapk made the average output response more linearly related to (i.e., proportional to) the input18. research in both biology and engineering 17,1922 has also suggested or shown that negative feedback can increase the signal-to-noise ratio in system output and decrease the sensitivity of output to variation in properties of system components (see supplementary information 8 for further discussion). these observations suggested to us that negative feedback might mediate dose-response alignment and improve information transmission in the yeast pheromone response system. prior work showed that the pheromone response system quickly establishes dose-response alignment; the accumulation of reporter gene expression in cells increased linearly from 15 minutes to three hours after stimulation (see fig. 2 in 5), and at all times the normalized dose-response of downstream output aligned with the receptor-ligand binding curve. these facts suggested that the molecular mechanisms that bring about and stabilize dora occurs in the first 15 minutes of pheromone stimulation. however, no quantitative measurements of system activities in this time frame existed to indicate the action of negative feedback to align dose-responses. we therefore developed tools to measure the early dynamics of molecular events that the system uses to operate before, during, and after establishment of dora. we developed reporters and methods to measure real-time signal transmission in single cells, at the membrane, and in the nucleus, and supplemented these data with biochemical measurements. we then measured system outputs (i.e., system activities at different stages in the signaling pathway, see fig. 1) after stimulating cells with 100 nm pheromone, a concentration that produces maximal downstream transcription reporter response (fig. two membrane-proximal system outputs, g-protein activation and ste5 recruitment to the membrane, peaked and declined rapidly. to follow g-protein activation in single cells over time, we measured loss of fluorescence resonance energy transfer (fret) between cyan fluorescent protein (cfp)-tagged gpa1 and yellow fluorescent protein (yfp)-tagged ste18 by image cytometry 23 (supplementary information 2) in a derivative of a strain developed by yi and coworkers6. loss of g-protein fret rapidly peaked in the first minute and declined (fig. s2), consistent with lower time resolution, single time point population measurements in an earlier study 6. we then measured, also in single cells and at sub-minute intervals, a subsequent membrane-proximal signaling event, the recruitment of ste5 to the membrane. to do this, we measured the redistribution of yfp-ste5 from the nucleus and cytosol to the membrane (supplementary information 2). membrane recruitment of ste5 was rapid (fig 3b). within 5 seconds of stimulation with high pheromone, individual cells showed an increase in yellow fluorescence at the cell membrane, and a corresponding depletion of fluorescence from the cell interior; no change in fluorescence was observed in unstimulated cells or cells with unlabeled ste5 (fig. average membrane recruitment reached near-maximal values within seconds and peaked by 20 seconds, before declining toward a plateau in later minutes, similar to the dynamics of g-protein loss of fret. we then assessed intermediate system output further downstream by measuring the dynamics of mapk activation. using quantitative immunoblotting, we measured phosphorylation of fus3 residues thr180 and tyr182, which is required for fus3 activity and pheromone response 24. the amount of phosphorylated fus3 relative to total fus3 increased rapidly, reaching a maximum in 2.5 min before dropping to a plateau level in approximately 57 min (fig. 3c and fig. we then measured nuclear mapk activity dynamics in single cells over time. to do this, we developed a fret reporter to measure pheromone-induced changes in the association between the transcription factor ste12 and one of its inhibitors dig1 25. we deleted native ste12 and dig1 genes and chromosomally integrated versions of these proteins fused to cfp and yfp, respectively (see supplementary information 5). we then measured changes in fret between cfp and yfp 26 in the nucleus by image cytometry 23 (see fig. s7b), but did require both ste5 (fig s7c) and map kinase activity (fig s7d), consistent with the interpretation that loss of fret directly reported pheromone-induced, map kinase-mediated derepression of ste12. 3d), and the overall signal dynamics were very similar to those of fus3 phosphorylation (compare fig. this fast signal transfer from fus3 activation to ste12 derepression is consistent with the idea that fus3 moves quickly in and out of the nucleus, as shown in studies of changes in fus3 localization by fluorescence recovery after photobleaching (frap)27. we confirmed that the timing of ste12 de-repression measured by loss of ste12-dig1 fret was consistent with the dynamics of pheromone-induced mrna transcription. using ribonuclease protection assays mrna levels peaked at 5 minutes following pheromone stimulation before declining (fig. 3e and fig. the maximum rate of increase in mrna occurred between 3 and 5 minutes, consistent with the time of maximum loss of ste12-dig1 fret. all measurements of signal-relaying events showed a consistent pattern of rapid peak-and-decline toward a plateau after pheromone stimulation (fig. 3f), which suggested the action of one or more fast-acting negative feedbacks that might modulate the dose-dependence of the signal to achieve dora. a number of previous works suggested that the mapks fus3 and kss1 might mediate rapid negative feedback. our previous study of regulated cell-to-cell variation in system output revealed a fus3-dependent reduction in variation, suggesting an autoregulatory negative feedback mediated by fus3 5. gartner et al. showed that levels of phosphorylated fus3 were higher in cells bearing a kinase-dead mutant version of fus324. showed that ste5 t287a mutant cells, in which the ste5 carries a lesion in a site of threonine phoshorylated by fus3 on peptides in vitro exhibited increased reporter expression 28, albeit with no change in the ec50 of the dose-response. finally, phosphoproteomic studies of pheromone response system proteins29 have uncovered numerous sites of phosphorylation on pheromone response system proteins, whose levels change upon pheromone stimulation, many of which lie in consensus map kinase target sequences (r. maxwell and o. resnekov, personal communication). we therefore hypothesized that the signal decline at different measurement points depends on non-translational, fast-acting negative feedbacks mediated by fus3 or kss1. to test if fus3 or kss1 were sources of negative feedback on system activity, we compared the baseline system response, at system points up to and including fus3 phosphorylation, with system response after selective inhibition of either fus3 or kss1 kinase activity. to do this, we first modified reporter strains by replacing either fus3 or kss1 with the corresponding purine analog-sensitive allele 30. we did this by changing the gatekeeper residue in each kinase s atp binding pocket (q93 in fus3, n94 in kss1) to an alanine. the mutant fus3-as2 and kss1-as2 kinases were active, as measured by fluorescent protein reporter gene output (fig. s9a), and10 m 1-nm-pp1, a cell-permeable adenosine analogue, inhibited the activity of mutant kinases without inhibiting wild-type kinases (fig. we then quantified fus3 phosphorylation by quantitative immunoblotting after stimulation with pheromone, either with or without simultaneous inhibition with 1-nm-pp1 (fig. fus3 phosphorylation levels did not peak and decline to a plateau when we inhibited fus3-as2, but, rather, remained high, near peak levels. by contrast, when we inhibited kss1-as2, fus3 phosphorylation levels were unaffected (fig. these results indicated that fus3 kinase activity mediated one or more negative feedbacks in this system. we then studied where in the system the fus3-dependent feedback acted to diminish signal amplitude. yi et al. showed that the decrease in g-protein fret within 30 seconds of stimulation depended on sst2 6. this finding suggested that the fus3-dependent negative feedback might upregulate the gtpase-activating protein (gap) function of sst2, which would increase g protein reassociation and decrease downstream signal. we tested if fus3-as2 inhibition affected the observed decline in both g-protein dissociation and ste5 recruitment. 4c), but surprisingly had no effect on the decline in g-protein dissociation in a g-protein fret reporter strain carrying fus3-as2 (fig. 4d and fig. these results indicated that fus3-mediated negative feedback acted downstream of mechanisms regulating g-protein association. to confirm that fus3 acted downstream of g-protein activation, we measured ste5 recruitment after deleting sst2. we expected deletion of sst2 to have no effect on fus3-mediated signal decline, since sst2 is required for efficient g-protein inactivation and, as we showed above, fus3-mediated negative feedback does not reduce g-protein dissociation levels. unexpectedly, when we deleted of sst2, we completely disrupted fus3-mediated signal decline; unlike sst2+cells, inhibition of fus3 did not cause an increase in ste5 recruitment (fig. furthermore, the ste5 recruitment (with or without fus3-mediated feedback) peaked and declined, similar to the baseline response of sst2+cells (compare squares and circles in fig. this finding showed that signal peak-and-decline is the default behavior in the absence of sst2. since a sustained non-declining signal is only evident in sst2+cells in the presence of fus3-as2 inhibitor, these results also indicate that sst2 promotes ste5 membrane recruitment, a hitherto unknown function of the rgs protein family, and that fus3 negatively regulates this novel signal-promoting function (fig. we then investigated which portions of the sst2 protein might be involved in promoting ste5 membrane recruitment. during analysis of sst2 point mutants, we found that ste5 recruitment in a fus3-as2 strain that carried sst2-t134a instead of wild-type sst2 peaked-and-declined in the presence and absence of fus3 inhibitor (fig. the pheromone-induced growth-inhibition of sst2-t134a cells reported by halo assays was close to wild-type (fig. s11a), and the average number of sst2-t134a protein molecules per cell was similar to sst2 abundance in the parent strain, (fig. s11b), suggesting that the t134a mutation disrupted a significant fraction of the fus3-dependent, signal-promoting function of sst2 without disrupting the bulk of its signal-reducing gap activity. t134 lies within the n-terminal dep domains of sst2, which are required for localization of sst2 to the membrane by binding the cytosolic tail of ste2 31. these results indicate that the dep domains in sst2 might aid ste5 membrane recruitment, perhaps by providing additional membrane-proximal interaction surfaces, and suggest that mechanisms that regulate localization of sst2 to the membrane, such as disruption of sst2-ste2 interactions by yck1/2-mediated phosphorylation after longer periods of pheromone stimulation 31, might consequently regulate ste5 membrane recruitment. finally, we tested if dose-response alignment between receptor-pheromone binding and downstream activities required fus3 activity. in principle, fus3-mediated negative feedback might scale system activity by a dose-independent factor, and therefore cause no shift in the normalized dose-response curve. for example, the ste5 t287a mutation increases the magnitude of system output relative to wild-type cells without changing the pheromone concentration yielding half-maximal response (see fig. 5 in 28). we measured dose-responses of fus3 phosphorylation in a fus3-as2 strain with and without inhibitor 15 minutes after pheromone stimulation, the time when the amount of fus3 phosphorylation had declined to a steady-state level (fig. 3c). inhibiting fus3 kinase activity shifted the dose-response of fus3 activation, lowering the pheromone concentration needed for half maximal response by 20-fold (fig. moreover, inhibiting fus3 kinase activity doubled the dynamic range of the output (fig. these results showed that fus3-mediated negative feedback was required for dose-response alignment in the yeast pheromone response system. we found that mapk fus3 mediates rapid negative feedback that aligns the dose-responses of upstream and downstream system activities in the pheromone response system. we propose that dose-response alignment improves information transmission through this and other signaling systems. furthermore, we found that fus3 negatively regulates a novel signal-promoting function of the rgs protein sst2. our results demonstrate that rgs proteins, present in many eukaryotic signaling systems (the human rgs family, for example, contains more than 35 members 32), can function in signal transduction systems by increasing signal in addition to accelerating g-protein inactivation, possibly (as in the case of pheromone response) by facilitating recruitment of mapk scaffolds to sites of activity. the idea that dose-response alignment increases the amount of transmitted information has practical implications for drug discovery and design. for example, consider a drug that increased sensitivity of cells to a naturally occurring antagonist of cell proliferation, analogous to the downstream dose-response shift we observed upon fus3 inhibition in the pheromone response system (fig., the dose-response misalignment could reduce the amount of transmitted information about the signal. the decrease in transmitted information could increase cell-to-cell variation in response, causing a larger number of cells fall below a threshold in antagonist response and continue proliferation. it is possible some existing drugs that allosterically modify gpcr signaling systems downstream of ligand binding (see 33, fig. 3) and those that target mid-system signaling molecules such as pkc 34 and akt 35 may decrease dose-response alignment and increase response variation, whereas drugs that specifically affect the affinity of receptor-ligand binding (see 33, fig. we propose here that the fidelity with which a cell responds to different input concentrations of a ligand depends on a systems-level quantitative behavior, dose-response alignment, found in many other cell signaling systems. for biological systems, a deeper understanding of key quantitative behaviors will likely depend on articulating appropriate analytical frameworks and metrics. information theory36 defines a framework for quantifying the relationship between system input and output (see supplementary information 9 for further discussion), and has enabled researchers to quantify, for example, the amount of information that an axon of a single sensory neuron can transmit 37 and the amount of information about morphogen gradient that a transcription factor can transmit to a downstream effector 38,39. much as concepts from classical electromagnetism provide rigorous means to describe and understand the determinants of behaviors of electrical circuits, we expect that concepts from information theory will enable more rigorous and quantitative understanding of how genes (and the proteins they encode) of more complicated signaling systems interact to sense and transmit information into the cell. we constructed yeast strains and plasmids by standard methods 40,41 essentially as described (5 and supplementary information 1). by doctrine, we expressed all reporter constructs from native promoters integrated into the chromosome, and verified that the level of expressed protein was similar to the native level. with the exception of strains used for g-protein fret experiments, we constructed all strains were from otherwise- isogenic bar1- w303a reference parent strain, acl 379 5, by the steps described. we stimulated exponentially-growing cells with the indicated concentration of pheromone and/or other reagents (such as the inhibitor 1-nm-pp1) in one of two ways. for image cytometry, we affixed the cells to the bottom of wells in a glass-bottom 96-well plate, as described in 5 and in supplementary information 2.1. using custom fluidic hardware, we evacuated medium from the well, injected fresh medium containing the indicated concentration of pheromone and/or inhibitor, and proceeded to record images over time. for mapk phosphorylation, fus1 mrna, and flow cytometry experiments, we stimulated cells by using a micropipette to mix a small volume of pheromone and/or inhibitor into the cell suspension to the final concentration (as indicated, typically 100 nm pheromone and 10 m 1-nm-pp1). we performed image acquisition essentially as described in 5,23, with modifications as detailed in supplementary information. for image cytometry, we extracted values for parameters of interest from images using cell-id 1.0 23. we analyzed image and flow cytometric data using physics analysis workstation (paw; see 42) and custom scripts, depending on the type of image, described in the text and in supplementary information. supplementary information contains further details on plasmids, strains, construction methods, materials, and experimental methods.

haploid saccharomyces cerevisiae yeast cells use a prototypic cell signaling system to transmit information about the extracellular concentration of mating pheromone secreted by potential mating partners. the ability for cells to respond distinguishably to different pheromone concentrations depends on how much information about pheromone concentration the system can transmit. here we show that the mapk fus3 mediates fast-acting negative feedback that adjusts the dose-response of downstream system response to match that of receptor-ligand binding. this dose-response alignment, defined by a linear relationship between receptor occupancy and downstream response, can improve the fidelity of information transmission by making downstream responses corresponding to different receptor occupancies more distinguishable and reducing amplification of stochastic noise during signal transmission. we also show that one target of the feedback is a novel signal-promoting function of the rgs protein sst2. our work suggests that negative feedback is a general mechanism used in signaling systems to align dose-responses and thereby increase the fidelity of information transmission.

PMC2716709

pubmed-1234

it is known that breast density is a risk factor of breast cancer. a woman who has more than 75% breast density is 46 times more likely to have breast cancer than a woman who has a breast density of less than 25%1. guidelines for breast cancer screening have been suggested, and women over 50 years old are generally recommended to undergo mammography. one study even says women in their 40s can also increase their lifespan through mammography, recommending they too should undergo mammography2, 3. not only does higher breast density mean a higher chance of having breast cancer, but breast density can also cause false negatives and increase re-examination rates by lowering mammography s sensitivity4, 5. breast density is affected by race, age, bmi, and the level of female sex hormones. in particular, body mass index (bmi) is a risk factor for breast cancer in women who are in menopause. it has also been shown in several pilot studies that abdominal obesity and weight increase, even after correcting for bmi, have some connection with the increased rate of breast cancer after menopause6, 7. as the amount of body fat grows due to obesity, so does the amount of fatty tissue generally, and eventually the density in mammography decreases. as mentioned above, some factors related to breast density have been reported, but they are only basic characteristics. the study subjects were 230 patients who underwent mammography and biochemical marker tests between march 1 and october 1, 2014, at soonchunhyang university hospital. all the subjects signed a written informed consent form approved by the institutional review board of soonchunhyang university hospital. subjects who may have had abnormal bone density levels, such as diabetic patients, fasting glucose 126 mg/dl, 7.0 mmol/l, or those who had taken hormone treatment for over a year were excluded, and we chose only post-menopausal women. six diabetic patients, 14 before menopause, and 10 hormone treatment patients were excluded, leaving 200 subjects whose average age was 48.6410.92 yrs. to perform mammography, we used a lorad elite trex mammo (trex-lorad, usa) at 26 kvp and 80 mas. the american college of radiology, breast imaging reporting and data system (acr bi-rads, usa) categorizes breast parenchymal pattern density from mammography into four grades: grade 1, almost entire fat; grade 2, fibroglandular densities; grade 3, heterogeneously dense; and grade 4, extremely dense (fig. a) represents almost entirely fat, b) represents fibroglandular densities, c) represents heterogeneously dense, and d) represents extremely dense). height and weight were measured with an automatic height and weight scale, and body mass index (bmi) was computed using the formula weight (kg)/height squared (m). mammography. a) represents almost entirely fat, b) represents fibroglandular densities, c) represents heterogeneously dense, and d) represents extremely dense subjects fasted for at least 12 h before blood and urine collections for biochemical marker tests. fifty items were assayed: erythrocyte sedimentation rate (esr), white blood cell (wbc), red blood cell (rbc), hemoglobin, hematocrit, mean corpuscular volume (mcv), mean corpuscular hemoglobin (mch), mean corpuscular hemoglobin concentration (mchc), platelet count, red cell distribution width (rdw), platelet distribution width (pdw), mean platelet volume (mpv), neutrophil (%), lymphocyte (%), monocytes (%), eosinophil (%), basophil (%), neutrophil, lymphocyte, monocytes, eosinophil, basophil, hba1c, protein, albumin, glucose, total bilirubin, direct bilirubin, aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), urea nitrogen, creatinine, uric acid, calcium, phosphrus, gamma-glutamyl transferase (gt), lactate dyhydrogenase (ldh), amylase, triglyceride, total cholesterol, high-density lipoprotein (hdl-cholesterol), low-density lipoprotein (ldl-cholesterol), iron (fe), total iron binding capacity (tibc), c-reactive protein (crp), rheumatoid factor (ra factor), hiv combo, hbsag, antihbs, -fetoprotein (afp), carbohydrate antigen 19-9 (ca 19-9), cancer antigen 125 (ca-125), free thyroxine (free t4), thyroid-stimulating hormone (tsh), anti-hepatitis-c (anti hcv). simple correlation analysis was performed to investigate correlations among breast density, general characteristic, and biochemical markers. then, multiple regression analysis was performed to evaluate the factors that showed a significant correlation. spss software (ver.18.0, chicago, usa) with a significance of 0.05 was used. the simple correlation analysis of breast density, general characteristics, and biochemical markers before and after menopause is shown in table 1table 1.simple correlation analysis of breast density, general characteristics, and biochemical markersvariablebreastdensityvariablebreastdensityvariablebreastdensityvariablebreastdensityage0.55neutrophil% 0.05ast (got)0.22tibc0.00height0.28lymphocyte% 0.07alt (gpt)0.27crp0.13weight0.30monocytes% 0.06alp0.28ra factor0.03bmi0.48eosinophil% 0.04urea nitrogen0.07hiv combo0.04esr0.07basophil% 0.03creatinine0.10hbsag0.09wbc0.12neutrophil0.06uric acid0.16antihbs0.06rbc0.01lymphocyte0.04calcium0.09afp0.03hemoglobin0.14monocytes0.05phosphorus0.06ca 19-90.02hematocrit0.15eosinophil0.02gt0.18ca 1250.04mcv0.13basophil0.08ldh0.05free t40.11mch0.19hba1c0.10amylase0.03tsh0.05mchc0.05protein0.02triglyceride0.30antihcv0.12platelet count0.04albumin0.04cholesterol, total0.16rdw0.15glucose0.15hdl-cholesterol0.23pdw0.10bilirubin, total0.07ldl-cholesterol0.29mpv0.07bilirubin, direct0.09iron0.00bmi: body mass index, esr: erythrocyte sedimentation rate, wbc: white blood cell, rbc: red blood cell, mcv: mean corpuscular volume, mch: mean corpuscular hemoglobin, mchc: mean corpuscular hemoglobin concentration, rdw: red cell distribution width, pdw: platelet distribution width, mpv: mean platelet volume, ast: aspartate aminotransferase, alt: alanine aminotransferase, alp: alkaline phosphatase, gt: gamma-glutamyl transferase, ldh: lactate dyhydrogenase, hdl-cholesterol: high-density lipoprotein, ldl-cholesterol: low-density lipoprotein, tibc: total iron binding capacity, crp: c-reactive protein, ra factor: rheumatoid factor, afp: -fetoprotein, ca19-9: carbohydrate antigen 19-9, ca-125: cancer antigen 125, free t4: free thyroxine, tsh: thyroid-stimulating hormone, anti hcv: anti-hepatitis-c. items exhibiting significant correlations with breast density were: age, 0.55; height, 0.26; weight, 0.30; bmi, 0.48; hematocrit, 0.15; mch, 0.19; rdw, 0.15; glucose, 0.15; ast(got), 0.22; alt(gpt), 0.27; alp, 0.28; uric acid, 0.16; gt(ggt), 0.18; triglyceride, 0.30; total cholesterol, 0.16; hdl-cholesterol, 0.23; and ldl-cholesterol, 0.29 (p<0.05). the results of multiple regression analysis using breast density-related variables are shown in table 2table 2.multiple regression analysis of each variable related to breast densityvariablebseage0.040.00height0.040.01weight0.030.01bmi0.130.02hematocrit0.010.00mch0.060.02rdw0.080.04ast0.010.00alt0.020.00alp0.000.00uric acid0.030.01gt0.010.00triglyceride0.000.00cholesterol, total0.000.00hdl-cholesterol0.020.00ldl-cholesterol0.010.00bmi: body mass index, mch: mean corpuscular hemoglobin, rdw: red cell distribution width, ast: aspartate aminotransferase, alt: alanine aminotransferase, alp: alkaline phosphatase, gt: gamma-glutamyl transferase, hdl-cholesterol: high-density lipoprotein, ldl-cholesterol: low-density lipoprotein. significant factors were: age, 0.040.00, height, 0.040.01; weight, 0.030.01; bmi, 0.130.02; t-score, 0.130.05; hematocrit, 0.010.00; mch, 0.060.02; rdw, 0.080.04; ast(got), 0.010.00; alt(gpt), 0.020.00; alp, 0.000.00; uric acid, 0.030.01; gt(gtp), 0.010.00; triglyceride, 0.000.00; total cholesterol, 0.000.00; hdl-cholesterol, 0.020.00; and ldl-cholesterol, 0.010.00 (p<0.05). bmi: body mass index, esr: erythrocyte sedimentation rate, wbc: white blood cell, rbc: red blood cell, mcv: mean corpuscular volume, mch: mean corpuscular hemoglobin, mchc: mean corpuscular hemoglobin concentration, rdw: red cell distribution width, pdw: platelet distribution width, mpv: mean platelet volume, ast: aspartate aminotransferase, alt: alanine aminotransferase, alp: alkaline phosphatase, gt: gamma-glutamyl transferase, ldh: lactate dyhydrogenase, hdl-cholesterol: high-density lipoprotein, ldl-cholesterol: low-density lipoprotein, tibc: total iron binding capacity, crp: c-reactive protein, ra factor: rheumatoid factor, afp: -fetoprotein, ca19-9: carbohydrate antigen 19-9, ca-125: cancer antigen 125, free t4: free thyroxine, tsh: thyroid-stimulating hormone, anti hcv: anti-hepatitis-c bmi: body mass index, mch: mean corpuscular hemoglobin, rdw: red cell distribution width, ast: aspartate aminotransferase, alt: alanine aminotransferase, alp: alkaline phosphatase, gt: gamma-glutamyl transferase, hdl-cholesterol: high-density lipoprotein, ldl-cholesterol: low-density lipoprotein of the many risk factors that cause breast cancer, the best known is long-term exposure to female sex hormones8,9,10,11,12. it has been reported that as estrogen levels increase, so does the risk of breast cancer13. additionally, breast density has a high correlation with breast cancer; as breast density increases, women are more likely to have breast cancer8. factors that affect breast density include race, age, bmi, and levels of female sex hormones6, 7. in this study, the factors related to breast density were age, height, weight, bmi, hematocrit, mch, rdw, ast, alt, alp, uric acid, gt, triglyceride, total cholesterol, hdl-cholesterol, and ldl-cholesterol. breast tissue degenerates due to the decline in estrogen and progesterone, and breast density starts to decrease due to an increase in fatty tissue14. bone density level, breast density, and breast cancer are related. in this study, studies of western women reported bmi as one of the major factors that affecting breast density15, 16. according to those studies, when bmi was low, even after correcting for age, it indicated high density breasts. the proportion of dense breast was 60% in low-weight women under age 40, similar to the results of our present study. a limitation of this study was that, because there were a small number of subjects, it was difficult to identify correlations between breast density and biochemical markers objectively. thus, an effort is needed to make more precise risk evaluations of breast density by conducting a large-scale prospective study of korean women.

[ purpose] the aim of this study was to identify biochemical markers related to breast density. the study was performed with 200 patients who received mammography and biochemical marker testing between march 1, 2014 to october 1, 2014. [subjects and methods] following the american college of radiology, breast imaging reporting and data system (acr bi-rads), breast parenchymal pattern density from mammography was categorized into four grades: grade 1, almost entirely fat; grade 2, fibroglandular densities; grade 3, heterogeneously dense; and grade 4, extremely dense. regarding biochemical markers, subjects underwent blood and urine tests after a 12-h fast. we analyzed correlations among breast density, general characteristics, and biochemical markers. [results] breast density-related factors were age, height, weight, body mass index (bmi), hematocrit, mch, rdw, ast, alt, alp, uric acid, gt, triglycerides, total cholesterol, hdl-cholesterol, and ldl-cholesterol. [conclusion] the results can be used as basic and comparative data for the prevention and early control of breast cancer.

PMC4540825

pubmed-1235

platinum, ruthenium, rhodium, and iridium (platinum group metals) and gold together with silver (copper group metals) are known as noble metals. we discuss the ones that are already used in oncology or have a therapeutic potential. platinum (pt), with atomic number 78, atomic mass 195.08, density 21.45 g/cm, and melting point 1772c, is quite rare in nature and usually is found together with ruthenium, rhodium, and palladium. in chemical compounds it is mainly found in its oxygenation state of ii, iii, iv, and vi; it has very good properties of electric and thermal conductivity. platinum was first found in columbia in 1735, named from the spanish platinum, meaning small silver because of its relative lack of usefulness compared to silver, which is called in spanish plata. platinum is mined in russia, in the republic of south africa, canada, and in the usa. rosenberg et al. used cisplatin firstly for the suppression of escherichia coli growth. the most commonly used cytostatics from this group are cisplatin, carboplatin, and oxaliplatin (used from 1978, 1980, and 1988, respectively). they are used in the therapy of many malignant tumours: ovarian cancer, testicular cancer, lung cancer, oesophageal cancer, stomach cancer, prostate cancer, bladder cancer, squamous cell carcinoma of head and neck, cervical cancer combined with radiotherapy, colorectal cancer and non-hodgkin lymphoma, multiple myeloma, neuroblastoma, melanoma, and mesothelioma. cisplatin is built from one atom of platinum, two chloride ions, and two molecules of ammonia. carboplatin has one atom of platinum, two molecules of ammonia, and a cyclobutanedicarboxyl ligand with oxygen atom from the carboxyl group. oxaliplatin, however, is a complex compound of platinum with 1,2-diaminocyclohexane and an oxalate group. the addition of new ligands to platinum atom has been done with the aim of breaking chemoresistance to platinum compounds, which is observed during therapy. this may be a result of the quantity and type of new dna adducts and also of the reduction of treatment side effects [4, 5]. cytostatics that are platinum derivatives are alkylating agents. by making stable cross bindings with dna (bonds with nucleophilic nitrogen atom n7 of two neighbouring guanines) dna replication the change of dna structure is recognised by specific proteins, including the hmsh2 protein form the high mobility group (hmg), and may cause damage repair; other proteins may be a signal triggering apoptosis [69]. gong et al. found that cisplatin may also induce cell death in parallel by activation of suppressor protein p53 and protein p73. there is, however, evidence that there are other (independent from p53 cell death) pathways in cisplatin based therapies. cisplatin induces activation of the c-abl tyrosine kinase, which may induce proapoptotic protein p73 [1114]. according to the information enclosed with the drug, the most common side effects (more than 1 out of 10 patients) after cisplatin treatment are: leukopenia, thrombocytopenia, anaemia, hypernatremia, impairment of hearing, decreased appetite, nausea, diarrhoea, kidney malfunction, and fever. in more than 1 out of 100 patients but in less than 1 out of 10 patients peripheral neuropathy side effects of carboplatin include kidney damage (less often than cisplatin), alopecia, fatigue, and elevated liver enzyme activity. oxaliplatin causes peripheral neuropathy, fatigue, hypernatraemia and hypokalaemia, anaemia, thrombocytopaenia, and leukopaenia. gold (au), with atomic number 79, atomic mass 197.0, density 19.3 g/cm, and melting point 1063c, is found in its free state and in minerals. in chemical compounds it is mainly in oxygenation states i and iii; it has very good properties of electric and thermal conductivity. the history of gold mining is more than 6000 years old, its colour and timeless value were well known in art and architecture of ancient civilisations. the main producer of gold is the republic of south africa, although it is also mined in other regions of the world. its complex auranofin is used in rheumatism treatment, and its anticancer potential is also described in both in vitro and in vivo models [1517]. in recent years nanotechnology using structures of 1 nm to 150 nm also involves the use of gold. gold particles are stable and non-toxic; they can bind drugs, antibodies, or antigens, they can have transport and active substance release capability. they are safe for healthy tissues because they do not disintegrate during transport [15, 18, 19]. they reduce tumour mass also in platinum-resistant cancers, and they have lower toxicity, especially for the kidneys [2022]. the anticancer activity of gold complexes is not known, although some research shows that they may have inhibiting activity towards enzymatic protein complexes like thioredoxin (trx) and thioredoxin reductase (trxr); they may also inhibit proteasome activity a mechanism linked with carcinogenesis [15, 2327]. thioredoxin and thioredoxin reductase protect the cell from reactive oxygen species and apoptosis, and they are involved in cell growth and proliferation. it is one of the factors that is linked to cell chemoresistance; it is also linked to cells invasive and metastatic potential. thioredoxin also causes expression of hypoxia-induced factor 1 (hif-1) and increases production of protein products from the vegf gene promoting neoangiogenesis in tumours [15, 28, 29]. thioredoxin and thioredoxin reductase are found in two forms: cytoplasmic and mitochondrial (trxr1 and trxr2). gold complexes inhibit thioredoxin reductase in mitochondria by reducing membrane potential, leading to apoptosis [15, 21]. it is thought that the enzymatic complex of thioredoxin thioredoxin reductase can be a target in cancer therapy [26, 28]. this is a complex responsible for degradation of ubiquitin marked proteins that may cause selective apoptosis of cancer cells [16, 20]. stat pathway is a transcription activator that plays a role in proliferation and has an antiapoptotic effect on cancer cells. recent research has shown evidence anticancer activity of gold complexes both in vitro and in vivo. it was proven that auranofin in human ovarian cancer cell lines induces apoptosis by inhibition of thioredoxin reductase; it was also proven on cisplatin-resistant cell lines. a similar action of gold (iii) complex was observed on human ovarian cancer stem lines, and in one study it was compared with cisplatin and oxaliplatin. antiproliferative and proapoptotic actions of gold complexes were stronger than those of platinum derivatives. in another study gold complexes were assessed for their antiproliferative potential in ovarian cancer cell lines and embryonic cell lines a study on human breast cancer showed that the use of auranofin also has an anticancer effect; the inhibition mechanism was linked to the stat pathway and telomerase. it was found that the effectiveness of pegylated gold nanoparticles (aunps) combined with docetaxel on prostate cancer cell lines was 50% only half of cells survived, others were damaged. an even higher effectiveness of gold nanoparticles was observed in xenograft of human prostate cancer in mice with the use of particles having gold au isotope. gold nanoparticle complex was developed, and this complex was linked to the cetuximab antibody and gemcitabine, which was administered to animals that had hepatocellular carcinoma (hcc) heterografts. heterografts entered apoptotic pathway, they were found to be necrotic, their proliferative potential was diminished, and healthy tissues were not damaged. an interesting study on auranofin use in transgenic mice with chronic lymphocytic leukaemia (cll) was made. this type of leukaemia has a high remission rate after first-line chemotherapy, although relapses are linked to chemoresistance. it seems that gold complexes and nanoparticles, because of their anticancer activity, will find their way into clinical trials, not only experimental models. argentum), with atomic number 47, atomic mass 107.86, density 10490 kg/m, and melting point 961.78c, is found in nature in its free state and in minerals like argentite. it is a silver-white metal, with very good thermal and electrical conductivity. greeks coated plates and cups with silver to stop disease spreading, and they put silver coins into water buckets to extend the water's freshness. they also gave silver spoons to children for sucking, which was believed to protect them from illnesses. the very first silver compound that was used for treatment was silver nitrate, which was discovered by basilius valentinus back in the 15 century. in the 19 century, for the first time, 0.2% solution of silver nitrate was used for burn wound care. in 1874 t. billroth proved the antiseptic properties of silver by using its antibacterial effect on staphylococcus aureus. later on the antibacterial properties of silver were proven against the following bacteria: streptococcus, pseudomonas and escherichia. in the early 1990s it was observed that people with low concentrations of silver as a trace element often undergo bacterial, viral, or fungal infections. nowadays many surgical instruments are silver coated, as well as other instruments like dialysis catheters. everywhere where risk of infection is present, silver can be used. in the last few years oncological research the first report of the cytotoxic effect of silver nanoparticles (agnps) was proven against mcf-7 breast cancer cell line in 2013. kathiravan used the extract of melia dubia leaves (a tree growing in india), and sathishkumar used extract from dendrophthoe falcata. silver nanoparticles developed in this way were proven to have an anticarcinogenic effect on mcf-7 breast cancer cell line. vasanth, with the use of agnps, stopped replication of a cervical cancer cell line (hela) by induction of apoptosis. to produce silver nanoparticles he used extract from the bark of moringa oleifera. chinese researchers have proven the cytotoxic effect of silver nanoparticles against aml (acute myeloid leukaemia) cell lines such as shi-1, thp-1, dami. he proved that those complexes inhibit proliferation by inducing cell cycle cessation in phase g1 and s in hepatocellular carcinoma cell line (hepg2). he observed in vivo tumour reduction when a silver nanoparticle was combined with alisertib (selective kinase inhibitor). iridium (ir), with atomic number 77, atomic mass 192.217, density 22.56 g/cm, and melting point 2466c, is found in nature as osmiridium (an alloy of osmium and iridium). in chemical compounds it is mainly in an oxygenation state of iv; oxygenation states ii, iii, and vi are also possible, and it has good properties of electric and thermal conductivity. discovered in 1803 by smithson tennant iridium was named after the greek rainbow goddess iris because of its different coloured salts. annual production of iridium is only 3 tonnes; it is one of the rarest elements in nature. at, iridium has been used together with platinum as a component of electrodes for stimulation. iridium oxide was used instead of ag/agcl in dry electrodes with microtips that could be used without gel eeg. because of the high price of iridium there was an attempt to produce electrodes with titanium-iridium oxide. in oncology iridium has found its way as ir isotope in brachytherapy. they can be used in after-loading systems that protect medical staff from radiation. it is one of the basic isotopes used in brachytherapy. in the field of gynaecological oncology it is used mainly in plesiobrachytheraphy in cervical and uterine cancer [48, 49]. research was made on the use of iridium in flt4 (also known as vegfr3) kinase inhibitor [50, 51]. ruthenium (ru), with atomic number 44, atomic mass 101.07, density 12.45 g/cm, and melting point 2334c, is found in nature as sulphide, iron, and chrome ores. in chemical compounds some researchers claim that ruthenium was first discovered in 1808 by jdrzej niadecki; he called this element vestium in honour of the discovery of the planet vesta. the first ruthenium compound used in clinical practice was nami-a. a is used together with gemcitabine as second-line chemotherapy in the treatment of metastases in non-small cell lung carcinoma. other compounds used in clinical practice are kp1019, and soluble salt kp1339, which has finished phase i of a clinical trial in neuroendocrine carcinomas. electroporation of kp1339 was tested in slovenia, and this method was found to be effective in vivo because of its extra antiangiogenic properties. it may also spread the idea of electrochemotherapy, which is based on local injection of chemotherapeutics accompanied by electrical impulses. developed dohuru, hothyru, and tothyru, which are aziru derivatives, and together with phospholipids they created stable nanoaggregates. the antiproliferative activity of nanocompounds towards cell lines widr, c6, and mcf-7 was also studied.

worldwide research groups are searching for anticancer compounds, many of them are organometalic complexes having platinum group metals as their active centers. most commonly used cytostatics from this group are cisplatin, carboplatin and oxaliplatin. cisplatin was used fot the first time in 1978, from this time many platinum derivatives were created. in this review we present biological properties and probable future clinical use of platinum, gold, silver, iridium and ruthenium derivatives. gold derivative auranofin has been studied extensively. action of silver nanoparticles on different cell lines was analysed. iridium isotopes are commonly used in brachyterapy. ruthenium compound new anti-tumour metastasis inhibitor (nami-a) is used in managing lung cancer metastases. electroporation of another ruthenium based compound kp1339 was also studied. most of described complexes have antiproliferative and proapoptotic properties. further studies need to be made. nevertheless noble metal based chemotherapheutics and compounds seem to be an interesting direction of research.

PMC4631304

pubmed-1236

alzheimer s disease (ad) is a progressive and the most prominent old-age debilitating disease which had a notable epidemic growth in recent years. according to statistical analyses, it is estimated that the number of people suffering from ad will double every twenty years; and by 2050, the number of sufferers from ad in the world would reach 115 million (1, 2). one-eighth of people aged 65 and older and half of people aged 85 and older were diagnosed with ad, approximately; its death toll from 2000 to 2008 has increased by 66% (3). the costs for this disease are so high that one percent of global gdp is spent on ad s consequences (4). diagnosed patient with ad usually dies after 5 to 7 years (2). in iranian traditional medicine (itm) manuscripts, diseases in which forgetfulness is the main symptom are categorized under nesyan label. in itm manuscripts, one of the types of nesyan which originates from coldness and dryness of the brain has the same symptoms as ad. forgetting recent events and remembering old happenings, cognitive disturbance, problems in constant talking (language disability), and insomnia are of its most important signs (5, 6) which are similar to ad s according to nincds-adrda criterion (7). itm takes nourishment as one of the six pillars of health (8, 9); therefore, itm manuscripts have dedicated a considerable part of their content to nutritional recommendations. in the first volume of canon of medicine, avicenna (970-1051 a.d.), the great persian scholar, described those in detail (10). he urged elderly people not to take foods producing black bile (sowd) such as lentil, eggplant, beef, dried meat, and salted foods; and phlegmatic foods such as fish, watermelon, melon, and cucumber. on the other hand, taking milk, shirberenj (an iranian dish consists of milk, rice, sugar, and rosewater), honey milk, and milk with ginger are recommended. apart from preventive recommendations, nutritional therapeutic recommendations are included to boost medication and shorten illness-period; and nesyan is no exception. muhammad ibn zakariy rz (rhazes) (865-925 a.d.), another great persian scholar, in his book al-hawi recommended to have poultry (such as chicken, dull-yellow partridge, hoopoe, pheasant, and sparrow meat), their brains, and yolks for people affected to nesyan. at the same time the patients were prohibited from having beef, mutton, goat meat, camel meat, and rabbit meat because they produce black bile; and fish as well for producing phlegm (balgham). moreover, having nuts such as almond, hazelnut, coconut, and walnut were recommended (12). it is worth mentioning that some other persian scholars such as aghili korasani and chishti had the same idea as rhazes (5, 6). there have been remarkable either observational or experimental studies on the role of diet and nutrients in prevention and treatment of dementia, especially ad. studies on effects of different kinds of fat such as saturated fatty-acids (sfa), unsaturated fatty-acids (ufa), and cholesterol on ad have been conducted recently. unsaturated fatty-acids and cholesterol play an important role in the brain; 60 percent of dry weight of the brain is consisted of fat which 20% of it is of unsatu-rated fatty-acids (13). although the brain is only 2% of the total body mass, it carries 25% of cholesterol of the whole body (14). in the light of the fact that most of the brain is consisted of fat and it uses nutrients as other organs, types and amounts of fat consumption could be vitally important in the brain s health. studying multi-nutrient diets (instead of one or more nutrients) has a notable importance. one of the multi-nutrient diets which have been frequently studied is the mediterranean diet (table 1) (15). studies have shown that it has preventive effect on ad (16, 17). taking types of fat into consideration, this diet has a great deal of ufas and lesser amounts of sfas and cholesterol. ufas positive impact has been reported in several other researches (18). while the results of studies on therapeutic role of ufas (especially omega-3) on ad are controversial (19, 20), ufas multi-nutrient diets like the mediterranean diet which contains high levels of ufas have abated the incidence of ad (16, 17); the abatement is 13% according to sofi et al. study on the relationship between the mediterranean diet and health status in 2008 (17). about cholesterol, it plays a vital role in cell membranes, yet its functions in neurons are more prominent. cholesterol is amassed within lipid rafts in the neuronal cell membrane and is involved in the formation and maintenance of synaptic connections (21). several studies on the effect of cholesterol on ad had ambiguous results. according to the importance of diets especially fats-in ad, this study attempted to analyze the fat content of itm recommended diet for nesyan (itm equivalent of alzheimer s disease). since traditional physicians used to recommend distinctive diets based on their own clinical experiences, analyzing these diets would eventuate in new nutritional regimen to improve ad sufferers quality of life. in order to study the traditional recommended diet for nesyan, itm prominent books including al-hawi (rhazes, 10th century), canon of medicine (avicenna, 11th century), zakhire-ye-khawrazmshahi (jorjani, 12th century), kholasat-ul-hekma (aqilikhorasani, 18th century), and exir-e-azam (chishti, 19th century) were searched for recommended and abstinent foods and diets traditionally prescribed for patients afflicted by senescence and/or nesyan (5, 8, 11, 12, 22). to quantify the results, the content of fatty components of each food (including polyunsatu-rated (pufa), monounsaturated (mufa), and saturated fatty acids (sfa) and also cholesterol) were extracted from the database of the department of agriculture of the usa (usda) (23). the content of fatty elements per 100 gr of each recommended food was compared with that of the abstinences and statistically analyzed by mann-whitney test via spss (version 16). about the diet, the sum of the nutrients of recommended and abstinent diets for daily consumption was also measured. ad dietary recommendations and abstinences which currently published in medical journals were also searched via scopus. finally, traditional and new dietary suggestions were compared with each other and the results presented as tables and figures. the results show that the recommended foods (presented in the first column of table 2) are fattier than the abstinent ones (presented in the first column of table 3) (p<0.001). there are also meaningful differences between unsaturated fatty acids (p<0.001), saturated fatty acids (p<0.001), and cholesterol (p<0.05) of recommended foods and abstinent foods (fig. these foods form recommended and abstinent diets for nesyan. this recommended diet is fattier than the abstinent diet (4.5 times) (see the last row of table 2 and 3); polyunsaturated fatty acids of recommended diet it is the same story for monounsaturated fatty acids and cholesterol (11 and 1.4 times more than abstinent diet, respectively) (fig. 2). fatty components in 100 gr of each food traditionally recommended or forbidden for people afflicted by nesyan.*error bars indicate sem comparison between nutritional factors of recommended and abstinent diet in nesyan the results of searching scopus database show that recent studies emphasize on diets with high amounts of unsaturated fatty acids for ad sufferers. a considerable number of papers but not all of them-emphasizes on probable positive role of cholesterol on ad. nutritional factors of daily diet recommended for patients affected with nesyan in iranian traditional medicine. the last row of the table presents the amounts of nutrients in daily recommended diet nutritional factors of daily diet forbidden for patients affected with nesyan in iranian traditional medicine. the last row of the table presents the amounts of nutrients in daily abstinent diet. iranian traditional medicine has nutritional recommendations to help curing nesyan (ad equivalent in traditional medicine). in this study, evaluating the ingredients of the recommended foods highlights that it is ufas-rich because of having nuts. the types of meat in the recommendations are mostly poultry (chicken, pheasant, and sparrow); their remark is their greater amount of cholesterol compared to the abstinences. some recent studies substantiate that cholesterol is a boost to improve memory function (24, 25). when cholesterol is consumed with ufas, it will turn into hdl (26, 27). in 2004, reitz and colleagues have concluded that there is no connection between serum cholesterol level and ad (28). however, some other studies have shown that the higher the serum cholesterol level in the elderly, the better memory functions (24, 25). some studies also have shown the decline in serum total cholesterol levels is associated with increased dementia risk (29, 30). furthermore, in a cohort study conducted on 1130 people (published 2010), reitz et al. conclude that high hdl levels in elderly individuals may be associated with a decreased rate of ad (31). prior to reitz, singh-manoux and colleagues (2008) indicated that a low level of hdl could be considered as a risk factor of memory dysfunction (32). bear in mind, there has been no study on hdl destructive effect on ad, so far (33). therefore, prescribing more amounts of cholesterol along with ufas in itm is in accordance with recent studies. on the other side, recent studies have shown that cholesterol and all of its precursors in cerebrospinal fluid (csf) and its precursorslanosterol, lathosterol, and desmosterol in plasma abate in ad sufferers (34, 35). in 2010, klsch et al. determined csf and plasma levels of cholesterol and its precursors in ad diagnosed people compared to those of healthy individuals. the results showed that csf levels of cholesterol and all of its precursors and also plasma levels of two of the precursors named lanosterol and lathosterol are lower in ad diagnosed people than those of healthy individuals (34). sato et al. showed in 2012 that plasma levels of desmosterol, another precursor of cholesterol, are also lower in alzheimer s sufferers than the levels in healthy ones (35). this casts doubt whether the cholesterol reducing agents have preventive effect on ad or not. there are some studies highlighting that cholesterol reducing agents such as statins are ineffective on ad incidence (36, 37). in a prospective study conducted by zandi et al. (2005) on 5000 elderly people in the usa, the relationship between statins consumption and the onset of dementia or ad has been violated (36). along the lines of zandi s study, mcguinness and colleagues (2009) co-evaluated two randomized double blind clinical trials containing 26 thousand participants which resulted that there is no relationship between statins consumption and the risk of ad incidence (37). also, reduction in serum total cholesterol could be considered as a risk factor of dementia in the elderly (29, 30). there are other ingredients in the abstinences which are not considerable in having fats and cholesterol. pumpkin, lettuce, melon, garlic, onion, cabbage, beans, broad beans, and mushroom are of its examples. according to iranian cuisine in the past, there were foods in itm that have no place in eating habit nowadays; so there is not much information about them. hoopoe, lark, dull-yellow partridge meat and their brain are of the examples. this diet contains high amounts of unsaturated fatty acids and cholesterol to boost treatment of nesyan (traditional equivalent of alzheimer disease). according to new scientific findings, this kind of diet could be useful as a complementary therapy of alzheimer disease. ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.

abstractbackgrounddietary notifications have been introduced recently for alzheimer disease (ad). in iranian old medical manuscripts, there are some nutritional recommendations related to nesyan (ad equivalent). the aim of this article was to compare dietary recommendations of iranian traditional medicine (itm) with novel medical outcomes. methods1) searching for dietary recommendations and abstinences described in itm credible manuscripts; 2) extracting fatty components of itm diet according to the database of the department of agriculture of the usa; 3) statistical analysis of fatty elements of traditionally recommended foods via mann-whitney test in comparison with elements of the abstinent ones; 4) searching for ad dietary recommendations and abstinences which currently published in medical journals; 5) comparing traditional and new dietary suggestions with each other. results1) traditionally recommended foods are fattier than abstinent ones (p<0.001). there are meaningful differences between unsaturated fatty acids (ufas) (p<0.001), saturated fatty acids (p<0.001), and cholesterol (p<0.05) of recommended foods and abstinent ones. 2) traditionally recommended diet is also fattier than the abstinent diet (4.5 times); ufas of the recommended diet is 11 times more than that of the abstinent one; it is the same story for cholesterol (1.4 times); 3) recent studies show that diets with high amounts of ufas have positive effects on ad; a considerable number of papers emphasizes on probable positive role of cholesterol on ad; 4) traditional recommended diet is in agreement with recent studies. conclusionitm recommended diet which is full of unsaturated fatty acids and cholesterol can be utilized for complementary treatment of ad.

PMC4441938

pubmed-1237

left ventricular hypertrophy (lvh) and increased left ventricular mass (lvm) are strong risk factors for cardiovascular disease and morbidity. cardiac hypertrophy is characterized by increased cell size, cardiac remodeling of myofilaments, and increased expression of fetal genes. lvm results from a complex of interaction between genetic, environmental, and lifestyle factors. increased knowledge concerning genes involved in the modulation of lvm will lead to a better understanding of the etiopathogenesis of lvh. calcium (ca) is arguably the most important messenger in cardiac muscle and plays a central role in regulating contractility, gene expression, hypertrophy, and apoptosis. it has been well described that ca transient movements regulate the transcription and gene expression that characterize the hypertrophic response of cardiomyocytes [2, 3]. the levels of ca are precisely controlled. a major sensor and mediator of intracellular ca transient movements is calmodulin (cam). the cacam complex binds and activates enzymes, including protein kinases, protein phosphatases, phospholipases, nitric oxide synthases, and endonucleases. three ca calmodulin dependent enzymes have significant roles in cardiac function: ca calmodulin-dependent protein kinase (camk), protein phosphatase 2b (calcineurin, can), and myosin light-chain kinase (mlck). camk and can have been shown to play key and often synergistic roles in transcriptional regulation in cardiomyocytes. it has been suggested that camk regulates gene expression via activation of several transcription factors [5, 6]. ca-cam-dependent kinase ii (camkii), a major cam target protein, is a uniquely regulated multifunctional regulatory enzyme. there are several studies indicating the major role of camkii involvement in cardiac hypertrophy and heart failure. in hypertrophic myocardium of animal models, experimental studies have demonstrated that transgenic mice overexpressing nuclear camkii have increased incidence of cardiac hypertrophy. inhibition of nuclear camkii activity causes transgenic mice to have smaller hearts than their nontransgenic littermates. thus, any genetic variants that directly affect cam gene expression or function are promising as candidates involved in modulating lvm. cam is encoded by a multigene family consisting of three members: calm1, calm2, and calm3. discovered that 2622a>g and 3001g>a polymorphism, both located in intron 1, may be associated with osteoarthritis in the japanese population. liu et al. indicated that calm2 is a candidate gene for primary open-angle glaucoma. to date, only vasan et al. have demonstrated, in meta-analysis, the correlation between calm2 polymorphism rs7565161 and echocardiographic diameter lvm in adults. the guanine to adenine transition at nucleotide position 474955027 (g.474955027 g>a, rs7565161) of human chromosome 2p21 is intergenic, adjacent to the calm2 gene. however, there are no reports which have focused on the association of intergenic adjacent calm2 polymorphisms with left ventricular mass in newborns. the factors influencing heart development during fetal life or first days of life, when external environmental factors such as diet, lifestyle, smoking or diseases have not yet had a marked impact, are still being sought. we hypothesize that adjacent intergenic calm2 polymorphism could potentially modify lvm during fetal life and in the first period of life in newborns. in the present study, the relationships between g.474955027 g>a (rs7565161) being adjacent intergenic calm2 gene polymorphism and lvm in a population of polish newborns have been investigated. the population included 206 consecutive healthy polish newborns (92 females and 114 males), born after the end of the 37th week of gestation (from 37 to 40 weeks). mothers in this study were healthy without any complications such as preeclampsia or eclampsia, and there was no fetal growth restriction. newborns in this study were appropriately grown for their gestational age (defined as birth mass above the 10th centile). exclusion criteria were twins, intrauterine growth restriction, chromosomal aberrations and/or congenital malformations, or small for gestational age, that is, below the 10th centile body length (bl), birth weight (bw), or head circumference (hc). at birth, cord blood (500 l) of neonates was obtained for isolation of genomic dna. the gender of the newborn, bl, bw, and hc were taken from standard hospital records. body surface area (bsa) was calculated using the following equation: (1)bsa=[bl(cm)bw(kg)3600]. a diascope oscillometer (artema) was used to determine systolic and diastolic blood pressure (sbp or dbp, resp.), and only one of the investigators performed all of the blood pressure (bp) measurements using a standardized protocol. the smallest cuff size that covered at least two thirds of the right upper arm and encompassed the entire arm was selected. newborn measurements were taken at least one and a half hours following their last feeding or medical intervention. an appropriately sized cuff was applied to the right upper arm, and the newborn was then left undisturbed for at least 15 minutes or until the infant was sleeping or in a quiet awake state. echocardiographic measurements in newborn on the 3rd day after delivery were made by one pediatric cardiologist. two-dimensional m-mode echocardiography was performed using an acuson sequoia 512 unit (usa), equipped with a 24 mhz imaging transducer. measurement techniques were consistent with the american society of echocardiography conventions. in a parasternal long-axis view, lvidd-left ventricular internal diameter-diastolic, lvids-left ventricular internal diameter-systolic, lvpw-left ventricular posterior wall thickness at end diastole, ivs-thickness of interventricular septum at end diastole, lad-left atrial diameter, aod-aortic diameter, pad-pulmonary artery diameter, lvv-left ventricular volume, and lvef-left ventricular ejection fraction were measured (using m-mode formulas). the left ventricular masses (lvm) were calculated from the echocardiographic left ventricular dimension measurements, using the penn convention with the equation modified by huwez et al. (1994) as follows:(2)lvm=1.04[(ivst+lvpwt+lvid)3lvid3], where ivst, lvpwt, and lvid denote interventricular septal thickness, left ventricular posterior wall thickness, and left ventricular internal dimension, respectively. to accurately determine and standardize the left ventricular mass, the lvm was indexed with respect to body length (lvm/bl (g/m)), body weight (lvm/bw (g/kg)), and body surface area (lvm/bsa (g/m)), respectively. genomic dna from cord blood was isolated using the qiaamp blood dna mini kit (qiagen, germany), according to the manufacturer's protocol. for the analysis of the intergenic g>a calm2 (rs7565161) polymorphism, a polymerase chain reaction-restriction fragment length polymorphism (pcr/rflp) method was designed with the following primer pair: forward 5-agggcctgcaatctaat-3 and reverse 5-atataatccccaccttcag-3 (tib mol biol, pozna, poland). the calm amplicons were subsequently digested with the acii restriction enzyme (mbi fermentas, vilnius, lithuania). the pcr product of 417 base pairs (bp) was cut into fragments of 258 bp, 137 bp, and 22 bp in the presence of the g allele and into fragments of 395 bp and 22 bp in the presence of the a allele. restriction fragments in each case were electrophoretically separated and visualized in midori green-stained (nippon genetics) 3% agarose gels. to verify the results, each calm2 amplicon was cleaned with genelute pcr clean-up kit (sigma). sequencing was performed according to the dideoxy sanger method in a geneamp pcr system 9700 thermal cycler (applied biosystems), using bigdye terminator v3.1 cycle sequencing kit (applied biosystems). afterwards, samples were purified (bigdye xterminator purification kit, applied biosystems), and 20 l deionized formamide (applied biosystems) was added. the sequencing results were read using sequencing analysis software v5.1 (applied biosystems). in each case, the result obtained with pcr-rflp method was identical with that appropriate one from sequencing. the divergence of calm2 genotypes frequencies from hardy-weinberg equilibrium was assessed using tests, and the distribution of each quantitative variable was tested for skewness. quantitative data were presented as means sd and analyzed either by student's t-test or by one-way anova. left ventricular mass indexes (lvmis) were tested for association with genotype using multivariate analysis (ancova) in order to adjust for possible confounding factors: neonatal (gestational age, gender, sbp, and apgar at three minutes) and maternal (age, bmi at the beginning and the end of the pregnancy, smoking status, and hypertension status). all data were analyzed with statistica (data analysis software system, version 10.0, statsoft, inc. characteristics of the newborn cohort (n=206) are shown in table 1. the distribution of these characteristics in our cohort approached normality (skewness<2 for all variables). mean bw and bsa values in boy newborns were significantly higher than those in girls. 69 gg calm2 homozygotes (33.5%), 95 ga heterozygotes (46.1%), and 42 aa homozygotes (20.4%) were identified. there were no significant differences in calm2 genotype or allele distributions between boys and girls (p=0.273, and p=0.107, resp.). the calm genotype distributions conformed to the expected hardy-weinberg equilibrium (p=0.396). lvmi measurements were tested for association using multivariate analysis (ancova) in order to adjust for possible confounding factors, after adjusting for newborn (gestational age, gender, sbp, and apgar at three minutes) and maternal (age, bmi at the beginning and the end of the pregnancy, smoking status, and hypertension status) parameters. we revealed a significant association between lvmis (lvm/bw in recessive and additive modes and the calm2 polymorphism). the carriers of the g allele of the calm2 polymorphism had significantly higher lvm/bw values, when compared with newborns homozygous for the a allele (3.1 g/m versus 2.5 g/m, padjusted=0.036, resp.). the ag genotype of calm2 was associated with the highest values of lvm/bw, exhibiting a pattern of heterozygote advantage (2.9 g/kg versus 3.1 g/kg versus 2.5 g/kg, padjusted=0.037) (figures 2 and 3). carriers of the a allele did not differ in lvm indexes (figure 1). an association was observed between genotype and dbp 90 percentile (p=0.027). carriers of the allele a of the calm2 gene had an increased incidence (%) sbp 90 percentile (p=0.027, 76.2% versus 23.8%). lastly, the calm2 polymorphism was significantly correlated with maternal history of gestational age (p=0.019). genetic factors are estimated to be responsible for between 30% and 70% of cardiac mass variance. studies in twins [20, 21] and populations [22, 23] showed that lvm is under genetic control. the present study in a cohort of newborns has demonstrated for the first time the significant association between variants of the intergenic adjacent calm2 polymorphism and increases in lvm indices in newborns. therefore, to minimize the disparities, we carefully selected homogenous group of full-term newborns. to accurately determine lvm, we used lvm in relation to bsa, bl, and bw, which are reported to be more appropriate. it should be emphasized that confounding factors such as especially gestational age may play a role in the development of lvm in fetus. the fetal programming hypothesis states that, for example, birth mass in newborns may be partially related to maternal factors. in this study, the ag genotype of intergenic adjacent calm2 polymorphism was associated with the subtle higher values of lvmi, exhibiting a pattern of heterozygote advantage in results. what is important, in our study, the carriers of the g allele have higher lvm than the carriers of the a allele. these results were similar to those of a large cohort of adults, who were studied by vasan et al.. in this meta-analysis of echocardiographic data associated with interindividual variation in cardiac dimension, it should be mentioned that total sample included those with coronary heart disease, peripheral vascular disease, valvular heart disease, stroke, and circulation heart failure. current results exhibit a pattern of heterozygote advantage, as heterozygote newborns had significantly higher lvmi than the carriers of homozygote genotypes. the heterozygote advantage hypothesis attributes heterosis to the superior fitness of heterozygous genotypes over homozygous genotypes at a single locus. some studies suggest that heterozygote advantage is a favorable process, the positive selection over evolution, as a natural consequence of adaptation role of variation in gene [2628]. however, in light of vasan's study, the feature that may be potentially beneficial in early life may lead to predisposition to increase or hypertrophy left ventricular in adults. williams suggested antagonistic pleiotropy theory, which assumes that some genes responsible for increased fitness in the children, fertile organism contribute to decreased fitness in adults. we conclude that this theory may be relevant here. we hypothesized that genetic variation in the intergenic adjacent calm2 gene polymorphism, analogously to the other common polymorphisms in developmental genes, may cause minor changes in the development or modulation of lvm in newborns. we continue observing our population and consider conducting follow-up, which will show in later years whether the heterozygotes have a predisposition to develop left ventricular hypertrophy or not. however, our results require confirmation in further independent large studies. the connection between calmodulin and modulating cardiac contractile function and growth is well documented [30, 31]. otherwise, in an experimental animal study, the protein level of cam was shown with a relatively high level of calmodulin appearing on gestational days 14-15, followed by a steady but significant decrease at birth and during the first week of postnatal life. it is reported that specific elevation of cam levels directly affects the rate of cell proliferation. also, gillett et al., in animal study (fetal sheep), showed that increased calm2 mrna expression levels may reflect an important role for calmodulin in expansion-induced fetal lung growth. a study performed in human showed that genes encoding calmodulin (calm1, calm2, and calm3) are involved in increasing proliferation [35, 36]. although such knowledge indicates the important role of calmodulin-dependent protein kinases and phosphatases in regulating cardiac hypertrophy, the role of genetic variation in cam in the physiology of the development human heart has not been clarified. our results suggest that genetic variation of calm2 may be partly involved in regulating myocardial cell proliferation and growth, during embryogenesis and in the first days of life. it is possible that genetic variation in cam may have been involved in regulating the activity or/and levels in serum kinases and phosphorylases (e.g., camkii, calcineurin) during fetal life. in the current study, we investigated healthy newborns born at full term. our previous studies reported that ras (renin-angiotensin system) or bmp4 (bone morphogenetic protein 4) and bmpr1a (bone morphogenetic protein, receptor type 1a) genetic variation may partially account for subtle variation in lvm or parameters or heart parameters in newborns [37, 38]. to the best of our knowledge, the recent results have never been replicated, and therefore the replication of the study findings in different population is needed. additionally, an association between calm2 polymorphism, and dbp and map was found, but the mechanism by which this might act is not clear. blood pressure is regulated by multiple neuronal, hormonal, renal, and vascular control mechanisms, as well as genetic and environmental factors. it is also dependent, inter alia, on the force of contraction of the heart muscle which is connected indirectly to the left ventricular mass. there are many known candidate genes that have huge influence on the blood pressure or development of hypertension [3941]. however, the mechanisms of interaction intensifying effects of these genes are still researched. it is known that changes in signaling mechanisms in the endothelium of vascular smooth muscle (vsm) cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increase in vascular resistance. vascular tone that is a component of regulating blood pressure can be controlled indirectly by different genes activity. an experimental study demonstrated findings that expression levels of several cam-related proteins are changed in vascular tissues and suggested that cam-related proteins might be at least in part related to the pathogenesis of hypertensive vascular diseases. a recent study reported that camkii inhibitor inhibited the ang ii-induced vascular smooth muscle cell hypertrophy. however, the role of cam-related protein in vascular pathophysiology is not yet fully clarified., we have shown that the intergenic adjacent calm2 polymorphism is associated with left ventricular mass in newborns. this might be the consequences of variation in cell proliferation and growth, and this finding may indicate an important role for genetic variation of calm2 in expansion-induced heart growth in fetal life.

calmodulin ii (calm2) gene polymorphism might be responsible for the variation in the left ventricular mass amongst healthy individuals. the aim was to evaluate the correlation between left ventricular mass (lvm) and g.474955027g>a (rs7565161) polymorphism adjacent to the calm2 gene. healthy polish newborns (n=206) were recruited. two-dimensional m-mode echocardiography was used to assess lvm. polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism and sequencing analyses. the carriers of the g allele of the calm2 polymorphism had significantly higher left ventricular mass/weight (lvm/bw) values, when compared with newborns homozygous for the a allele (3.1 g/m2 versus 2.5 g/m2, padjusted=0.036). the ag genotype of calm2 was associated with the highest values of lvm/bw, exhibiting a pattern of overdominance (2.9 g/kg versus 3.1 g/kg versus 2.5 g/kg, padjusted=0.037). the results of this study suggest that g>a calm2 polymorphism may account for subtle variation in lvm at birth.

PMC3835711

pubmed-1238

according to the international continence society (ics), overactive bladder (oab) is a condition characterized by frequency (> 8 micturitions per day) and nocturia (waking one or more times at night to void) with or without urge incontinence (involuntary emptying of the contents of the bladder). many epidemiological and clinical studies have shown that oab affects the physical and mental health of patients and is an economic and social burden to patients. epidemiological and clinical studies have surveyed the symptoms and causes and the prevalence of oab. estimates of oab prevalence differ, but almost all studies have shown that its prevalence increases with age. there is also some evidence of an association with depression. many previous studies on oab have been limited to the elderly or to subjects with incontinence [4-6]. we conducted a cross-sectional study of subjects aged over 40 years in an urban and a rural region to measure the prevalence of oab in the community and assessed the risk factors for the condition. the study participants were 1,226 subjects aged over 40 years in guri city and yangpyeong county, south korea, who were approached in october and november 2010. the response rate was 74.2% (940 responders out of 1,226), and a few participants with incomplete questionnaire were excluded (n=14). therefore, a total of 926 subjects were included in the final analysis (fig. the protocol of this study was developed by the department of preventive medicine, hanyang university college of medicine. the survey was conducted with the overactive bladder symptom score (oabss) questionnaire (table 1). oab was defined according to the ics as urgency with or without urge incontinence, and usually with frequency and nocturia. in this study, oab was defined as over 2 points for the urgency score and 3 points for the sum of total scores in the oabss questionnaire. they included dwelling place (rural or urban area), marital status, education, behavioral factors (smoking, drinking, etc), and self-reported medical history, including hypertension, stroke, myocardial infarction, angina, hyperlipidemia, diabetes, osteoporosis, arthritis, tuberculosis, asthma, rhinitis, atopic dermatitis, cataract, hepatitis b, and depression. the individuals surveyed were divided into three groups in relation to smoking: a never smoking group (no history of smoking), an ex-smoking group, and a current smoking group. in the same way, individuals were classified in three categories with regard to drinking: the never-drinkers (no history of drinking), the ex-drinkers, and the current drinkers. categorical variables were analyzed by using a logistic regression model and were adjusted for age by using a logistic regression model. the characteristics of the subjects are shown in table 2. of the 926 subjects, 403 subjects (43.52%) were male and 81 were female (15.5%). a total of 130 subjects (14.1%) were diagnosed with oab, including 49 males (12.2%) and 81 females (15.5%). a total of 694 of the responders lived in the rural area (yangpyeong county), and 79 of them were diagnosed with oab; 332 responders lived in the urban area (guri city), and 51 of them were diagnosed with oab (p=0.20). the distribution of oab among individuals with various other characteristics is presented in table 2. according to educational status, the prevalence of oab was below 10% in those with an educational status of high school graduate or above; in those with less than a high school level of eduction, the prevalence of oab was over 90%. there were 28 people with a history of stroke, and the prevalence of oab with a history of stroke was 9/28 (32.1%). there were 83 patients with a history of osteoporosis, and the prevalence of oab with a history of osteoporosis was 23/83 (27.7%). there were 27 patients with a history of asthma, and the prevalence of oab with a history of asthma was 9/27 (33.3%). there were 100 subjects with cataract, of whom 32 (32.0%) met the criteria for oab. the prevalence of oab was not related to other risk factors except for those mentioned above. we used age-adjusted p-values to assess the risk factors for oab. educational status (p<0.0487), stroke (p<0.0414), osteoporosis (p<0.0208), asthma (p<0.0091), rhinitis (p<0.0150), and cataract (p<0.0008) were significantly associated with oab (table 3). other factors (dwelling place, marital status, smoking, drinking, hypertension, diabetes, hyperlipidemia, myocardial infarction, angina, tuberculosis, atopic dermatitis, hepatitis b, and depression) were not significantly associated with oab. the prevalence of oab reaches 16.6%, and increases to 22.1 to 41.9% in people over 70 years of age [7-10]. as part of the national overactive bladder evaluation program in the united states, a large-scale telephone survey was conducted of 5,204 respondents of both sexes aged 18 years. the total prevalence of oab reached 857/5204 (16.5%) and attained 25% or more in people aged over 65. the prevalence of oab with urge incontinence was 319/857 (6.1%) and the prevalence of oab without urge incontinence was 538/857 (10.4%). in addition, in japan, many epidemiologic surveys and clinical studies have shown that the prevalence reaches 12.4%; in individuals aged 70 to 79 years, it reaches 22.6%, and in those over 80 it reaches 36.8% [11-13]. the prevalence of oab was found to increase with age in countries including korea [14-16]. in our study, oab prevalence was a little higher than in the korean european prospective investigation into cancer and nutrition (epic) study. however, the study populations differed slightly between the korean epic study and our study; our study population consisted of community-based subjects. in particular, the korean epic study showed that storage lower urinary tract symptoms (luts) were more prevalent than voiding or post-micturition luts. in our study also, storage luts were more prevalent than other voiding symptoms. we obtained the same result for age dependence and also identified other risk factors, though age was the most important risk factor. the differences in oab prevalence between countries may be related to cultural factors including race and cultural circumstances. stewart et al. showed that the prevalence of oab without urge incontinence increased more steeply with age in men than in women and was significantly different by sex. in men, oab without urge incontinence increased approximately three-fold, whereas oab without urge incontinence gradually increased in women less than 44 years of age and reached a plateau in women over the age of 44 years. in our study, the prevalence of oab by sex was not significantly different. this was probably because of differences between the populations studied in the united states and those studied in korea. studies of other risk factors in japanese persons over 70 found that depression (multiple adjusted odds ratio=2.07), a recent drinking history, and obesity (body mass index) were significantly linked to the prevalence of oab. in our study these variables were not risk factors, probably because of differences between the populations studied in japan and in korea, especially in terms of age. in our study, the age-adjusted p-value for depression was 0.0671. other studies have examined the relationship between oab and lifestyle, especially smoking, alcohol use, and diet, and reported that controlling these factors improved or prevented oab. in the present study, however, residential area, marital status, smoking, alcohol consumption, hypertension, diabetes, hyperlipidemia, myocardial infarction, angina, tuberculosis, atopic dermatitis, and hepatitis b were found to be unrelated to the risk of oab. this difference may be due to differences in diet and national make-up between the groups surveyed. previous studies have, like ours, found a clear relationship between lower educational status and risk of oab. there is also a close relationship between socioeconomic status and educational level and it is the former that probably relates most directly to the risk of oab. persons with a higher educational level are more likely to seek better health behaviors and may have healthier lifestyles, whereas persons with a lower educational level may have a higher prevalence of smoking, poor diet, more labor, and exposure to toxins. the prevalence of oab in the general population is sizeable; considering the odd ratio, it could be over-estimated. second, the oab group identified by the questionnaire did not receive any clinical follow-up. despite these limitations, however, our study has clarified the prevalence of oab in korea and has identified several associated risk factors. this study aimed at surveying the prevalence of oab in the community and the multiple risk factors for oab. analyzing the risk factors for oab may help in the diagnosis of oab. use of these risk factors may be helpful for creating new criteria for oab with multiple risk factors. the prevalence of oab in the community in korea was 14.1% and the identified risk factors for oab were age, educational status, stroke, osteoporosis, asthma, rhinitis, and cataract. knowledge of these risk factors should facilitate the diagnosis and also the treatment of oab.

purposeto evaluate the risk factors for overactive bladder (oab) in a population aged 40 years and over in the community. materials and methodswe conducted a community-based survey of oab in a population aged 40 years and over in guri city and yangpyeong county, south korea, by use of the overactive bladder symptom score (oabss) questionnaire. a total of 926 subjects were included in the final analysis. the definition of oab was more than 2 points for the urgency score and 3 points for the sum of scores. in addition, the subjects were asked about age, dwelling place, marital status, educational status, behavioral factors (smoking, drinking, etc), and medical history. categorical variables were analyzed by using the logistic regression model and were adjusted for age by using the logistic regression model. resultsoverall oab prevalence was 14.1% (130/926), made up of 49/403 males (12.2%) and 81/523 females (15.5%). oab prevalence increased with age (p<0.0001). risk factors for oab were educational status (age-adjusted p=0.0487), stroke (p=0.0414), osteoporosis (p=0.0208), asthma (p=0.0091), rhinitis (p=0.0008), and cataract. other factors (dwelling place, marital status, smoking, drinking, hypertension, diabetes, hyperlipidemia, myocardial infarction, angina, tuberculosis, atopic dermatitis, hepatitis b, and depression) were not associated with oab. conclusionsthe prevalence of oab in our study was about 14.1% and the risk factors for oab were educational status, stroke, osteoporosis, asthma, rhinitis, and cataract. knowledge of these risk factors may help in the diagnosis and treatment of oab.

PMC3427838

pubmed-1239

phlegmon is a spreading diffuse inflammatory process associated with the formation of a suppurative exudate or pus. phlegmonous infection may involve any gastrointestinal tract site, although the stomach is most frequently involved (1). however, phlegmonous involvements of the esophagus, small bowel, or colon are rare (1-3). therefore, strong suspicion and recognition of this disease is a key to the diagnosis and prompt management of patients with acute symptoms. however, the role of surgery has been questioned for the diffuse disease form (2). the authors report a rare case of acute diffuse phlegmonous esophagogastritis. in this case, proper radiologic diagnosis with typical chest computed tomography (ct) findings (4,5) enabled appropriate treatment and timely surgical intervention. a 48-yr-old man presented with left chest pain, abdominal pain, and dyspnea of three days duration. five days before admission, he had been involved in a minor motorcycle accident, but was asymptomatic for two days. however, his vital signs were stable; heart rate 70/min, respiration 37/min, blood pressure 110/80 mmhg, and body temperature 36.8. a physical examination also revealed no remarkable finding with normal bowel sounds and a soft, flat abdomen with no general or rebound tenderness. laboratory tests revealed; wbc 3,200/l, c-reactive protein 31.68 mg/dl, and serum glucose 201 mg/dl, and chest radiography on admission showed mediastinal widening and bilateral pleural effusion (fig. the patient underwent endoscopy on the admission day to exclude esophageal rupture, and diffuse thickening of mucosal folds with decreased distensibility and an 1 cm sized mucosal ulcer in upper thoracic esophagus were observed with scattered patches of hemorrhage in the gastric mucosa of the body and antrum. on the evening of first hospital day, the patient was became febrile with a body temperature of 39, and thus, empirical treatment with broad spectrum antibiotics was immediately started under the suspicion of empyema or secondary infection. on the second hospital day, the dyspnea worsened and the amount of left pleural effusion increased on chest radiography. left closed thoracostomy was performed with pus drainage. on the forth hospital day, a contrast-enhanced chest ct scan was performed and showed diffuse and marked circumferential wall thickening of the entire thoracic esophagus, extending to gastric cardia and associated with diffuse intramural low density and a peripheral enhancing rim (fig. a ct diagnosis of acute phlegmonous esophagogastritis was suggested and bilateral open thoracotomies were performed immediately. pleural fluid analysis revealed exudates and a surgery was decided due to worsening of clinical condition of the patient and radiologic findings. during surgery, bilateral multiloculated pleural effusions were evacuated through open thoracotomies and the esophagus was freed from adjacent tissue. the adventitial and muscular layers of the esophagus were intact and no perforation was identified. several separate esophageal myotomies were performed and the submucosal layer was found to have been filled with thick, cheesy materials, which were removed from the mucosa by scraping. blood and sputum cultures of the patient and a microbiologic examination of pleural fluid demonstrated klebsiella pneumoniae. a follow-up chest ct scan performed on the 49th postoperative day showed reduced diffuse esophageal wall thickening and bilateral pleural effusion (fig. 2c). phlegmonous infection can affect any site of the gastrointestinal tract, although the stomach is most frequently involved (1-3). involvements of other sites have been rarely reported, but the simultaneous involvement of esophagus and stomach has only been reported in a very limited number (1, 2). phlegmonous infection usually involves the submucosa and not the mucosa (1-3). diffuse esophagogastric involvement of phlegmonous inflammation was evident in our case by chest ct and during surgery. in its localized form, an area of acute inflammation in submucosa phlegmonous infection may also present as a mass in the gastric wall (6). the inflammation produced may involve the muscularis mucosa and the serosa, and lead to perforation or even peritonitis (1). in its diffuse form, phlegmonous infection can involve the entire stomach, but it rarely extends beyond the cardia or pylorus (6). reported predisposing factors (4) include immune suppression, alcoholism, peptic ulcer disease, chronic gastritis or some other gastric mucosal injury, achlorhydria, infection, connective tissue disease, and malignancy. presumably, these conditions predispose the stomach to infection by eliminating various defense mechanisms, such as, inherent gastric cytoprotection or the bactericidal effect of gastric acid (1, 6). nevertheless, approximately 50% of reported cases were previously healthy and had no significant anteceding risk factors (6). we consider that uncontrolled diabetes mellitus and a recent history of chest trauma in combination with excessive alcohol consumption played an important role in the development of the disease in our patient. histopathologically, the submucosa is thickened and infiltrated by neutrophils and plasma cells with intramural hemorrhage, necrosis, and thrombosis of submucosal blood vessels (1). the most common pathogens are streptococcus species, staphylococcus species, escherichia coli, haemophilus influenzae, proteus, and clostridia (6). streptococcus accounts for approximately 70 to 75% of cases, and it is also the organism most commonly associated with death caused by phlegmonous gastritis. the causative pathogen of phlegmonous infection in our patient is believed to be klebsiella pneumoniae, based on positive culture results on blood, sputum and pleural fluid. phlegmonous gastritis has rarely been diagnosed before surgery, because it is seldom considered in the differential diagnosis of an acute abdomen (3). other symptoms include nausea, vomiting, hematemesis, hiccups, prostration, and fever (6). on the other hand, when the esophagus is involved, odynophagia, dysphagia, and chest pain are the most common symptoms (1). phlegmonous infection is usually diagnosed at surgery or at autopsy (3), and because there are no pathognomonic signs or symptoms, phlegmonous gastritis is rarely diagnosed before surgery. endoscopy of the affected esophagus shows diffuse luminal narrowing with poor distensibility and ulcer-like lesions (3). endoscopic ultrasonography (eus) findings in previous case reports were diffuse thickening with hypoechoic lesions in the submucosal layer (1, 6). the endoscopic examination of our patient also revealed similar findings of diffuse mucosal fold thickening, a mucosal ulcer in the upper thoracic esophagus, and scattered hemorrhagic patches in gastric mucosa. the reported ct findings of acute phlegmonous esophagitis or gastritis include diffuse esophageal and stomach wall thickening with circumferential intramural low attenuation surrounded by a peripheral enhancing rim. the intramural low attenuation represents severe inflammation and abscess localized to the submucosa and muscularis layer (4, 5). within the thickened wall, contrast-enhanced chest ct in our patient showed findings typical of acute phlegmonous inflammation with simultaneous esophageal and stomach involvement. the radiographic differential diagnoses included a dissecting intramural hematoma and tubular duplication of esophagus and emphysematous esophagitis or gastritis (5, 7). however, the clinical symptom of a dissecting intramural hematoma is chest pain with no evidence of infection or inflammation, and patients with tubular duplication are likely to have no symptoms or signs. the overall mortality of phlegmonous gastritis in a review of 36 reported cases was 42%, and the mortality rates of the 10 patients that underwent surgical resection as compared with the 26 patients treated conservatively were 20% (2/10) and 50% (13/26), respectively (6). during the last 50 yr, some reports have described patients with phlegmonous gastritis successfully treated with medical therapy alone. overall, the mortality rate for patients with medially treated localized disease was 17%, whereas that for diffuse disease was 60%. thus, antibiotic therapy and surgical drainage are effective treatments for acute phlegmonous esophagitis depending on the clinical situation (1, 3, 8). in cases of phlegmonous esophagogastritis, protracted conservative treatment result in surgical resection due to the possibilities of esophageal necrosis, esophageal stricture, gastric mucosal atrophy, and complicated peritonitis (1, 8, 9). our patient was initially treated with broad spectrum antibiotics and left closed thoracostomy with empyema drainage. however, surgical intervention was decided upon due to a worsening of his condition, the duration of his clinical symptoms, and radiographic findings, which included a proper ct diagnosis of acute phlegmonous esophagogastritis. we consider that combined medical treatment and timely surgical intervention played an important role in the cure achieved in our patient, who experienced no major post-operative complications. in conclusion, although acute phlegmonous esophagogastritis is rare and a preoperative diagnosis is difficult, awareness of this disease entity and prompt diagnosis based on typical chest ct findings are major key factors to successful treatment.

acute phlegmonous infection of the gastrointestinal tract is characterized by purulent inflammation of the submucosa and muscular layer with sparing of the mucosa. the authors report a rare case of acute diffuse phlegmonous esophagogastritis, which was well diagnosed based on the typical chest computed tomographic (ct) findings and was successfully treated. a 48-yr-old man presented with left chest pain and dyspnea for three days. chest radiograph on admission showed mediastinal widening and bilateral pleural effusion. the patient became febrile and the amount of left pleural effusion is increased on follow-up chest radiograph. left closed thoracostomy was performed with pus drainage. a ct diagnosis of acute phlegmonous esophagogastritis was suggested and a surgery was decided due to worsening of clinical condition of the patient and radiologic findings. esophageal myotomies were performed and the submucosal layer was filled with thick, cheesy materials. the patient was successfully discharged with no postoperative complication.

PMC2946669

pubmed-1240

a lot of research has been done in the past, and still research is going on to explore tools and techniques for regeneration of lost tissues as a result of the disease process. the use of various grafts and recent tissue engineering techniques including stem cell research are testimony to the ever increasing need for most suitable treatment option to replace/repair lost tissues due to various pathologic processes. the use of autogenous periosteum in general medical treatment has been extensive and has shown promising results [13]; on the contrary in dentistry, the use of periosteum as a regenerative tool has been limited and highly underrated; therefore, the purpose of this paper is to highlight the current status of use of periosteum in dentistry as well as suggesting its future use in various treatment options related specifically to dental field. the periosteum is a highly vascular connective tissue sheath covering the external surface of all the bones except for sites of articulation and muscle attachment (figure 1). the periosteum comprises of at least two layers, an inner cellular or cambium layer, and an outer fibrous layer. the inner layer contains numerous osteoblasts and osteoprogenitor cells, and the outer layer is composed of dense collagen fiber, fibroblasts, and their progenitor cells; osteogenic progenitor cells from the periosteal cambium layer may work with osteoblasts in initiating and driving the cell differentiation process of bone repair characterized by the development of the initial fracture callus and subsequent remodeling. periosteum can be described as an osteoprogenitor cell containing bone envelope, capable of being activated to proliferate by trauma, tumors, and lymphocyte mitogens. research on the structure of periosteum has shown that it is made up of three discrete zones. zone 1 has an average thickness of 1020 um consisting predominantly of osteoblasts representing 90% of cell population, while collagen fibrils comprise 15% of the volume. the majority of cells in zone 2 are fibroblasts, with endothelial cells being most of the remainder. zone 3 has the highest volume of collagen fibrils and fibroblasts among all the three zones. the morphology of fibroblasts is variable across the three zones (figure 2). it is thicker, more vascular, active, and loosely attached as compared to adults where it is thinner, less active, and firmly adherent. in all age groups, the cells of the periosteum retain the ability to differentiate into fibroblasts, osteoblasts, chondrocytes, adipocytes, and skeletal myocytes. the tissues produced by these cells include cementum with periodontal ligament fibers and bone. the periosteum has a rich vascular plexus and is regarded as the umbilical cord of bone. the vasculature system of the periosteum was first studied in detail by zucman and later by eyre-brook .bourke's studies showed that the capillaries supplying blood to bone reside within the cortex linking the medullary and periosteal vessels; a recent study has even shown that periosteal cells release vascular endothelial growth factor which promote revascularization during wound healing. recently, studies have reported the existence of osteogenic progenitors, similar to mesenchymal stem cells (mscs), in the periosteum [12, 13]. under the appropriate culture conditions, the periosteum can be easily harvested from the patient's own oral cavity, where the resulting donor site wound is invisible. owing to the above reasons, the periosteum offers a rich cell source for bone tissue engineering; hence, the regenerative potential of periosteum is immense. developing bone substitutes for bone defect repair has inspired orthopedic surgeons, bone biologists, bioengineering researchers to work together in order to design and develop the promising products for clinical applications. duhame in the year 1742 can be considered the first investigator to study the osteogenic potential of periosteum and published his findings in the article sur le development et la crueded os des animaux. a century later, another french surgeon, ollier, discovered that the transplanted periosteum could induce de novo bone formation. one of the earliest experimental studies to demonstrate osteogenic potential of periosteum was that of urist and mclean who reported that periosteum produced bone when transplanted to the anterior chamber of the eye of the rat. skoog subsequently introduced the use of periosteal flaps for closure of maxillary cleft defects in humans; he reported the presence of new bone in cleft defects within 36 months following surgery. since then, surgeons have reported the successful use of maxillary periosteal flaps [18, 19] as well as periosteal grafts from the tibia or rib. melcher observed that new bone is laid down in parietal bone defects of rats and was deposited by periosteum that had not been previously elevated or disturbed in any other way, while other investigators have suggested that the contact between the periosteal flap or graft and the underlying bone is crucial to stimulation of osteogenesis [21, 22]. more recently, the osteogenic/chondrogenic capacity of periosteum and related mechanisms have been confirmed, and the underlying biology is better understood through a number of studies [2340]. various research papers have been published explaining the osteogenic potential of human periosteal grafts [41, 42]. the use of periosteum as a gtr has been suggested by many studies [4346], although long-term results are still awaited to establish the regular and the most effective use of periosteal grafts as barrier membranes. the need for a graft, which has its own blood supply, which can be harvested adjacent to the recession defect in sufficient amounts without requiring any second surgical site and has a potential of promoting the regeneration of lost periodontal tissue is a long-felt need. the adult human periosteum is highly vascular and is known to contain fibroblasts and their progenitor cells, osteoblasts and their progenitor cells, and stem cells. in all the age groups, the cells of the periosteum retain the ability to differentiate into fibroblasts, osteoblasts, chondrocytes, adipocytes, and skeletal myocytes. the tissues produced by these cells include cementum with periodontal ligament fibers and bone; in addition the presence of periosteum adjacent to the gingival recession defects in sufficient amounts make it a suitable graft. recent papers published have shown promising results with the use of periosteum in the treatment of gingival recession defects (figure 3) [47, 48]; moreover, with the advancement in tissue engineering techniques the periosteal derived stem cells have been grown effectively to reconstruct lost tissues. periosteum-derived progenitor cells may serve as an optimal cell source for tissue engineering based on their accessibility, ability to proliferate rapidly, and capability to differentiate into multiple mesenchymal lineages. the periosteum is a specialized connective tissue that forms a fibrovascular membrane covering all bone surfaces except for that of articular cartilage, muscle, and tendon insertions and sesamoid bones. cells residing within the periosteum may be excised from any number of surgically accessible bone surfaces; in addition, when properly stimulated, the periosteum has the potential to serve as a bioreactor supporting a dramatic increase in the progenitor cell population over the course of a few days. further, once the cells are removed from the periosteum, they have the potential to proliferate at much higher rates than bone marrow, cortical bone, or trabecular bone-derived progenitor cells. in addition to their robust proliferation aptitude, it is well established that periosteum-derived progenitor cells have the potential to differentiate into both bone and cartilage. further, their potential for regenerating both bone and cartilage constructs is superior to that of adipose-derived progenitor cells and comparable with that of bone marrow-derived mesenchymal stem cells. a recent study by de bari et al. indicates that periosteal progenitor cells are able to differentiate not only into bone and cartilage cells but also into adipocyte and skeletal myocyte cells. there is a growing requirement for dentists to regenerate alveolar bone as a regenerative therapy for periodontitis and in implant dentistry. concerning the donor site, it is easier for general dentists to harvest periosteum than marrow stromal cells, because they can access the mandibular periosteum during routine oral surgery; also the regenerative potential of periosteum has been effectively used in osteodistraction which has the benefit of simultaneously increasing the bone length and the volume of surrounding tissues. although distraction technology has been used mainly in the field of orthopedics, early results in humans indicated that the process can be applied to correct deformities of the jaw. these techniques are now utilized extensively by maxillofacial surgeons for the correction of micrognathia, midface, and fronto-orbital hypoplasia in patients with craniofacial deformities. the use of periosteum can revolutionize the success of various dental treatments which require either bone or soft tissue regeneration; particularly the future use of periosteum must be explored in periodontal and implant surgical procedures. although the regenerative potential of periosteum has been proved by numerous studies, till date the use of periosteum-derived grafts has still not become a standard tool in the armamentarium of dental surgeons, and it may still need some time, and further research before the full regenerative potential of periosteum is utilized .

the ultimate goal of any dental treatment is the regeneration of lost tissues and alveolar bone. under the appropriate culture conditions, periosteal cells secrete extracellular matrix and form a membranous structure. the periosteum can be easily harvested from the patient's own oral cavity, where the resulting donor site wound is invisible. owing to the above reasons, the periosteum offers a rich cell source for bone tissue engineering; hence, the regenerative potential of periosteum is immense. although the use of periosteum as a regenerative tool has been extensive in general medical field, the regenerative potential of periosteum is highly underestimated in dentistry; therefore, the present paper reviews the current literature related to the regenerative potential of periosteum and gives an insight to the future use of periosteum in dentistry.

PMC3179889

pubmed-1241

infections are currently one of the major causes of morbidity and mortality in patients undergoing hematopoietic stem cell transplantation (hsct). one of the most prevalent bacterial infections in these patients is due to gram-positive organisms which have been rising during last decade. hence many of these patients require empirical antibiotic with aerobic gram positive coverage when developing neutropenic fever. recent update of clinical practice guideline by the infectious diseases society of america (idsa) for the use of antimicrobial agents in neutropenic patients with cancer, does not recommend vancomycin (or other agents active against aerobic gram positive cocci) as a standard part of the initial antibiotic regimen for fever and neutropenia. these agents have been suggested as an integral part of the empirical management of febrile neutropenia for specific clinical indications, including suspected catheter-related infection, skin or soft-tissue infection, pneumonia, or hemodynamic instability. adequate empirical antibacterial therapy in febrile neutropenia after hsct may reduce infection-related morbidity and mortality. when deciding to use an antibiotic active against aerobic gram positive cocci as an empirical treatment in febrile neutropenic patients who have underwent hsct, vancomycin is often used concerning its availability and cost. serum vancomycin concentrations should be monitored to minimize the risk of development of microorganism resistance and to avoid potential concentration-dependent adverse events. it is especially important in hsct patients who often receive vancomycin for longer duration and are under therapy with other nephrotoxic drugs. optimal vancomycin dosing regimen for empirical treatment of febrile neutropenia in these patients has not been defined and therefore managing the clinical use of vancomycin in this population with complicated medical problems, is very challenging. pharmacokinetic studies in patients with cancer have shown an increase in volume of distribution (vd) and clearance (cl) of vancomycin. moreover such pharmacokinetic changes during neutropenia and fever necessitate higher vancomycin doses and routine dosing regimen would be sub optimal in many of these patients. on the other hand, recent recommendations by a consensus statement from three groups, the american society of health-system pharmacists, idsa, and the society of infectious diseases pharmacists consisted increasing vancomycin doses to form elevated target trough levels (15 20 mg/l) especially in severe infections like pneumonia and bacteremia which are common during febrile neutropenia in high risk hsct patients. determining initial vancomycin dosing regimen in this population of patients is one of the mentioned challenges. furthermore a single vancomycin dosing regimens can not be applied to all patient populations and it becomes more important to initiate regimens with a good understanding of population-specific pharmacokinetic parameters. to the best of our knowledge, despite widespread use of vancomycin in hsct, there is just one study regarding adult patients who underwent autologous hsct. even though, there are some studies evaluating the pharmacokinetic of vancomycin in cancer and hematological malignancies. the purpose of this study was to investigate vancomycin pharmacokinetic parameters in hsct patients and to evaluate current dosing regimen based on trough vancomycin concentration measurement. this prospective study included patients who were treated with vancomycin for neutropenic fever after hsct, in the adult (> 15 yrs) hsct unit at hematology-oncology and stem cell transplant research center/ tehran university of medical sciences (shariati hospital), between december 2012 and april 2013. the inclusion criterion was receiving at least 3 successive doses of vancomycin (fixed dose and dosing interval) as empiric treatment of febrile neutropenia. patients, for whom vancomycin was discontinued prior to achieving a steady state, were excluded. blood samples (5 ml) were collected from central vein and sent to the laboratory within two hours of collection. first steady-state trough vancomycin serum concentrations were measured in blood samples which were drawn within 30 minutes prior to the administration of the fourth dose (css trough or pre-dose sample). samples collected 60-180 minutes after end of vancomycin infusion were used for determination of css peak (post dose sample). random steady state vancomycin serum concentrations were measured in some patients, instead of determining peak levels. serum concentrations of vancomycin were analyzed by fluorescence polarization immunoassay (fpia) (cobas integra 400 system from roche diagnostics, switzerland). the lower detection limit of this assay was 0.74 g/ml, and the coefficients of variation (cv%) were 3.0% at 8.70 g/ml, 2.2 at 26.3 g/ml, and 3.3% at 54.6 g/ml. for each patient, the data including concomitant medications, patient weight, height, sex, age, daily laboratory data (such as serum creatinine, bun and albumin), vancomycin dosage and serum sampling histories, including the date, time, dosage, and duration of infusion were registered. creatinine clearance was calculated using the cockcroft and gault equation using the ideal body weight. only the first course of therapy was analyzed in patients who received more than one course of therapy with vancomycin. the akin definition was used to identify acute kidney injury (aki) during vancomycin therapy. severity of kidney injury in patients who developed aki was staged according to the akin criteria. individual vancomycin pharmacokinetic parameters including elimination rate constant (k, in hour-1), elimination half-life (t1/2, in hour), apparent volume of distribution at steady state (vd, l/kg) and clearance (cl, l/h/kg) were determined assuming a one-compartment model using the following equations: ke=(ln cssmax-ln cssmin)/-t cl=kev v=d/t(l-e-ket)ke[cssmax-(cssmine-ket)] ke is the elimination rate constant (in hour-1), css max and css min are peak and trough concentrations (in mg/l) at steady-state as described above, vd is apparent volume of distribution at steady state in l, d is the administered vancomycin dose (mg), t and are the infusion time and dosage interval (hr). in cases that trough and a random concentration were measured following equation was used to calculate css max and then above equation were used to calculate parameters. cssmax=c1/e-ket) c1 is the random steady-state concentration, ke is the elimination rate constant, and t is the time between c1 and css max. correlations between patients demographic and clinical characteristics and vancomycin pharmacokinetic parameters were investigated using bivariate correlations procedure including pearson's correlation coefficient or spearman's rho based on data distribution. calculated parameters between males and females were compared by mann-whitney u test or independent-sample t test. median and inter-quartile of pharmacokinetic parameters range are also reported. to compare the values of this study with other reports, mean and 95% confidence interval of the mean pharmacokinetic parameters were determined. total of 46 patients (mean age of 32.9 12.45), 30 men and 16 women, were included in the study. patients demographic data patients clinical characteristics of the 46 patients, 13 (28.2%) were more than 30% above their ideal body weight. among 20 patients in whom pharamacokinetic parameters calculated, 7 (35%) patients were more than 30% above their ideal body weight (ibw). the most popular dosing regimens were 1000 mg q12hr in 32 patients (69.6%) and 1000 mg q8hr in 8 patients (17.4%). mean (sd) vancomycin total daily dose was 31.9 (10.5) mg/kg. a total of 76 vancomycin serum concentration (46 trough, 18 peak and 2 random levels) were measured of which, 18, 2 and 26 patients had both peak and trough samples, both random and trough samples and only trough samples respectively. median (inter-quartile range) steady-state peak and trough concentrations in mg/21 patients (45.7%) had trough concentrations above 10 mg/l. of these, 9 patients (19.6% of all patients) had trough concentrations above 15 mg/l, 5 of whom (10.9% of all patients) had trough concentrations above 20 mg/l. 25 (54.3%) patients had trough concentrations of<10 mg/l and 6 patients (13%) had trough levels of<more than 90% of measured vancomycin trough concentrations were outside the range of 15-20 mg/l. about 38.9% of measured peak levels were either greater than 40 mg/l or lower than 20 mg/l. for 20 patients who had peak (or random) and trough measurements, summary of pharmacokinetic parameters calculated for 20 patients of the study vancomycin pharmacokinetic parameters did not differ significantly between males and females. values of pharmacokinetic parameters with assumption of one-compartment pharmacokinetic model for vancomycin in this study and different studies on similar populations are shown in table 4. 95% confidence interval for mean of vancomycin cl and vd were calculated and compared between our results and other studies on similar population to evaluate differences. mean vancomycin vd in our patients is smaller than those observed in two studies on patients with cancer and hematological malignancies but mean vancomycin cl does not differ significantly (table 4). one-compartment pharmacokinetic parameters (mean standard deviation (95%ci of mean)) of vancomycin from some studies on cancer patients versus this study (boldface) sixty-five percent of patients were leukemic, and the other patients were from other clinical units. correlation of different demographic and clinical factors with vancomycin pharamacokinetic parameters in enrolled patients was investigated. creatinine clearance of patients on day of vancomycin sampling was correlated with vancomycin clearance (p<0.01). 38 of 46 patients (82.6%) were on nephrotoxic drugs concurrent with vancomycin. among them, 16 patients (42.1%, 34.8% of all included patients) and 22 patients (57.9%, 47.8% of all included patients) received one and two nephrotoxic drug(s) concomitant with vancomycin respectively. of 46 patients, 21 patients (45.7%) developed acute kidney injury (aki) during vancomycin therapy. among patients who developed aki, 4 and 15 17 patients did not develop aki, even though they were also on nephrotoxic drugs. of 21 patients who developed aki, 19 patients were akin stage one and 2 patients were akin stage two respectively. two patients who developed akin stage two aki were on cyclosporine and amphotericin b concurrent with vancomycin therapy. of 19 patients who developed akin stage one aki, 2 of them did not receive concurrent nephrotoxic drug but remainder were on concurrent nephrotoxic drugs. 13 of these 17 patients were on 2 nephrotoxic drugs, cyclosporine and amphotericin b, concurrent with vancomycin. we conducted our study in order to evaluate vancomycin pharmacokinetics in patients undergoing hsct and to determine if the changes in pharmacokinetic parameters seen in previous studies in cancer and febrile neutropenic patients in different countries are evident in our patients. several studies showed that vancomycin cl and vd tends to be higher in patients with malignancies and during febrile neutropenia., have shown that patients with neutropenia have an increased total clearance of vancomycin compared with both intensive care unit and control patients, and an increased vd compared with controls. these results were confirmed by le normand et al., who further found that the elimination half-life of vancomycin in patients with neutropenia was twice as short as in healthy individuals. buelga et al., also reported greater vd (26 to 42%) and cl of vancomycin in patients with hematological malignancies relative to other adult patients population. in a study by al-kofide et al., on comparison of vancomycin pharmacokinetics in cancer (88% leukemic) and non-cancer patients, both vd and cl were significantly higher in the cancer group. based on these results, teramachi et al., also reported that cl and vd were significantly greater in the malignancy group than non-malignancy group in japanese patients. however, in their report some patients in the malignancy group showed similar values of cl and vd to those in the non-malignancy group. mean vancomycin cl in our patients is similar to results of above studies and nearly 70-80% higher than mean cl observed in adult medical&surgical patients. but vancomycin vd in our patients is lower than what were shown in studies on patients with cancer, hematological malignancies or neutropenic fever and is near to what observed in other medical patients. mean total body weight of our patients was not significantly different with patients included in above studies. in our study, patients were heterogeneous as they had different types of hematological diseases and malignancies. since our patients with hematological malignancies were in remission when they were admitted for hsct, they may affect vancomycin distribution and clearance during febrile neutropenia in a different way from patients who receive induction chemotherapy for their malignancy and develop neutropenic fever. moreover little is known how underlying non-malignant hematological diseases like thalassemia would affect vancomycin pharmacokinetics. some of the studies on patients with hematological malignancies that developed neutropenic fever included few patients who underwent autologous hsct but they were not evaluated separately. to the best of our knowledge, there is no data about vancomycin pharamcokinetics in patients who underwent autologous and allogeneic hsct. in order to investigate factors affecting vancomycin pharmacokinetics in our patients, some demographic and clinical characteristics of patients such as age, total body weight, gender, creatinine clearance, diagnosis and transplantation type were analyzed, and no significant correlation or effect on vancomycin pharmacokinetic parameters was found except for creatinine clearance, which were correlated with vancomycin clearance. this correlation would be expected in vancomycin that its main way of elimination is renal and was shown in other studies on cancer and hematological malignancy patients. non significance of other expected or presumed correlation may be due to heterogeneity in our patients underlying disease and small number of patients. al-kofide et al., recommended several theories regarding why vancomycin cl is significantly increased in this subgroup of patients: (1) glomerular filtration is the main mechanism of vancomycin elimination but there may be some tubular secretion which have been proved in previous trials on vancomycin pharmacokinetics, as this pathway may be enhanced in cancer patients leading to higher cl than expected; (2) vancomycin has some hepatic metabolism mainly through conjugation and this pathway of vancomycin deactivation may be increased in cancer patients leading to lowered vancomycin levels; (3) as a result of high amount of intravenous fluid given to those patients, urine flow may have increased leading to decrease in the re-absorption of vancomycin and enhancing its clearance. high vancomycin clearance that is observed in our patients could be resulted from these proposed mechanisms. median (inter-quartile range) and mean (sd) steady-state trough concentration in our patients were 9.59 (6.67) and 11.2 (7.4) respectively and 25 (54.3%) patients had trough concentrations of<10 mg/l. based on evidence suggesting that s. aureus exposure to trough serum vancomycin concentrations of<10 mg/l can produce intermediate resistant strains, recent idsa, ashp and sidp consensus guideline on vancomycin tdm, recommends that trough serum vancomycin concentrations always be maintained above 10 mg mg/l in patients with serious infection like pneumonia, bacteremia, meningitis and osteomyelitis. it is not clear whether this level would be recommended when vancomycin is used as empiric treatment of neutropenic fever and target vancomycin trough level for this indication is not defined in guidelines on neutropenic fever management in cancer patients. but in many febrile neutropenic patients who fulfill the criteria of starting vancomycin empirically, suspected infection is serious enough to dose vancomycin aiming at steady-state trough concentrations of at least 15 mg/l. in a vancomycin drug utilization review done by hayatshahi et al., in our center, it was shown that among patients in whom vancomycin administration was justified, 42.3% received appropriate dose. but in this study, vancomycin concentrations were not measured and clinical outcomes were not evaluated. in another study of vancomycin utilization evaluation at hematology-oncology ward of a teaching hospital that was conducted by vazin et al. this trough levels seem to be higher than our patients but comparison of 95% ci of means shows that this difference is not statistically significant. although patients included in above study received fix doses of 1000 mg q 12 hr, mostly as empirical treatment of neutropenic fever, only 3.6% of patients, far from our results, had trough vancomycin concentration less than10 mg/l. mean (sd) total daily dose of vancomycin was 31.9 (10.5) mg/kg/day in our study and it seems to be higher than 14.7 mg/kg/day that was administered in mentioned study. furthermore, mean sd (95% ci of mean) tbw of patients in above study is 68.05 12.26 kg (68.89-71.20) that seems to be lower than our patients with 74.83 16.6 kg (69.89-79.77) but this difference is not significant. on the other hand more than half of their patients had supra-therapeutic trough level which is shown in 10.9% of our patients. by the way, broad spectrum of trough levels among included patients despite fixed equal doses in the study done by vazin et al., which is shown to some extent in our study, confirms inter and intra individual variability of vancomycin pharmacokinetics which necessitates using individual or same population based pharmacokinetic approach in dosing vancomycin. based on these findings, we consider that pharmacokinetics of vancomycin may change in hsct patients from other patients and our patients need its unique population based pharamcokinetic approach in vancomycin dosing. furthermore, considering the observed inter individual variability of vancomycin pharmacokinetics, dosage should be adjusted and individualized based on drug concentrations. the most frequent dosing regimen in our patients was 1000 mg q12hr (69.6%) which is usually determined based on 15-20 mg/kg q 8-12 hr and often lower doses were chosen due to concerns about nephrotoxicity. patients who undergo hsct usually receive concurrent nephrotoxic drugs especially after transplantation and during neutropenia. this becomes more important in allogeneic hsct in which, patients receive a calcineurin inhibitor, most of the time cyclosporine in our center, as prophylaxis and treatment of graft versus host disease (gvhd). calcineurin inhibitors are nephrotoxic drugs and cyclosporine is more nephrotoxic than tacrolimus. in hsct patients if serum creatinine rises with any reason and becomes stable, might lead to changes in cyclosporine dosing regimen which can put the patients under the risk of acute gvhd. another nephrotoxic drug which might be administered in these patients is amphotericin b which induced aki in a dose dependent manner. although vancomycin is not considered a nephrotoxic drug, it can aggravate nephrotoxicity of other drugs. concomitant nephrotoxic agents can increase the incidence of vancomycin-associated nephrotoxicity by up to 35%. but it is shown in a recent systematic review that higher doses administered in order to achieve new target trough levels (15-20 mg/l) recommended by guideline in recent years, increases the risk of aki. but it is reported to be dependent on vancomycin therapy duration (mostly occurs after 7 days of therapy) and reversible. aki occures in 45.7% of our patients which seems to be higher than vancomycin induced aki rate reported in literature. on the other hand 36% of our patients most of patients who develop aki in our study were receiving concurrent cyclosporine and amphotericin b and this high rate of aki can not be related absolutely to vancomycin. moreover aki is a common early complication after hsct. in a study by saddadi et al., on aki in hsct patients in our center it is reported that 37.6% developed aki and higher frequency of aki was observed in patients who received cyclosporine a (40%), patients with allogeneic hsct(42.1%), and those who developed gastrointestinal gvhd (47.3%). schrier et al., showed the frequency of aki increased significantly from autologous hsct (21%) to non-myloablative allogeneic hsct (40%) to myeloablative allogeneic hsct (69%). correlation between vancomycin dose or concentration and aki rate and influence of concurrent nephrotoxic drug could not be shown in our study may be due to small number of patients. with regard to indeterminacy about optimal trough vancomycin concentration in hsct patients with neutropenic fever and existence of many predisposing factor to aki in these patients, we suggest that clinical and microbiological outcome and safety of dosing regimen versus different target trough vancomycin concentration (10-15mg/l or 15-20 mg/l), be assessed in a randomized clinical trial on these patients. on the other hand, not only therapeutic drug monitoring and dose adjustment is necessary in our patients population and is recommended by mentioned guidelines, but also initial dosing regimen determination method needs to be changed. this study had several limitations, as small sample size and including patients with heterogeneous underlying diseases and different hsct type. since blood sampling is limited in the clinical setting, therefore only one-compartment model could be used for pharmacokinetic analysis and this could be another limitation of the study. in summary, conventional vancomycin dosage regimens could not lead to recommended therapeutic serum concentrations in our patients although, optimal trough vancomycin concentration in febrile neutropenia in hsct patients needs to be defined. large variation in vancomycin pharmacokinetic parameters observed among patients of this study along with the difference of vancomycin pharmacokinetics between our patients and other similar studies further explain the need for level monitoring and individualization of vancomycin dosing. a population pharmacokinetic approach in determining vancomycin dosing for these patients needs to be described.

backgroundvancomycin is used abundantly in patients undergoing hsct, especially during neutropenic fever. despite its widespread use little is known about vancomycin pharmacokinetics in hsct patients. we conducted this study to investigate vancomycin pharmacokinetic parameters in our hsct patients and to evaluate current dosing regimen based on trough vancomycin concentrations measurement. methodsvancomycin serum concentration at steady-state was determined prospectively in 46 adult hsct patients who received vancomycin as empirical treatment of neutropenic fever. individual steady-steady pharmacokinetic parameters were also determined in 20 patients who had two vancomycin levels from an administered dose, assuming one-compartment model. acute kidney injury was also evaluated in our patients during vancomycin therapy. resultsmean (sd) apparent volume of distribution (l/kg) and clearance (ml/min) were 0.6 (0.33) and 109.7 (57.5) respectively. with mean (sd) total daily dose of vancomycin 31.9 (10.5) mg/kg/day that was administered, more than 90% of measured vancomycin trough concentrations were outside the range of 15-20 mg/l and 54.3% of patients had trough concentrations below 10 mg/l. of 46 patients, 21 patients (45.7%) developed acute kidney injury (aki) during vancomycin therapy; among them 19 patients were receiving nephrotoxic drug(s) concomitantly. conclusioncurrent vancomycin dosage regimen could not lead to recommended therapeutic serum concentrations in our patients. large variation in vancomycin pharmacokinetic parameters observed among patients of this study along with difference of vancomycin pharmacokinetics in our study and other similar studies further explain the need for therapeutic drug monitoring and individualization of vancomycin dosing.

PMC3915428

pubmed-1242

inflammatory bowel disease (ibd) is a group of chronic conditions of the colon and small intestine, consisting of crohn's disease (cd) and ulcerative colitis (uc), characterized by acute pain, vomiting, and diarrhea symptoms followed by remission. a single etiology has not been identified, but rather the pathogenesis of ibd is very complex and involves the external environment, genetic makeup, intestinal microbial flora, and immune system. although new and powerful medical treatments are available, many are biological drugs or immunosuppressants, which are associated with significant side effects, in particular infection and increased risk of malignancy, and elevated costs which require optimal medical treatment adjustment. as a result, major attempts have been made at identifying clinical characteristics, concurrent medical therapy, and serological and genetic markers as predictors of response to biological agents. only few reports exist on how mucosal/tissue markers are capable of predicting clinical behaviour of the disease or its response to therapy. due to its ability of interfering with intestinal barrier function and stimulating local and systemic inflammation, dextran sodium sulfate (dss) is often used as a mouse model of colitis which can mimic clinical and histological features of ibd with uc characteristics. most commonly, experimental colitis is induced by heparin-like polysaccharide dss because of its capacity of inducing colonic lesions.. showed that oral administration of 5% of dss in drinking water of balb/c mice was able to induce a chronic colitis after several cycles of dss. this study was followed by a report from cooper et al. who induced chronic colitis by (a) 7 days of oral dss followed by 7 days of h2o (for 1, 2, and 3 cycles) and (b) 7 days of oral dss followed by 14 and 21 days of h2o. the results of this study showed that chronic colitis induced after only 7 days of dss may serve as a useful model to study the effects of pharmacologic agents in human inflammatory disease and mechanisms of perpetuation of inflammation and gave an extensive description of histological lesions, showing that the main histological changes consisted of focal crypt loss, which was followed by signs of both acute and chronic inflammation. the present study aimed to investigate the effect of fr-91 on the attenuation of the chronic experimental colitis induced by dss in swiss mice. furthermore, we investigated if the chronic phase was characterized by a regulation in the expression of apoptotic genes and by a dysregulation of t helper 1 (th1)/t helper 2 (th2) balance and how this would relate to mucosal regeneration. twenty-six specific pathogen-free swiss cd1 female mice (7 weeks old; santiago de compostela's university animal breeding core, spain) were maintained (two or three per cage) in isolator plastic cages with shavings under standard laboratory conditions (sterilizable diet, 50% humidity, 23-24c temperature, and 12-h light/dark cycle). all mice were quarantined 3 weeks after arrival and then randomized by body weight into experimental and control groups. all mice were permitted free access to a commercial diet and treatment or normal drinking tap water in individual bottles. all procedures conformed to the guidelines established by the european communities council directive of 24 november 1986 (86/609/eec) and by the spanish royal decree 1201/2005 for animal experimentation and were approved by the ethical committee of ebiotec. the design of the present study was focused primarily on inducting colitis-associated dysplasia and/or ulcerative hallmarks by administering synthetic dextran sulfate sodium (dss) to mice and then treating them with different fr91 (standardized lysate of microbial cells belonging to the bacillus genus) dilutions as shown in figure 1. at the age of 7 weeks, the animals were divided into two control groups (1 and 2, n=4 each group) and three experimental groups (35, n=6 each group). along the entire experimental procedure, distilled water containing different fr91 dilutions (5% in group 3, 10% in 1 and 4, and 20% in 5) onwards, distilled water containing 20 g/l (2%) synthetic dextran sulfate sodium (dss; mol mass 5000; d4911, sigma-aldrich; mo, usa) was also administrated to animals of groups 25. for comparison, control groups 1 and 2 received dss or the fr91 treatment alone, respectively, as untreated control. all mice were sacrificed at the end of the experiment (8th week), at the age of 15 weeks. experimental colitis was induced to mice of groups 2, 3, 4, and 5, by repeated administrations of 2% (wt/vol; 20 this dose was empirically reported to induce moderate to severe colitis while minimize mortality in mice. none of the mice died before the termination of this experiment study at day 50. mice were deeply anesthetized with ether and intracardially perfused with saline buffer and then fixed by 4% paraformaldehyde in 0.1 m phosphate buffer (ph 7.4). the entire colorectum (from colocecal junction to the anal verge) was removed, measured, examined macroscopically, washed with saline buffer, and immediately fixed by immersion in the same fixative for 48 h. part of the colon was divided into three equal segments (proximal, middle, and distal), and portions were determined under a dissecting stereomicroscope (leica, m125). intestine portions were cryoprotected with 30% sucrose in 0.1 m phosphate buffer, embedded in oct compound (tissue tek, torrance, ca), and frozen with liquid-nitrogen-cooled isopentane. parallel series of transverse sections of 1416 m thick were obtained on a cryostat (starlet 2212, bright, uk) and mounted on superfrost plus (menzel-glser) slides. routine histological examination was performed on hematoxylin and eosin (h&e)-stained sections, where different morphological alterations that occur during inflammation- associated colorectal carcinogenesis (such as cryp abscess, mucosal dysplasia, adenomas, and adenocarcinomas), were identified and diagnosed according to previous published studies, see keohane et al.. to detect the expression of colorectal histopathological markers such as catenin-, p53, bcl-2, mlh1, and apc, we used immunohistochemical techniques. the sections were pretreated with h2o2 to eliminate endogenous peroxidase, rinsed twice in phosphate-buffered saline (pbs) at ph 7.4 (10 minutes each), and then sequentially treated with nonspecific binding blocked solution (0.1 m pbs containing 0.2% tween 20 and 15% normal goat serum from dako; glostrup, denmark) for 1 h, primary rabbit policlonal antibodies such as anti-catenin-, anti-p53, anti-bcl-2, anti-mlh1, and anti-apc antibodies (rabbit antibodies that were affinity purified from rabbit antiserum by affinity chromatography using mouse epitope-specific immunogen; bioworld technology, mn, usa; cat. bs3603, bs3736; bs1511, bs2418, bs1017 respectively, dilution 1: 200) overnight, pbs (two 10-min rinses), goat anti-rabbit igg serum biotinylated (dako, dilution 1: 100) for 1 h, pbs (two 10-min rinses), vectastain abc kit (vector laboratories, burlingame, ca, usa) for 1 h, and pbs (two 10-min rinses). as a negative control, omission of the primary, secondary or tertiary antibodies were used, and no immunostaining was observed. at the last step, the immunoreaction was developed with 0.005% diaminobenzidine (dab; sigma-aldrich) and 0.003% h2o2. all dilutions were made in pbs containing 0.2% tween 20, and incubations were made in a humid chamber at room temperature. finally, the sections were dehydrated, mounted, and coverslipped. antibody characterization and specificity. according to the technical information supplied by the manufacturer (bioworld technology, mn, usa), the primary antibodies used were raised against denatured mouse epitopes from rabbit antiserum, and they were affinity purified by chromatography using epitope-specific immunogen with purity higher than 95% (by sds-page). its specificity has been assessed by western blot; it recognizes a single-protein band of approximately 8690 kd (-catenin), 4345 kd (p53), 2628 kd (bcl-2), 8486 kd (mlh1), and 270280 kd (apc). moreover, antibodies have wide species cross-reactivity and were used for demonstrating their expression in mouse, rat, and human. lesions were classified as positive for catenin-/bcl-2/mlh1/apc if cytoplasmic/nuclear staining was detected, and p53 was considered positive if nuclear expression was detected. two different observers evaluated individually and independently the experimental group slides in a double-blind manner and achieved a high level of concordance. three sections of each mouse colorectal segment were coded and scored for lesions according to the extent ulceration (0, not present and 1 present), severity of lesions, hyperplasia, and area involved, graded as follow: 0, normal; 1, mild; 2, moderate; 3, severe. the sections were photographed with an olympus microscope (bx50) equipped with a color digital camera (dp10). the photographs were converted to gray scale and adjusted for brightness and contrast with corel draw (corel, ottawa, canada), and the plates composed with corel photo paint. quantitative detection of gm-csf, ifn-, il-1, il-2, il-4, il-5, il-6, il-10, il-17, and tnf- was performed by using a flowcytomix mouse th1/th2 10plex (bms820ff) from bender medsystems which allowed to measure multiple analytes in a single 50 microliter aliquot of mice sera. in brief, microparticle beads were dyed with differing concentrations of two fluorophores to generate distinct bead sets. captured analyte was detected using a biotinylated detection antibody and streptavidin-phycoerythrin (s-pe). a facscan flow cytometer from becton dickinson was used to acquire samples. for calculation of results, the mice that received repeated administrations of 2% dss and lower or absent levels of fr91 (groups 2 and 3) showed bloody stools during the second half of the experiment, (from the fifth week onwards), whereas no such internal inflammation feature was observed in the other mice groups. macroscopically, we have identified numerous gross inflammatory polypoid lesions in mice of groups 2 (6/6; 100%) and 3 (5/6; 83.3%), mainly on the middle and distal portions of the colorectal segment, and very few were observed in group 4 (1/6; 16.6%). remarkably, none of the mice group 5 (dss/20%fr91) showed any ulcer formation in the colorectal segment analyzed, as well as the treatment control mice group 1 (10%fr91) that was also free of colitic ulcerations. these macroscopical observations were confirmed by histological analysis of the intestinal morphology emphasizing the alterations regarding the integrity and inflammation of colonic mucosa and submucosa, dysplastic epithelium, and the presence of ulcers. this histological examination of transverse sections staining with h&e (figure 2) showed ulcers with moderate-to-severe morphological alterations (multifocal areas of inflamation in the submucosa or ulcers that covered large mucosal areas), mild-to-severe crypt hyperplasia (lining epithelium was two to three times normal thickness, marked hypercromasia of cells, and multiple crypts with arborizing pattern), epithelial dysplasia (alteration in the differentiation of epithelial cells that may progress to invasive carcinoma), and large affected areas of crypt loss. however, severity of these colorectal lesions differed significantly among mice groups, being the mice group 2 (dss) the one with a high severity level, while the mice group 3 (dss/5%fr91) showed a mild-to-moderate severe level, and the mice group 4 (dss/10%fr91) presented a few mild scattered lesions. no colitis characteristic lesions were observed in the colorectal segment of mice group 5 (dss/20%fr91) or in the treatment control mice group 1 (10%fr91), (figure 2), as described previously in the macroscopical exam. histological scoring data obtained from the colorectal lesions examination are presented in table 1. immunohistochemical techniques used to identify cell markers of the colorectal lesions showed expression of catenin- (cellular adhesion regulator), bcl-2 (apoptotic regulator), mlh1 (dna-mismatch repair), apc, and p53 (tumour suppressor proteins) in all colonic mice lesions observed and described above. catenin- antibody detected endogenous expression levels of catenin- protein mainly in the cytoplasm of adenocarcinoma and dysplastic cells. intense catenin- expression was observed in the proximal and middle (figure 3(f)) colon of mice group 2 (dss) whereas a moderate-to-intense immunoreactivity was also observed in the colorectal segments (figure 3(k)) of mice group 3 (dss/5%fr91). catenin--immunoreactive (-ir) cells were localized in the dysplastic criptal cells and adenocarcinoma cells, at the internal criptal layers (figures 3(f) and 3(k)). colorectal portions of mice groups 1, 4, and 5 showed a weak or absent catenin--positive reaction in their cryptal cells (figures 3(a), 3(p), and 3(u)), considered as catenin- cellular basal expression. bcl-2-ir and p53-ir cells were observed at colorectal portions of mice groups 2 (figures 3(g) and 3(j)) and 3 (figures 3(l) and 3(o)), showing an intense staining in adenocarcinoma and cryptal cells, particularly strong in mice group 2. immunoreactivity to bcl-2 and p53 was absent in colorectal section of mice groups 1, 4, and 5 studied (figures 3(b), 3(q), 3(v), 3(e), 3(t), and 3(y)). strong staining mlh1-ir cells were observed at colorectal segments of mice groups 2 (figure 3(h)) and 3 (figure 3(m)), showing an intense staining in adenocarcinoma and cryptal cells of the dysplastic epithelium, particularly intense in mice group 2. mlh1-ir cells were absent in colorectal section of mice groups 1, 4, and 5 studied (figures 3(c), 3(r), and 3(w)). apc immunoreactivity was intensely observed in all colorectal portions of mice groups 2 (figure 3(i)) and 3 (figure 3(n)) although cryptal cells of mice group 2 showed a stronger positivity. colorectal portions of mice groups 1, 4, and 5 showed a weak apc immunoreactivity in their cryptal cells (figures 3(d), 3(s), and 3(x)). the incidence of colon lesions showed by these cell markers in the group 5 was 8.2% (4%), whereas it was 38.2% (6%), 33.3% (6%), and 20.4% (3%) in groups 2, 3, and 4, respectively (see figures 4 and 5). similar histological incidence was observed in mice group 5 when compared with negative control group 1 (7.8%, 2%). two-factor anova of data for each immunohistological markers in the colorectal portion showed significant differences among experimental groups 1/5 and 2/3/4, (figure 5). group 5 was the most resistant mice group to dss-induced lessions in the colorectal portion, as indicated by the lowest value for each marker, similar to that observed in the control group 1. groups 2, 3, and 4 showed more susceptibility to dss-induced lessions, consistent with high values. significant group differences were found between group 1/5 and 2/3/4 in dss susceptibility, being correlated with differences in the fr91 mean consumption per group. we next considered potential mechanisms which might underlie the colitis exhibited by dss treatment and the effect of fr-91 in the treated mice groups. as shown in table 2, dss treatment increased only the production of ifn- proinflammatory cytokine. no significant changes were detected in il-1, tnf-, il-6, il-10, and il-17 in both treated and untreated groups. more than 20,000 bioactive compounds synthesized by microorganisms have been identified, and over 10,000 of these secondary metabolites are produced by actinomycets, representing 45% of all bioactive metabolites discovered. among actinomycetes, around 8,000 compounds are synthesized by streptomyces species. in addition, many of these compounds, such as anthracyclines (aclarubicin, daunomycin, and doxorubicin), peptides (bleomycin and actinomycin d), aureolic acids (mithramycin), enediynes (neocarzinostatin), antimetabolites (pentostatin), carzinophilin, mitomycins, and others [9, 10] have also been tested for the inhibition of chemically induced carcinogenesis in both in vitro and in vivo animal models. in the present study, we investigated the effect of fr91, a standardized lysate of microbial cells belonging to the bacillus genus which has been previously shown to have significant immunomodulatory effects tested on human tumor cell lines, on colonic inflammation induced by 8-week exposure of 2% dss in the drinking water and, in particular, whether fr91 affects colorectal inflammation. we found that the six-week treatment resulted in a slightly reduction of colorectal lesions at lower dosis (5% fr91) and a moderate-to-complete reduction at higher dosis (1020% fr91). moreover, histopathological data showed that fr91 has no pathological effect on the morphological organization of the mice colon tissues, as observed when administering fr91 alone during the entire experimental study. the data obtained suggests that fr91 may be an important chemopreventive agent against intestinal inflammation in mice colon. in our experiment, we induced a wide range of colorectal lesions to better evaluate the effects of an anticancer agent (fr91) on chronic ulcerative colitis in mice. the repeated administration of dss as an inductor of ulcerative lesions in mice models of colitis was extensively reported [1318], being essential in advancing our understanding of the complex interactions between the environment, genetics, and epithelial barrier dysfunction in the human-related inflammatory bowel disease [1922]. in this study, the use of dss in drinking water during five weeks resulted in epithelial damage and a robust inflammatory response, obtaining a valid mice model acute injury colon to test an eight-week treatment with fr91, as reported in previous studies [2326]. our results demonstrated that the optimal dose response was the 20% fr91 concentration tested in mice group 5, where no histological alterations or mild lesions were observed. routinary histological staining [20, 23, 24, 27] and pretumoral cell markers [18, 20, 23, 28] used to evaluate the severity of lesions confirmed the protective effect of fr91 against the inflammation effect of dss. these pretumoral cell markers such as apc, p53, mlh, bcl-2, catenin-, and cytokines, among others, take part in the molecular pathogenesis pathway of chronical colorectal inflammation that has been reported to derive at further stages in the development of sporadic colorectal carcinoma and colitis-associated colon cancer [22, 2931]. the present results show the same significant interaction of these genetic markers in the pathologic characterization of the dss-induced lessions observed. as reported in similar studies, a dosis-dependent effect is normally associated with the chemoprevention process in dss-induced colitis [25, 26]. therefore, we tested other lower fr91 concentrations in drinking water, obtaining gradual colorectal lesions as the fr91 concentrations decreased. moreover, we also tested the effects of fr91 in mice during a large period of time (8 weeks), obtaining similar histological results as the nontreated mice, which indicates a chemopreventive action against carcinogenesis without interfering with healthy epithelial structures of the mice colon. chemopreventive effects of a wide range of compounds on colonic tumors induced in mice have been reported previously, such as organosulfur compounds, n acetylcysteine, tetrandrine, phytosteryl ferulates, and dibenzoylmethane derivatives. the dss-induced lessions reported in these studies are similar to that observed in the preset work, where generally predominated in the midcolon [7, 35] and distal [24, 36] intestinal portions. these particular locations have been attributed to several factors such as the selective uptake of dss in certain colonic portions, the presence of weak intestinal barrier regions, and different distribution of macrophages populations along the mice colon. although the real impact of these findings in the pathogenesis of ibd remains a controversial issue, as it is unclear whether they are primary or secondary factors involved in the regulation of the mucosal intestinal immune system, they can be considered as markers in the differentiation of groups of patients. in active ibd, an unbalance between regulatory and effector cells has been described, which mainly involves effector t cells (th1 and th2) and regulatory t cells (tregs, th3). cd is associated with a th1 t-cell cytokine profile, including ifn-, tnf-, and il-12, whereas uc is associated with a modified th2 type response cytokine profile including il-15 and il-10. in addition, these findings have been recently complemented by the discovery of the il-23/il-17 axis, that is, part of the effector t-cell immunological response and seems to be involved in ibd. levels of expression of il-23 and il-17 are increased in patients with active ibd. although specific determination of the fr91 metabolic action in the colitis prevention and treatment will require further investigations, we showed that fr91 prevented ulcerative lesions in mice models of colitis, inhibiting the development of colorectal tumors. fr91 has proved to be an interesting and promising investigational agent for studying chemoprevention of carcinogenesis.

one of the main treatments currently used in humans to fight cancer is chemotherapy. a huge number of compounds with antitumor activity are present in nature, and many of their derivatives are produced by microorganisms. however, the search for new drugs still represents a main objective for cancer therapy, due to drug toxicity and resistance to multiple chemotherapeutic drugs. in animal models, a short-time oral administration of dextran sulfate sodium (dss) induces colitis, which exhibits several clinical and histological features similar to ulcerative colitis (uc). however, the pathogenic factors responsible for dss-induced colitis and the subsequent colon cancer also remain unclear. we investigated the effect of fr91, a standardized lysate of microbial cells belonging to the bacillus genus which has been previously shown to have significant immunomodulatory effects, against intestinal inflammation. colitis was induced in mice during 5 weeks by oral administration 2% (dss). morphological changes in the colonic mucosa were evaluated by hematoxylin-eosin staining and immunohistochemistry methods. adenocarcinoma and cryptal cells of the dysplastic epithelium showed cathenin-, mlh1, apc, and p53 expression, together with increased production of ifn-. in our model, the optimal dose response was the 20% fr91 concentration, where no histological alterations or mild dss-induced lesions were observed. these results indicate that fr91 may act as a chemopreventive agent against inflammation in mice dss-induced colitis.

PMC3348609

pubmed-1243

diabetic retinopathy (dr) is a one of the most common microvascular complications of diabetes. in 2012, there are more than 371 million people suffering from diabetes, and it is being projected that the number of diabetic patients will reach 550 million in 2030 (http://www.eatlas.idf.org/; assessed 29-nov-2012). diabetes can be generally divided into two types: type 1 (insulin dependent) and type 2 (insulin independent), although patients of both types will have hyperglycemia. a study reported that about one-third of the diabetic patients have signs of dr and about one-tenth of them even have vision-threatening retinopathy. nearly 60% and 35% of dr patients progress to proliferative dr and severe vision loss in 10 years, respectively. clinically, dr can be classified into nonproliferative (npdr) and proliferative (pdr). npdr can be further graded into mild, moderate, and severe and is characterized by the presence of microaneurysms, hemorrhages, hard exudates (liquid deposits), cotton wool spots, intraretinal microvascular abnormalities, venous beading, and loop formation. npdr may develop into pdr, where hallmarks of neovascularization of the retina and vitreous hemorrhage are found. moreover, maculopathy, including macular edema and ischemia, can occur at any stage of dr; it accounts for the majority of the blindness due to dr. in fact, the growing number of diabetic patients and a longer life span in the aging population imply an increase in patients suffering from dr, which not only affects the quality of life of the individuals and their families but also increases the medical and economical burden to the society. as a consequence, effective therapy is urgently needed. in order to develop effective drugs, detailed understanding of the pathophysiological progression of dr is required. over half a century ago, histological studies have been performed in postmortem retinas of diabetic patients. in retinal vessels and capillaries, selective endothelial and mural cells loss, presence of mural cell ghosts, endothelial clusters, acellularity, and microaneurysms were found to be increased in diabetic patients [4, 5]. basement membrane thickening, presence of hemorrhage in the inner nuclear layer (inl), and outer plexiform layer (opl) as well as eosinophilic exudates in the opl were also reported. nowadays, immunological studies evidenced an increased glial fibrillary acidic protein (gfap) expression in the mller cell processes throughout the inner and outer diabetic retina, suggesting that these cells were hypertrophied. there was also increased apoptosis in diabetic retina. abu el-asrar et al. further showed that proapoptotic molecules were expressed in ganglion cells, together with the activation of glial cells, which expressed several antiapoptotic molecules. elevated vascular endothelial growth factor (vegf) immunoreactivity was found in retinal blood vessels in diabetic humans with preproliferative or no retinopathy, further consolidated the role of vegf in angiogenesis and vascular permeability. alternation in other factors, including somatostatin, cortistatin, a and b-crystallins, advanced glycation end products (ages), and receptor for age (rage) as well as apolipoprotein a1 (apoa1), were also observed in the postmortem tissues. somatostatin and cortistatin, which are neuropeptides with a very similar structure, were both downregulated in diabetic retinas, and their expression levels are inversely correlated with glial activation and apoptosis. on the other hand, upregulations of a-, b-crystallin, age, rage, and apoa1 in the diabetic retinal tissue were reported. these morphological studies provide a better picture, yet without the mechanistic pathway, of the pathogenesis of dr at a cellular level. moreover, advanced technologies further allow us to study the mrna or protein expressions of various chemokines [1417], cytokines [1518], inflammatory markers [16, 19, 20], angiogenic factors [16, 17, 1921], and other factors [19, 22, 23] in aqueous humor, serum, or urine from diabetic patients, thereby predicting the pathological pathways of dr. with the aid of the sophisticated computerized equipments and technologies, clinicians would even be able to monitor or predict the progression structural lesion [2429] as well as functional defects [30, 31] in live patients with dr. although a lot of important information or clues on the development of dr can be obtained from human studies, the mechanisms of dr development still can not be elucidated. emergence of animal models, therefore, not only enables us to have a more comprehensive understanding of the etiology of dr at a molecular level in a controlled manner, but also fulfills the need for drug screening tools. with these models, we may even be able to discover the early markers for dr in the body fluids from diabetic patients. this not only allows a faster and more convenient screening but also serves as an alarm for diabetic patients before the presence of cellular or functional lesion. until now, many studies on the pathogenesis of dr have been carried out in animal models. a cascade of events, including oxidative stress, inflammation, protein kinase c (pkc) activation, accumulation of age and sorbitol, and upregulation of rennin-angiotensin system (ras) and vegf, contribute to retinal vascular endothelial dysfunction as a result of hyperglycemia. based on the mechanistic studies, drugs targeting different molecules in the cascade are being developed. in order to evaluate the effect of the drug properly, reliable and appropriate animal models are required. throughout the years, many animal models of dr have been developed; however, none of them can mimic the entire pathophysiological progression as observed in human. while most animal models only show the early symptoms of dr, some show the late stage proliferative angiogenesis. researchers have to select an appropriate model or models which can compensate each other in order to address their research questions. in this review, we focus on the animal models of dr that researchers have used, briefly describe how the models were generated, highlight the morphological and functional changes in the retina, and finally discuss the strengths and weaknesses of each model. in general, mice have been routinely used in many in vivo studies since they are small in size and therefore easy to handle and inexpensive to house. indeed, mechanistic studies of dr have been carried out extensively in mice as these models share similar symptoms of early dr as in human. more importantly, the availability of a collection of transgenic and knockout mice allows researchers to study the role of particular genes, which may even be cell type specific, in the development and pathophysiological progression of dr. there are three main types of mouse models to study dr; the first two involve mice with hyperglycemia development either via pharmacological induction or inbreeding of mice with endogenous mutation while the third type focuses on pathological angiogenesis found in transgenic animals or induced by experimental procedures, in mice without diabetes. type 1 diabetes can be induced in mice by injection of chemicals, including streptozotocin (stz) and alloxan, both of which are toxic to and therefore destroy the -cells in the pancreatic islets. stz-induced mice have been routinely used as a dr model in a lot of mechanistic studies and therapeutic drug testing for a long period of time owing to the abundant reports on the phenotypes. depending on the injection dosage, the onset of diabetes can be achieved within a few days after injection in the wild-type animals, making it a popular diabetic model. nevertheless, there are many variations in the injection protocol in terms of dosage, route of injection, and with or without insulin compensation that are usually based on the practice in individual laboratories; nonetheless, all mice end up with hyperglycemia in 1 to 4 weeks after stz injection. for an easy reference, a summary of dr studies using stz-induced mice in the past five years is presented in table 1. this table aims to provide the researchers with a general idea of the appropriate dosage or injection method in mice of different age and/or with genetic background. amongst these methods, intraperitoneal injection of single high-dose injection of 150 mg/kg and multiple low doses of 50 mg/kg for 5 consecutive days to c57bl/6 mice without insulin compensation are the standard protocols recommended by the animal models of diabetic complications consortium (http://www.diacomp.org/; accessed on 9-dec-2012). in the stz-induced diabetic mice, transient astrocyte activation as well as increased astrocyte number gfap upregulation in glial cells and reactive gliosis were also evidenced at the same time. retinal ganglion cells (rgcs) are reduced starting from 6 weeks [34, 35] while thinning of inl and onl were observed at 10 weeks of hyperglycemia. apoptosis of rgcs and vascular cells can be identified at 6 weeks and 6 months of hyperglycemia, respectively. yet, some studies showed that there is no significant in ganglion cell death even after a long time, up to 9 to 10 months of hyperglycemia [3639]. increased leukocyte number, together with leukostasis, was reported at 8 weeks of hyperglycemia. for vascular pathogenesis, upregulation of vascular permeability was being observed as early as 8 days of hyperglycemia, resulting in vessel leakage at 2 months. after 17 weeks of hyperglycemia, thickening of capillary basal lamina and neovascularization were reported. acellular capillaries and pericyte ghosts were found in retina in mice after 6 to 9 [32, 37] months of hyperglycemia. decreased retinal arteriolar and venular rbc velocities, retinal arteriolar and venular blood flow rates, and arterial velocity although decreased arteriolar and venular diameters were also reported in mice after 4 weeks of hyperglycemia, it is controversial in other studies. functional defects were described in some electroretinography (erg) studies including decreased op3 and ops, prolonged implicit time of op2-3 at 4 weeks of hyperglycemia [49, 50]; decreased a-waves and b-waves at 6 months of hyperglycemia; and decreased pattern erg amplitude at 7 weeks of hyperglycemia. the variations in the above observations may be due to difference in mouse strains, stz dosage, or observation time points. moreover, individual animals may be resistant to stz and fail in hyperglycemia induction; therefore, it is essential for the experimenters to confirm the blood glucose level of the animals and exclude those without hyperglycemia development. on the other hand, alloxan is less commonly used in mice, which may be due to the absence of the inducible cellular and vascular lesions associated with hyperglycemia. morphologically, the dendrites of microglial cells were found to be shortened without any ganglion cell apoptosis after 3 months of hyperglycemia. at the same time functional abnormalities, however, were being reported in erg, in which b-wave amplitude [129, 130] and b/a wave amplitude ratio were found to be decreased at 3 weeks and 3 months of hyperglycemia, respectively. nevertheless, cellular and vascular lesions are possibly detectable after a longer period of hyperglycemia as suggested by the presence of functional defects in this model., the blood aldohexose concentration is elevated in the animal without affecting other metabolic abnormalities, such as alterations in concentrations of insulin, glucose, fatty acids, and amino acids. this allows the researchers to study the consequential retinal complications solely due to the elevation of hexose concentration. galactose-fed mice have a longer life span than other diabetic models; therefore, an extended monitoring period of up to 26 months can be allowed [131133]. endothelial cell loss and increased acellular capillaries were observed starting from 15 months of hyperglycemia. for a further 6 months of hyperglycemia, other morphological lesions including the presence of pericyte ghosts, saccular microaneurysms as well as basement membrane thickening of retinal capillaries were also evidenced [131133]. amongst the mouse models currently available, the animals in this model have the least mortality at around 2 years old of age, which allows a longer period of hyperglycemia, and therefore phenotypes associated with increment of hexose concentration can be targeted. researchers should be aware that it takes a relatively longer period of time to develop retinopathy in these mice, which in turn leads to a higher cost. apart from injection or intake of chemicals, spontaneous hyperglycemia can be found in mice carrying endogenous mutation. by inbreeding the mutated mice with the wild-type animals, researchers can further expand the colonies and use them as mouse models for diabetes studies. although breeding is a time-consuming process, further manipulations, such as injections and feeding with specialized chow, are avoided. retinopathies in terms of morphological and functional lesions have been observed in a few type 1 and type 2 diabetic models, including ins2, nonobese diabetic (nod), db/db, and kka mice. in these animals, onset of hyperglycemia takes place spontaneously as a result of the presence of the transgene or mutation; a relatively consistent phenotype as well as a higher success rate in induction of hyperglycemia can be obtained. these mice carry a point mutation in the insulin2 gene, which causes a conformational change in the protein that accumulates in the pancreatic -cells, and ultimately leads to cell death. cellular lesions, such as reactive microglia, are evidenced as early as 8 weeks of diabetes. by 12 weeks of onset of hyperglycemia, immunological studies showed abnormal swelling in somas, axons, and dendrites of rgcs, and the number of these cells was reduced in the peripheral region; while more dendritic terminals, increased total dendrite length, and greater dendritic density were observed in the on-type rgcs. it has been reported that the number of rgcs was significantly reduced in the peripheral regions after 22 weeks of hyperglycemia; yet, another study showed that there was no ganglion cell death in these mice even up to 10 months of hyperglycemia. morphological change in astrocytes was also observed where they had short projections and became in less contact with the vessels. moreover, the ipl and inl became thinner, which may be due to the decrease of the cholinergic and dopaminergic amacrine cells as evidenced in retina after 6 months of hyperglycemia. vascular lesions such as increased of leukocyte number are already found in mice upon 8 weeks of hyperglycemia and retinal vascular permeability was increased in 12 weeks of hyperglycemia. the presence of acellular capillaries and neovascularization were described after 8 to 9 months of hyperglycemia. abnormal vascular functions were also reported in a study which showed that the arteriolar and venular rbc velocity, shear rate, and retinal blood flow rate were significantly decreased in the diabetic animals with 26 weeks of hyperglycemia. a decrease of a- and b-wave amplitudes in the erg after 8 month indicates functional problem associated with the cellular defects or degeneration. although the diabetic animals have an average life span of 305 days, they provide a stable induction of hyperglycemia while projecting the early and some of the late dr symptoms in human. thus, this model could be very useful in drug screening, and it has received more attention in the field of dr. the nod mice are another model of type 1 diabetes, in which pancreatic -cells were destroyed via an autoimmune process by the cd4 and cd8 cells. the onset of hyperglycemia in these animals is 12 weeks of age, and the frequency of having hyperglycemia at the age of 30 weeks old is about 80% in female and less than 20% in male. owing to the low induction rate and inconsistency in the male mice, female nod mice were commonly used. using transmission electron microscopy, ultrastructural changes including apoptosis of pericytes, endothelial cells, and rgcs, perivascular edema, and retinal capillary basement membrane thickening were reported as early as 4 weeks of hyperglycemia, and these retinopathogeneses became more obvious after 12 weeks of hyperglycemia. at about 4 months of hyperglycemia, vascular abnormalities were described in the nod mice. vasoconstriction or degeneration was observed in some of the major vessels, together with the presence of poorly defined microvessels and disordered focal proliferation of the new vessels. the presence of these vascular pathologies and the etiology of the development of type 1 diabetes in the nod mice are relatively similar to those of human, making this model unique. however, there is a big variation in the time of onset of diabetes; frequent and regular monitoring of the blood glucose levels is therefore required. more importantly, since female animals were used in these studies, estrogen, which plays a role in regulation of metabolism, may have a protective function in dr. this further complicates the mechanistic studies or contributes unknown effects in drug screening, thereby affecting the accuracy. the db/db mice spontaneously develop type 2 diabetes owing to the deficiency in the leptin receptor. hyperglycemia and obesity were observed in the homozygous mice at 4 to 8 weeks old. reduction of rgc number and thickness of the central total retina, inl, and photoreceptor layers were identified in the histological sections of mice after 6 weeks of hyperglycemia. after 18 weeks and 13 months of onset of hyperglycemia, pericyte loss and glial reactivation were reported, respectively. vascular lesions including capillary basement membrane thickening, the presence of acellular capillaries, increased vessel density in the inl, and vessel leakage were observed in the diabetic retina. compared with other mouse models, the reported glial reactivation and vessel leakage occurred relatively late in db/db mice, which could be explained by a relative late time point chosen in a long-term study. the use of db/db mice to study dr is not very popular, potentially due to a low birth rate resulting from the unsatisfactory mating performance in the male homozygotes and failure to reproduce in the female homozygotes (http://jaxmice.jax.org/; accessed on 15-dec-2012). kka mouse is a combined model made by the introduction of the yellow obese gene, a, into the kk mouse, in which moderate diabetic traits are thought to be inherited by polygenes. these mice spontaneously develop diabetic characteristics, such as hyperglycemia, hyperinsulinemia, and obesity, at around 6 to 8 weeks of age, but revert to normal at the age of 40 weeks. of the limited dr studies using this mouse model, increased apoptosis of the retinal neuronal cells was found in the rgc layer and the inl in mice after 4 weeks and 3 months of hyperglycemia, respectively. owing to the limited retinopathologic findings and the uncertain etiology of this model, this model is not popularly used in dr studies. in order to compensate for the lack of proliferative pathogenesis in the retinal vasculature in most of the above diabetic mouse models, researchers developed a number of nondiabetic models, which allow them to specifically target neovascularization in the ocular region. when using these models, however, researchers should be aware of the fact that the etiology of the progression of vascular abnormalities is different owing to the absence of classical systemic characteristics as seen in diabetes. proliferative retinopathy can be achieved mainly from two approaches: the first one is via introduction of ischemia to the eyes, such as oxygen-induced retinopathy (oir) and retinal occlusion; the second one is by direct injection or genetically induction of the angiogenic factor, vegf, into the ocular region. owing to the presence of neovascularization, this model is also adapted for studying the angiogenesis as seen in proliferative dr. in brief, postnatal day 7 (p7) neonatal mice were put into a 75% oxygen chamber for five days and then returned to room air [151, 152]. vessel loss in the central area of retina, which is associated with hypoxic challenged, is observed immediately at this time. neovascularization extending from the inner retina into the vitreous begins at two days after the return to room air, peaks on p17, and is gradually regressed and spontaneously resolved by p25. a comprehensive study was carried out in this model, in which mice at p18 were analyzed. the total retinal thickness was reduced in the midperipheral region, while the ipl and the outer segment length were reduced in the central and midperipheral regions. the number of vessels was also reduced in the ipl in the central region; and in the deep plexus in the central and midperipheral regions. retinal function was examined by erg, in which reductions in the amplitudes of a-wave, b-wave, op3, and op4, and delayed b-wave implicit time were revealed. mouse oir model showed a number of vascular, neuronal, and glial changes in the retina; however, the spontaneous regression of the neovascularization within a week confines its application in therapeutic drug research. since dr can also be considered as an ischemic disorder in the retina, retinal occlusion models, such as unilateral ligation of pterygopalatine artery (ppa) and external carotid artery (eca), branch retinal vein occlusion, and elevating the intraocular pressure (iop), were also applied in studies of vasculature abnormalities. in these occlusion models, increased apoptotic cells, reduced thickness of retinal cell layers, reduced a-wave, b-wave, and ops amplitudes of the erg were evidenced. nevertheless, the acute induction of ischemia, particularly those followed by reperfusion, to the retinal tissue makes these models less appropriate for studying dr, in which chronic ischemia is persistently involved. kimba (trvegf029) is a transgenic mouse model of neovascularization, as a result of transient overexpression of human vegf165 in the rhodopsin-expressing cells with which it peaks at p10 to p15 and declines at p20. characterization studies have been carried out as early as at p7. at that time, reduced thickness of the rgc layer, inl, outer nuclear layer (onl), and the total retinal layer was observed. by p28, such reduction was found in ipl, the outer segment, and the total retinal layer. vascular leakage was also observed at p28, but it ceased at 9 weeks of age potentially due to the absence of over-expression of vegf. increased adhered leukocytes were found in veins and capillaries, together with increased acellular capillaries by 6 weeks of age. topological and fractal analysis of retinal vasculature showed that vessel-covered area, vessel length, and crossing points in the 9-week-old kimba mice were reduced. varied degrees of the pathogenesis were being reported, which were separated into two groups based on the assessment of fundus fluorescein angiography. this transgenic mouse is not commercially available, it is not a popular model in dr. in order to generate an ideal mouse model to study dr, a new mouse model of dr was created by crossing the kimba mice with the ins2 mice, named akimba. these mice inherit the key systemic diabetic phenotypes from their parental strains, making it a unique model. at 8 weeks of age retinal edema and reduced photoreceptor layer thickness, together with retinal detachment, were observed. abnormal microvasculature, including microaneurysms, capillary dropout, hemorrhage, neovascularization, venous loops, vessel tortuosity, vessel beading, vascular dilatation, and vascular leakage, were also evident in mice of 8 weeks old, although the leakage stopped by 20 weeks of age. the akimba mouse model displays a number of vascular changes; however, more complete mechanistic studies are essential before its utilization in therapeutic drug studies. a summary of the earliest reported morphological and functional lesions in different mouse models of dr is shown in table 2. although rats have a slightly bigger size than mice, they are still easy to handle with a low cost of maintenance, making them to be another popular animal frequently used in in vivo studies. the use of rats in dr study is particularly common owing to a relatively larger tissue size, with which functional assessment and morphological and molecular analyses can be done. similar to dr studies in mice, three types of rat models were used; these include pharmacological induction of hyperglycemia, spontaneous diabetic rats, and models of angiogenesis without diabetes. a summary of the morphological and functional lesions in different rat models of dr is shown in table 3. similar to mice, hyperglycemia can be induced in rats by injection of stz or alloxan or by ingestion of galactose. compared to mice, rats are more susceptible to the toxicity of stz; therefore, usually a much lower dosage of stz is used. in order to minimize the mortality, insulin complementation table 1 summarized most of the induction method of stz in the past 3 years and served as a reference for the researchers to select the most appropriate dosage, injection paradigm, and rats with different genetic background for their studies. amongst the methods, a single dose of 6065 mg/kg of weight is the most popular one. variation of the retinal lesions was reported, which can be explained by the genetic background. indeed, a comparative study showed a strain difference in the rate of developing early dr symptoms in rats upon stz challenge. after 8 months of hyperglycemia, lewis rats displayed accelerated degeneration of retinal capillaries and rgc loss, whereas wistar rats only showed the capillary degeneration and sprague-dawley (sd) rats showed no morphological defects to a significant level. increased apoptotic cells after 1 month of hyperglycemia, the number of astrocytes in the peripheral region was reduced; however, the number of mller cells and microglial cells is increased [160, 161] with the accompany of microglial hypertrophy. the density of astrocyte was further reduced in the central region, together with the reduction of astrocyte processes in the peripheral region after 6 weeks of hyperglycemia. decreased total retinal thickness, as well as decreased number of cells in the rgc layer, the onl, and the inl was also reported. regarding the vascular changes, blood-retinal-barrier (brb) breakdown was evident at 2 weeks of hyperglycemia [161, 163]. increased adherent leukocytes and arterial or venous capillaries basement membrane thickening retinal function was affected from 2 weeks after the onset of hyperglycemia as reflected by the erg. reduced b-wave, ops, and a-wave amplitudes were progressively found at 2 weeks, 8 weeks, and 10 weeks of hyperglycemia. at around the same time, morphological and functional studies suggested that stz-induced diabetic rats only showed early dr symptoms, which is comparable to those in stz-induced mice. the use of alloxan in dr studies is not very common nowadays, and the existing morphological studies were mainly focused on the vascular lesions. neovascularization was already observed from 2 months of induction of hyperglycemia starting from the midperiphery region and progressing to the whole retina after 9 months of hyperglycemia. extravascular macrophage accumulation and capillary endothelial cells swelling were also identified after 2 months and 5 months of hyperglycemia, respectively. by 8 months, increased cell death in the retinal microvasculature was evident. acellular capillaries, basement membrane thickening, and pericyte loss were also reported upon 12 months of hyperglycemia. nevertheless, appearance of the lesions varied between studies; it would be due to the different time points selected by the authors or different dosage of alloxan being injected. reported lesions with the earliest onset similar to mice, dr can be studied in rats fed with galactose, with the equivalent advantage of longer life span [172, 198]. other vascular lesions, such as acellular capillaries and basement membrane thickening as well as pericyte loss, were observed after 12 months of feeding with galactose. a long-term study demonstrated cellular lesions, including gliosis and disruption of retinal layers, together with vascular abnormalities, capillary dilation and microaneurysm formation in the inner plexiform layer (ipl) and the inl, in rats fed with galactose for 28 months. owing to the differences in galactose concentration and time points selected by various studies, the earliest onsets of the lesions were listed in this review. these include type 1 diabetic model: biobreeding (bb) as well as type 2 diabetic model: wistar bonn/kobori (wbn/kob) rats, zucker diabetic fatty (zdf) rats, otsuka long-evans tokushima fatty (oletf) rats, nonobese goto-kakizaki (gk) rats, and nonobese spontaneously diabetic torii (sdt) rats. like nod mice, the bb rats spontaneously develop polygenic autoimmune type 1 diabetes, in which the pancreatic -cells were selective destroyed [175, 199]. after 4 months of hyperglycemia, absence of infolding and derangement of the basal plasma lemma of the rpe were also observed. the number of pericyte and the pericyte to endothelial cell ratio were reduced [174, 175] after 8 months of hyperglycemia. lesions associated with the retinal microvasculature, including capillary dilation and basement membrane thickening, were found from 2 months and 4 months of hyperglycemia, respectively. microinfarctions with areas of nonperfusion were evident whereas no neovascularization was detected up to 11 months. several inbred and outbred lines, such as bb/wor, bb/e, and bb/ph, have been produced and named based on the origin of the breeding colony. since genetic variations and potential differential phenotypes the wbn/kob rats are a type 2 diabetes model owing to endo-exocrine pancreatic insufficiency, and only male offspring develop diabetic symptoms. retinal degeneration was already observed before the animal becomes hyperglycemic at around 9 to 12 months of age. thickness of outer segments and onl was reduced in wbn/kob rats at 5 months of age whereas high blood glucose was evident in these rats at 10 months old. about 2 months after becoming hyperglycemic, these rats also showed reduction in the visual cells, the opl, and the total retinal layer. vascular lesions were also identified, and capillaries clustered into small tortuous knots after about 1 month of hyperglycemia. after 5 to 6 months of diabetes, capillary basement membrane thickening, increased capillary loop, and reduced number of capillary were also observed. after a prolonged hyperglycemia of 12 months, some rats showed increased proliferation of fibrovascular element in the vitreous, intraretinal neovascularization, and hyalinization of intraretinal vessels. wbn/kob rats, which display the symptoms of the progressive dr, may serve as a model for testing therapeutic drug targeting angiogenesis. however, the early onset of neuronal degeneration (before hyperglycemia commencement) suggests that the etiology of retinal degeneration may not be the same as that in human; therefore, further studies need to be carried out before this can be used as a model for dr. they carry an inherited obesity gene mutation, which results in impairment of glucose tolerance and insulin resistance (http://www.criver.com/sitecollectiondocuments/zdf.pdf; accessed on 19-nov-2012). excessive body weight gain was observed in male zdf rats in the first 6 months of life, but the weight decrease to a level similar to the lean controls afterwards. hyperglycemia starts at 6 to 7 weeks of age and maintains high throughout their life. thickening of the capillary basement membrane and increased capillary cell nuclear density were reported in rats after 5 months of hyperglycemia [180, 181]. apoptosis of endothelial cells and pericytes was higher in these rats compared to the lean controls, together with an increased number of acellular capillaries and pericyte ghosts upon 6 months of diabetes. retinal functional analysis and long-term morphological studies of the retinal neuronal and glial cells of these rats remain to be elucidated. another type 2 diabetic rat model is oletf rats; they significantly gained more weight from 1 to 6 months of age, but they lost weight from 9 to 10 months of age. elevated blood glucose was observed from 5 months of age and it maintained high [182, 183]. after about 6 months of hyperglycemia, despite no significant difference in the number of acellular capillaries and pericyte ghosts, oletf rats with 9 months of hyperglycemia showed reduced ratio of pericyte area to the total capillary cross-sectional area and damaged endothelial cells. by 14 months of hyperglycemia, the inl and the photoreceptor layer became thinner, accompanied with shortening of the rpe height and poorly developed basal infoldings. relatively early microvessel-related symptoms were reported in these rats, in which leukocyte entrapment was evident in rats after 6 weeks of hyperglycemia. other abnormalities, including thickening of capillary basement membrane, tortuosity, microaneurysms, loop formation in capillary, caliber irregularity and narrowing of arteries, were also described in rats after 9 to 12 months of diabetes [182, 183, 185]. no hemorrhages, emboli, and exudates were found in these rats up to 14 months of diabetes. moreover, erg revealed that oletf rats fed with sucrose for 8 weeks had a prolonged peak latency of ops. the absence of acellular capillaries as well as the late onset of diabetes and the related symptoms diminished the popularity of using this model to study dr. the gk rat is a spontaneous model of non-insulin-dependent diabetes without obesity. these rats are originated from normal wistar rats and they were selected via repeated inbreeding exercise using glucose intolerance as a selection index [186, 204]. rats at 46 weeks of age develop hyperglycemia [186, 187]; they also showed reduction of retinal segmental blood flows and prolonged retinal mean circulation time after 1 month of hyperglycemia. increased brb permeability and endothelial/pericyte ratio were also evident in 3 months and 7 months after the onset of hyperglycemia owing to the limited publications on the retinopathology in the gk rats, further characterizations on the non-vascular-related lesions need to be performed. the sdt rat, which is a substrain of the sd rat, is another model of nonobese type 2 diabetes. glucose intolerance and impaired insulin secretion were demonstrated in the male sdt rats at 14 weeks, followed by hyperglycemia and glucosuria at 5 months of age. these rats showed a sexual differentiation in the development of diabetes that the cumulative incident is about 100% in males at 40 weeks of age and only about 33% in females up to 65 weeks of age [190, 205]. retinal dysfunction was observed after 4 weeks of hyperglycemia, as evident by delayed peak latency of the ops. the amplitudes of a-wave, b-wave, and ops were significantly reduced with prolonged implicit times at 24 weeks of hyperglycemia. at the same time, leukostasis and the number of apoptotic cells in the gcl and the inl were increased in the retinas of sdt rats. vascular lesions, such as acellular capillaries and pericyte loss, have been described by kakehashi et al.. advanced lesions, including leakage of fluorescein around the optic disc as well as distortion of retina and protruded optic disc, were also observed after 48 weeks of hyperglycemia. more importantly, a few studies showed that proliferative dr can be detected in some of the aged sdt rats, which have been exposed to hyperglycemia for more than 48 weeks [190, 191, 205, 206]. the reported symptoms include retinal hemorrhages, tortuous vessels, capillary nonperfusion, neovascularization, and tractional retinal detachment with fibrous proliferation. amongst the diabetic rat models mentioned in this review, the sdt rat is the only one that shows severe ocular complications similar to those seen in human. although some common phenotypes in human dr, such as microaneurysms and development of avascular area, are rare in this model, this is a unique model to study proliferative dr [205, 207]. similar to the mouse models for studying angiogenesis, oir and occlusion models were also applicable to rats. owing to the relevance of ischemic-induced neovascularization, we will focus on the oir in this section. the basic principle of oir in rats is very similar to that in mice, which involves the induction of neurovascularization in nondiabetic animals. different from the standard protocol of oir in mice, several paradigms with varied oxygen concentrations and duration of the exposure period have been applied in rats. in brief, the newborn pups are exposed in alternative hyperoxia-hypoxia cycles for 11 to 14 days and then returned to room air [194, 195, 208210]. peripheral astrocyte degeneration was observed soon after the rats were exposed to room air. at p18, the number of astrocyte was reduced almost throughout the whole retina with prominent mller cell reactivity in the regions that are devoid of intraretinal blood vessels. reduction of thickness in the inl, the ipl, and the total retinal layers was evident; the outer segment layer also became thinner and disorganized. while the number of pericytes was comparable to the room air control, the pericyte-endothelial interactions were impaired. intravitreal neovascularization, incomplete development of the outer vascular plexus and extension of the abnormal endothelial functional lesions were also studied using erg, in which the a- and b-wave amplitudes were reduced. this model is useful in therapeutic drug screening or in the study of the mechanisms in angiogenesis, yet special equipments are required. moreover, strain-dependent difference in the degree of retinal vascularization and abnormalities in vascular morphology have been reported. the albino sd, the pigmented dark agouti, and hooded wister rats were more prone to the hyperoxia-hypoxia challenge, and they showed severe vascular attenuation following the oxygen exposure as well as severe vascular pathologies when compared to other strains. in summary, rodents are very popular models to study the pathogenesis and examine the efficiency of therapeutic drugs of dr in laboratories. they have the advantage of being small in size which allows easier handling; however, this also makes in vivo examinations, such as fundus photography, fundus fluorescein angiography, and optical coherence tomography, difficult. despite the lack of proliferative dr symptoms as described in most of the models mentioned above, researchers also focus on other animals in order to obtain the most representative model of dr, which ideally displays the comparable dr symptoms as seen in human patients. higher mammals not only can serve as a platform for easier examinations but also allow easier treatment particularly those involving sophisticated surgical procedures. in these animals, sampling of body fluid, for example, vitreous and blood, can also be performed routinely. similar approaches have been applied to rabbits to induce dr; these include pharmacologically induced and dietary-induced diabetic models as well as vegf-induced angiogenesis in the retina without affecting the blood glucose level. hyperglycemia can be induced in rabbit by stz, although this method is not very frequently used. a study showed that intravenous injection of stz (100 mg/kg) in rabbits can elevate their blood glucose level. fundus examination was done after 19 weeks of hyperglycemia and all eyes showed certain degree of retinopathy, of which 50% showed proliferant retinopathy; 40% showed serious vasculopathy with serious retinal and preretinal hemorrhages, vascular lesions, hemovitreous and venous thrombosis; and the remaining 10% showed moderate vasculopathy with hard or soft exudates and widespread hemorrhages. early dr can be found in rabbit models of diet-induced impaired glucose tolerance plus hyperlipidaemia. rabbits were fed with standard chow with 10% lard, 40% sucrose, and 0.10.5% cholesterol for a period of 24 weeks. the blood glucose level slightly elevated in the animals after feeding with 12 weeks of the special diet, and they became hyperglycemic by the end of the study period. histological findings suggested that increased microaneurysms and hyperfluorescent dots were already present before the rabbit becomes hyperglycemic, while those pathological symptoms further progressed with prolonged feeding. although this model mimics the natural development of type 2 diabetes in human, the drawback is the slow progression of dr symptoms. in brief, a polymeric pellet containing human recombinant vegf was implanted into the vitreous cavity of the rabbit. after 7 days of implementation, increased dilation and tortuosity of retinal vessels were observed. during 14 to 21 days after implementation, fluorescein angiography further showed profuse leakage of dye, together with the presence of numerous small tortuous blood vessels, suggesting induction of neovascularization. however, such vascular changes stopped afterwards and neovascularization almost totally regressed after 35 days of implementation. the authors suggested that the regression of vessels may be due to depletion of the vegf, implying that choosing the experimental endpoint is crucial when screening therapeutic agents in this model. therefore, another group generated a similar model, in which human recombinant basic fibroblast growth factor (bfgf) was also incorporated into the polymeric pellet besides the human recombinant vegf. in this model, similar retinopathologies were observed but they only required approximately half of the time to develop when compared with the vegf-induced model previously described. in addition, hemorrhage from the new vessels and even total traction retinal detachment were also observed. moreover, differential retinal angiogenic response to vegf/bfgf was reported in different rabbit strains, where dutch belt rabbits are more susceptible than the nzw/black satin cross rabbit. it is evident that vascular retinopathy could be observed in the rabbit models mentioned above; however, researchers should be aware of the fact that retinal vasculature in the rabbit differs from those in other species. in rabbit, the optic artery branches into major blood vessels in a bidirectional horizontal manner; they further arborized into capillaries, forming a ring-like network. moreover, the visual streak of rabbit is located below this region; functional defects may not be able to be detected if the lesion site is in the medullary ray where the blood vessels are. as compared with other animals, such vascular system is only present in a small area of the retina in rabbit; therefore, the global deleterious contributions by the vessels may be underestimated. on the other hand, if researchers aim to study the vessel-to-cell interaction at a molecular level, this model provides an alternative choice with an additional advantage of a bigger eyeball size than rodents. therefore, more precise and delicate experiments can be performed, but the problem of limited housing space needs to be attended to. the majority of the dr studies in cats are induced by pancreatectomy with or without alloxan injection. the animal will become hyperglycemic 1 to 2 weeks after the surgery [216218]. capillary basement membrane thickening was first described from 3 months of pancreatectomy, where no change was observed in the number of endothelial cells and pericytes as well as the contacts between endothelial cells and pericytes up to 10 months. a case report showed that microaneurysm was first observed in one eye in a diabetic cat after 5 years of pancreatectomy; and by 6.5 years, both eyes showed microaneurysms and small intraretinal hemorrhages in the area centralis. region of capillary nonperfusion and intraretinal microvascular abnormalities (irma) were also evident from 7.5 years. at 8.5 years, presence of small foci of neovascularization was suggested. cotton-wool spots, venous beading, extensive preretinal neovascularization, or microvascular changes were not found in the peripheral retina. on the other hand, another study showed that only one out of two experimental diabetic cats showed microaneurysms, but not hemorrhages or area of nonperfusion, after 7 years of pancreatectomy, while the other diabetic cat did not show any microaneurysm or hemorrhage in the retina. cats only showed mild cataracts upon diabetes, thereby allowing visualization of the fundus angiography and erg. however, the studies of dr in cat are very limited and the described phenotypes are less consistent. a long follow-up period for the development of retinal pathology and lack of reagents in molecular studies may be the drawbacks for using this animal model. attempts of using dogs for studying dr have also been made, in which most of them are about inducible hyperglycemia either by injection of stz or alloxan or feeding the animals with galactose. it has been suggested that galactose-fed dog is the animal model that shares the retinal lesions morphologically and clinically as those developed in human diabetic patients. induction of diabetes in dogs by intravenous injection of stz and alloxan resulted in basement membrane thickening in 3 years of injection, and it was recognized in some vessels from the first year. loss of pericytes and smooth muscle cells was observed in the retinal arterioles from 4 years of postinjection; no microaneurysm was noted towards the end of this 5-year study. moreover, a comparative study showed that increased microaneurysms, acellular capillaries, pericyte ghosts, endothelial cells to pericytes ratio, and basement membrane thickening were evident in the dogs after 5 years of galactosemia than those of alloxan-induced diabetes. in the galactose-induced dr model cellular lesions such as presence of pericyte ghosts and uneven distribution of endothelial cells were observed in retina of dogs after feeding with galactose for 19 and 24 months, respectively, followed by the formation of microaneurysms [222, 223]. dot and blot hemorrhages were found from 33 months, which became more confluent, progressing to the preretinal and intravitreal regions after 66 months of feeding. nonperfusion was evident in dogs from 37 months of feeding [223, 224], and the area was broadened with time. after 36-month feeding of galactose, acellular capillaries and endothelial cells to pericytes ratio were increased. other vascular lesions, such as abnormalities in intraretinal microvessels, occlusion of arterioles, presence of large arteriovenous shunts, and node formation on arterial and arteriolar walls, were also reported after feeding for about 5 years. at about the same time, presence of soft exudates and gliosis in the nerve fiber layer was reported. further advanced retinopathy of neovascularization was described in dogs being galactose fed for 68 to 84 months [222, 224]. it has been suggested that the onset of dr symptoms is age dependent in galactose-fed dogs; younger animals develop dr symptoms earlier than the older ones. the biggest advantage of using dogs as a model is that they develop similar retinal morphological lesions as compared with human. routine in vivo vasculature assessments, however, were impeded owing to the spontaneous diabetic cataract, particularly in the galactose-fed model; additional lensectomy is necessary [219, 223, 224]. moreover, high maintenance cost, long-term follow-up period, and lack of molecular reagents, such as antibodies, make this model less commonly used for studying dr. pig eye has become a useful tool in eye research because of its close similarities in the size as well as the basic retinal structure and vasculature to the human eye. a number of models have been generated in order to study the retinopathy in swine upon diabetes, which include alloxan- and stz-induced type 1 diabetic models. there is also a recently developed model of proliferative vitreoretinopathy that involved surgical procedures and intravitreal injection of retinal pigment epithelial (rpe) cells. there are only limited reports on the retinal morphology of the chemically induced diabetic pigs. instead, researchers make use of the large amount of specific retinal cells and vitreous available in the pig eyes for in vitro experiments [227, 228]. nevertheless, reactivation of mller cells was evident from the increased gfap immunoreactivity from the onl extending to the outer limiting membrane in 2 to 3 months after onset of alloxan-induced diabetes. at around 4 months of hyperglycemia, pericyte degeneration in parallel to reduced the total number of brb capillaries and capillary collapse were also observed. retinal vascular lesions, such as basement membrane thickening [230, 231] and rarefaction, were reported in pigs after 18 weeks of stz-induced diabetes. development of hyperglycemic cataract was also reported in this animal after 32 weeks of hyperglycemia that constrain the visualization of the vasculatures, such as fundus angiography. recently, a new swine model of proliferative vitreoretinopathy has been described. in brief, vitreal and retinal detachments were initially induced by vitrectomy and injection of subretinal fluid, respectively, prior to injection of cultured rpe cells into the vitreous cavity. formation of contractile membranes on the inner retinal surface as well as localized tractional retinal detachments was evident and maintained after 14 days of induction while the retina reattached in the control animals at 3 days after the surgery. further characterization of this model needs to be carried out before its use in therapeutic drug screening. although pig is a valuable model for disease study in human, high maintenance cost, requirement of special housing facilities, and lack of biochemical reagents make this model less commonly being used. monkey, a nonhuman primate, is considered to be a potential model in eye research owing to its structural similarity to human and, in particular, the presence of macula. the studies of dr in monkey can be divided into 3 groups: type 1 diabetic model, type 2 diabetic model, and model of vegf-induced neovascularization. in an attempt to produce dr in monkeys, monkeys with type 1 diabetes that developed spontaneously as well as that resulted from total pancreatectomy or stz injection were being used. unexpectedly, 37 out of 39 of these monkeys did not show any significant dr within 5 years of hyperglycemia. animals with hyperglycemia of 6 to 15 years only showed mild disruption of the blood-retinal barrier. on the other hand, spontaneous or pharmacological induction of hypertension in the hyperglycemic monkeys, either by stz injection or with spontaneous diabetes, resulted in ischemic retinopathies, such as cotton-wool spots which were found in the peripapillary region, microaneurysms, capillary dropout, capillary dilatation, focal intraretinal capillary leakage spots, arteriolar and venular occlusions, and atrophic macula, between 6 and 15 years of diabetes. the authors suggested that the fluctuating blood glucose levels and systemic blood pressure, but not hyperglycemic alone, play a role in the pathogenesis of dr. dr studies have also been carried out in monkeys that spontaneously develop type 2 diabetes. while moderate retinal lesions can be identified in a case of monkey with 3 years of diabetic history, no detectable retinopathy was reported in a monkey with 15 years of diabetes. the presence of these lesions was variable in individual animals, making it hard to deduce the precise onset of symptoms based on the diabetic duration. among those showing retinopathies, cotton-wool spots, intraretinal hemorrhages, and nonperfused areas were the early observations. progressive lesions, such as growing nonperfused area, which are associated with the formation small irmas and microaneurysms, as well as macular edema were also evident. similar observations were also reported in another case study in which the subject is a monkey with at least 5 years of diabetes. the authors have mentioned other histological abnormalities, including reduction of the thickness of the onl and the inner and outer segments of the photoreceptor layers. functional lesions were suggested by a loss in the amplitudes in the multifocal erg, and they were virtually correlated to the nonperfused areas. progressive reduction of amplitudes and delayed a-waves were also observed in the dark-adapted ganzfeld erg, suggesting a loss of function in the both inner and outer retina and reduced sensitivity in the photoreceptors, respectively. moreover, it is reported that the occurrence of retinopathy is correlated with hypertension, which is coincidently similar to the descriptions in the type 1 diabetic monkey model. vegf-induced proliferative retinopathy has also been carried out in nonhuman primates. in brief, a pellet containing human recombinant vegf was implanted into the vitreous cavity of the animal. at 2 weeks after the implementation, severe brb breakdown was noted. apart from the variations in the onset of morphological abnormalities and the absence of advanced retinopathies, low birth rate, high cost, long duration of study, and the heightened ethical concern make this model unfeasible for the purpose of drug screening. despite the enormous ethical concern in the laboratory use of the mammals mentioned above, in particular the primates zebrafish is extensively used in the study of visual development and impairments owing to its similarity to those seen in human. the distinctive pattern of the mammalian retinal cell layers, ranging from ganglion cell layer to retinal pigment epithelium, is observed in zebrafish. blood supply to the retina is supported by the optic artery, which branches into four to nine major blood vessels. these vessels further arborize into smaller vessels towards the peripheral of the retina where anastomosis between the neighboring capillaries is present. this radial vascular network covers the entirely inner surface retina with direct contact with the gcl. oxygen-deprived blood is collected in the circumferential vein surrounding the retina where the cilliary marginal zone is. dr can be studied in zebrafish via direct elevation of glucose in the surrounding as well as angiogenesis without the involvement of glucose. in brief, zebrafish was exposed to freshwater with alternation between 2% and 0% glucose in every 24 hours for 30 days. hyperglycemia can be achieved in the animal in 1 day of immersion in the 2% glucose freshwater and maintained for at least 30 days with repeated hyperglycemic spikes every time after the removal from the glucose-freshwater. after 28 days of persistent hyperglycemia, the thickness of the ipl was significantly decreased, and yet no other abnormality has been observed. the mechanism of glucose uptake in zebrafish is regulated by osmoregulation, in which influx of water, together with glucose, goes into their body as a result of high internal salt concentration. it has been reported that teleosts also have endocrine islet tissue containing hormone-producing cells which converge in the fish body, and the secretory teleost insulin is functional and is homologous to the human insulin. this further validates the potential use of glucose-induced diabetic zebrafish in studying the retinopathy. yet two models to study angiogenesis in zebrafish are described below, namely, environmentally and transgenic-induced models. retinal neovascularization can be achieved by keeping the zebrafish in hypoxic aquaria where the air saturation is gradually reduced to 10% (820 bbp) over a course of 48 to 72 hours and maintained for 12 days [240, 241]. in these studies, fli-egfp-tg zebrafish, which is a transgenic line that overexpresses egfp in the vascular endothelium, after 12 days of hypoxic challenge, neovascularization was observed in the retina evident by increased number of branch points, sprouts, and vascular area as well as reduced intercapillary distance. this model can be useful for studying the development of angiogenesis or possibly for screening antiangiogenic pharmacological agents. zebrafish carrying vhl mutation displays an upregulation of hypoxia-inducible factor, which in turns triggers vegf production and expression of the vegf receptors. by 5.75 days after fertilization (dpf), increased hyaloids and choroidal vascular networks were observed, followed by vascular leakage at 7.25 dpf. excessive blood vessels were evident in the ipl, together with severe macular edema and retinal detachment at 7.5 dpf. however, this model is not commercially available, which limits its use in the field even though severe neovascularization and proliferative retinopathy are observed. zebrafish is very small in size; therefore, its maintenance is simple, convenient, and inexpensive. they have a short life span and a large breeding size, which in turn allow a shorter experimental turnover time. moreover, a number of studies showed that genes of interest can be specifically induced, deleted, or overexpressed in zebrafish, allowing mechanistic studies of diseases. as a consequence, researchers have developed certain zebrafish models in order to study dr, including glucose-induced diabetic model and models specifically of angiogenesis. however, the retinal cells layers differ in thickness and thereby the number of cells, the findings may under- or overestimate the contribution of a specific cell type in regard to the pathogenesis of dr. in terms of the vasculature in zebrafish, the growth of the tertiary plexus of blood vessels in the inl is absent and the venous system is different from those in human. therefore, researchers should be aware that using zebrafish may lead to potential discrepancy in cellular and vascular aspects and may not truly reflect the pathological development of dr in patients. moreover, owing to the limited supply of tissue from a single animal, skillful techniques and a large quantity of eyeballs are required in dissection and for molecular analysis. a summary of the temporal morphological and functional lesions of the animal models, other than rodents, described above is shown in table 4. animal models are very important in understanding the pathogenesis of diseases in human, defining novel therapeutic targets as well as screening of novel therapeutic drugs. in this review, a number of animal models of dr have been described and compared, ranging from different species to different induction methods of diabetes or angiogenesis, together with their corresponding temporal morphological and functional lesions. up to date, there is no single model which can mimic the development of dr as in human, that is, from the very early cellular and vascular abnormalities to the proliferative stage, and subsequently retinal detachment, as a result of prolonged hyperglycemia. rodents have been extensively used in dr studies owing to their small size and the ability to develop retinopathies within a relatively short period of time. the availability of a collection of transgenic mice further aids in elucidating the role of target molecules in dr. since the stz-induced diabetic rodents are the most frequently used models in studying the associated retinopathy, we have summarized the administration dosage and paradigm published in the recent years as a reference to other researchers. nevertheless, a majority of the diabetic rodent models only demonstrated the early symptoms of dr, which restricts their applications in mechanistic studies and drugs screening targeting the early progression of dr. some higher-order animals showed relatively advanced retinopathies, such as neovascularization, upon induction of diabetes, yet they still can not imitate the later stage of dr as seen in human. moreover, high maintenance cost, long duration of study, and lack of molecular reagents, such as antibodies, as well as ethical concern further limit their use in studies. zebrafish is another model that emerged recently in studies of dr; however, further characterization needs to be done. the presence of neovascularization is controversial in some animal models; such variation may come from animals of different strain and age, individual variation, and/or even the detection methods. therefore, we suggested that researchers should have a bigger sample size, use at least two detection methods, such as fluorescence angiography, immunohistochemical staining of blood vessel marker on retinal flat mounts or cross-sections in combination with molecular analysis, in order to have a more convincing claim. furthermore, overexposure of the fluorescence staining and cleanness of the section, particularly in the flat mounts, are other issues that researchers should be aware of. although neovascularization can be observed in animals overexpressing vegf, either via transgenic approach or direct introduction, the development of neovascularization is not caused by prolonged hyperglycemia. therefore, using these models in studies of the etiology of the disease or the development of preretinal neovascularization should be avoided. another approach for induction of neovascularization is by hypoxic challenge in rodents; however, regression was reported within a few days, which may limit the duration of the drug treatment versus the formation of new vessels. as outlined in this review, individual model of dr has different strengths and weaknesses; careful consideration should be made in choosing appropriate models to address the research questions.

diabetic retinopathy (dr) is a microvascular complication associated with chronic exposure to hyperglycemia and is a major cause of blindness worldwide. although clinical assessment and retinal autopsy of diabetic patients provide information on the features and progression of dr, its underlying pathophysiological mechanism can not be deduced. in order to have a better understanding of the development of dr at the molecular and cellular levels, a variety of animal models have been developed. they include pharmacological induction of hyperglycemia and spontaneous diabetic rodents as well as models of angiogenesis without diabetes (to compensate for the absence of proliferative dr symptoms). in this review, we summarize the existing protocols to induce diabetes using stz. we also describe and compare the pathological presentations, in both morphological and functional aspects, of the currently available dr animal models. the advantages and disadvantages of using different animals, ranging from zebrafish, rodents to other higher-order mammals, are also discussed. until now, there is no single model that displays all the clinical features of dr as seen in human. yet, with the understanding of the pathological findings in these animal models, researchers can select the most suitable models for mechanistic studies or drug screening.

PMC3826427

pubmed-1244

the design of efficient water oxidation catalysts (wocs) based on nonprecious materials remains an important challenge for achieving a clean and sustainable solar fuels-based energy economy. we have previously shown that active wocs can be formed by anodic electrodeposition of metal-oxides from neutral and near-neutral buffered aqueous solutions of cobalt, nickel, and recently, manganese. in particular, the cobalt oxygen-evolving catalyst (co-oec) has been studied in detail, resulting in an understanding of the electrochemical kinetic mechanisms of its formation, catalysis, and charge transport. the structural and electronic properties of co-oec have been clarified using xas, x-ray pdf, epr, and x-ray gid. these studies have revealed that the electrodeposited catalyst films comprise molecular to nanoscale-sized metalate clusters composed of edge-sharing coo6 octahedra with a mixed valence co(iii/iv) resting state. the development of soluble molecular wocs based on co as well as other transition metals, such as ir, ru, cu, and fe have also been a subject of intense focus. molecular wocs are attractive research targets because they provide a tractable means to characterize catalytic mechanisms and to identify reactive intermediates, thus forming the basis for the continued development of new wocs. however, the true identity of the active catalyst must be clarified prior to a detailed interrogation of the woc mechanism. indeed, some molecules that were thought to be wocs have subsequently been shown to be precursors of heterogeneous or colloidal materials, which are the active catalysts. proper catalyst identification is especially challenging for the study of molecular cobalt wocs because extremely small amounts of co-oec may be produced from the decomposition of the molecular catalyst. an exemplar of this challenge is the all-inorganic cobalt polyoxometalate [co4(h2o)2(pw9o34)2] (co4pom), which was suggested as a woc. re-examination of the molecule showed that electrochemically driven oxygen evolution arose from the formation of co-oec on glassy carbon (gc) electrodes at 1.1 v vs ag/agcl. because the co4pom was unstable at higher potentials, water oxidation activity could not be conclusively attributed to the co4pom, as opposed to its role as a molecular precursor to co-oec. the co4pom has now been suggested to exhibit water oxidation activity but under specific photochemical conditions where ru(bpy)3 is the oxidant. (left) molecular structure of co4o4 cubane structure 1 and (right) thermal ellipsoid representation at the 50% probability level of the one-electron oxidized cubane, 1[pf6]. atoms are color-coded: gray (carbon), blue (nitrogen), red (oxygen), dark blue (cobalt), green (fluorine), and yellow (phosphorus). against this backdrop, cubane co4o4 clusters, such as co4o4(oac)4(py)4, (1, figure 1), have come under investigation as a class of molecular cobalt complexes that are potential wocs. we had previously investigated 1, first synthesized by das and co-workers, and a related co4o4 cubane of christou, in order to gain valuable insights into the electronic characteristics and proton-coupled electron transfer (pcet) behavior of co(iii/iv) in a co-oec environment. the structure of 1 has been previously reported (figure s1, supporting information); the crystal structure of the oxidized cubane 1 was known as a perchlorate salt and is now obtained as a pf6 salt, as shown in figure 1. in our studies, we did not find any evidence that these cubanes were active wocs. motivated by the recent reports to the contrary and subsequent computational work outlining a detailed mechanistic pathway for 1 as a woc, we renewed our investigation of these molecules. a comparison of the h nmr spectra of (black line) 10 mm crude 1 and (red line) 10 mm pure 1 in d2o. herein, we report that a co(ii) impurity in as-synthesized cubane 1 is primarily responsible for the reported catalytic water oxidation activity. we present a series of experiments that are useful for determining whether a small amount of a co(ii) impurity may lead to formation of a heterogeneous woc. we further emphasize the utility of differential electrochemical mass spectrometry (dems) for clarifying how anodic potentials affect the decomposition of glassy carbon electrodes, which are commonly used in the study of wocs. the reported experiments are aimed at establishing a standardized approach to evaluate the presence of co(ii) impurities in molecular complexes under investigation as water oxidation catalysts. we synthesized and isolated 1 by precisely following the one-pot procedure developed by others. despite satisfactory elemental analyses for 1 (table s1), we determined that this as-synthesized material, which was isolated by concentrating a dichloromethane (dcm) extraction, was not pure. the presence of impurities was indicated by the observation of many small peaks in the h nmr spectrum (figures 2 and s2s4) and by the presence of slowly moving bands that eluted behind the product band on a silica thin layer chromatography (tlc) plate (figure s5). on the basis of the tlc result, purification of the compound was performed by column chromatography on silica, eluting with a gradient of 210% meoh in dcm. along with the slowly moving green bands, a comparison of h nmr spectra for crude (i.e., as-synthesized) and purified 1 is shown in figure 2. several peaks that are observed in the aromatic region in the nmr of the crude sample are absent in the nmr of the purified sample. molecular impurities are also indicated by many peaks in the m/z range of 300700 in the esi-ms of crude 1; these peaks are absent in the purified sample (figure s6). a structural variant, 1-coome, was also synthesized according to das original procedure; the final product was isolated by precipitation and filtration. no diamagnetic impurities were detected in the h nmr spectra of the precipitated 1-coome. however, to remove possible paramagnetic impurities, the precipitated 1-coome was subject to further purification by chromatography. interestingly, the same h nmr spectrum was obtained for precipitated and chromatographed 1-coome (figure s7), though the former was observed to have impurities that were not removed by precipitation. background corrected cvs of crude (black dotted) and purified (red solid) samples of 1 (0.852 mg/ml) in 0.2 m kpi buffer, ph=7. two scans are presented for the crude sample demonstrating the loss of activity upon the second scan. the reported water oxidation activity of 1(36,39) could not be replicated using purified samples. figure 3 compares the cvs of crude and purified 1 (0.852 mg/ml, 1 mm assuming 100% purity). the catalytic current, peaking at 1.3 v (all potentials are referenced to ag/agcl), in the crude sample is consistent with the woc activity that has been previously reported of 1 in the presence of proton accepting electrolytes. however, a similar catalytic wave in the purified sample is completely absent; only a reversible co(iii)3co(iv)/co(iii)4 couple centered at e1/2=+ 1.05 v is observed. interestingly, the catalytic current detected with the crude sample is only prominent in the first scan of the cv. a similar behavior is observed for 1-coome where precipitated samples exhibit a large catalytic current in the cv and chromatographed samples show only the reversible co(iii)3co(iv)/co(iii)4 couple, as shown in figure s8. the e1/2 of the reversible couple is at a more positive potential than for 1, due to the electron withdrawing nature of the methyl ester substituents on the pyridine ligands. the crude sample also showed a catalytic current (ep=1.4 v, figure s9), which was absent in the purified sample. the only observed difference between the cvs in carbonate and phosphate electrolyte is that the catalytic peak current of the crude sample occurs at a more positive (80 mv) potential in carbonate electrolyte. to confirm that the catalytic current in the crude sample was associated with the oxygen evolution reaction, electrochemical oxidation was performed in a dems experimental setup, which allows for the immediate and simultaneous detection of all gaseous products formed at the electrode surface. the catalytic current from an unpurified sample shown in the red trace of the top of figure 4a is accompanied by the production of o2, as shown in the middle panel of figure 4a. purified 1 and 1-coome were also investigated using dems under the identical conditions employed for that of the crude sample. as shown in the top panels of figure 4b, c for purified 1 and 1-coome, respectively, the faradaic current density decreases by over an order of magnitude from that of the crude sample. the waveform of the cvs in figure 4 are different than those of cvs taken on stationary gc electrodes (e.g., figure 3, red trace) owing to the flow conditions of the dems experiment; similar waveforms are observed, for instance, at rotating disk electrodes where there is forced solution flow across an electrode surface. the signal from the mass channel of o2 for the purified samples (middle panels in figure 4b, c) shows no o2 production for applied potentials below 1.4 v; at potentials of 1.4 v or greater, an extremely small amount of o2 is observed (pa intensities as opposed to na intensities of crude samples). we note that for all three samples, the mass channel of co2 exhibits a sizable signal when the electrode potential surpasses 1.2 v. the high level of evolved co2 is observed even in the background scans of blank gc electrodes (black lines in the bottom panels in figure 4a c). dems experimental data for three samples: (a) crude 1-coome, (b) purified 1 and (c) purified 1-coome. top panels display the faradaic current density vs potential; middle and bottom panels display the current collected for mass channels 32 (o2) and 44 (co2) m/z, respectively. red lines are representative data from the samples, and black lines are the data from the corresponding blank gc electrodes. we sought to place a limit on the level of o2 produced by the cubane cluster within the error of our measurements. the middle panel of figure 4b indicates that there is a small but non-negligible amount of o2 produced in purified samples of 1 at applied potentials>1.4 v. we therefore wished to quantify the amount charge passed with the current associated with the slight downturn in the red cv trace at potentials above 1.4 v in figure 3. three separate voltammograms (using three independently prepared gc electrodes) were collected with a sample of purified 1 (black traces in figure s10b d). a simulated cv (figure s10a) was subtracted from the background corrected raw data to remove the current that is due to the reversible co(iii)3co(iv)/co(iii)4 couple, thus leaving only the current that may be attributed to oxygen evolution (red traces in figure s10b d). from these data, the average current density was 0.11 0.04 ma/cm at 1.5 v. assuming that all of this current leads to the production of o2, then a tof of 0.06 mol o2/mol catalyst is calculated at an overpotential of 0.89 v (see si for details). this low current density and tof is consistent with catalysis from ppb concentrations of co(ii) produced from decomposition of the cubane (see discussion). solution [o2] measurements during illumination of crude samples of 1 (black), purified 1 (red), and without added 1 (green). photochemical reactions were performed in the presence of 0.5 mm ru(bpy)3, 35 mm na2s2o8, and 100 mm kpi ph=7 buffer. the concentration of crude and purified 1 was 0.33 mm, assuming 100% purity for the crude material. cvs of 2 mm (assuming 100% purity) crude 1 and [edta] =0 (black), 0.10 (red), 0.25 (blue) and 0.50 (green) mm in 0.2 m kpi (ph=7). arrow and cross indicates the initial point and direction of scan. to exclude the possibility of chemistry specific to a 1:gc interaction, pt, au, and fto a similar behavior was obtained as for the gc experiments: the cvs of the crude 1 showed significant water oxidation current, which was absent in the cvs of the purified material (figure s11). in addition to electrochemical woc activity, photochemical water oxidation has been reported for as-synthesized samples of 1 using the ru(bpy)3/s2o8 sacrificial oxidant system. the photochemical assay was performed in triplicate according to the literature procedure, with the exception that phosphate buffer was used instead of carbonate (see si for details). the concentration of o2 was measured for samples of crude 1, purified 1, and without added catalyst. a fluorescence-based o2 sensor was immersed into n2 purged solutions containing [ru(bpy)3]=0.5 mm, [s2o8]=35 mm, and =0.33 mm, and the cuvettes were photolyzed with a hg/xe arc lamp (exc>400 nm). the yield of o2 over 400 s of photolysis decreased from 167 15 m for the crude samples to 31 6 m for the purified samples (figure 5). (a) p nmr spectra of the phosphate signal of a 0.5 mm solution (0.426 mg/ml) of purified 1 in 0.2 m kpi (ph=7) with added co(ii) at the indicated concentrations. (b) the measured full-width at half-maximum (fwhm) of the phosphate p nmr signal is linearly dependent on the concentration of added co(ii). the equation of the linear calibration curve is fwhm={(936 8) [co(ii)]}+(7.2 0.4). (c e) p nmr spectra of the phosphate signal for three separate batches of 0.426 mg/ml of crude 1 in 0.2 m kpi at ph=7. using the calibration curve of (b), the amount of line broadening corresponds to a co(ii) concentration of 0.086 0.004 mm, 0.091 0.008 mm, and 0.065 0.006 mm for samples (c), (d) and (e), respectively. to identify and quantify the impurity found in the crude samples of 1, a series of spectroscopic and the epr spectrum of a solid sample of crude 1 reveals a broad paramagnetic signal over the range g=10 to 2, which is absent in the purified sample (figure s12). to confirm the presence of a co(ii) impurity, edta was titrated into a cv solution of the crude sample. figure 6 shows the cvs for the addition of edta (00.5 mm) into a 2 mm solution (assuming 100% purity) of crude 1 in 0.2 m kpi ph= 7. nearly complete suppression of the catalytic current was observed at 0.5 mm edta addition. as a control, a 50 m solution of purified 1 was treated with 10 mm edta in 0.2 m kpi at ph =7 for 1 h, and no changes in absorbance were observed (figure s13), confirming that 1 is kinetically stable in the presence of edta. the cv wave of the co(iii)/co(iv) couple of purified 1 with addition of edta (figure s14) is fully reversible, indicating that 1 is also stable to edta on the time scale of the cv experiment. the amount of co(ii) introduced by dissolving the crude preparation of 1 in aqueous media could be quantified by applying p nmr line broadening analysis, which we previously employed to quantify the self-healing properties of co-oec. a calibration curve was constructed by adding increasing amounts of a 1:1 mixture of co(oac)2:pyridine to a 0.5 mm solution (0.426 mg/ml) of purified 1 in 0.2 m kpi buffer (figure 7, see si for experimental details). this calibration curve was used to determine the amount of co(ii) in batches of crude 1. although cv experiments were performed with 1 at a concentration of 0.852 mg/ml, at this concentration of crude 1, the broadening of the phosphate signal is too great to construct a calibration curve over a wide enough range. thus, we performed p nmr line broadening experiments at half the concentration used for cv experiments. figure 7 shows the p nmr signals of phosphate upon dissolving 0.426 mg/ml of crude 1 for three separately prepared batches. per the calibration curve, we determine that the co(ii) ion concentration in solution is [co(ii)]=0.086 0.004 mm, 0.091 0.008 mm and 0.065 0.006 mm for samples (c), (d) and (e), respectively ([ co(ii)]avg=0.08 0.01 mm). translating this result to the concentrations used for cv experiments, a sample of 0.852 mg/ml of crude 1 introduces an average concentration of [co(ii)]=0.16 0.02 mm into solution. the results of the p nmr experiments were confirmed by an electrochemical titration, in which [co(ii)] was correlated with the catalytic current observed by cv (figure s15). with increasing [co(ii)], the peak current of the catalytic wave at 1.3 v increases linearly. a calibration curve was again constructed and used to assess [co(ii)] in the three batches of crude 1 at the concentration used for cv experiments. the results of this assay shows excellent agreement with the p nmr experiment, albeit with larger error bars, giving 0.153 0.019 mm, 0.178 0.020 mm, and 0.120 0.016 mm for the three samples, with a [co(ii)]avg=0.15 0.03 mm. because of the insolubility of co3(po4)2 in aqueous media, the measured co(ii) concentration could be diminished due to loss of cobalt in the form of a co3(po4)2 precipitate. however, at these low [co(ii)], precipitation of co(ii) by phosphate is negligible due to the slow kinetics of formation of the co3(po4)2 on the time scale of the electrochemical or photochemical experiments, which take minutes to complete. to experimentally verify that no co3(po4)2 formed under our experimental conditions, a 0.15 mm solution of a 1:1 mixture of co(oac)2:pyridine in the presence of 0.2 m kpi at ph=7 was monitored by p nmr line broadening over a 4 h period (figure s16). the p nmr spectrum establishes that the concentration of co(ii) in solution does not significantly decrease over this time period. the p nmr line broadening experiment is also a sensitive measure of compound stability. solids of purified 1 can be stored on the benchtop for at least 25 days without decomposition. the p nmr line broadening analysis of 0.5 mm 1 in 0.2 m kpi buffer solution shows that the presence of co(ii) ions after 25 days is negligible (figure s17). in addition, comparison of the h nmr spectra of 1, hours after purification and after 25 days are identical (figure s18). since crude 1 introduces co(ii) into the solution, we would expect that at anodic potentials, co-oec will be deposited. indeed, bulk electrolysis of a 1 mm solution of crude 1 at 1.2 v for 5 min resulted in the deposition of co-oec material on the electrode surface, which was readily observed by scanning electron microscopy (sem) and energy dispersive x-ray spectroscopy (eds) (figures s19a and s20a, respectively). bulk electrolysis of the crude sample at a higher potential of 1.4 v results in significantly less co-oec detected on the electrode surface (figures s19c and s20c), despite more charge being passed (figure s21). per the dems experiment, current is redirected from water splitting (o2 production) to degradation of the gc electrode (co2 production at higher potential). although bulk electrolysis performed over 300 s of a purified sample at 1.2 v resulted in an eds spectrum that is indistinguishable from that of a blank sample (figure s20b, d), the sem images of the pure and blank samples showed a subtle difference. the density of light contrast material was increased in the pure sample as compared to that of the blank sample. we therefore pursued further characterization of the electrode surface by xps analysis, which is more selective to analysis of surface materials than eds. a comparison of high-resolution co 2p xps spectra of crude, pure, and blank gc electrodes after 300 s of bulk electrolysis at 1.2 v is shown in figure s22. a trace signal at the co 2p3/2 peak of the pure sample is barely distinguishable over background, whereas a large co 2p3/2 signal is observed for electrodes removed from bulk-electrolyzed solutions of crude 1. as-synthesized samples of 1 contain significant amounts of impurities in two forms. the many aromatic peaks in the h nmr spectra of crude 1 (figure 2) and the slowly eluting bands on tlc plates are likely co(iii) clusters of smaller nuclearity, which are known to be stable compounds. of greater significance, as demonstrated by epr spectroscopy (figure s12), electrochemical measurements in the presence of the ion scavenging edta (figure 6) and p nmr line broadening analysis (figure 7), a co(ii) impurity is present in crude samples of 1. electrochemical titration experiments and p nmr line broadening experiments quantify significant amounts of co(ii) in as-synthesized preparations of 1. repeated experiments on different batches of as-synthesized 1 show that the concentration of co(ii) is 16% of the expected concentration of the cubane molecule, 1. because the co(ii) impurity is soluble in dcm, the ligation of the co(ii) ion likely involves solubilizing organic groups, such as the acetate or pyridine reactants of 1, as salts of co(ii) with outer-sphere anions, such as acetate or nitrate, are unlikely to have significant solubility in dcm. ligation of the solubilizing groups appears to be sufficiently weak that co-oec is easily formed (vide infra). as a cautionary note, the absence of line broadening in the h nmr spectra of 1 does not provide sufficient evidence that co(ii) is not present in solution. the lack of significant line broadening in the h nmr spectra upon titrating 1:1 co(oac)2:pyridine into a sample of purified 1 (figure s23) indicates that this is not a sensitive measure of paramagnetic impurities, presumably because 1 (a neutral, weakly basic molecule) does not interact significantly with the co(ii) ion. as figure 7 demonstrates, the phosphate p nmr signal is a much more sensitive measure of the presence of co(ii) impurities. the co(ii) ion impurities do not elute on silica and thus are easily removed from 1. the same behavior is observed for 1-coome, where silica gel chromatography can be used to remove co(ii) impurities from as-synthesized or precipitated samples. the co(ii) impurity acts as a source for the formation of the known water oxidation catalyst, co-oec. the formation of heterogeneous co-oec occurs from solutions of co(ii) with any proton accepting electrolyte, as long as the concentration of the electrolyte is sufficiently high to control ph. moreover, co-oec will be formed from co(ii) either electrochemically or (photo)chemically as long as the potential is sufficient to oxidize co(ii) to co(iii) in the presence of electrolytes such as phosphate or carbonate. consistent with the formation of co-oec, the catalytic wave in figure 3 has the same peak potential and onset current as found for a cv of co(ii) solutions from which co-oec electrodeposits (figure s24). however, unlike a well-behaved catalytic process, as is typical of co-oec on fto, a peak response is observed in the cyclic voltammogram. a peak in the catalytic wave will result from either depletion of substrate or catalyst deactivation. since the solvent, h2o, is the substrate, ph is maintained by a high concentration of phosphate, and current densities are low, we can safely rule out substrate depletion as the cause for the peak in figure 3. however, a peak will result if the catalyst were to be removed from the electrode in a parasitic side reaction, or as in this case, if oxidative degradation of the electrode is significant (vide infra). once the impurities are removed by column chromatography, the large catalytic waves in cvs of solutions of unpurified 1 (figure 3) and 1-coome (figure s8) disappear completely. this behavior is observed on other electrode materials (pt, au, and fto, figure s11) as well. crude 1 shows higher currents at anodic potentials than purified 1, providing further evidence that an impurity is responsible for the woc, as opposed to spurious activity arising from a specific deleterious interaction between the cobalt cubane molecule, 1, and a gc electrode. sem, eds, and xps support the formation of a heterogeneous co catalyst, which we attribute to co-oec, which deposits on electrodes from bulk electrolyzed solutions of crude 1. even in purified samples of 1, xps indicates that indeed a small amount of cobalt can be detected on the electrode. the production of co-oec from purified 1 explains the small amount of o2 observed in the dems experiment (figure 4b, middle panel) and the minute amount of current beyond background (figure 3, red trace) at potentials above 1.4 v vs ag/agcl. if all the current at 1.5 v goes to the production of o2, the tof at this potential would be 0.06 mol o2/mol of 1. however, only an extremely small amount of cobalt in the form of co-oec is needed to support the current density associated with this tof. using the tafel slope and the known dependence of the exchange current density on the thickness (i.e., co content) of films of co-oec, it was determined that only 70 ppb of 1, with its 4 cobalt atoms, would need to decompose to furnish enough cobalt to form co-oec and produce this observed current density (see si for details of the calculation). however, we note that the amount of co-oec and o2 produced is negligible as compared to the co-oec formed from as-synthesized samples of 1. at potentials above 1.4 v, the dems results show that the observed current is predominantly due to the production of co2 when a gc electrode is used as the anode. as eds and xps results show, the process is so efficient at 1.4 v, that the current is largely redirected from co-oec production from the co(ii) impurity to oxidative degradation of the electrode. importantly, the direct evidence of co2 formation (figure 4a, bottom) under conditions that thermodynamically favor the formation of co-oec argues against the possibility that the co-oec catalyst is unstable at these high potentials. if the potential is such that the rate of degradation of the gc surface is rapid, as stracke et al. have noted, one can not interpret the absence of deposited heterogeneous material after electrolysis as evidence of actual molecular catalysis, since surface catalyst will be lost upon degradation of the underlying electrode. consistent with this argument, sem and eds analysis show a decrease of observable co-oec on the electrode for bulk electrolysis experiments performed at 1.4 v vs 1.2 v (figures s19 and s20). any carbon material (e.g., graphene, carbon nanotubes. etc.) may be compromised due to degradation at high anodic potentials and thus water oxidation experiments performed on carbon-based anodes should be subject to dems or other mass spectrometric analysis to ensure that the current is not due to electrode oxidation to co2. as in electrochemical experiments, removing the co(ii) impurity from photochemically driven woc also leads to a dramatic reduction in the amount of o2 observed (figure 5). in the photolysis experiment, persulfate (s2o8) is used as a sacrificial oxidant to form ru(bpy)3 upon irradiation. the reduction potential of ru(bpy)3 is 1.06 v vs ag/agcl. at ph=7, co-oec is formed from co(ii) at potentials in the range of 0.750.80 v vs ag/agcl. therefore, under the conditions of the photolysis experiment, co(ii) can be oxidized to co-oec by ru(bpy)3. furthermore, the onset of woc by co-oec is 0.900.95 v vs ag/agcl, and so ru(bpy)3 is thermodynamically capable of driving catalyst turnover. in addition, the quenching reaction of ru(bpy)3 by persulfate to produce ru(bpy)3, also produces so4 as a potential oxidant, which has ample overpotential to drive water oxidation (e 2.2 v vs ag/agcl). thus, the major pathway giving rise to water photooxidation activity with as-synthesized 1 is consistent with the formation of co-oec from the in situ oxidation of co(ii) ions. although all photochemical studies have used as-synthesized 1, and thus water oxidation may be supported by co-oec, the present study shows the photochemical oxidation of purified 1 also results in the production of measurable quantities of o2 (31 6 m) over 400 s of photolysis, leading to a tof=2.3 10 s. at the potential of ru(bpy)3, which is within the co(iii)3co(iv)/co(iii)4 wave (figure 3), no o2 is produced as measured by dems (figure 4b). therefore, ru(bpy)3 is not a potent enough oxidant to turn over 1; a greater overpotential is required, if 1 is indeed a molecular catalyst under these specific photochemical conditions. as noted above, the protocol of the photochemical experiment produces the strongly oxidizing species so4. this species is free not only to react directly with ru(bpy)3 but also to react with 1 because the concentrations of [ru(bpy)3]=0.5 mm and =0.33 mm are similar. therefore, the observed o2 emanating from the photolysis conditions used for purified 1 in figure 5 is likely due to the interaction of 1 with so4, which has a considerably more positive reduction potential than ru(bpy)3. h bond (bdfe=123 kcal/mol) of water directly to produce the radical, oh. h bonds of the ligands, and thus the molecule itself, to be thermodynamically stable with respect to hydrogen atom abstraction given the extreme potentials provided by the electron accepting so4 and proton accepting phosphate buffer species. if the cubane were to decompose, co-oec is a likely product of the decomposition pathway. alternatively, computational investigations into the mechanism of woc by 1 suggest that two oxidations of 1 to the level of co(iii)2co(iv)2 and an acetate ligand dissociation were required prior to water attack and subsequent o o bond formation. we can not confirm if so4 is capable of oxidizing 1 because the electrochemical window limits the range of potentials for investigating the behavior of 1 at potentials beyond 1.5 v. if a higher oxidized cubane is capable of water oxidation activity, it occurs at extremely high overpotentials. without purification by silica chromatography, the co(iii) oxo cubanes can be contaminated with co(ii) impurities, which are responsible for the observed water oxidation activity reported for these molecules. we have shown that an edta titration can be used to test for the presence of co(ii) and a p nmr experiment can be used for the co(ii) quantification; these experiments are more definitive than h nmr spectroscopy for identifying paramagnetic co(ii) impurities. beyond co(ii) as an impurity, the use of any co(ii) complex should be assessed as an authentic woc versus precursors for heterogeneous catalysts such as co-oec owing to the proclivity of co(ii) complexes to undergo rapid ligand substitution. we note that water oxidation activity of a catalyst should not depend on whether an anodic potential is supplied electrochemically or (photo)chemically for mechanisms involving outer sphere electron transfers. in instances where homogeneous and heterogeneous o2 evolution experiments do not concur, it is appropriate to consider whether other species are responsible for catalytic activity. finally, when inspecting carbon-based electrode surfaces for the deposition of heterogeneous catalysts, care must be exercised in the choice of oxidizing potentials, as extreme values can give rise to spurious current that is associated with co2 evolution and electrode degradation as established by dems.

the observed water oxidation activity of the compound class co4o4(oac)4(py x)4 emanates from a co(ii) impurity. this impurity is oxidized to produce the well-known co-oec heterogeneous cobaltate catalyst, which is an active water oxidation catalyst. we present results from electron paramagnetic resonance spectroscopy, nuclear magnetic resonance line broadening analysis, and electrochemical titrations to establish the existence of the co(ii) impurity as the major source of water oxidation activity that has been reported for co4o4 molecular cubanes. differential electrochemical mass spectrometry is used to characterize the fate of glassy carbon at water oxidizing potentials and demonstrate that such electrode materials should be used with caution for the study of water oxidation catalysis.

PMC4277775

pubmed-1245

in recent years, there is increasing evidence to suggest that prokaryotes adopt primitive organelle-like structures called bacterial microcompartments or nanocompartments, depending on their size. such assemblies localize and compartmentalize multiple enzymes and substrates involved in specific metabolic pathways. the ability to mimic and understand enzymatic activity in confinement would provide ground-breaking insight into these assemblies and in organelles in general. one of the main challenges is to controllably package and coencapsulate different enzymes noncovalently within the same compartment as exemplified by nature. there has been some success using coiled-coil helices, peptide tags, and protein protein fusion constructs to direct enzymatic cargo encapsulation into protein cages. however, such approaches often lead to covalently connected protein cargo or inefficient loading (i.e., formation of empty cage assemblies). to circumvent these problems, we seek a versatile approach that would promote a noncovalent co-encapsulation of enzymes within a single protein cage in vitro. the cowpea chlorotic mottle virus (ccmv) is an ideal candidate to mimic bacterial nanocompartments, owing to its size and biocompatibility. the ccmv capsid is 28 nm in diameter and is based on a t=3 lattice (t, triangulation number), with 12 pentamers and 20 hexamers of identical monomers of capsid protein (cp) organized as 90 dimers. similar to bacterial compartments, it has multiple pores in the capsid shell (around 2 nm), which allows molecules and substrates to diffuse in and out. the cp n-terminal region is enriched in positively charged residues, termed the arginine-rich motif (arm), that face the capsid inner surface. after removal of native single-strand (ss) rna cargo, the arm can trigger reassembly of capsid protein dimers in the presence of an appropriate negatively charged template, resulting in the formation of monodisperse virus-like particles (vlp). encapsulation of enzymes in vlps has been shown to stabilize and protect the enzymes, and its ease of modification enables new applications. in this contribution, single- and complementary-stranded dna tags are chemically attached to the exterior of chosen enzymes, resulting in negatively charged complexes that induce the co-encapsulation inside ccmv capsids. unlike covalent interactions, electrostatic interactions between the dna tags and the interior of the capsid provide a tunable system, enabled, for instance, by changing the salt concentration or by varying the length of the dna chains. hence, this makes our system a model for natural bacterial compartments (e.g., the encapsulins) where the confined enzymes are not covalently bound but rather included in the protein cage by noncovalent, multivalent interactions. noncovalent encapsulation mediated by nucleic acid tags has been reported with the use of genetic engineering and/or only focused on a single enzyme. using this strategy, we were able to confine two separate cascade systems in vitro, for which the glucose oxidase (gox), a 160 kda dimeric enzyme, is chosen as the primary enzyme for both encapsulated cascades. gox catalyzes the oxidation of glucose into gluconolactone (which undergoes spontaneous hydrolysis into gluconic acid) and produces hydrogen peroxide as the side product. in the first cascade system, hydrogen peroxide produced by gox is consumed by the so-called dnazyme, a peroxidase-mimic formed in situ by a specific sequence of ssdna in the presence of hemin (figure 1a). in the second cascade system, in the presence of atp and nadp, gluconic acid produced by gox is consumed by a secondary enzyme, gluconokinase (gck) that is coencapsulated inside the ccmv-like particles, followed by a nonencapsulated tertiary enzyme, 6-phosphogluconate dehydrogenase (6-pgdh), to form ribulose-5-phosphate and nadph, the latter of which can be monitored spectroscopically (figure 1b). therefore, the nucleic acid tags in this work are useful both as a secondary biocatalyst (in cascade system i) and as negatively charged tags to trigger the encapsulation of the enzyme(s) (in cascade systems i and ii). schematic representation of the enzyme pathways (encapsulated processes shown in gray boxes). gox oxidizes glucose to gluconic acid and produces h2o2, which dnazyme uses for subsequent reaction with abts inside ccmv capsid. the conversion of glucose to d-gluconate-6-p occurs at the interior of the ccmv capsids, whereas the conversion of d-gluconate-6-p into ribulose-5-p occurs at the exterior of the ccmv capsid catalyzed by tertiary enzyme, 6-pgdh. we anticipated that functionalization and subsequent hybridization of gox and gck with (complementary) single-stranded dna strands should promote their co-encapsulation into ccmv capsids (figure 2). we suspect that the size of the enzyme cargo as well as the number and spatial distribution of negative charges anchored to the enzyme surface might play a role in determining the efficiency of the encapsulation. to modify and hybridize the relevant enzymes, the lysine residues of gox and gck were functionalized with a heterobifunctional linker using sulfo-nhs coupling followed by maleimide thiol chemistry on the single stranded dna (ssdna) or its complementary sequence (csdna), respectively (see figures s1 and s2). all hybridized complexes were purified initially by spin-filtration to remove excess dna. furthermore, size-exclusion chromatography (sec) was used for the gox gck dual-enzyme complex to remove nonhybridized gck and gox prior to encapsulation (figure s3). encapsulation of different enzyme dna hybrids inside ccmv capsids (gray) at ph 7.5. encapsulation of (a) ssdna in yellow, (b) gox, in blue, functionalized with ssdna, (c) gck, in green, functionalized with the complementary ssdna in red, (d) gox conjugated to gck. the specific sequence of ssdna is catalytically active in the presence of hemin. encapsulation of gox ssdna, gck csdna, or hybridized gox gck in ccmv at ph 7.5 led to the formation of stable capsid-like assemblies, which were purified by sec (figure 3a d). the elution volume (12 ml), together with the relative absorbance ratio (260 nm/280 nm)>1, are characteristic features of intact ccmv capsids containing dna-based cargo. control experiments with no dna tags (i.e., nonfunctionalized gox and/or gck) confirmed that enzymes lacking ssdna or csdna can not be encapsulated (figure s4). as anticipated, the dna strands provide the required negative charges for reassembly into virus-like particles. this further ensures that no empty particles are obtained with this strategy and that formed particles always contain a negatively charged cargo. size-exclusion chromatograms for ccmv containing (a) ssdna, (b) gox ssdna, (c) gck csdna, and (d) gox gck, with monitoring at =260 (red), 280 (blue), and 450 nm (black), for dna, ccmv, and flavin (gox), respectively. negatively stained transmission electron microscopy of (e) ssdna, (f) gox ssdna, (g) gck csdna, and (h) gox gck encapsulated ccmv assemblies. the assembled ccmv-like particles were characterized by negative staining transmission electron microscopy (tem) (figure 3e h) and dynamic light scattering (dls, figure s5), showing spherical structures of around 20 nm in diameter. the size of around 20 nm indicates the formation of t=1 icosahedral symmetry that is composed of 60 identical capsid subunits. furthermore, co-encapsulation of gox and gck in a single particle was confirmed with sds-page and western blot analyses (figures s6 and s7). additionally, their concentrations and relative ratios were estimated by gel densitometry, which suggested a gox/gck/capsid protein ratio of approximately 1:1.4:60. since the capsid protein is composed of 60 identical subunits, we estimate that only a single gox ssdna is confined inside ccmv-like particles for cascade system i and a hybrid of 1 gox dna and 1 or 2 gck csdna is confined for cascade system ii. to confirm the assembly of ccmv-like particles with t=1 icosahedral symmetry, we analyzed the gox ssdna-loaded ccmv-like particles with cryo-electron microscopy (cryo-em) to calculate their native three-dimensional reconstruction (3dr) (figure 4a). the sample contained particles with spherical and elongated profiles as well as irregular assemblies (figure 4a, inset). two-dimensional classification followed by a three-dimensional classification using relion software resulted in two sizes of icosahedral capsids with t=1 architecture. whereas class i capsids were 214 in diameter (figure 4b), class ii capsids were 226 (figure 4c). three-dimensional cryo-em reconstructions of gox ssdna-loaded ccmv capsids. (a) cryo-electron micrograph of gox ssdna-loaded ccmv capsids. two-dimensional class averages derived from the final 15481 particle data set (inset). (b) surface-shaded representation of the outer surface of the class i t=1 capsid (diameter 21.4 nm) viewed along a 2-, 3-, and 5-fold axis of icosahedral symmetry (top to bottom). models of the class i t=1 capsid, with the front half of the cargo and protein shell removed (right). (c) surface-shaded representations of the outer surface of the class ii t=1 capsid (diameter 22.6 nm) (as in b). capsids i and ii made up 50% of the total particles in the sample (70% class i, 30% class ii). the cryo-em images analyzed for processing are in fact snapshots of the dynamic states of the sample; the ratio observed could be due to displacement of dynamic equilibrium toward class i t=1 capsids (70% class i, 30% class ii). the two particle sizes might be related to the reported dynamic swelling of the t=3 ccmv native capsid, as the size difference of 5% involves a 7 outward radial expansion and widening of the pores, hinting at possible structural breathing. in both t=1 capsids, the pentamer bases were strongly connected to the underlying gox ssdna cargo, although the capsid surface pores were distinct. whereas class i capsid pentamers barely left any space between their lateral contacts, those of class ii capsids were clearly separated and left large pores, especially at the icosahedral 2-fold axes (figure 4b, c, arrows). docking of ccmv capsid protein (cp) dimer into the cryo-em density maps of gox-ssdna-loaded t=1 vlp showed major structural differences of the two classes (figure 5a, b). connecting densities between pentamers and cargo were mediated by residues 4250 of the cp n-terminal region (figure 5c, dark blue), although the preceding region (residues 2741) could also be involved (figure 5c, pink). the cp c-terminal ends were responsible for cp dimer assembly in class i ccmv t=1 capsids (figure 5a, arrows; figure 5d, red). the hinge angle formed between cp dimers in gox ssdna-loaded t=1 class i capsids was 60 (figure 5d); it resembles that found at the quasi-2-fold axes of the swollen t=3 ccmv capsid and in other cp dimers such as the phthalocyanine-loaded t=1 vlp. ccmv cp dimers are the building blocks of native t=3 virion capsids as well as of in vitro assembled structures such as tubes and icosahedral capsids with t=1 (containing 30 cp dimers), t=2 (60 dimers), and t=3 (90 dimers) architecture. the outward expansion of the class ii capsid pentamer entailed the disappearance of or a great reduction in dimeric contacts (figure 5b, arrows) and indicated that these interactions contribute much less to class ii capsid stability than to that of class i capsids. assuming the same building block is involved, the class ii capsids are based on pentamers bound weakly by the cp c-terminal ends (figure 5e, red), which adhere strongly to the polyanionic cargo. pseudoatomic model of gox ssdna-loaded ccmv capsids. (a) t=1 class i capsid viewed down a 3-fold axis from outside, with docked ccmv cp atomic coordinates. (b) class ii t=1 capsid viewed down a 3-fold axis from outside (as in b). (c) pentamer contacts with the cargo mediated by residues 4250 in the n-terminal region (dark blue, bottom view). the n-terminal region residues 2741 might also contribute to cargo side view (top), top view (bottom). the hinge dihedral angle is indicated. cp monomers in the class ii dimer are 6.5 further apart than class i dimers (n-terminal 2732 region is omitted). after imposing icosahedral symmetry in class i and ii capsids, we observed the packed cargo as a hollow sphere (9.6 10 and 1.2 10, respectively), with numerous connections to the t=1 capsid inner surface. based on the atomic model of gox (pdb 1gal), several copies of gox could be encapsulated in the capsid, although our biochemical analyses indicated the presence of a gox dimer only. this discrepancy is probably due to the chemical modification of accessible lys residues of gox that are covalently bound to ssdna. capsid connections observed in the 3d cryo-em maps probably represent the interaction of the arm region with negatively charged dna strands. gox ssdna packaging resulted in a slightly disordered icosahedral capsid (also reflected in a limited map resolution), but this cargo enabled structural polymorphism with weak cp interactions in the dimer. both t=1 capsids coexist in dynamic equilibrium, probably enabled because the cp ssdna interactions are more flexible (or less well-defined) than the cp ssrna interactions. to our knowledge, this is the first demonstration of the formation of a t=1 ccmv-like structure templated by a biological soft material that also displays an extreme capsid swelling. following structural characterization of enzyme dna complexes inside t=1 ccmv-like particles, we proceeded to monitor the enzymatic activity of both cascade systems to examine whether the encapsulated complexes were still catalytically active. for cascade system i (figure 1a and figure 6a), we deliberately chose the ssdna sequence coupled to the gox to be that of a hemin-binding dna quadruplex, the so-called dnazyme. in the presence of hemin, the ssdna spontaneously forms a scaffold that mimics the catalytic properties of horseradish peroxidase (hrp). dnazyme was monitored via the production of abts at =410 nm upon addition of glucose to the system. the activity plot obtained for encapsulated gox dnazyme shows that both gox and dnazyme remained catalytically active after encapsulation (figure 6b and figure s8a). an increase in both km values (2.2-fold) and kcat values (1.7-fold) is observed when the system is encapsulated (summarized in table s1). (a) schematic representation of cascade system i in the presence of a competing enzyme, catalase (encapsulated processes shown in gray boxes). (b) kinetic measurements of cascade system i; the production of abts was monitored at =410 nm at different glucose concentrations. (c) kinetic measurements of cascade system ii; the production of nadph was monitored at =340 nm at different glucose concentrations. (d) kinetic measurements for the production of abts in the presence of and after ph inactivation of the competing enzyme, catalase. for cascade system ii consisting of gox, gck, and 6-pgdh (figure 1b), we monitored the formation of the end product, nadph at =340 nm, upon addition of glucose to the system. both enzymes (gox and gck) are therefore required for the reaction and its visualization at 340 nm. the activity profile in figure 6c and figure s8b confirms that both enzymes were present in the system and still active upon hybridization and subsequent encapsulation. while the km values remain similar for both systems, the kcat values show a 2-fold increase for the encapsulated system (summarized in table s1). based on the recurring trends, a slightly higher turnover number (kcat) upon pathway encapsulation is estimated, although the protein concentration determination by gel densitometry is expected to have a large deviation and consequently also the kcat. an eventual increase might be the result of a local enhancement in effective molarity due to confinement or of the channeling effect when multiple enzymes in a cascade pathway are brought to a close proximity inside a confined system. the enzymatic activities observed for both cascade pathways indicated that the substrate glucose was able to diffuse into the capsid shell. we further investigated whether the intermediate of the cascade could also diffuse freely or was trapped inside the cagelike structure during the reaction. in order to confirm the state of the intermediate, h2o2 is broken down to water and oxygen by the enzyme catalase, and it can therefore act as an external competitor with the dnazyme. in contrast, if the h2o2 intermediate is trapped inside ccmv (as proposed for the bacterial microcompartments), the kinetics of abts production should remain unaltered. instead, we observed almost complete suppression of abts production in the presence of catalase (figure 6d). only upon lowering the buffer to ph 4 and hence inactivating catalase (t=60 min) could the h2o2 conversion by dnazyme be restored while maintaining the stability of the particles, as confirmed by sec and tem analyses in figure s9. taken together, in agreement with a previous report, we also observed that the ccmv capsid shell is permeable to small molecules such as h2o2, which can diffuse out of the capsid shell and react with the competing enzyme. nevertheless, it should be noted that the dnazyme is likely to exhibit lower catalytic efficiency and lower affinity to h2o2 compared to catalase, which could also lead to the diffusion of h2o2 out of the ccmv capsid. we have presented a highly effective strategy of using single-stranded dna for the controlled noncovalent packing of enzyme cascades in a single protein capsid assembly. to demonstrate the versatility of this strategy, two different cascade systems based on glucose oxidase were assembled inside the protein shell of ccmv at ph 7.5 and this encapsulation strategy resulted in icosahedral structures of approximately 20 nm, which were further analyzed with 3d cryo-em. the resulting 3d reconstruction provides the first-time demonstration of t=1 structured assemblies of ccmv around a biological soft matter template. in addition, an extra-swelling phenomenon was indicated on the basis of the coexistence of two differently sized particles of similar structure and origin. the method presented for assembling virus-like particles can provide a structural and functional basis to analyze bacterial protein organelles and will further improve our understanding of their containment properties and biochemical function. the ssdna (5-hs-(ch2)6-gggtagggcgggttgggtttt-3) and csdna (5-hs-(ch2)6-aaaacccaacccgccctaccc-3) oligonucleotide sequences were synthesized by eurofins mwg operon. for the coupling of dna to enzymes, the bifunctional cross-linker sulfo-emcs (n-[-maleimidocaproyloxy] sulfosuccinimide ester) was purchased from pierce. d-gluconate/d-glucono--lactone assay kit was purchased from megazyme and used as provided. all other reagents were purchased from sigma-aldrich or fluka unless stated otherwise and were used without further purification. a stock solution of hemin (5 mm) was prepared in dmso and stored in the dark at 4 c. hcl, 500 mm nacl, 50 mm mgcl2, 1 mm dtt, ph 7.5) to obtain ccmv dimer coat proteins (ccmv-cp) (500 m). gck, and gox gck were buffer exchanged against milli-q water using amicon ultra centrifugal filters (30 kda or 10 kda mwco). gck and ccmv cp (in assembly buffer) were mixed in a 4:1 (v/v) ratio and incubated for 2 h at 4 c before purification by size-exclusion chromatography (sec) using a superose 6 10/100 gl column, eluting with 50 mm tris hcl, 100 mm nacl, 10 mm mgcl2, 0.5 mm dtt at ph 7.5. protein fractions were collected and analyzed by sds page, agarose gel, and western blot analysis. for the complete procedure, samples (5 l) were applied onto formvar carbon-coated grids. uranyl acetate (5 l, 1% w/v) was added and the excess liquid was drained after 20 s and dried for 30 min at room temperature. the samples were examined on a feg-tem (phillips cm 30) operated at 300 kv acceleration voltages. ssdna-loaded vlp (5 l) were applied to one side of quantifoil r 2/2 holey grids, blotted, and plunged into liquid ethane in a leica em cpc cryofixation unit. the grids were analyzed in a tecnai g2 electron microscope equipped with a field emission gun operating at 200 kv, and images were recorded under low-dose conditions with a fei eagle ccd at a detector magnification of 69,444x (2.16 /pixel sampling rate). image processing operations were performed using xmipp and relion, and graphic representations were produced with ucsf chimera. the xmipp automatic picking routine was used to select 15481 particles, and defocus was determined with ctffind. images were 2d-classified using the appropriate relion routine and 7932 isometric particles were selected. the structure of phthalocyanine-loaded ccmv t=1 capsid was filtered out to 30, and the cargo density was masked. this map was used as an initial model for 3d classification of spherical particles, using relion to select 5572 (class i) and 2318 (class ii) particles; these data sets were used to obtain the final 3drs using the relion autorefinement routine. resolution was assessed by gold standard fsc between two independently processed half-data sets. applying a correlation limit of 0.5 (0.3), the resolution for class i and ii 3d maps was 22.7 (22.2) and 25.6 (21.3), respectively. the chimera fitting tool was used to dock the atomic structure of a whole pentamer from the x-ray structure of ccmv (pdb entry 1cwp) into the cryoem maps. substrate solutions containing various glucose concentrations (01 m, 180 l) were prepared. an enzyme solution containing either (1) free gox gck (60 l) or (2) encapsulated gox 6-pgdh (55 u/ml, 2 l) and 16.1 mm nadp+ containing 69.4 mm atp were added to each reaction mixture at ph 7.5, according to the manufacturer s instructions (megazyme kit). the reaction was started upon addition of glucose (120 l) to enzyme (82 l), and formation of reduced nadph was monitored at =340 nm in 100 s time intervals over 2 h at 27 c. stock solutions containing both substrates glucose (ranging from 01 m) and 4 mm abts were freshly prepared at room temperature. dnazyme (30 l) and hemin (30 l, 5 m) or (2) encapsulated gox dnazyme (30 l) and hemin (30 l, 5 m) were prepared and incubated at rt for 2 h. to each enzyme containing solution (60 l) was added the substrate solution containing both glucose and abts (120 l), and the reaction was monitored immediately at =410 nm for the conversion of abts to abts at 27 c in 100 s time intervals over 2 h. control experiments containing hemin, glucose, and abts were performed under the same reaction conditions. experimental data were corrected for background absorbance (using the control experiment as a reference). the concentration of abts or nadph was determined using the lambert beer law, assuming extinction coefficients of abts (410 nm=36000 m cm) or nadph (340 nm=6300 m cm) before plotting concentration (m) vs time (min) curves, from which the velocity (v) was determined (m/min). a dilution factor (df) relative to the enzyme (gox) and a proportionality factor of abts to substrate consumption (p=1/2) were used to correct the velocity values as described in eq 1.1

the packaging of proteins into discrete compartments is an essential feature for cellular efficiency. inspired by nature, we harness virus-like assemblies as artificial nanocompartments for enzyme-catalyzed cascade reactions. using the negative charges of nucleic acid tags, we develop a versatile strategy to promote an efficient noncovalent co-encapsulation of enzymes within a single protein cage of cowpea chlorotic mottle virus (ccmv) at neutral ph. the encapsulation results in stable 2122 nm sized ccmv-like particles, which is characteristic of an icosahedral t=1 symmetry. cryo-em reconstruction was used to demonstrate the structure of t=1 assemblies templated by biological soft materials as well as the extra-swelling capacity of these t=1 capsids. furthermore, the specific sequence of the dna tag is capable of operating as a secondary biocatalyst as well as bridging two enzymes for co-encapsulation in a single capsid while maintaining their enzymatic activity. using ccmv-like particles to mimic nanocompartments can provide valuable insight on the role of biological compartments in enhancing metabolic efficiency.

PMC5330652

pubmed-1246

castleman s disease, giant lymph node hyperplasia, is an autoimmune, lymphoproliferative disease that shows itself with the enlargement of lymph nodes and varied clinical presentations. castleman s disease commonly involves mediastinum and hence it is thoracic in most of the reported cases. it is a rare disease that presents itself by hyperplasia of lymph nodes with no malignant origin. this disease is known by other names such as angiofollicular lymph node hyperplasia or giant lymph node hyperplasia. simply, the pathology of this disease can be attributed to the hypervascular of lymph nodes and hyalinization of vessels. from a pathological perspective, three variants of this type of tumor are recognized: hyaline vascular cd type, plasma cell type, and mixed type. from a clinical standpoint, this tumor is found in two types of unicentric and multi-centric, in which the unicentric type is much more common. previous studies have presented it as an unusual finding in retroperitoneal ct imaging. in this study, we aim at introducing one patient with castleman s disease who had referred with clinical picture of occasional abdominal pain. a 34-year-old woman complaining of occasional abdominal pain referred to the surgery clinic. there was no remarkable point in physical examination and the patient did not have any other clinical symptoms. laboratory findings only reported microcytic anemia (mch: 18.5, mcv: 63, hemoglobin 10.2 g/di). imaging results were chest and abdominal x-ray without any remarkable point. in abdominal ultrasonography, a solid and firm tumor with 12.25.36.6 cm was reported in patient s retropritoneum. for more evaluation, the tumor was not attached to the walls of the intestines and it did not cause a blockage. with coordinates obtained by ct scan and ultrasonography (us), a big tumor was found in the retropritoneum that was solid and firm but was not attached to the walls of the intestine or to the lymph nodes of that area. in the operation area, the tumor was completely removed in order to treat patent and more reviews. from the macroscopic point of view, the tumor was shaped like an egg, covered by a fibrous thick capsule, with no bizarre appearance and without visible bleeding on its surface (figure 1). in cutting points, its surface was homogeneous, chocolaty and in terms of consistency, hard. although it was similar to lipoma in early studies, but the initial pathologic study reported its lymphoid origin that rejected the possibility of malignancy. macroscopic view of the resected castleman s tumor (the tumor was shaped like an egg, covered by fibrous thick capsule, with no bizarre appearance and bleeding). more histopathological investigation of tumor was an extensive lymphatic tissue containing hyperplastic follicles that was placed in the frame of a lymphoid tumor. mantle zones of follicles had spread and small germinal centers were seen (figure 2). microscopic view; expanded mantle zone and the small germinal centers of the lymphoid follicles have shown apparently. hyaline vascular type revealed castleman s disease (h&e, x400). among these germinal centers, interfollicular stroma as hyperplasia is defined as post-capillaries venules, in which combination of plasma cells and eosinophils are seen. histopathology of these samples gave a definitive diagnosis of angiofollicular lymph node hyperplasia, which in this case was hyaline vascular type. a chest ct scan was performed at the end of treatment and its review implied the absence of a similar tumor elsewhere. the patient did not have any particular postoperative problems and was discharged on day-7 after surgery. patient s follow up was done until 9 months after discharge for symptoms of recurrence or any kind of clinical abnormalities. our study was approved by the medical ethics committee, according to the helsinki declaration. castleman s disease is a kind of rare pathology, usually benign, with unclear etiology and prevalence. it is reported in childhood and adolescence periods in much lower numbers. in most of the reported cases, the disease arises in the chest and especially in the mediastinum. from other involved areas, we can mention mesentery, armpits, neck and in very rare cases in retroperitoneum. despite the fact that the etiology of this disease remained unknown, we can cite autoimmune disease, and some viral diseases as predisposing factors for this disease, abnormality of test results and clinical examinations. none of the above diseases was seen in this reported case, even until the end of the follow-up period. therefore, these immunity factors are discussed as predisposing factors and not as a main etiological factor. as mentioned, the castleman s disease is clinically divided into two groups of unicentric and multi-centric. in unicentric type, that is more common, the possibility of invasion is lower and is seen with hyaline vascular type. therefore, it is difficult and sometimes impossible to diagnose only based on disease symptoms before the operation and pathology study, especially in such diagnosis. among clinical tests, it is possible that complete blood to be along with anemia with small changes, similar to what was observed in our patient, while this finding is not specific. in some case reports, lack of result from these can not be a reason to reject castleman s disease. some studies started their diagnostic procedures with endoscopic ultrasound-guided fine-needle aspiration (eus-fna). in this study, we used ultrasonography after abdominal x-ray leading to tumor detection and locating its exact location by using an abdominal ct scan. of course, since no study with a high sample size has been conducted in this field, sensitivity and specificity of none of these diagnostic methods were exactly identified. histopathology of tumor tissue after surgery is the only way for tumor (and its type) diagnosis. most of the castleman s tumor is seen as homogeneous and hypoechoic tumor in the view of the sonography. in color doppler view, it can be concluded that the radiological view is not specific enough to detect this disease and the observed views could be mistaken with each type of benign or malignant lymphomatous tumor. in this study, similar to most other published case reports, the tumor was of the unicentric type while multi-centric type is less common and should be noted with a broader therapeutic approach. it should be noted that in this disease, the localized type often responds to surgical treatment alone. complete removal of tumor and its margin will suffice with the treatment of laparotomy and even laparoscopy. these kinds of tumors do not have invasive behavior and are completely benign. on the other hand, castleman s disease is a kind of rare vascular hyperplasia that has often benign and with noninvasive behavior. there is no reliable diagnostic method and its definitive diagnosis is based on histopathology report. it is often found in the chest, especially in the mediastinum and is asymptomatic in most cases.

castleman s disease, giant lymph node hyperplasia, is a kind of benign lymphoproliferative disease with gentle behavior. its etiology and prevalence are unclear. this rare disease is usually found in mediastinal area asymptomatically and incidentally. it is also rare to see this tumor in the retroperitoneum. in this study, we have introduced a 34-year-old woman who referred just with occasional abdominal pain caused by compressive symptoms. laboratory findings only reported microcytic anemia (mch: 18.5, mcv: 63, hemoglobin 10.2 g/dl). chest and abdominal x-ray imaging showed no remarkable point. in abdominal ultrasonography, a solid and firm tumor with 12.25.36.6 cm was reported in patient s retroperitoneum. patient s surgery was done and the tumor (covered by a fibrous thick capsule, with no bizarre appearance and bleeding) was completely removed. pathologic examination indicated a castleman s tumor, type of unicentric and hyaline-vascular. this item had been one of the rare reported items of castleman s disease in the retroperitoneal space.

PMC4567609

pubmed-1247

breast cancer is a leading cause of death and disability among women, especially young women, in low- and middle-income countries. though incidence and overall mortality rates continue to be lower than in most high-income countries, case fatality rates from breast cancer are very high. these high case fatality rates are likely due to a lack of awareness of the benefits of detection and treatment and a scarcity of adequate facilities for detection and diagnosis, as well as poor access to primary treatment. remarkable improvements have been achieved in the probability of survival for women diagnosed with breast cancer in the usa as compared to 60 years ago. early detection through the use of mammography, high-quality surgery, and adjuvant therapies including chemotherapy and targeted therapies, such as hormonal therapy and, more recently the her2-directed agent trastuzumab, can be credited for much of the recent improvement in outcome for women with breast cancer in the usa. however, even prior to the routine use of mammography or adjuvant therapy, significant improvements were made in breast cancer survival, and these can be traced to relatively low-cost interventions that are still in use in high-income countries. understanding which healthcare interventions were available and how they resulted in improvements in the probability of survival could be important, especially for designing programs in resource-constrained settings where breast cancer case fatality is high and many of the most costly and technology-intensive diagnostic and therapeutic options are not available. in many developing countries, the incidence of breast cancer is now rising sharply due to changes in reproductive factors, lifestyle, and increased life expectancy. today, more than half of incident cases occur in the developing world [14, 15]. combined with still high case-fatality rates, this means that mortality from breast cancer is a leading cause of death among adult women in developing countries, as well as in the developed world. in mexico, for example, breast cancer is now the second leading cause of death among women aged 30 to 54 and the leading cause of tumor-related death among adult women of all ages. the high probability of dying from breast cancer the case fatality rate, which is approximated by the ratio of mortality to income across the developing world further reflects the inequities in early detection and access to treatment [1, 17]. the number of deaths as a percentage of incident cases in 2008 was 48% in low-income, 40% in low-middle-income, and 38% in high-middle-income countries, while it was 24% in high-income countries according to the most recent globocan/iarc data. available evidence on stage at diagnosis, though scarce, indicate that a very high proportion of cases in the developing world are detected in late stages [1, 3]. (table 1) in many underserved populations, a majority of women present with advanced disease; the figure is as high as 78% in black women in south africa. in contrast, in the united states the majority of cases are detected in localized stages of the disease (stages i and ii), a third is regionally advanced (stage iii), and only 5% are distant-stage metastatic (stage iv). many reasons are given for the advanced stage at presentation and resultant poor survival rates in low- and middle-income countries: the stigma of breast cancer and the associated societal implications of its treatments (especially mastectomy) discourage women from seeking care early on; lack of knowledge about breast health; scant options for early detection due to limited access to routine care and examinations; and lack of access to mammography and to affordable, high-quality treatment options. in the short term, mammography and other expensive and technologically complicated resources and therapies will not likely be available to many of the world's women. though we must continue to work at all levels to bring diagnostics and therapeutics with a proven impact on outcomes to these women as soon as possible, there are ways closer at hand to improve the immediate outlook for women in these settings. figure 1 shows the incidence and mortality rates for breast cancer in the usa between 1940 and 2000. from the late 1940s, thus mortality-to-incidence ratios decreased dramatically, even before the generalized use of mammography or adjuvant chemotherapy and antiestrogen therapy that commenced in the mid- to late 1970s. table 2 presents the ratio of mortality over incidence, as an approximation of the case-fatality rate, in 5-year increments between 1950 and 1975. between 1950 and 1975 incidence nearly doubled, increasing from 66.6/100,000 women to 119.2/100,000, while mortality remained relatively constant, 28/100,000 and 31.6/100,000, respectively. thus, during this time period, the ratio of mortality over incidence (an approximation of the case-fatality rate) fell from 0.42 to 0.27 representing a 36% decline: this suggests that more women were surviving their cancers in 1975 as compared to 1950 and is true for both whites and blacks. further, the reduction in case-fatality rates is at least as large as the improvement evidenced since the introduction of mammography and adjuvant therapy. these findings suggest considerable room for reducing the high mortality-to-incidence ratio found in many developing countries even without mammography or adjuvant therapy. the increases in incidence and survival for breast cancer in the usa between 1950 and 1975 can not be attributed only to detection of in situ cancers that would not have progressed. the proportion of in situ cases in known-stage cases in the connecticut tumor registry in that period was very small and increased from only 0.3% in 19501954 to 1.9% in 19701974, and it was largely unaffected by improved reporting and a reduction in unknown-stage tumors. thus, the reduction in the mortality-to-incidence ratio must largely reflect outcomes for patients with invasive cancers. from 1940 to 1970, regional and advanced stages fell from 58% to 54% between 19401944 and 19501954, and to 45% in 19701974. the period from 1940 to 1974 was a time in the usa when evidence-based medicine became more widespread, and healthcare became more generally available, including increased use of routine gynecologic and general physical examination. for example, the american cancer society began promoting self-examination for breast cancer in 1950 and routine screening by cervical cytology starting in 1952. further, the era of oral contraceptives in the 1960s contributed to greater interactions between healthy women and their healthcare providers. authors who analyzed data prior to 1974 assign the improvements in survival to more effective breast education programs, increased breast cancer awareness, detection of tumors palpable with self or breast-clinical examination, and better diagnostics [19, 20]. thus, the increase in survival rates in the usa prior to 1975 strongly suggests potential to improve breast cancer outcomes in developing countries more quickly than we will be able to make routine mammography and adjuvant therapy available. recent studies, showing breast physical examination and breast self-examination to be unhelpful in reducing stage at diagnosis [2326], have considered only developed countries or urbanized areas of developing countries where routine healthcare is generally available, breast cancer awareness and education are high, and mammography is more routinely accessible. these data, and hence the findings, are likely to be less applicable to a population where breast cancer education and awareness are low, access to the healthcare system severely restricted, and the vast majority of patients present with advanced disease. while reducing the incidence of breast cancer is an ideal goal, the options for achieving this are limited and longer term, particularly for the developing world. healthy lifestyle, including limiting alcohol consumption, maintenance of ideal body weight, regular physical activity, and avoidance of postmenopausal hormone replacement therapy, can have an important impact on breast cancer incidence [15, 27]. every effort should be made to limit these risk factors and thus breast cancer risk. yet, even with strong efforts aimed at prevention, the incidence of breast cancer is likely to increase in most developing countries due to changes in reproductive patterns including later first pregnancies, reductions in parity, and shorter duration of lactation; as well as, declines in physical activity and increased life expectancy. earlier detection and timely, adequate surgery would likely result in substantial improvements in survival in much of the developing world. education about breast cancer, advocacy around curability, and increased coverage of basic healthcare including skilled breast physical examinations could produce improvement in survival rates as occurred in the usa between 1950 and 1975. education efforts need to address the reality that many women, particularly those with less income and education, may not seek care when they feel a breast mass, because they are unaware of what it represents, are concerned about the stigma of cancer and being rejected by their community and their partners, fear the potential loss of the breast, or believe there are no effective therapies for the disease especially if all the women they have known with breast cancer died. hiv a stigma-laden disease, that if untreated is universally fatal by contrast, it has been demonstrated that by combining education, with better and more accessible healthcare facilities, trained medical personnel, and effective therapy, patients do seek and comply with treatment and benefit from it [2830]. the ability to provide adequate affordable access to physical exams by healthcare workers is not a trivial obstacle. an essential first element is the existence of a functioning primary care system staffed by providers trusted by their community. while many countries continue to battle with a weak primary infrastructure, examples, such as the oportunidades program and seguro popular in mexico and partners in health in rural africa and haiti, provide important lessons for strengthening primary healthcare including, and often especially, interventions to improve the health of women [16, 31, 32]. these interventions are essential parts of overall health system strengthening and can help with the prevention and treatment of many diseases in addition to breast cancer. clinical breast exams do not need to be performed by physicians or nurses. in settings where community healthcare workers have learned to care for patients with diseases as complex as hiv, multidrug resistant tuberculosis, and malaria, they could be trained to effectively perform breast exams. large-bore core needle biopsy is a reliable method to obtain tissue for diagnosis and can be performed by trained personal in relatively simple ambulatory settings. pathology services must be available to process the specimens but can be located regionally or outsourced globally. in many developing countries, surgery is available in regional centers, although additional training of surgeons in appropriate techniques may be needed, and women will require financial support and transportation. where radiation therapy is not available, as is the case in many low-income countries given the high proportion of hormone receptor positive cancers, tamoxifen can be effectively combined with surgery. unlike many treatments for breast cancer, generic tamoxifen is low cost, taken orally, and in the vast majority of patients is well tolerated and does not generate unmanageable side effects or require additional medications or care to control symptoms. options exist to greatly expand low-cost alternatives for earlier detection and treatment of breast cancer in developing countries. guidelines have been developed and have been stratified according to the resources available in specific countries and health systems [3336]. many of the basic interventions focus on education, awareness building, the health of women, and expanding capacity at the primary and community healthcare levels, and thus and also contribute to overall health system strengthening. education to improve breast health awareness, breast self-examination, and clinical breast exam are relatively inexpensive and can be incorporated into existing primary health infrastructures. surgery and hormone therapy based on tamoxifen are cost effective, especially with early detection, and implementable in poor-resource settings. focusing on providing these interventions in locations where they do not currently exist could dramatically improve survival. in no way does this abrogate the responsibility to eventually provide resources such as mammography, adjuvant chemotherapy, and advanced targeted therapies such as trastuzumab in these settings. however, great benefit can emerge from basic breast cancer education and awareness, integrating breast exams into primary healthcare infrastructure, and adequate surgery combined with tamoxifen. implementation of these interventions should proceed as quickly as possible, while the more complex and costly interventions, such as mammography, are being made more available. the provision of better primary healthcare, education, and better medical outcomes will provide a solid foundation for reducing stigma and fear that will make more effective the introduction of complex technologies, such as mammography or adjuvant therapy. there is no reason not to immediately strive for the implementation of basic interventions for breast cancer care and control in all settings. all authors have completed the unified competing interest form at http://www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare that all authors had: (1) no financial support for the submitted work from anyone other than their employer, (2) no financial relationships with commercial entities that might have an interest in the submitted work, (3) no spouses, partners, or children with relationships with commercial entities that might have an interest in the submitted work, (4) no nonfinancial interests that may be relevant to the submitted work.

breast cancer survival in the usa has continually improved over the last six decades and has largely been accredited to the use of mammography, advanced surgical procedures, and adjuvant therapies. data indicate, however, that there were substantial improvements in survival in the usa even prior to these technological and diagnostic advances, suggesting important opportunities for early detection and treatment in low- and middle-income countries where these options are often unavailable and/or unaffordable. thus, while continuing to strive for increased access to more advanced technology, improving survival in these settings should be more immediately achievable through increased awareness of breast cancer and of the potential for successful treatment, a high-quality primary care system without economic or cultural barriers to access, and a well-functioning referral system for basic surgical and hormonal treatment.

PMC3021855

pubmed-1248

the consumption of high levels of fructose in humans and animals causes insulin resistance, lipid abnormalities, obesity, hypertension, and renal changes.1-5 the combination of these metabolic and cardiovascular alterations observed in fructose-fed subjects is collectively known as metabolic syndrome (ms). to model the development of ms experimentally, long-term fructose overload in rats has been used.1,2 statins (or hmg-coa reductase inhibitors) have been shown to lower arterial pressure (ap) in borderline hypertensive dyslipidemic humans. this favorable effect of statins may be a result of both lipid-based mechanisms and non-lipid-based mechanisms affecting endothelial vasoregulation and the sympathovagal balance in the disease state.6 experimental studies have shown that simvastatin improves baroreflex sensitivity (brs).7 findings from several studies have strongly suggested that simvastatin normalizes the autonomic function in individuals with heart failure, inhibiting the central mechanisms of angiotensin ii and, consequently, the superoxide production pathway.8 moreover, simvastatin may improve left ventricular function8 and reduce vascular dysfunction in mice with dyslipidemia.9 despite these positive results, the effects of statin therapy on autonomic function have not been established to date, particularly in females with ms. it is important to emphasize that significant advances in the management of cardiovascular disease and ms have been made in recent years.1,2,10,11 however, cardiovascular diseases remain the leading cause of death among women in the most developed areas of the world,12 exceeding the number of deaths in men and the combined number of deaths due to the next seven causes in women.10 because autonomic dysfunction leads to cardiometabolic disorders11 and because statins have demonstrated neuroprotective effects,12-15 we hypothesized that chronic simvastatin administration in female rats submitted to long-term fructose overload (18 weeks) would improve cardiac autonomic control and reduce the cardiometabolic risk. therefore, the aim of the present study was to investigate the effects of simvastatin on the metabolic, cardiovascular and autonomic changes induced by fructose overload in female rats. experiments were performed using 24 female wistar rats (70 days old, approximately 50 g) that were obtained from the animal shelter of sao judas tadeu university in sao paulo, brazil. the rats received standard laboratory chow (nuvital, colombo, brazil) and water ad libitum. the animals were housed in individual cages in a temperature-controlled room (22c) with a 12-h dark-light cycle. all surgical procedures and protocols were in accordance with the ethical care guidelines for experimental animals and the international animal care and use committee and were approved by the sao judas tadeu university ethical committee (protocol number 058/2007). three experimental groups were used in this study: control (c; n=8), fructose (f; n=8), and fructose+simvastatin (fs, n=8). fructose overload was induced via dilution of d-fructose in the drinking water (100 simvastatin (5 mg/kg/day) treatment was performed by gavage for the last two weeks of fructose overload. after 18 weeks of fructose overload, the blood glucose and triglyceride concentrations were measured using a roche device (accutrend gct, roche, sao paulo, brazil) after four hours of fasting at the end of the protocol. for the insulin tolerance test (itt), the rats were fasted for two hours and then anesthetized with thiopental (40 mg/kg body weight, ip). a drop of blood was collected from the tail to measure the blood glucose concentration using the accucheck system (roche, sao paulo, brazil) before and 4, 8, 12, and 16 minutes after insulin injection (0.75 u/kg). the constant rate of decrease of the blood glucose concentration (kitt) was calculated using the 0.693/t/2 formula. the t/2 for blood glucose was calculated from the slope of the least squares analysis of the blood glucose concentrations during the linear phase of decline.16,17 after metabolic measurements, two catheters filled with 0.06 ml of saline were implanted in anesthetized rats (ketamine 80 mg/kg+xylazine 12 mg/kg) into the carotid artery and jugular vein (pe-10) for direct measurements of the ap and for drug administration, respectively. one day after the catheter placement, the rats were conscious and allowed to move freely during the experiments. the arterial cannula was connected to a strain-gauge transducer (blood pressure xdcr, kent scientific, litchfield, ct, usa), and ap signals were recorded over a 30-min period by a microcomputer equipped with an analog-to-digital converter board (codas, 2-khz sampling frequency, dataq instruments, inc., akron, oh, usa). the recorded data were analyzed on a beat-to-beat basis to quantify the changes in the mean ap (map) and the heart rate (hr).16,18 the vagal and sympathetic effects were studied by injecting methylatropine (3 mg/kg iv, sigma-aldrich, st. louis, mo, usa) and propranolol (4 mg/kg iv, sigma-aldrich) in a volume of 0.1 ml/100 g of body weight. the resting hr was recorded while the rats were in their cages in an unrestrained state. methylatropine was injected immediately after the recording. because the hr response to these drugs reaches its peak within 10 to 15 minutes, this time interval was allowed to elapse before the hr measurement was taken. on the next day, the sequence of the injections was inverted, and propranolol was injected before methylatropine.16 the sympathetic effect was determined by calculating the difference between the basal hr and the lowest hr after the administration of propranolol. the vagal effect was obtained based on the difference between the maximum hr after methylatropine injection and the basal hr. the data were expressed as the meanssem and were compared using one-way analysis of variance (anova) or repeated one-way anova followed by the student newman-keuls test. the body weight was not different between the studied groups at the beginning (c: 486 g, f: 435 g, and fs: 451 g, p>0.05) and at the end of the protocol (c: 28510 g, f: 2949 g, and fs: 2888 g, p>0.05). the fasting glucose levels were increased in f (922 mg/dl) and fs (932 mg/dl) rats compared with that in c rats (822 mg/dl, p<0.05). the blood triglyceride concentration was also higher in the f and fs groups (14212 and 18722 mg/dl, respectively) compared with that in the c group (1015 mg/dl, p<0.05). the constant rate of the plasma glucose disappearance (kitt) was reduced in the f group (3.40.32%/min) compared with that in the c group (4.40.29%/min, p<0.05) during itt, which is indicative of the insulin-resistant state in the fructose-fed rats. the simvastatin treatment increased the kitt (fs: 5.40.66%/min, p<0.05) in f rats. the f group exhibited increases in the systolic, diastolic, and mean arterial pressures (p<0.05). simvastatin treatment did not change the ap in the f rats (p>0.05) (table 1). the resting hr showed similar values among groups (p>0.05) (table 1). the cardiac vagal effect was similar between the c (499 bpm) and f groups (465 bpm, p>0.05). simvastatin treatment increased the vagal effect in fructose-overloaded animals (fs: 847 bpm) compared with that in c and f animals (p<0.05) (figure 1a). the sympathetic effect was enhanced in the f group (737 bpm) compared with that in the c group (487 bpm, p<0.05). this effect was normalized by simvastatin treatment (fs: 318 bpm, p<0.05) (figure 1b). the aim of this study was to determine the effects of simvastatin treatment on the metabolic, cardiovascular, and autonomic modulation in an experimental model of ms that was induced by long-term fructose overload (18 weeks) in female rats. although simvastatin treatment did not change the blood metabolic parameters or the ap, this pharmacologic approach improved insulin resistance, reduced the exacerbated cardiac sympathetic effect and increased the vagal effect to the heart. additionally, our findings in female rats corroborated previous data that have been obtained in male animals submitted to fructose overload; these male rats exhibited enhanced blood glucose and triglyceride levels, insulin resistance, increased ap and sympathetic activation. we should emphasize that the protocol used in our research differed from those of previous studies. because fructose overload was performed in animals starting from their 70 day of life through their adult phase previous investigations have covered a shorter time-span and gathered data from the acute (2-24 h) or mid-term (1-9 weeks) phases of fructose administration.2-5,16, the rationale behind our design choice lies in the fact that metabolic and cardiovascular disorders take years to manifest as clinical alterations due to the stepwise compensatory behaviors, physiological adaptations, and new equilibrium levels. the fructose drinking model in rats resembles a state of early insulin resistance in humans, which is associated with mild hypertension.16,23 previous studies have demonstrated that increased body weight is not common in male rat or in fructose-rich chow-fed mouse models. however, fructose-rich chow-fed mouse models can develop hypertriglyceridemia, increased blood glucose concentrations, glucose intolerance or insulin resistance and hyperinsulinemia.3,5,16, in our study, the fructose-overloaded female rats displayed increased blood glucose and triglyceride concentrations and insulin resistance. although simvastatin treatment did not alter the fasting blood glucose and triglyceride levels in our study, it increased kitt in fructose-fed rats. most studies investigating the effects of high fructose consumption on the basal ap in male or female rats have used tail-cuff plethysmography, which is an indirect measurement of ap that can only measure systolic ap.19-22 increased fructose consumption leads to increases in the ap in male20,21 and female rats16 and in male mice during the dark period.5 farah et al.5 have demonstrated an increase in the low-frequency component of systolic ap in fructose-overloaded (eight weeks) male mice. these results show that increased ap is associated with sympathetic modulation in the circulation that is limited to the dark (active) period. in the present study, we observed an increase in the ap and in the sympathetic effect in the heart in fructose-fed female rats after 18 weeks of fructose consumption. the ap change in fructose-fed animals is mediated by activation of the sympathetic nervous system,5,24 impairment of the cardiac parasympathetic tonus16 and of endothelium-dependent relaxation25 and dysfunction in the angiotensin-renin system.5 our results show that the simvastatin treatment normalizes the cardiac sympathetic effect and insulin resistance. in addition, simvastatin treatment increased the cardiac vagal effect in fructose-fed female rats. however, simvastatin treatment did not change the basal ap or the blood triglyceride level. these results suggest that these statin-induced improvements might result from a pleiotropic effect that is independent of the drug's classical effect on lipids. pliquett et al.26 have also demonstrated improvements in the brs in rabbits with heart failure after statin treatment without changes in the total plasma or high-density cholesterol levels. additionally, if decreased ap was observed in the present study in simvastatin-treated fructose-fed rats, the autonomic improvement observed in these animals may be attributed, at least in part, to this change. previous studies have reported lower aps in borderline hypertensive dyslipidemic humans who were treated with statins and have attributed this favorable alteration to both lipid-based mechanisms and non-lipid-based mechanisms affecting endothelial vasoregulation and the sympathovagal balance in the disease state.6 based on these findings, we hypothesize that the role of simvastatin in the autonomic nervous system is vast and includes enhancing no synthesis in the endothelium27,28 and reducing angiotensin ii induced injury, at1 receptor expression,29,30 and eta receptor expression.31 these functions indicate a potential role for statins in regulating sympathetic and vagal outflow in the central nervous system and improving the afferent or efferent arms of the cardiovascular autonomic reflexes. these autonomic pleiotropic effects of statins may account for patient outcomes and require further characterization. the results of the present study demonstrate that fructose overload in female rats induces increases in the ap and the cardiac sympathetic response, which are associated with insulin resistance. these findings reinforce the role of autonomic dysfunction in the development of early cardiometabolic disorders that are induced by a high fructose diet in female rats. importantly, we demonstrated that a short-term simvastatin treatment may improve insulin sensitivity and cardiac autonomic control in an experimental model of ms in female rats. these effects were independent of improvements in the classical plasma lipid profile and of reductions in the ap, reinforcing the hypothesis that statins reduce the cardiometabolic risk in females with ms. however, additional studies are needed to confirm the pleiotropic effects of long-term statin treatment on autonomic dysfunction and on the outcome of women with ms. this study was supported by the conselho nacional de desenvolvimento cientfico e tecnolgico (pibic-usjt) (107977/2006-0), the fundao de amparo pesquisa do estado de so paulo (05/60827-0, 05/60828-6, 07/57595-5, 11/11267-2) and capes (capes- prosup).

objective: because autonomic dysfunction has been found to lead to cardiometabolic disorders and because studies have reported that simvastatin treatment has neuroprotective effects, the objective of the present study was to investigate the effects of simvastatin treatment on cardiovascular and autonomic changes in fructose-fed female rats. methods:female wistar rats were divided into three groups: controls (n=8), fructose (n=8), and fructose+simvastatin (n=8). fructose overload was induced by supplementing the drinking water with fructose (100 mg/l, 18 wks). simvastatin treatment (5 mg/kg/day for 2 wks) was performed by gavage. the arterial pressure was recorded using a data acquisition system. autonomic control was evaluated by pharmacological blockade. results:fructose overload induced an increase in the fasting blood glucose and triglyceride levels and insulin resistance. the constant rate of glucose disappearance during the insulin intolerance test was reduced in the fructose group (3.40.32%/min) relative to that in the control group (4.40.29%/min). fructose+simvastatin rats exhibited increased insulin sensitivity (5.40.66%/min). the fructose and fructose+simvastatin groups demonstrated an increase in the mean arterial pressure compared with controls rats (fructose: 1242 mmhg and fructose+simvastatin: 1263 mmhg vs. controls: 1122 mmhg). the sympathetic effect was enhanced in the fructose group (737 bpm) compared with that in the control (487 bpm) and fructose+simvastatin groups (318 bpm). the vagal effect was increased in fructose+simvastatin animals (847 bpm) compared with that in control (499 bpm) and fructose animals (465 bpm). conclusion: simvastatin treatment improved insulin sensitivity and cardiac autonomic control in an experimental model of metabolic syndrome in female rats. these effects were independent of the improvements in the classical plasma lipid profile and of reductions in arterial pressure. these results support the hypothesis that statins reduce the cardiometabolic risk in females with metabolic syndrome.

PMC3180142

pubmed-1249

oral melanoma is an extremely rare tumor arising from uncontrolled growth of melanocytes found in the basal layer of oral mucous membrane. it occurs between 30 and 90 years of age, with a higher incidence in the 6th decade with a mean age of 56 years. it is having a higher prevalence in yellows, blacks, japanese, and indians of asia due to more frequent finding of melanin pigmentation in oral mucosa of these races. green et al. described criteria for diagnosis of primary oral melanoma which includes demonstration of melanoma in the oral mucosa, presence of junctional activity, inability to demonstrate extraoral primary melanoma. a total of 80% to 90% of oral malignant melanoma arises in the mucosa of maxillary jaw with a majority occurring on the keratinized mucosa of hard palate and gingiva. clinically, it is easy to diagnose them as these are pigmented ones and have irregular shape and outline. these are mostly asymptomatic and detected only when there is ulceration or hemorrhage of the overlying epithelium. the delayed detection may be the cause for the poor prognosis with a 5-year survival being between 15% and 38%. the purpose of this article is to present a case of oral malignant melanoma, as well as to emphasize the necessity for early recognition and treatment of this lesion. a 48-year-old male patient reported to the department of oral medicine and diagnosis with chief complaint of pain and swelling in the upper right gums. the clinical examination revealed a large mass of 8 3 cm in dimension on buccal aspect of right maxillary alveolus involving marginal, attached, and interdental gingiva [figure 1]. anteriorly, it extends from the gingiva of mesial surface of 22, to the gingiva in relation to 17 posteriorly. 13 was missing and 12 was displaced laterally; while, 11, 12, and 21 exhibited mobility. primary oral malignant melanoma extending from 22 to distal aspect of 17 the palpatory findings revealed a firm consistency of lesion with mild pain. a complete examination of the lesion was done and no other primary site of the lesion was found. correlating all clinical features, diagnosis of primary malignant melanoma of oral cavity was made and the patient was referred for further investigations. a computed tomography examination of neck, chest, abdomen, and bone scanning and ultrasounds of liver and kidney were normal excluding any diagnosis of distant metastasis. an incisional biopsy was done for the lesion under local anesthesia and the specimen was sent for histopathologic examination. the gross examination of tissue revealed a mass of 2 mm 3 mm 1 mm in size, which was black in color and firm in consistency. the hematoxylin and eosin-stained section showed a melanin-producing tumor, consisting of atypical irregularly elongated spindle and oval-shaped melanocytes, exhibiting uniformly dark, enlarged and irregular nuclei [figure 2]. in the superficial layers of the tissue, a junctional nevus with pigmentation the diagnosis of an invasive melanoma arising most likely from a pre-existing junctional nevus was made and the patient was referred to the oral and maxillofacial surgery clinic for required therapy. as per the traditional approach, partial maxillectomy of the right side was performed. to reduce the defect and to reconstruct alveolus, microvascular fibula flap was used. the orbital floor near to maxilla was reconstructed with the help of premolded titanium mesh. the histologic examination of the specimen confirmed the initial diagnosis of an invasive melanoma of the oral mucosa. the patient has been followed-up with no evidence of recurrence or metastasis either clinically or radiographically, 11 months after the tumor's resection. the hematoxylin and eosin stained section shows melanoma with invasive pattern showing large cells with pleomorphic vesicular nucleus and brown pigment (40) the hematoxylin and eosin stained section shows stratified squamous keratinized epithelium with in situmelanotic pigment growth (10) it has no known predisposing factors and is difficult to diagnose and manage. differentiating it from a metastatic melanoma is often challenging. the first symptoms of oral melanoma described by berthelsen were those of asymptomatic swelling and occasional bleeding, where he found that only 2 (14%) patients had a pain. because most of the melanomas are painless in their early stages, the diagnosis is often unfortunately delayed until symptoms resulting from ulceration, growth, or bleeding are noted. the pain may be the later manifestation in melanoma as in our case that again could cause delay in seeking treatment. on gross appearance, the tumors on the palate are usually flat, with a varying degree of thickness. microscopically, the tumor cells present themselves as densely packed, large epithelioid cells with eosinophilic cytoplasm. the melanin pigment, which is located intra or extracellularly, may be abundant, but may be sparse or even absent at the light microscopic level. the prognostic value of various levels of invasion, as established in the clark's classification, does not apply for mucosal melanoma because of the absence of histologic landmarks, which are analogous to the papillary and reticular dermis. the staging systems that are applied to cutaneous melanoma are not applicable to mucosal melanomas. the american joint committee on cancer the generally followed guideline is a clinical classification stage i clinically localized disease, stage ii regional lymph node disease, and stage iii-distant disease. the tumor thickness is a reliable prognostic indicator for survival. in this case, the patient was in stage i. westbury describes a clinical classification as follows: 1only primary tumor present, and 2metastasis present, 2a adjacent skin involved, 2b adjacent lymph nodes involved, and 2ab adjacent skin and lymph nodes involved. thus, according to these systems specification, our patient was in stage i. the etiology of malignant melanoma is essentially unknown. tobacco use and chronic irritation from ill-fitting dentures have been considered as possible risk factors, but the evidence is weak. the cause could also be related to our patient as he was having the history of smoking from last 15 years. but most of the malignant melanomas arise de novo, from apparently normal mucosa, and about 30% are preceded by oral pigmentations for several months or even years. some melanoma-associated antigens become expressed during transformation process from a benign melanocytic nevus to melanoma; the majorities of these are related to the melanin production process and most are hla restricted. a recent study demonstrates that the loss of heterozygosity at 12p13 and p27kip1 protein expression contributes to melanoma progression. cytogenetic analysis and evaluation of melanocyte-specific gene-1 (msg-1) appears to be very helpful for understanding the pathogenesis of oral malignant melanoma. the current guidelines for the surgical management of primary cutaneous melanoma recommend a diagnostic excisional biopsy of the lesion followed by a wide local excision where the diagnosis is proved. however, in oral cavity, the size of the lesion or anatomic limitations, particularly the presence of teeth, may preclude the taking of excisional biopsy. younes et al. proposed to take an excisional biopsy with a 12 mm margin for small lesions in amenable locations, but incisional biopsy, through the thickest or the most suspicious part of the tumor, in case of a large lesion or a location in sites where an excisional procedure would involve extensive and militating surgery. usually oral malignant melanoma can be diagnosed with confidence on hematoxylin and eosin stained sections. if pigment is completely absent (amelanotic melanoma), immunohistochemical stains are of significant help. useful markers include s-100 protein, gp 100 (hmb-45), and mart-1 (melan-a). it has always been suggested that cutting into malignant neoplasm during incisional biopsy could result in accidental dissemination of malignant cells within adjacent tissues or blood or lymphatic stream with subsequent risk of local recurrence or regional or distant metastasis. did find a somewhat reduced survival rate in patients with melanoma who had incisional biopsies but against the studies done by lederman and sober where they found no correlation in patient's prognosis with incisional and excisional biopsies. distant metastasis to the lungs, brain, liver, and bones are frequently observed. the treatment of oral malignant melanoma is still controversial and there is no census regarding the best therapeutic approach. data from several studies indicate radical resection of the primary as the treatment of choice. regression in melanoma is a well-recognized phenomenon and may account for many of the cases of metastatic melanoma with occult primaries. partial regression of melanoma is relatively common, but complete regression is quite rare and relatively few cases are well documented. a further feature of regression is its association with poor rather than a good prognosis. the rather nonspecific features of regressed melanoma (apparently inflammatory nodules, depigmented patches, and flat or slightly depressed scars) are easily missed or discounted unless the patient had noticed the regression. from the prognostic point of view, clinical stage at presentation is probably the most important factor in determining the outcome. it has been found by liu et al. that thickness of the tumor, cervical lymph node metastasis, presence or absence of ulceration, and the anatomic sites are all independent risk factors. it has been calculated that nodal metastasis reduces the mean survival time from 46 to 18 months. furthermore, a tumor thickness greater than 5 mm, presence of vascular invasion, necrosis, polymorphous tumor cell morphology, and inability to properly resect the lesion with negative margins have been associated with poor survival in patients with primary melanomas of head and neck region. despite the improvement of surgical techniques and the introduction of new chemotherapeutic agents, prognosis of this malignancy remains poor. the generally advanced stage of the tumor at initial diagnosis leads to a poorer survival of patients with mucosal melanomas as compared with patients with cutaneous melanomas and presence of vertical growth phase are associated with median survival rate. analysis of published cases and recognition of new ones may be helpful in establishing definite classification and proposing clinical features that would facilitate its early diagnosis as a prerequisite for timely treatment and better prognosis of this rare pathology.

primary oral melanoma is a rare neoplasm of melanocytic origin, accounting for 0.5% of all oral malignancies. the chameleonic presentation of a mainly asymptomatic condition, rarity of this lesion, poor prognosis, and the necessity of a highly specialized treatment are factors that should be seriously considered by the involved health provider. here is a case report presenting a malignant melanoma of oral mucosa in 48-year-old male patient on maxillary gingiva. the lesion was removed by partial maxillectomy and patient is disease free after 11 months of regular followup. this case provides an example of how dental clinicians play a major role in the identification of pigmented lesions of oral cavity and also emphasize on the fact that any pigmented lesion detected in the oral cavity may exhibit potential growth and should be submitted to biopsy to exclude malignancy.

PMC3469906

pubmed-1250

in physical therapy for patients of cerebrovascular, cardiovascular, respiratory or musculoskeletal diseases, assessments of muscle strength and the movement of joints are essential for treatment planning and also for lifestyle guidance. in order to evaluate patient performance, the range of motion test (rom-t) is used to assess movement impairment of the joints1, and the manual muscle test (mmt) is used to assess muscle impairment2. both of these test methods are fundamental skills required of every physical therapist, which they have to master in training schools. the mmt is a manual maneuver to evaluate the strength of the prime mover muscles for a given motion of a joint. the procedures employed in the mmt were determined mainly based on our present knowledge of kinesiology and electromyography (emg). not only in diagnostic tests, the mmt has also been used for muscle training and maintaining muscle strength2. the muscles involved in finger function are one of the most important tasks for humans, since the function of the hands is strongly related with the quality of life. the mmt of the forearm muscles are also based on emg. however, it is quite difficult to employ emg for muscles located deeply beneath the skin. it is also difficult to discriminate the actual source of the emg detected by surface electrodes3,4,5,6. recently, magnetic resonance imaging (mri) has been used to monitor muscle activities by using the t1, t2, and water content values of skeletal muscles that increased after muscle exercise7,8,9. intensive studies have been conducted on the determination of agonist muscles using the increase in t2 values10, 11, and takamori detected increases in the t2 weighted mr (t2w-mr) image intensity of the extensor digiti minimi muscle after extension exercise of the mp joint of the digitus imnimus12. in this study, among the mmt for the forearm muscles, we selected 3 mmt for the primary mover muscles confirmed by the emg. in order to confirm the reliability of the mr images, 1) the increase in the t2w-mr image intensity was detected after the mmt of the supinator muscle or the pronator teres muscle13, 14, and 2) we also aimed to detect the mmt of a smaller muscle of the forearm, the extensor indicis muscle15. three healthy male volunteers participated in this study. the age, height, and weight of the subjects averaged 36.4 12.3 years, 172.3 6.9 cm, and 72.3 17.0 kg (means sd), respectively. the left forearms of the subjects were studied, and the mmt was applied by physical therapists with more than 10 years of experience. the procedures, purpose, and risks associated with the study were explained to all of the subjects, and written consent was obtained prior to the commencement of the study. the study was approved by the human research review board at the dokkyo medical university school of medicine (no. mr images of each forearm were obtained with a 0.2 t compact mri system (mrtechnology, tsukuba, japan) equipped with an oval h solenoidal radiofrequency probe12. the transverse t2w-mr images were measured using multi-slice spin-echo mr image sequences with a 200 200 mm field-of-view, a 128 128 data matrix, a 39 ms echo-time with a 2,000 ms repetition time, 11 slices with an interval 10 mm, a 9.5 mm slice thickness, 1 accumulation and a total image acquisition time of 4 min 16 s. each left forearm was fixed by a shell-type holder of the forearm12, and a t2w-mr image in the resting condition was measured at first. then, manipulative isometric contraction exercise (5 sec duration) was applied to the supinator muscle, the pronator teres muscle or the extensor indicis muscle using borg s rating of perceived exertion (rpe) scale of 151716. immediately after the exercise, the arm position was restored to the original position, and another t2w-mr image was measured. the increase in the t2w-mr image intensity of muscle was evaluated by 3 physical therapists (m.t., s.a. and k.y.) with 45 years of experiences with 0.2 t mri. the epimysium of the muscle was traced by hand, and used for the same trace for the t2w-mr image of the resting muscle. the image intensity of a circular region of interest (roi) with 25 pixels was measured by imagej software 1.46r (nih, bethesda, usa). in each slice, 3 rois were set in the muscle without overlapping each other, and the average image intensity (cm) was obtained. in the same slice, an roi was set in the background and sd (sdair) was obtained. the image intensity of the muscle was represented by the signal-to-noise ratio (snr): i=cm/ sdair17. an image intensity outside of the 99.9% of confidence interval (ir 3.3) was considered as a significant increase (cl=99.9%), and an image intensity within 99.9% of the confidence interval was considered not significant (n.s.), where ir was the snr for resting muscle. t2w-mr images of the supinator muscle (a), the pronator teres muscle (b) and the extensor indicis muscle (c) of the subject #1. (i) t2w-mr images before the exercise, (ii) t2w-mr images after the exercise, (iii) the trace around muscle (*). represents a typical result of the mmt of the supinator muscle sliced at one-third of the length of the ulna from the olecranon. the subjects were sitting, arm at the side, with the elbow flexed to 90, and the forearm in full pronation to neutral. then, the examiner supported the elbow, and applied resistance with the heel of the hand over the dorsal (extensor) surface at the wrist (fig. the subjects #1, #2 and #3 did the exercise 82, 40 and 40 times until they reached borg s rpe scale of 1517, respectively. the image intensity of the t2w-mr image increased more than 1.7 times compared with the resting muscle, showing a significant increase (cl=99.9%) (table 1atable 1.t2w-mr image intensity before and after exercisesubject#1#2#3a) supinator musclebefore exercise19.813.516.8after exercise33.530.343.5b)pronator teres musclebefore exercise14.416.725.4after exercise30.930.646.3c)extensor indicis musclebefore exercise15.514.920after exercise29.230.236.6image intensity was represented by the signal-to-noise ratio. no significant changes were shown in the image intensities for the rest of the muscles. transverse t2w-mr images of the supinator muscle (a), the pronator teres muscle (b) and the extensor indicis muscle (c) of the subject #1. (i) t2w-mr images before the exercise, (ii) t2w-mr images after the exercise, (iii) the trace around muscle (*). figure 1b represents a typical result of the mmt of the pronator teres muscle sliced at one-third of the length of the ulna from the olecranon. the function of the pronator teres muscle is to pronate the forearm with the pronator quadratus muscle. the subjects were siting, arm at the side with the elbow flexed to 90 and the forearm in supination. then, the examiner supported the elbow, apply resistance with hypothenar eminence over radius on the volar (flexor) surface of the forearm at the wrist (fig. the subjects #1, #2 and #3 did the exercise 45, 40 and 50 times until they reached borg s rpe scale of 1517, respectively. image intensity of the t2w-mr image increased more than 1.8 times compared with the resting muscle, and represented significant increase (cl=99.9%) (table 1b). no significant changes were shown in the image intensity of the rest of the muscles. figure 1c represents a typical result of the mmt of the extensor indicis muscle sliced at two-third of the length of the ulna from the olecranon. the extensor indicis muscle is a deep-layer, narrow skeletal muscle and its function is the extension of the index finger. the subjects were sitting with the forearm in pronation, the wrist in neutral. and the mp and ip joints in a relaxed flexion posture. then, the examiner stabilized the wrist in neutral, and placed the index finger of the resistance hand cross the dorsum of all proximal phalanges just distal to the mp joints, and applied resistance in the direction of the flexion (fig. the subjects #1, #2 and #3 did the exercise 40, 40 and 40 times until they reached borg s rpe scale of 1517, respectively. the image intensity of the t2w-mr image increased more than 1.8 times compared with the resting muscle, showing a significant increase (cl=99.9%) (table 1c). no significant changes were shown in the image intensity of the rest of the muscles. in this study, the t2w-mr image intensity of the supinator muscle, the pronator teres muscle and the extensor indicis muscle increased (cl=99.9%) with the mmt for these muscles with borg s rep scale of 1517. we did not detect any increases in the image intensity of the rest of the muscles. since the first report by lovett in 191518, the mmt has been applied to evaluate the musculoskeletal and nervous systems, and the reliability and validity of the mmt is well established19. the mmt depends on manual procedures for joint motion, such as flexion of the mp joint. although the prime movers of a movement can be identified, secondary or accessory movers may be equally important, but definitive studies remain incomplete2. compared with emg, mri can image all of the muscles in a single fov in the arm. moreover, we can observe 11 slices simultaneously along the longitudinal axis of the forearm using a multi-slice spin-echo mr sequence. thus, mri can detect secondary or accessory movers located even distant from the primary movers in the same fov. therefore, even with a low number of subjects, the 3 in this study, we can conclude that muscle activity was not detected in other than the primary mover muscles of the 3 mmt used in this study. it can be considered that the strength of the manual resistance of the mmt is important. in a previous report12, the mmt for the extensor digiti minimi muscle was applied until the subjects were unable to continue the extension of the digitus minimus. as a result, we detected other muscle activity in the extensor carpi ulnaris muscle that acts to extend and adduct at the carpus, in addition to the extension of the digitus minimus. therefore, in this study, we adjusted the strength of the exercise to borg s rpe scale of 1517, as that might be suitable for the mmt of the extensor indicis muscle. the results of this study suggest that mr imaging could be useful in training physical therapists in using the mmt, because muscle activity can be visualized by mr images. we plan to study using the mmt for these muscles, such as the flexor carpi radialis muscle and the flexor carpi ulnaris muscle. in these muscles, we can expect activity due to not only the primary mover muscles, but also accessory muscles. it can be considered that mri technique will be useful to analyze mmt results, since we need not predict specific candidate muscles. thus, mri techniques should be useful to increase the reliability of the mmt.

[ purpose] in order to detect muscle activity with manual muscle testing, t2-weighted magnetic resonance (t2w-mr) images were detected by a 0.2 t compact mri system. [subjects and methods] the subjects were 3 adult males. transverse t2-weighted multi-slice spin-echo images of the left forearm were measured by a 39 ms echo-time with a 2,000 ms repetition time, a 9.5 mm slice thickness, 1 accumulation and a total image acquisition time of 4 min 16 s. first, t2w-mr images in the resting condition were measured. then, manipulative isometric contraction exercise (5 sec duration) to the supinator muscle, the pronator teres muscle or the extensor indicis muscle was performed using borg s rating of perceived exertion (rpe) scale of 1517. the t2w-mr images were measured immediately after the exercise. [results] t2w-mr image intensities increased significantly in the supinator muscle, the pronator teres muscle and the extensor indicis muscle after the exercise. however, the image intensities in the rest of the muscle did not change. [conclusion] using t2w-mr images, we could detect muscle activity in a deep muscle, the supinator muscle, and a small muscle, the extensor indicis muscle. these results also support the reliability of the manual muscle testing method.

PMC5361000

pubmed-1251

bis-quinolizidine alkaloids produced by lupine species have generated much interest because of their valuable pharmacological properties. both pharmacological and toxicological properties of these alkaloids are well known [1, 2]. sparteine appears to offer protection to plants from leguminosae family against insects and grazing mammals [3, 4]. several bis-quinolizidine alkaloids (sparteine, lupanine, 17-oxosparteine, 13-hydroxylupanine, angustifoline, etc.) show antihypertensive, antipyretic, anti-inflammatory, antiarrhythmic, diuretic, hypoglicemic, hypotensive, antidiabetic, respiratory depressant and stimulant, and uterotonic properties [5, 6]. the mass spectrometry study of bis-quinolizidine alkaloids has been stimulated by the evidence of the method's ability to distinguish their stereoisomers, metamers, and positional isomers [714]. the main characteristic of the so-called hard electron-impact induced ionization (ei) of mass fragmentation of bis-quinolizidine alkaloid molecular ions is the dependence of the fragmentation pathway of the bis-quinolizidine skeleton on the stereochemistry of the a/b and c/d ring junctions. the stereochemical effects that are encountered with dissociations of stereoisomers incorporating saturated heterocycles rings are due to the ability of chemical bonds to be broken or formed. mass spectrometry includes a broad range of techniques that have allowed us to prove the detailed structures of organic compounds in a variety of ways. fast atom bombardment ionization (fab) is classified as a soft ionization technique in mass spectrometry and is well suited to organic compounds which contain a basic functional group. those compounds tend to run well in positive ion mode. in the positive fab technique a high velocity, rare gas atom molecular beam was produced in the ionization source, and directed onto the sample which was in solution (in the matrix) on a target, thus causing desorption of protonated molecular ions from the sample. generally, positive fab produces protonated molecular ions m+h with a little fragmentation, and there are some limitations because the presence of matrix gives rise to matrix-related ions. if, by chance, the sample give rise to ions at anyone of the m/z values of matrix, than the matrix should be changed. there are many references in the literature for different matrices where their molecular formulae and masses, their most frequently encountered m/z ions, and their uses have been summarized. as a continuation of our previous study it seems reasonable to extend investigation over fab mass fragmentation of isomeric oxo-substituted sparteine derivatives. fab-ms gives useful information for the chemical characterization of different types of alkaloids [16, 17]. fast atom bombardment (fab) mass spectral behaviour of oxo-substituted sparteine derivatives has not been reported in the literature. in a previous work we described the fragmentation routes of seven bis-quinolizidine alkaloids under fast atom bombardment conditions. we have shown that positional isomers of sparteine derivatives can be differentiated on the basis of their fab mass spectra. the aim of this study was to explain the fab mass fragmentation of isomeric sparteine lactams, for example, 2-oxosparteine (lupanine) 1, 15-oxosparteine 2, 17-oxosparteine 3, 2,17-dioxosparteine (17-oxolupanine) 4, 2,13-dioxosparteine (13-oxolupanine) 5, 2-oxo-13-hydroxysparteine (13-hydroxylupanine) 6, and 2-oxo-17-hydroxysparteine (17-hydroxylupanine) 7 (figure 1). these compounds consist of four rings, two of which (a/b) form a sofa/chair system of trans quinolizidine, the second trans system (c/d) form a boat/chair conformations. on the basis of the analysis of the fragmentation processes of 17 in the fab conditions we wished to establish whether it would be possible to distinguish the positional isomers (13; 4,5; 6,7) (figure 1). compounds 17 were obtained in the form of free basis according to literature methods [1824]. their spectral characteristics were consistent with the literature data [1824]. the fab spectra were produced using 3-nitrobenzyl alcohol (m-nba) as matrix. these spectra were recorded in positive mode on an amd-intectra gmbh harpstedt d-27243 model 604 two-sector mass spectrometer. for the collision-induced dissociation (cid) experiments, helium was used as a collision gas in the first field-free region (1ffr) at a pressure corresponding to 50% attenuation of the precursor ion signal. the mass spectrometric behaviour of isomeric 17 was investigated in details by the positive fab mass spectrometry combined with cid. the relative abundances of characteristic peaks of even-electron as well as matrix-derived ions are presented in table 1. on the basis of fab and fab/cid mass spectra of 17, the fab mass fragmentations of these compounds are shown in scheme 1. in the fab spectra of 17 apart from the expected protonated [m+h] a ions, there are also fragment ions. fragmentation of the cyclic m+h a of 1, 2, and 3 (scheme 1, table 1) proceeds by the cleavages of two bonds of ring b and c of the sparteine skeleton. the cleavages of c6-c7 and c9-c10 bonds of ring b (for 1) or c9-c11 and c7-c17 ones of ring d (for 2 and 3) lead to the even-electron fragment ions d [c9h15n+h], at m/z 138. the cleavages of c7-c17 and n16-c17 bonds of ring c (for 3) lead to the even-electron fragment ion b [c14h24n2+h] [m+h-co], at m/z 221. it should be pointed out that the origin of the even-electron fragment ions b and d has been confirmed by the fab/cid mass spectra of 13. the even-electron fragment ions a [m+h] gives the base peaks for the spectra of 2 and 3. in the fab mass spectrum of 1 fragmentation of the cyclic protonated molecule m+h of 3 (table 1) proceeds also by the cleavages of n16-c17 and c17-c7 bonds of ring c with elimination of the neutral molecule of carbon monoxide and leads to the even-electron fragment ion b [c14h24n2+h] [m+h-co] at m/z 221. it ought to be pointed out that in the molecules 2 and 3 in the bis-quinolizidine skeleton a and b rings form a trans double-chair system that is relatively resistant (for thermodynamic reasons) to conformational-configurational changes than a/b trans-fused rings with sofa/chair conformation of the molecule of 1. this suggests that the structure of 2 and 3 increases the stability of m+h ions of these compounds in comparison with that of m+h ion of 1. on the other hand the localization of oxo groups in the different position in bis-quinolizidine skeleton, that is, at c2 (ring a; 1), at c15 (ring d; 2) and c17 (ring c; 3) influences clearly on the elimination of the neutral molecule of carbon monoxide from m+h ion of 3. such ejection of co has been seen previously in the ei mass fragmentation of the molecular ion of 17-oxosparteine. the differences in the relative abundances of ions a in the spectra of 13 depend clearly on the differences in the conformations of bis-quinolizidine skeleton of these compounds. in the light of these data isomeric compounds 1, 2, and 3 can be distinguished from each other on the basis of the differences in the relative abundances of ions a and d (table 1, scheme 1) as well as the presence of the even-electron fragment ion b [m+h-co] in the fab mass spectrum of 3. the common characteristic features of the fab fragmentation of the protonated molecules [m+h] a of 4 and 5 are the cleavages of bonds of rings b and c (c6-c7 and c9-c10 as well as c7-c17 and c9-c11, resp.). the fragment ions d [c9h12no+h] at m/z 151 were obtained in this way by fab fragmentation (table 1, scheme 1). it should be pointed out that the even-electron ions [m+h] are the base peaks in the fab spectra of 4 and 5. in the fab fragmentation of 5 (table 1, scheme 1) the elimination of a neutral molecule of water from the enol-tautomeric form of the [m+h] molecule of 5, that is, the cleavage of c13-o bond of ring d leads to the even-electron fragment ion c. isomers 4 and 5 can be distinguished by the presence of even-electron fragment ion [m+h-h2o] c [c15h20n2o+h] at m/z 245 in the fab spectrum of 5. the absence of the elimination of water in the fab mass fragmentation of m+h a ion of 4 is probably causes by neighbouring heteroatom participation in the elimination of this small molecule. protonated 4 shows no fragmentation by loss of water than does protonated 5. in 4 the proton forms a hydrogen-bridge between oxygen a nitrogen atom of enol-tautomeric form of 4 thus stabilizing the ion. no such stabilization is possible in enol-tautomeric forms of 5 which undergoes fast elimination of water (figure 2). the common characteristic features of the fab fragmentation of 6 and 7 are the cleavages of c7-c17 and c9-c11 bonds of ring c of the lupanine skeleton. the fragment ions d [c9h12no+h] at m/z 151 are obtained in this way of fab fragmentation. in the fab mass spectra of 6 and 7 there are also even-electron fragment ions c [c15h22n2o][m+h-h2o] at m/z 247. in the fab mass spectrum of 6 the base peak is ion [m+h] a and in the fab mass spectrum of 7 the base peak is ion c. the differences in the relative abundances of ions a, c, and d in the fab spectra 6 and 7 allow differentiation of these positional isomers. it ought to be pointed out that the water loss is much more favourable in the fab mass fragmentation of 7 than 6 because after this elimination in the even-electron fragment ion c of 7 the charge is probably situated on the annular nitrogen atom n16, and in the case of 6 the charge is probably situated on the carbon atom c13. identification and structural characterisation of isomeric bis-quinolizidine alkaloids is an important problem in their analysis. mass spectrometry is a powerful tool for unambiguous determination of the structure of these compounds. in the literature that is no information about the fab mass fragmentation of bis-quinolizidine alkaloids. the present study has demonstrated that fab mass fragmentation of isomers of lactams of sparteine sparteine 17 (13; 4, 5; 6, 7) could be expressed as follows. (1) the fab mass fragmentation of the protonated molecules m+h of 17 proceeds mainly by the cleavages of bonds in b (c6-c7 and n1-c10) ring and c (c9-c11, c7-c17) ring of the bis-quinolizidine skeleton (table 1, scheme 1). (2) the protonated molecules m+h of investigated isomers 17 follow the common fragmentation pathways, but with differences in the relative abundances of fragment ions (table 1). (3) the differences in the relative abundances of fragment ions depend mainly on the location of the carbonyl function in the bis-quinolizidine skeleton. (4) the differences in the relative abundances of fragmentation ions depend also on the stereochemistry of a/b and c/d ring junctions of investigated 17. (5) the main fab fragmentation of 3 involves also elimination of the neutral molecule of carbon monoxide [(m+h-co)] m/z 221 (table 1). (6) the main fab mass fragmentation of 6 and 7 involves also elimination of the h2o neutral molecule, yielding ions at m/z 245 [m+h-h2o] (5) and m/z 247 [m+h-h2o] (6, 7), respectively (table 1). (7) the differences in relative abundances of even-electron ions a and d (table 1) in the fab mass spectra of 1, 2, and 3 and the presence of even-electron ion b [m+h-co] in the fab mass spectrum of 3 allow differentiation of positional isomers 1, 2, and 3. (8) the presence of even-electron fragment ion [m+h-oh] c allow differentiation of positional isomers 4 and 5 (table 1). (9) the differences in the relative abundances of ions a, c, and d in the fab mass spectra of 6 and 7 allow differentiation of these positional isomers.

the unpublished in the literature fab mass spectral fragmentation of seven oxosparteines (i.e., 2-oxosparteine, 15-oxosparteine, 17-oxosparteine, 2,17-dioxosparteine, 2,13-dioxosparteine, 2-oxo-13-hydroxysparteine, and 2-oxo-17-hydroxysparteine) is investigated. fragmentation pathways, elucidation of which was assisted by fab/collision-induced dissociation (cid) mass spectra measurements, are discussed. the data obtained create the basis for distinguishing positional isomers.

PMC3132517

pubmed-1252

throughout life, we are constantly challenged by pathogens, and re-exposure to previously encountered viruses or bacteria happens frequently. homologous re-infections are often effectively controlled by neutralizing antibodies, but pathogens that express altered serological epitopes (heterosubtypic re-infection) can bypass antibody-mediated immunity and cause a secondary infection (corti and lanzavecchia, 2013). right at the beginning of such a re-infection, the t cell repertoire may already contain large numbers of pathogen-specific t cells, and some of these can immediately exert effector function (zhang and bevan, 2011). moreover, preexisting pathogen-specific but insufficiently neutralizing antibodies could strongly alter infection and replication kinetics (beura et al., 2016) of a pathogen, and this may significantly alter tissue tropism (rothman, 2011). it has been shown that cells of the innate arm of the immune system can respond more vigorously (sun et al., 2009), and memory cd8 t cells can even contribute to controlling pathogen load in the early phase of infection in a non-cognate, innate-like fashion (chu et al., 2013). thus, pathogen spreading, antigen presentation, t cell activation kinetics, and levels of inflammation and tissue destruction can significantly vary between primary and secondary infections. a particular feature of heterosubtypic re-infections is that many antigens are shared in the primary and secondary infection, but the second pathogen expresses epitopes, which are new to the immune system. such a situation of preexisting partial immunity to a pathogen can also occur in a primary infection when a pathogen happens to share an epitope with a previously encountered unrelated pathogen a phenomenon known as heterologous immunity (welsh et al., 2010, che et al., 2015). though highly relevant for understanding immunity in humans, interferences between past and present infections (or vaccinations) are rarely investigated under well-defined conditions. thus, we still have very limited insight into how existing immunity impacts t cell activation, expansion, and conversion into effector and memory t cells. similarly, pre-existing partial immunity can significantly alter the outcome of vaccination (frahm et al., 2012). it is also particularly important to consider that pre-existing immunity does not inevitably result in better immune protection. instead, certain re-infections are known to be accompanied by enhanced pathology, as occurs when a dengue-virus-immune individual is exposed to a different virus serovar (rothman, 2011). similarly, certain vaccination trials are suspected to have augmented the severity of subsequent infections (blanco et al., 2010, moore et al., 2008). the mechanisms underlying such disease enhancement are not clearly resolved, and it is not known how frequently this may occur. all of these points strongly underline the need to investigate immune responses in immune or partially immune individuals. here, we utilize well-controlled experimental systems to dissect and characterize how different levels of pre-existing immunity influence t cell responses. we observed that t cell activation thresholds differ substantially between primary and secondary infections. we report that only the highest-affinity ligands induce t cell expansion in heterosubtypic re-infections, whereas minor reductions in the levels of tcr stimulation fail to induce t cell expansion. importantly, we show that neither a shortened inflammatory response nor a globally altered antigen presentation pattern are responsible for raising the threshold. instead, we found that even a single shared epitope recognized by previously formed memory cd8 t cells is sufficient to increase the t cell activation threshold of naive t cells. we noted that this elevated threshold is mediated by a non-transferrable endogenous memory t cell population. together our data provide relevant insights into t cell differentiation mechanisms in secondary heterosubtypic infections and for the design of vaccines that are given multiple times. we previously reported that low-affinity t cells differentiate into effector and memory cells in primary infections (zehn et al., 2009). we established this using recombinant listeria strains, which express full-length ovalbumin [ova] that contains either the original high-affinity, h-2k-restricted ot-1 ligand siinfekl (ova357364) or different altered peptide ligands (apls) a2 >y3 >q4 >t4 apl (listed in decreasing ot-1 stimulatory potency). we then became interested in addressing the stimulation requirements in secondary heterosubtypic re-infections. to do so, we infected naive or wild-type listeria (lm-wt)-experienced mice with lm-ova- or apl-expressing listeria (see figure 1a). with this experimental setup, we mimic a frequently occurring situation where an individual is immune to some but not all antigens in a secondary infection. we observed that the high-affinity ot-1 ligand n4 induces similar expansion in mice with or without a previous lm-wt immunization. in contrast, ot-1 expansion in response to listeria expressing the slightly weaker sainfekl (a2) or siynfekl (y3) apl was decreased in lm-wt immune mice (figure 1b) and significantly impaired in response to q4 or t4. similar exclusions of low-affinity t cells were observed when we transferred ot-1 t cells before the primary lm-wt infection (figure s1a) and when memory ot-1 t cells were used instead of naive ot-1 t cells (figure s1b). notably, both n4 and t4 induce similar carboxyfluorescein succinimidyl ester (cfse) dilution in primary infections, but only n4 induces robust proliferation in immune mice (figure 1c). some ot-1 t cells proliferate in response to t4 in listeria-immune mice, but this did not increase total ot-1 t cells (data not shown). we therefore conclude that low-affinity stimulation leads to an abortive t cell response in heterosubtypic re-infections. to illustrate the relevance of our observation, we transferred low-affinity h-2k/ova-specific ot-3 tcr transgenic t cells (enouz et al., 2012) into naive or lm-wt immune rip-mova mice. the lm-wt immune mice were then infected with the same challenge as in figure 1a, whereas the naive rip-mova mice received a priming dose. using this setup, only naive mice showed diabetic blood glucose levels above 300 mg/dl (figure 1d). next, we wanted to define the difference in the affinity range of t cells responding in primary versus heterosubtypic infections. we previously showed that the low-affinity v4 ligand activates ot-1 t cells (zehn et al., 2009), even though it has a 1,000-fold lower ec50 than the n4 ligand (figure 2a). interestingly, the d4 apl has an ec50 value that is 10,000-fold below n4. to test the ot-1 t cell response to this very low-affinity ligand we confirmed that the lm-d4 strain is infectious and comparable to lm-n4 (figure s2a). unexpectedly, even lm-d4 induced rapid initial ot-1 proliferation (figure 2b). d4 activated ot-1 t cells secrete tnf and ifn (figure 2c) and produce granzyme b (figure 2d), and they differentiate into memory t cells that can undergo secondary expansion (figure 2e). we illustrated this by transferring ot-1 cells into mice that were either infected with lm-d4 or with lm-wt. four weeks later, we challenged the mice with vesicular stomatits virus expressing ova (vsv-ova). we noticed higher numbers of ot-1 cells in mice primed by d4 in comparison to lm-wt-primed mice that do not stimulate ot-1 t cells (figure 2e). t cells indicated that the signal 1 (tcr stimulation) activation requirement in primary infections is very low. in contrast, the difference between the normal n4 and the a2 or y3 apl is rather small (figure 2a). nonetheless, this level already strongly impairs t cell expansion in secondary or cross-reactive infections. we therefore conclude that there is a substantial stimulation threshold difference between primary and secondary infections. to further illustrate the significance of this difference, we marked the known boundaries for ligands which induce positive and negative selection of ot-1 t cells in figure 2a, whereby t4 is the threshold ligand between positive and negative selection (figure 2a) (daniels et al. g4 is known to induce positive selection in fetal thymic organ cultures (yachi et al., 2006). given that d4 with a potency much below g4 supports effector t cell differentiation, we wondered whether even the presumed endogenous positive selecting ligands for ot-1, isfkfdhl from the f-actin capping protein (cp1) and rtytyekl from -catenin (hogquist et al. 2002) would drive ot-1 expansion when expressed by listeria, but this was not the case (figure 2f). we confirmed that the lack of an ot-1 response is not due to a lack of bacterial replication and that an lm-cat infection is, in this respect, comparable to an lm-n4 infection (figure s2a). e1 ova (eiinfekl) is the only known apl that supports ot-1-positive selection in vivo (stefanski et al., 2001). the lack of a peripheral response to e1 (figure 2f) suggests that it more closely resembles the quality of ligands (cp1 and -catenin) that are thought to naturally support positive selection. given the activation threshold differences in primary versus heterosubtypic infections, we sought to define the responsible mechanism. depletion of cd4 t cells and nk cells from lm-wt primed mice did not restore low-affinity t cell activation (data not shown). taking a possible role of listeria-specific memory cd8 t cells into consideration, we set up a system that allowed us to control the size of the antigen-specific memory population by sharing only one cd8 t cell epitope between two consecutive infections. we constructed listeria strains that express n4 or t4 plus the lymphocytic choriomeningitis virus (lcmv)-derived gp33-41 epitope. we found that lm-gp33-n4 and lm-gp33-t4 have similar in vivo growth rates (figure s2a) and induce similarly sized populations of endogenous gp33-specific t cells (data not shown and figures s2b and s2c). t cells are primed by these listeria strains in naive or lcmv immune hosts (figure 3a), we observed a 2-fold reduction in ot-1 expansion to n4 and an essentially non-detectable response to t4 in lcmv immune mice (figure 3b). this indicates that pre-existing antigen-specific memory cd8 t cells effectively raise the t cell activation threshold and that only one epitope (or two if one also considers the h-2k-restricted gp34 epitope) (hudrisier et al., 1997) we considered that rapid listeria clearance in immune hosts might shorten the duration of antigen-presentation and lower the magnitude of concomitant inflammation (zehn et al., 2014, prlic et al., 2006). to address this point, we transferred cfse-labeled ot-1 t cells into naive or lm-wt-primed mice and challenged them with lm-n4. we observed similar cfse dilution in naive and immune hosts excluding major differences in antigen presentation kinetics in the two hosts (figures 3c and 3d). also anti-cd40-mediated dc maturation or administration of recombinant ifn to increase inflammation left the t4 response unchanged (data not shown). next, we established comparable levels of listeria induced inflammation and tissue destruction in lcmv immune and naive mice by co-injecting lm-wt and lm-gp33-t4 into lcmv immune mice. here, only lm-gp33-t4 are recognized by memory t cells, whereas the lm-wt infection propagates normally. this resulted in similar absolute listeria titers in lcmv immune and naive mice, but the response to t4 remained unchanged in immune mice (figure 3e). to control for an impact of the different listeria challenge doses in naive or immune mice (figures 1, 3a, and 3b), we used acta-deficient listeria. these strains can not spread from cell to cell and are rapidly cleared by the host, allowing c57bl/6 mice to be challenged with a high listeria dose. this creates a setup in which antigen levels are primarily dependent on the amount of the initial bacterial challenge dose, but not the in vivo expansion, of injected bacteria. we observed a lack of low-affinity t cell expansion in immune mice challenged with acta-deficient lm (figure 3 g). this excludes a principle shortage in antigen presentation and a lower inflammatory response as causes of the observed effect. to determine whether the presence of memory t cells is sufficient to raise the activation threshold, we transferred gp33-specific memory t cells into new c57bl/6 hosts along with cd45.1/2 congenic ot-1 t cells. contrasting our prior observation, the transferred memory p14 t cells reduced, but did not completely suppress, the ot-1 response to lm-gp33-t4. in an lm-gp33-n4 infection, the same p14 dose caused only a minor impact on the ot-1 response (figure 4a). similar outcomes were observed when p14 memory t cells were transferred 28 or 80 days after the lcmv infection (figure s3a). we considered the possibility that the number of transferred p14 t cells was too low to suppress the t4 response effectively and therefore increased the number of transferred p14 to 10 cells. again, the t4 response was incompletely blocked, but surprisingly, this p14 dose already caused a strong decline in the high-affinity ot-1 response (figure 4b). these data suggest that transferring memory t cells is not sufficient to recapitulate the selective and complete exclusion of low-affinity t cells, as observed when endogenous memory t cells respond to a pathogen expressing a shared antigen (figure 1). to exclude a sole ot-1-dependent phenomenon, we reversed the strategy and used the recently described gp33c6 low-affinity apl for p14 t cells (utzschneider et al., 2016). we transferred ot-1 memory and naive p14 t cells into naive b6 mice and infected them with listeria expressing ova and wild-type (lm-gp33-n4) or the low-affinity apl gp33c6 (lm-gp33c6-n4). again, the memory ot-1 had a minor impact on the p14 response to high-affinity ligands and a much stronger impact on the lower-affinity gp33c6 response (figure s3b). these data also show that a weak or strong response of de novo primed naive ot-1 or p14 t cells does not impact the response of memory p14 (figure s3a) or ot-1 t cells (figure s3b). we also examined the phenotype of p14 t cells before (endogenous situation) and after the transfer and noticed a larger population of klrg1 t cells among endogenous compared to transferred memory t cells and a higher proportion of cd43cd27 cells (figures 4c and 4d). both populations are thought to have immediate effector function (olson et al., 2013). moreover, listeria monocytogenes enters the spleen via the bloodstream and in a dendritic cell dependent fashion (neuenhahn et al., 2006). at this stage, they will likely be in contact with effector/memory t cells that are positioned in the spleen in close proximity to the bloodstream. by injecting fluorescently labeled anti-cd8 antibody into mice, we marked the fraction of p14 t cells that are in close contact with blood (galkina et al., 2005). interestingly, but in line with an earlier publication (olson et al., 2013), we saw that the klrg1 or cd43cd27 t cells (figure 4e), which show a low engraftment efficacy in our memory transfers, were enriched among the p14 cells with access to the blood stream. therefore, we favor the conclusion that a form of tissue-resident t cell population is more effective than transferred t cells in raising the activation threshold in heterosubtypic infections. we show a clear difference in the tcr stimulation threshold between primary and secondary infections. only very high-affinity ligands activate t cells in immune mice, whereas ligands such as a2 and y3, whose ec50 for inducing a half-maximum ifn response differed only 2- to 4-fold from the wild-type n4 peptide (figure 2a), trigger a much weaker response in immune compared to naive mice. this is in contrast to the situation in naive mice in which even ligands with 10,000-fold lower ec50 activate ot-1 t cells (figure 2a). our observations help to understand t cell responses in heterosubtypic re-infections caused for instance by different influenza strains or dengue virus serotypes. serial infections with the distantly related lymphocytic choriomeningitis virus and pichinde virus were shown to induce a narrowed pathogen-specific t cell repertoire because of antigens that are shared between these infections (cornberg et al., 2006, welsh et al., 2010). our data indicate that this exclusion is caused by a memory t-cell-mediated elevation of the stimulation threshold and that even minimum cross-reactivity is sufficient to cause this effect. the exclusion of low-affinity t cells by previously established memory t cells has consequences for designing vaccine strategies that rely on a prime-boost regimen and intend to elicit a broad t cell repertoire (such as hiv vaccine trials). several examples underline that optimum protection against pathogens is mediated by highly diverse t cell populations. this ensures cross-reactivity to pathogens expressing mutated epitopes, and it enables individuals to better handle a heterosubtypic infection with a related but non-identical pathogen. it also prevents the selection of escape variants (price et al., 2004, meyer-olson et al., 2004, t cell receptor diversity in the population of antigen-specific t cells is largely facilitated by expanding t cells, which suboptimally respond to antigen (low-affinity t cells) (zehn et al., 2009). the focusing of the t cell repertoire as we observed it may be beneficial for the ongoing secondary infection, but the narrow repertoire may severely diminish the ability to subsequently cross-react to related antigens. similarly, tumor immune responses rely on recruiting t cells of intermediate or low affinity given that high-affinity t cells are often eliminated by tolerance mechanisms (enouz et al., 2012). our data imply that vaccination strategies, which involve repetitive injections, will perform suboptimally if the vaccine vector contains previously experienced epitopes. in addition, pre-existing immunity to vaccine vectors were shown to strongly diminish the efficacy of inducing t cell immunity. specifically, adenovirus-seropositive vaccine recipients had lower hiv-specific responses to an adenovirus 5-vectored hiv vaccine in comparison to adenovirus-seronegative recipients (frahm et al., 2012). in line with this previous study, our data show that a minimal epitope overlap is sufficient to impact not only the quantity but also the breadth of the recruited t cell repertoire. in contrast, memory t cells do not seem to impact the clonal-like expansion of antigen-specific nk cells (johnson et al., 2016). our data suggest that populations of memory t cells, which are difficult to transfer, are more effective than transferrable memory populations in raising the stimulation threshold. these cells may impair the translocation of antigen-loaded dcs from the marginal zone into the t cell zone, and this barrier function may lead to altered antigen presentation and consequently a higher stimulation threshold. this is in line with a recent report that highlights the presence of a tissue-resident t cell population in the spleen (schenkel et al., 2014). nonetheless, it remains difficult to precisely distinguish between qualitative and quantitative causes for the observed differences between transferred and endogenous memory t cells. what supports the notion of a qualitative difference is that the transfer of a large number of memory p14 t cells, which already diminished a high-affinity response, did not resemble the effect seen with endogenous memory t cells. c57bl/6 mice were obtained from charles river, and ot-1, rip-mova, and cd45.1 congenic c57bl/6 mice were obtained from jackson laboratories. low-affinity ot-3 tcr transgenic mice were described previously (enouz et al., 2012). mice were bred in specific pathogen-free (spf) facilities and infected in spf or conventional facilities at the university of lausanne. mice that were at least 6 weeks old were used for experiments in compliance with the university of lausanne institutional regulations, and the experiments were approved by the veterinarian authorities of the swiss canton vaud. single-cell suspensions were obtained by mashing spleens through a 100 m nylon cell strainer (bd falcon). red blood cells were lysed with hypotonic ammonium-chloride-potassium (ack) lysis buffer. if cells were harvested less than 5 days after the infection, then the spleens were before mashing digested with 150 g/ml dnase i (roche) and 200 g/ml liberase tl (roche) for 40 min at 37c. the mouse cd8 t cell enrichment kit (miltenyi biotech) was used for cd8 t cell enrichment. memory p14 cells were isolated by using fluorescein isothiocyanate (fitc), phycoerythrin (pe), or biotin-conjugated cd45.1 antibodies followed by anti-fitc, anti-pe, or anti-biotin microbeads (miltenyi biotech) according to the manufacturer s instructions. cells were labeled in serum-free medium with 5 m cfse at 37c for 10 min. mice were infected intraperitoneally (i.p.) with 2 10 plaque-forming units (pfu) of lymphocytic choriomeningitis virus (lcmv strain 53b, armstrong) grown and titrated on vero cells (utzschneider et al., 2013). vesicular stomatitis virus expressing siinfekl (n4) (kim et al., 1998) was grown and titrated on bhk cells. recombinant listeria monocytogenes strains expressing ovalbumin that contain k/ova-derived apl were previously described (zehn et al., 2010). 2002), which contains native ovalbumin (aa134387) or ovalbumin with the siitfekl variant (zehn et al., 2009), were digested with sapi. oligonucleotides encoding kavynfatc (gp33) or kavyncatc (gp33c6) plus an alanine on both sides were ligated into the sapi restriction site. the constructs were inserted into wild-type or acta-deficient 10403s listeria by e. coli conjugation as previously described (lauer et al. listeria were grown in brain heart infusion broth (oxoid, thermo fisher) to mid-log phase. then, bacterial numbers were determined by measuring the od at 600 nm, and diluted stocks were i.v. naive mice received 1,0003,000 colony-forming units (cfu), mice previously infected with listeria received 5 10 cfu, mice previously infected with lcmv received 2 10 cfu, and, for infection with acta-listeria, 10 cfu were injected. bacterial loads were determined by lysing spleens in pbs supplemented with 0.1% tergitol np-40 (sigma-aldrich). serial dilutions were spread out on brain-heart infusion plates containing streptomycin (200 g/ml), and colonies were enumerated. a large pool of siinfekl-derived synthetic altered peptide ligands were tested for h-2 kb stabilization in rma-s cells and recognition by ot-1 t cells as previously described (zehn et al., 2009). the d4 peptide shows similar surface stabilization as the n4 peptide, but it is a weak agonist for ot-1 t cells (figure 2a). ot-1 cells were stimulated in rpmi (10% fcs, 100 iu/ml penicillin, 100 iu/ml streptomycin, 5 m 2-me, 5 mm hepes; invitrogen) with anti-cd3/cd28-coated beads (dynabeads, invitrogen) and cultured with 50 u/ml human il-2 (chiron) in 7% co2. 6 days after activation, 2 10 ot-1 cells and 1 10 rma cells were mixed in 96-well plates, and titrated concentrations of siinfekl or apl peptides (emc microcollections) were added. after 30 min, 7 m brefeldin a (sigma-aldrich) was added, and cultures were incubated for another 3.5 hr. up to 4 10 cells were plated in 96-well plates in pbs supplemented with 2% fcs and 0.01% azide. cells were surface stained for 20 min at 4c with fluorescently labeled anti-cd8 (536.7), cd127 (a7r34), klrg1 (2f1), cd27 (lg7.f9), cd43 glyco (1b11), cd4 (gk1.5), cd45.1 (a20), and cd45.2 (104) antibodies and fixed in pbs supplemented with 1% formaldehyde, 2% glucose, and 0.03% azide. for intracellular cytokine staining, cells were fixed in pbs 2% formaldehyde and permeabilized in pbs with 0.25% saponin and 0.25% bsa (perm buffer). cells were stained in perm buffer for ifn (xmg1.2), tnf (mp6-xt22), or granzyme b (gb12). fluorescently labeled mhc-i-gp33 multimers were used for detecting gp33-specific t cells (tc-metrix). for in vivo staining experiments, we injected mice i.v. with fluorescently labeled anti-cd8 antibodies. mice were sacrificed 3 min later, and harvested organs were processed as indicated above. flow cytometry data were obtained on a bd facs lsrii machine and analyzed using flowjo software (tree star). graphpad prism was used for graphic representation of data, calculation of ec50 values, and statistical calculations. p values 0.05 were considered significant (p <0.05; p <0.01; p <0.001), and p values >0.05 were considered non-significant (ns).

summarymany infections are caused by pathogens that are similar, but not identical, to previously encountered viruses, bacteria, or vaccines. in such re-infections, pathogens introduce known antigens, which are recognized by memory t cells and new antigens that activate naive t cells. how preexisting memory t cells impact the repertoire of t cells responding to new antigens is still largely unknown. we demonstrate that even a minimum epitope overlap between infections strongly increases the activation threshold and narrows the diversity of t cells recruited in response to new antigens. thus, minimal cross-reactivity between infections can significantly impact the outcome of a subsequent immune response. interestingly, we found that non-transferrable memory t cells are most effective in raising the activation threshold. our findings have implications for designing vaccines and suggest that vaccines meant to target low-affinity t cells are less effective when they contain a strong cd8 t cell epitope that has previously been encountered.

PMC5081394

pubmed-1253

uveal melanoma (um) is a rare intraocular cancer affecting the choroid, ciliary body, or iris. at diagnosis, almost all patients will present without evidence of metastatic disease. despite high rates of primary tumor control, metastatic lesions, predominantly to the liver, occur in approximately 2050% of um patients. primary tumor gene expression profile (gep) testing has shown that patients can be accurately and reliably classified into low-risk class 1 and high-risk class 2 with significantly different metastatic potentials [25]. these patient groups should be managed based upon their relative risk versus the overall population risk. for clinical care purposes, a prognostic tool should be highly accurate in its risk prediction (clinical validity), and its results should be implemented to inform patients ' subsequent management plans (clinical utility). clinical factors including age, extraocular extension, tumor size, and ciliary body involvement have been linked to higher risk for metastasis of um tumors [7, 8]. however, none of these have offered the prognostic accuracy or reproducibility required for clinical implementation. genetic analysis has led to the identification of metastasis-associated cytogenetic abnormalities on chromosomes 1, 3, 6, and 8 [914]. in particular, the presence of monosomy 3 in primary tumor cells is a significant factor for predicting metastatic risk [10, 15, 16], and fluorescence in situ hybridization (fish), comparative genomic hybridization (cgh), multiplex ligation-dependent probe amplification (mlpa), and loss of heterozygosity (loh) have been developed to identify chromosome abnormalities [1720]. however, to our knowledge, these molecular tests to detect gains and losses of chromosomes 1, 3, 6, and 8, including the commercially available mlpa platform [17, 21], have not been clinically validated in prospective, multicenter studies and published clinical utility is limited to high-risk patients. the decisiondx-um gep test is an accurate prospectively validated molecular classifier, and its results are highly correlative to metastatic potential [2, 5], as reported by the collaborative ocular oncology group (coog). notably, because decisiondx-um has been extensively validated exclusively on pretreatment um specimens and the training set, to which the machine-learning algorithm compares each patient sample to generate a class 1 or 2 result, consists of only such samples, nonmelanoma samples are inappropriate for prognostication with the gep panel, consistent with the exclusion criteria of the coog study. similarly, given the lack of validation on posttreatment tumors and the potential for radiobiological effects on tumor's genomics, irradiated samples are also ineligible [23, 24]. decisiondx-um's clinical utility has also been reported, indicating that the majority of ophthalmologists who order the test use the results to guide risk-appropriate treatment and management strategies for um patients. herein are the first results from an interim analysis of the ongoing, prospective, multicenter clinical application of decisiondx-um gene expression assay results (clear) registry. this study tracks decision impact (surveillance regimens and treatment referral patterns), as well as clinical outcomes for decisiondx-um patients. these results add additional prospective, multicenter evidence underscoring decisiondx-um as a highly accurate and clinically actionable assay for determining risk associated with primary um tumors. after irb approval of the study (number nct02376920; clinicaltrials.gov) at participating centers (mayo clinic, rochester mn; university of virginia, charlottesville, va; retinal consultants, sacramento, ca; retina specialists of michigan, grand rapids, mi), patient consent was obtained. data entry was performed at receipt of test results and semiannually (censor date: june 2015). physician-obtained fnab or ffpe um tumor specimens were submitted to a centralized cap-accredited, clia-certified laboratory for gep. frozen specimens, mostly fnab, were dispersed into rnase-free stabilization buffer immediately following biopsy, and all samples were shipped to the recipient lab on dry ice. rna isolation was performed with the picopure rna isolation kit (molecular devices, sunnyvale, ca). all ffpe samples were prepared from enucleated globes, and tumor sections on microscope slides were shipped at room temperature to the recipient lab. tumor tissue was macrodissected from slides using a sterile, disposable scalpel, deparaffinized in xylene, and processed for rna isolation with the ambion recoverall total nucleic acid isolation kit (life technologies corporation, grand island, ny). all rna was assessed for quantity and quality using the nanodrop 1000 system (life technologies corporation) and the agilent bioanalyzer 2100 and then converted to cdna (applied biosystems high capacity cdna reverse transcription kit; life technologies corporation). each cdna sample underwent a 14-cycle preamplification step and was then diluted 20-fold in tris-edta buffer. fifty microliters of each diluted sample was mixed with 50 l of 2x taqman gene expression master mix (life technologies corporation) and loaded onto a custom high-throughput microfluidics gene card containing primers specific for 12 class-discriminating genes and three endogenous control genes. each sample was run in triplicate on an applied biosystems ht7900 instrument (life technologies corporation). delta ct values were calculated by subtracting the mean ct of each discriminating gene triplicate from the geometric mean of the three endogenous control genes ' mean ct values. molecular class assignments were determined by comparing the 12 discriminating gene ct values from each sample to a well-characterized, proprietary um training set of low-risk class 1 and high-risk class 2 geps using a support vector machine- (svm-) learning algorithm. the predicted confidence is indicated by the discriminant score, which is inversely proportional to the proximity of the sample to the hyperplane established between class 1 and class 2 training set samples. surveillance regimens were not prespecified but instead were independently decided upon by each participating physician utilizing the decisiondx-um result and documented as part of the registry data entry. the intensity of surveillance for each patient was categorized based upon a previously reported study and determined by the frequency of imaging (ultrasound, pet/ct, or mri) and liver function tests (lfts) that a patient received in addition to their regular eye examination follow-up. a high-intensity schedule was characterized by imaging and/or lfts every 36 months, whereas a low-intensity schedule was characterized by annual imaging and/or lfts. the prospective, multicenter clear study was designed to assess (a) the management of patients according to their decisiondx-um results (clinical utility) and (b) their documented development of metastatic disease (clinical validity). seventy patients have been enrolled from four centers across the us (table 1). thirty-seven (53%) were class 1 and 33 (47%) were class 2. none of the patients who consented to be in the registry study had technical failures with gep testing. of the class 1 patients, 30 (81%) were class 1a, while 7 (19%) were class 1b. consistent with their high-risk gep, 12 (36%) class 2 patients experienced a metastasis, whereas only 2 (5%) class 1 patients experienced a metastasis (p=0.002 by fisher's exact test) with a median follow-up of 2.38 years. the median time to metastasis for class 2 patients was 1.4 years (table 2). class 2 patients had a significantly worse 3-year metastasis-free survival (mfs) rate of 63% (95% confidence interval=43%83%) compared to 100% in class 1 patients (log rank test p=0.003) (figure 1). the median rate of metastasis was 3.78 years for class 2 patients, while the median was not reached for the class 1 patients. the majority of the metastases were localized in the liver, but metastases were also found in the lungs, brain, and bone (table 2). of note, one of the class 1 patients who experienced metastasis had a large tumor, while the other patient did not undergo treatment of their primary tumor. nine out of the 12 class 2 patients who ultimately had metastases were treated by enucleation, while 2 were treated with plaque radiotherapy, and one was treated by transpupillary thermotherapy (ttt) (table 2). tumor nodal metastasis (tnm) staging by ajcc includes tumor diameter, thickness, ciliary body involvement, and extraocular extension of the tumor. in this cohort, neither largest basal diameter nor ciliary body involvement performed as statistically significant prognostic markers (figure 2). while tumor thickness did provide significant stratification of the patients, gep showed stronger prognostic significance in kaplan-meier and multivariate analysis (table 3), similar to previous studies [2, 28]. one primary objective of clear was to document clinical management differences that are implemented for class 1 compared to class 2 patients. of the 37 class 1 patients, the majority (n=30) were treated with low-intensity follow-up (imaging and/or lfts every year), while all 33 class 2 patients were treated with high-intensity follow-up (imaging and/or lfts every 36 months) (figure 3). two of the class 1 patients who received high-intensity surveillance had intermediate risk class 1b results. only 4 out of the 37 (11%) class 1 patients were referred to medical oncology. of note, two of these referred class 1 patients had ciliary body involvement, one of whom also had a large (22 mm diameter) tumor and displayed loss of chromosome 3. therefore, it is possible that the presence of these clinical features contributed to their medical oncology referrals. due to their high-risk disease, 6 out of 33 (18%) of class 2 patients were referred to medical oncology and 8 class 2 patients (24%) were referred to adjuvant clinical trials. importantly, four class 2 patients went on to receive systemic adjuvant therapy; three patients received combinatorial chemotherapy (tamoxifen, sunitinib, and cisplatin) within a clinical trial, while one received ivig immunotherapy. three of the four patients remained metastasis-free at last follow-up (mean follow-up: 4.48 years; median follow-up: 4.02 years). no class 1 patients were referred to clinical trials or had systemic adjuvant therapy. taken together, these results indicate that class 2 patients that have imaging and lfts are managed by medical oncology and are offered clinical trial participation significantly more often than class 1 patients (fisher's exact test for intensity of surveillance p<0.0001; for medical oncology/clinical trial referral p=0.04; table 1; figure 3). the national comprehensive cancer network (nccn) has emphasized the critical importance that all participants in the medical management of cancer patients, including the patients themselves, are provided with timely, reliable, and actionable education regarding molecular testing for diagnosis and treatment. as molecular biomarker tests are increasingly available for diagnostic, prognostic, and therapeutic purposes, there is a growing need for transparency in reporting clinical validity and utility to ensure confidence in the accuracy and clinical impact of their results [6, 29]. previous studies have reported the validity and utility of decisiondx-um to provide highly accurate prognostic information to guide individualized management of um patients [2, 5, 30]. herein, we report the initial results of the clear prospective registry study that confirms the clinical utility of decisiondx-um, since it became clinically available. as this trial is ongoing, a final analysis of 5-year survival rates the nccn recommends that the clinical utility of a biomarker test should be determined in a prospective clinical trial. the clear results represent a second independent, prospective, multicenter study making the test unique, not only in um, but in comparison to the majority of high-complexity advanced diagnostic tests. the multicenter coog study validated decisiondx-um's ability to accurately predict patient outcomes, reporting that metastatic events were observed in only 1% of class 1 cases versus 26% of class 2 cases after 50 months of follow-up time (median follow-up was 17.4 months). similarly, interim results from our prospective registry study to track decisiondx-um patients indicated a low-risk of metastasis for class 1 patients compared to class 2 patients (5% versus 36%, resp.; median follow-up of 27.3 months). taken together, the results from the clear registry add to the compelling evidence-based clinical validity of decisiondx-um. as has been demonstrated in other cancers for which prognostic and predictive testing are employed, tailoring a treatment strategy according to patient's individual tumor biology offers the potential for improved quality-of-life and more efficient utilization of healthcare resources [3134]. the clinical utility of decisiondx-um was initially reported following a review of medicare medical records that indicated the test significantly impacted patient management plans, resulting in less aggressive management of class 1 versus class 2 patients, and similar management impact results were observed from blinded surveys of ocular oncologists. the clear results also demonstrate that the clinical management of class 2 patients is associated with significantly higher surveillance intensity, including more frequent imaging, lfts, and referral to medical oncology compared to class 1 patients. the higher-intensity surveillance for class 2 patients is consistent with the goal of potentially identifying metastases earlier, thus permitting intervention, while the patient is asymptomatic and likely more amenable to treatment(s). conversely, unnecessary surveillance can potentially be avoided for patients in whom extraocular recurrence of disease is unlikely. it should be noted that it is possible, based on a class 1 result, for a patient who ultimately experiences metastasis to receive lower frequency management, thus potentially delaying the identification of metastatic disease by a few months (i.e., at 12 months versus 36 months with annual versus more frequent surveillance, resp.). however, given the consistency of the mfs rates between the coog study and our data presented here (table 4), we expect metastasis would occur in a small minority of class 1 patients. furthermore, the small percentage of class 1 patients who experienced metastasis (5%) that we report here is in line with previously reported metastasis rates reported for um patients who are identified as low-risk [21, 35, 36] and for breast and colon cancer patients classified as low-risk by other tests [37, 38]. the data reported in this study are important for um because numerous clinical, pathological, and genetic characteristics of the primary tumor have been proposed as being significantly prognostic for um metastasis, yet these methods have achieved neither adequate nccn level of evidence for clinical utility nor clinical validity. based upon multiple prospective and retrospective studies published to date, given its robust ability to identify high-risk patients, rational intervention trials to identify effective adjuvant therapies have been initiated [3942]. four class 2 patients within this cohort pursued adjuvant treatment for their high-risk disease; three of whom were metastasis-free at last follow-up, underscoring the importance of making more clinical trials accessible for high-risk um patients. the decision to enroll class 2 patients in clinical trials is directly related to the level of evidence for metastatic propensity that has been reported for the test. the continued clinical performance and utility demonstrated in this study contributes to the high level of evidence regarding the clinical validity and utility of the decisiondx-um test. this study demonstrates that the 15-gene expression assay decisiondx-um continues to accurately predict metastatic risk for um patients, thus enhancing the molecular test's established clinical validity. furthermore, this is the first prospective analysis of clinical utility of the assay, and this study demonstrates that the test results are being used to guide decision-making for physicians and patients in the clinic.

uveal melanoma management is challenging due to its metastatic propensity. decisiondx-um is a prospectively validated molecular test that interrogates primary tumor biology to provide objective information about metastatic potential that can be used in determining appropriate patient care. to evaluate the continued clinical validity and utility of decisiondx-um, beginning march 2010, 70 patients were enrolled in a prospective, multicenter, irb-approved study to document patient management differences and clinical outcomes associated with low-risk class 1 and high-risk class 2 results indicated by decisiondx-um testing. thirty-seven patients in the prospective study were class 1 and 33 were class 2. class 1 patients had 100% 3-year metastasis-free survival compared to 63% for class 2 (log rank test p=0.003) with 27.3 median follow-up months in this interim analysis. class 2 patients received significantly higher-intensity monitoring and more oncology/clinical trial referrals compared to class 1 patients (fisher's exact test p=2.1 1013 and p=0.04, resp.). the results of this study provide additional, prospective evidence in an independent cohort of patients that class 1 and class 2 patients are managed according to the differential metastatic risk indicated by decisiondx-um. the trial is registered with clinical application of decisiondx-um gene expression assay results (nct02376920).

PMC4944073

pubmed-1254

there have been numerous studies evaluating the anatomical distribution of vertebral fractures and consistently showing two prevalent peaks of vertebral fractures: the first one in the mid-thoracic spine region (t7/t8) and another one in the thoraco-lumbar junction (tlj)1,3,8,17,23,27). in these previous studies, approximately 4% of thoraco-lumbar vertebral compression fractures occur at l4 level1,3,8,17,23,27). furthermore, fractures of the 5 lumbar vertebra are quite uncommon, representing only 1.2% of overall spine fractures and 2.2% of thoraco-lumbar fractures9). on the other hand, according to the previous studies on percutaneous vertebroplasty (pvp) or kyphoplasty, the incidence of pvp at l3, l4 and l5 is about 9-13%, 5-9% and 2-5% respectively of all their thoraco-lumbar pvp or kyphoplasty procedures5,18,21,28,29). therefore, the approximate incidence of pvp at l3 is near to the sum of incidence of pvp at l4 or l5. as a result of performing over 200 pvp cases for compression fracture at a single institute annually, the authors identified distinct characteristics, clinical presentations and surgical outcomes of patients with lower lumbar compression fractures (l4 or l5). as the pioneers, deramond and galibert reported their first seven procedures in 198710), and pvp has been used to manage vertebral compression fractures15). there also have been studies on the factor, fracture level in thoraco-lumbar spine, that influences the result of pvp with regard to back motion pain2,11,14,25,28). however, most previous authors did not separate the clinical features and surgical outcomes of patients with l3 compression fractures from those with l4 or l5 compression fractures2,11,13,14,18,23,24,28). if the clinical feartures and the surgical outcomes of l3 fracture were significantly different from those of l4 or l5 fractures despite classifying l3, l4 and l5 as the lower lumbar spine, those of l3 level fracture, almost half proportion of the lower lumbar spine fractures, could make some confusion and ambiguousness in analysing those of l4 and/or l5 level fractures. the authors retrospectively investigated our patients data to elucidate whether those patients treated for l4 or l5 compression fractures by pvp were similar, in terms of demography, clinical features and surgical outcomes, to those treated for l3 fractures. we then reviewed the literatures to compare the surgical outcomes of patients with l4 or l5 compression fractures with those of patients with tlj level fractures. therefore, the purpose of this present study is to provide accurate understanding of clinical presentations and surgical outcomes as well as to identify the unique characteristics of lower lumbar osteoporotic compression fracture (ocf), which would enable physicians and patients to make more informed decisions about whether to perform the procedure and to develop more precise expectations of prognosis. between october 2008 and july 2012, a total of 762 patients with 948 symptomatic ocfs were consecutively treated with pvp at our single institution. all procedures were performed by the same team, who obtained a detailed and standardized history. preoperative clinical data were collected retrospectively from the medical records and assembled in database by one of the authors (sh). there were 105 l3 (11% of all procedures), 75/l4 (8%) and 46/l5 (4.8%) ocf patients respectively. of these cases, the patients with multiple fractures were excluded for the level homogeneity. hence, a total of 120 patients (17 male and 103 female) were investigated, in which there were 57 patients with l3, 40 patients with l4 and 23 patients with l5 ocfs respectively and they were all treated by pvp (no kyphoplasty). all patients demonstrated acute agonizing or chronic severe focal back motion pain, who did not respond to bed rest or analgesics at least for 2 weeks. preoperative data included the presence or absence of previous history of pvp or lumbar decompressive surgery, recent trauma history prior to occurrence of acute compression fracture, leg radiating symptoms and value of bone mineral density (bmd). postoperative data included some information about the presence or absence of cement leakage, subsequent fracture and additional lumbar decompressive surgery. on post-pvp plain images and magnetic resonance imaging (mri) after pvp) vertebral body compression ratio by calculating the height of anterior-posterior (ap) vertebral wall ratio on the lateral radiography19). therefore, a smaller ap ratio implies a greater degree of compression or wedge deformity. initially, the data from l5 ocf patients were compared with those from l4 ocf patients by use of chi-square statistics and students t-test (p<0.05). thereafter, the patients with l4 or l5 ocfs were grouped (group 1) and they were compared with those with l3 ocfs (group 2) for the insignificant variables from initial comparisons. the out-patients follow-up interview and examination were performed at 1 month after pvp. the postoperative back motion pains and leg symptoms were graded by the operating surgeons as improved, unchanged or worse. in addition, long-term follow-up data were obtained via telephone interview in 105 (87.5%) of 120 patients at a median value of 22 months (range, 2-47 mo.) after pvp. telephone interviews were performed by a third party, who was blinded and not involved in the treatment. the patients ' preoperative economic and functional statuses were compared with their current statuses using prolo economic and functional grading scale22)(table 1). patients were determined to have an improved score if either their economic or functional status, such as house-working or daily living activities improved after pvp or to have a worse score if either status was worse after pvp. furthermore, an additional long-term outcome of leg pain was graded by patients as improved, unchanged or worse. there was no statistically significant difference in incidence of multiple fractures between l3, l4 and l5, which were 46%, 47%, and 50% respectively (p>0.05). the characteristics of 63 patients with single level l4 or l5 ocf are listed in table 2. in the previous pvp histories, there was statistically significant difference between l4 and l5 (p<0.05) as well as l3 and l5 ocfs (p<0.05). in analysis of the medical chart review and follow up interview on the telephone, there was no statistical difference in postoperative back motion pain, leg radiating pain and functional or economic status between l4 and l5 ocf patients (p>0.05). 56% (13/23) of l5 ocfs and 30%(12/40) of l4 had not anterior wedge deformity but biconcave or uni-concave deformity at middle vertebral body portion. the characteristics of group 1 were compared with those of group 2 except the sole significant variable (previous pvp) from initial comparison at table 3. the causes of leg radiating pain were mostly combined with intra-canalicular stenosis, disc herniation or partly the encroachment of corresponding intervertebral foramen by height loss due to compression fractures. not all the cases with leg radiating pain required further lumbar decompressive surgery. in group 1, 64%(29/45) of cases with leg radiating symptoms needed further decompressive lumbar surgery, whereas 38%(9/24) of cases did in group 2. there were 5 patients who required the foraminal decompression surgery by paraspinal approach or fusion surgery during or after pvp in group 1 (2 patients with l4 ocf and 3 patients with l5 ocf), in which functional or economic status of 4 patients (80%) was improved and 3 patients (60%) showed leg radiating pain improvements on the long-term follow up interviews. however, all further lumbar surgeries were performed for the combined intracalicular pathologies, such as lumbar disc herniation or lateral recess stenosis in group 2. there were trends toward the higher frequency of cement leakage and needs for further pvp due to subsequent fractures in group 1 compared with those in the group 2 (41% versus 28%, 22% versus 11% respectively), however these did not reach statistical significance (p>0.05). 2 showed clinical success rates using prolo scale and there was statistically significantly worse long-term outcome in group 1 (p<0.05). only 56% patients showed improvement in economic or functional statuses in group 1, but 72% in group 2. there was no change of statuses in 40% of group 1 patients after pvp, on the contrary to those in 19% of group 2. the additional long-term follow up results for leg pain were illustrated in fig. 3. meanwhile, the long-term follow up results of patients with leg radiating symptom in group 1 showed leg pain improvements after further decompressive lumbar surgery in 50%(13/26) patients, whereas 27%(3/11) patients were improved without additional decompressive surgery. in group 2, 56%(5/9) patients with leg pain were improved after further decompressive lumbar surgery, on the other hand, 38%(5/13) patients showed improvement without additional decompressive lumbar surgery. however, there were no statistically significant differences between the two groups (p>0.05). osteoporotic vertebral compression fractures are not uncommon disease in the elderly people and can manifest as severe pain, functional deterioration and limited mobility. conventional treatments, such as bed rest, bracing, and physical therapy, can result in frequent adverse effects. furthermore, osteoporotic vertebral compression fractures can cause serious complications during surgery as well as after surgery, such as vertebral reconstruction or fusion operation under general anesthesia. therefore, pvp is considered the treatments of choice to relieve pain and to stabilize vertebrae. although it has been reported that pvp has a lot of advantage, the patient population that is most likely to benefit from this procedure is still uncertain1,13,20), and there have also been investigations on unfavorable outcomes and the levels of vertebral fractures as influencing factors2,11,14,25,28). however, most of these studies included l3 level as lower lumbar spine2,11,14,28). ryu and park25) divided the fracture levels into four groups (upper and lower parts in thoracic and lumbar vertebrae respectively), and categorized l4 or l5 into lower lumbar vertebrae. furthermore, their cases with lower lumbar fractures might involve many of cases with multiple fractures. most of accompanying fractures would be at tlj, and the excellent effects of pvp at tlj were well known and self-evident2,5,19,25,28,29,31). therefore, the inclusion of cases with multiple fractures can confuse the authentic outcomes of pvp for lower lumbar levels in spite of multivariate polytomous logistic regression analysis. recently, the most notable determining factor during pvp is the concept of cemented vertebral body fraction (cvbf)13,20). to be achievable volume of intravertebral cement is becoming progressively larger as increasing the volume of vertebral body in lower lumbar spine20). hence, the possibility to obtain an unfavorable outcome would also increase if an amount of cement was not decided in terms of the volume of fractured vertebra body and treated level. however, the ideal needle placement of our procedure was at the anterior third of the vertebral body, and the end point of cement injection during pvp was when the cement reached the posterior quarter of the vertebral body or when significant leakage occurred as described by jensen et al.12). therefore, cvbf alone can not explain our poor result of pvp for l4 or l5 compression fractures. the lower lumbar ocfs are unique in a number of ways: they are distinctly uncommon compared with fractures at tlj levels and their symptomatology frequently occurs without any traumatic events, which is combined with leg radiating pain, and outcome after pvp is less satisfactory than that of tlj fracture cases. there have been few studies in which the features of lower lumbar spine fractures have been divided into 2 parts, (1) l3 fracture and (2) l4 or l5 fractures, and the characteristics of their clinical presentations and surgical outcomes have been compared each other. our data indicate that l4 and l5 vertebral ocfs are different from those at the l3 level, and fractures at the l3 level are more similar to those occurring at tlj. therefore, the results of previously published reports on lower lumbar ocfs including a preponderance of l3 cases may mask the true features of l4 or l5 vertebral fractures. the origin of these differences is unclear, but those patients with l4 or l5 fractures may have their own spinal biomechanics which are different from those with tlj fractures. in orther words, l4-5 and l5-s1 segments have been reported to bear the highest loads and to undergo the most motion in the sagittal plane30), which means that it is resonable to seperate l3 from lower lumbar spine levels in this study. it is also supported by our findings that even though there were no statistical differences in demographic data and bmd between group 1 and group 2, the group 1 had the significantly higher incidence of non-traumatic fractures than that of group 2. this different biomechanics may prevent these patients from achieving the typical excellent outcome with regard to back motion pain experienced in pvp for tlj fractures. further clue to this unfavorable result of pvp for l4 or l5 compression fracture can be found in the previous literatures in which almehed et al.1) and el maghraoui et al.8) demonstrated and illustrated that compression severity was highest in the lower lumbar spine. also, alvarez et al.2) insisted that favorable result of pvp could be expected in patients with the vertebral height loss less than 70%. their study revealed that pvp for patients with more than 70% vertebral height loss was technically difficult to place the needle safely into the vertebral body with a high incidence of cement leakage into disc space, and the complete relief was limited to less than 30% of patients2). at last, our data demonstrated that group 1 had the higher incidence of cement leakage and rate of subsequent fracture than group 2, which did not reach statistical significance. however this relatively higher rate of subsequent fracture at l4 or l5 level also may contribute to the unfavorable long-term outcome of pvp, which is also suggested by our data that the long-term outcome via telephone interview of group 1 patients could be obtained in 11 patients of 14 patients who experienced the subsequent fracture, in which only 3 patients (27%) had improvement of their functional or economic status. thermal7), and chemical26) mechanisms have been proposed, however increasing strength and stiffness may be the key elements of its principle mechanism considering previous reports4,16). chung et al.6) used pvp for the treatment of severe leg radiating pain caused by lower lumbar ocf. they showed the excellent results in which all seven patients had experienced dramatic relief of leg radiating pain after cement injection into lower lumbar fractured body through the pedicle on the symptomatic side. therefore, they concluded that pvp may be an effective modality of ameliorating leg radiating pain caused by ocf combined with foraminal stenosis through local stiffness mechanism. however, they did not show the overall incidence or outcome of this unique symptom by lower lumbar ocf. furthermore, the outcome of pvp for leg radiating pain was disappointing level (27-56% of amelioration rate) irrespective of further decompressive lumbar surgery in our data. they suggested the clinical characteristics of those patients and described that objective evidence of radiculopathy was identified upon electromyography (emg) or neurological examination. however, the radiculopathy caused by foraminal pathology is not confirmed by clinical symptom, emg and neurological examination, but suspicious. thus, the definite, widely acceptable and uniform criteria are necessary to confirm this unique symptom of lower lumbar ocf. the limitations of this study were that there was no detailed investigation on functional outcomes such as short form-36 or oswestry disability index, which should be necessary for making this study more valuable. however, the retrospective nature of this study was limited thorough investigation on their functional outcomes. thus, we only investigated short term and long term postoperative prolo scale as clinical outcome. it is not possible to fully devaluate the usefulness of pvp for l4 and l5 ocf according to the long-term follow up results of this study reporting over 50% improvement, which is not quite good results to compare with those of previous studies2,5,19,25,28,29,31). therefore, the prospective randomized controlled clinical studies are needed to demonstrate whether pvp is effective at l4 or l5 level ocf and whether those level ocf may be a marker for progressive back pain, radiating pain and overall worse outcomes. the patients with l5 ocf patients treated by pvp were more likely to have had previous pvp, and the patients with l4 or l5 ocf were more likely to be non-traumatic, presenting leg radiating symptoms and requiring an additional decompressive surgery more often than l3 ocf group. the pvp outcome in terms of postoperative back motion pain is worse for compression fractures at l4 or l5 compared with those treated at l3 level.

objectivethe purpose of this study is to provide accurate understanding of clinical presentations and surgical outcomes as well as to identify the unique characteristics of lower lumbar osteoporotic compression fracture (ocf). methodsclinical data were collected from 120 patients who had l3, l4 or l5 percutaneous vertebroplasty (pvp) performed from 2008 to 2012 at the single institute. l4 or l5 pvp patients were classified into group 1 and group 2 was for l3 pvp patients. medical records were retrospectively investigated at 1 month after pvp. long term follow-up results were obtained at a median value of 22 months after pvp. results75% of the patients in group 1 were not associated with traumatic events, 71% presenting with leg radiating symptoms and 46% requiring an additional decompressive surgery, more often than those in group 2. these differences are statistically significant (p<0.05). the short term medical record review demonstrated that only 73% of patients in group 1 were ameliorated with regard to back motion pain, whereas those in group 2 reported 87.7% rates of amelioration in identical category (p<0.05). the long term follow up confirmed a significantly worse outcome in group 1, with only 55.7% of patients reporting amelioration in their pain or functional status, but 71.7% rate of amelioration in group 2. conclusionthe ocfs at the l4 or l5 level have different clinical characteristics from those at upper levels of the lumbar spine.

PMC4040645

pubmed-1255

venous malformation is defined as malformations comprised of slow-flowing, abnormal dilated veins, and venous network. clinically venous malformations (vm) are present at birth and tend to grow steadily in proportion to the somatic growth of the child. venous malformations are congenital lesions that affect boys and girls equally with a reported risk of developing other conditions within a specified period of time. the occurrence rate is 1-2 per 10,000 births and 0.1-1% of a population are found to have this condition. histologic and histochemical studies show that abnormalities are formed by small and large dysplastic post-capillary, thin-walled vascular channels with patchy deficiency of mural smooth muscle. a 28-year-old male was referred to the oral and maxillofacial clinic for evaluation of multiple swellings on the left side of the face and inside his mouth. the patient first noticed the swelling over the temple when he was 11-years-old. as the boy grew, the swelling also proportionately increased. multiple poorly defined swellings were noted on the left side of the head and neck region around the temple, cheek, and submandibular region [figure 1a and b]. similar lesion was noted in the supraclavicular and scapular region [figure 2a and b]. the swelling was pulsatile and increased in size the when patient was standing (dependent position). intraorally lobulated swelling was noted on the floor of the mouth in the left sublingual region. the lesion measured 3 4 cm, filling the entire floor of mouth, and purplish discoloration was also noted over the swelling [figure 3a and b]. (a) photograph (front view) shows swelling over the temple region and (b) photograph (side view) reveals well defined swelling over temple (large arrow) diffused swelling in the cheek, and submandibular region (small arrow). (a) photograph shows a well-defined swelling measuring 1 1 cm in the left supraclavicular region and (b) photograph of the scapular region shows a diffused swelling. (a) intraoral photograph shows swelling in the floor of mouth filling the entire floor of mouth (arrow) and a small swelling in the buccal mucosa that has a purplish hue (small arrow). (b) intraoral photograph of the labial sulcus reveals a purplish swelling (arrow). ultrasonography of the submandibular region showed a well-defined hypoechoic lesion having anechoic areas with septations, showing flow inside the lesion. ultrasonography of the submandibular region with 7 mhz small parts transducer shows heterogeneous hypoechoic lesion with slow flow and hyperechoic foci suggestive of calcifications (arrow). magnetic resonance imaging (mri)-t2-weighted fat suppression, post-contrast axial images showed hyperintense lesions, which are well-defined with no flow voids. all the lesions in the submandibular, cheek, and temple region had similar findings [figure 5]. direct puncture phlebogram of the left frontal scalp region revealed pooling of contrast into cavernous spaces draining into external jugular vein [figure 6]. (a) axial section at the submandibular region shows well-defined hyperintense lesion (arrow) in the floor of mouth. (b) axial section at the level of cheek shows well-defined hyperintense lesion in the cheek region (arrow). phlebography of the lesion in the temple region shows filling of the entire lesion and draining to the regional area (arrow). histopathological examination revealed several thin-walled venous channels lined by flattened endothelium, supported by a dense uninflamed fibrous connective tissue stroma. hematoxylin and eosin stained specimen (40) shows irregular venous channels (large arrow) lined by thin endothelium. venous malformation (vm) is the second most common vascular anomaly of the head and neck after hemangioma. venous malformations can occur anywhere in the body but are most frequently seen in the head and neck (40%), extremities (40%), and trunk (20%). vms are slow-flow vascular anomalies composed of ectatic venous channels that will continue to grow throughout the patient's lifetime. they grow slowly in size with age, but their growth may be exacerbated following trauma, sepsis, or hormonal changes and they do not regress spontaneously. both men and women are equally affected. vascular malformations are believed to be the result of a congenital error of vascular morphogenesis that occurs between the 4 and 10 weeks of intrauterine life. vascular malformations have a quiescent endothelium and are considered to be localized defects of vascular morphogenesis, likely to be caused by dysfunction in pathways regulating embryogenesis and vasculogenesis. these lesions vary in color depending on depth of involvement and range from mild detectable color change to deep purple color. these lesions fill when the patient is standing and are compressible, which helps to distinguish them from lymphatic malformations on physical examination. areas frequently involved in the head and neck region are masseter, temporalis, tongue musculature, as well as oral and airway mucosa. soft tissue lesions are most frequently facial in location, with the buccal region being the most common site followed by the mandibular space, sublingual space, tongue, and orbit. intraosseous calvarial involvement is most frequent in the frontoparietal region and the mandible is the most frequent location within the facial skeleton. there may, however, be no visible manifestations with deeper lesions. in our case, the first anatomopathologic classification of vascular lesions based on the microscopic appearance was developed by virchow and wegner. mulliken and glowacki (1982) developed a biological classification of vascular anomalies that included physical findings, clinical behavior, and cellular kinetics and classified them as hemangiomas and vms. a more recently updated classification of vascular anomalies by international society for the study of vascular anomalies is now widely used [table 1]. classification of vascular anomalies (issva 2007) plain radiographs have limited role in the investigations as they can identify only phleboliths. ultrasound (us) is often the initial investigation to evaluate vascular malformations and it may characterize and define the extent of more superficial lesions. on gray-scale imaging, venous malformations can appear as hypoechoic or heterogenous lesions with anechoic structures visible in 50% of cases. in addition, the doppler flow is generally monophasic low velocity flow, and in some cases flow is only discernible with compression and release of the lesion. they typically appear as isointense or hypointense lesions on t1-weighted images, but could be hyperintense depending on the presence of intralesional fat. lesions are typically lobulated, which gives them the characteristic bunch of grapes configuration. septations and rounded signal voids corresponding to phleboliths are additional distinguishing features. in t2-weighted or inversion recovery sequences, vms demonstrate high-signal intensity.this imaging modality is used to determine the full extent of the lesion and its relationship to adjacent vital structures. gradient echo sequences reveal areas of low signal corresponding to calcification or hemosiderin or thrombosis. t1-weighted post contrast imaging demonstrates homogenous or heterogeneous enhancement, and dynamic contrast-enhanced mri has increased the specificity of venous malformation diagnosis. vms are best demonstrated by direct phlebography, which fills the sinusoidal spaces and any anomalous veins, allowing assessment of the size and extent of the lesion. in the present study, phleboliths were seen on plain radiography and us features were suggestive of vascular anomaly with slow flow rate. whereas, the mri features were suggestive of venous malformation, which was confirmed by direct phlebography where the lesion was seen draining the regional vein. venous malformations usually are associated with syndromes like proteus syndrome and blue rubber bleb nevus (bean) syndrome. multiple treatment options exist for venous malformations, including conservative measures such as head of bed elevation and compression, laser therapy, sclerotherapy, and surgery. conservative management of venous malformations is usually reserved for smaller isolated asymptomatic lesions and is also important in controlling the growth and symptoms. elevation of the head of the bed is important as it decrease hydrostatic pressure in the malformation, which can lead to expansion and can also decrease symptoms of airway obstruction, swelling, and pain that are experienced. sclerotherapy remains a good option for the treatment of venous malformations in the head and neck. sclerotherapy involves percutaneous injection of a substance to induce inflammation and thrombosis of the lesion, which then will lead to more long-term fibrosis and hopefully decrease or eliminate the expansion of the lesion. sotradecol foam or ethibloc (glue), or the sclerosant is mixed with fibrin glue or ethyl cellulose, bleomycin (pingyangmycin) and picibanil (ok-432) have recently been used as sclerosants in asia with promising results. large cervicofacial venous malformations present a much greater challenge, and one must be prepared to use multimodal therapy to keep the lesion under control. these lesions generally can not be cured as doing so would leave devastating functional and cosmetic results. therefore, therapy is used to control growth, maintain cosmesis, and decrease symptoms. venous malformations are either superficial or deep veins that are abnormally formed and dilated. a thorough examination and investigation of the condition is needed to establish the exact extension of the condition and plan proper treatment.

vascular malformations are congenital lesions that are present at birth and do not regress. however, they often present later in life. they are subdivided into two categories: (1) slow- or low-flow and (2) fast- or high-flow malformations. low-flow malformations contain combinations of capillary, venous, and lymphatic components. venous malformations can occur anywhere in the body, but are most frequently seen in the head and neck (40%). these lesions present in a variety of ways, from a vague blue patch to a soft blue mass, which may be single isolated or may occur in multiple areas. treatment depends on the type of lesion, the location, degree of involvement, and the clinical symptoms. here we are report the imaging and histopathologic findings in a patient with multiple venous malformations affecting the left side of the face and trunk.

PMC3906654

pubmed-1256

ectopic thyroid tissue is a common abnormality and results from abnormal embryologic development and migration of the thyroid gland. such tissue is usually found along the path of descent of the thyroid gland in the anterior midline of the neck. a lingual thyroid is the most common presentation of thyroid ectopy along with thyroglossal duct remnants. ectopic thyroid tissue has been described in other parts of the head and neck such as the submandibular [3, 4] and parotid salivary glands. there are case descriptions of thyroid tissue identified in diverse locations such as the axilla, trachea [710], adrenal, small intestine, and porta hepatis. the most frequent noncervical location for ectopic thyroid tissue is the thoracic cavity [1418]., the authors describe a case of ectopic anterosuperior mediastinal thyroid mass excised through a cervical incision and the subsequent investigation to exclude malignancy. an 80-year-old nonsmoker female patient presented with an incidental finding of a mediastinal mass on mri scan for investigation of vertigo (figures 1 and 2). thyroid stimulating hormone (tsh) and free thyroxine (ft4) were within the normal range, and routine preoperative blood tests were normal. an anteroposterior chest radiograph demonstrated deviation of the intrathoracic trachea around the mass (figure 3). a subsequent ultrasound scan demonstrated a superior mediastinal mass separate from the inferior pole of the right thyroid lobe measuring 4.8 cm in maximal diameter (figure 4). the first sample was inadequate (thy1), the second sample demonstrated only colloid and a few follicular cells. since malignancy could not be definitively ruled out, the mass was excised through a cervical incision. under general anesthesia, the patient was placed in the supine position with the neck extended, prepared, and draped. endotracheal laryngeal nerve monitoring was used with nim contact emg endotracheal tube and nim-response 2.0 monitor (medtronic usa, inc.6743 southpoint drive north, jacksonville, florida, usa, 32216-0980). through a standard 5 cm midline cervical incision, the right thyroid lobe was dissected, found to be grossly normal, and excised. the right recurrent laryngeal nerve was identified in the normal position, confirmed functioning with nerve stimulation at 2 ma, and preserved. separate and inferior to the right thyroid lobe a discrete encapsulated mass was identified in the superior, mediastinum. this extended across the anterior surface of the trachea to the left side, adjacent to the thymus. the mass was separated from the thyroid gland by fat, in the same tissue plane but with no identifiable connection to the cervical region. the mass was excised through the cervical incision. during excision of the mass, a double vascular pedicle, arising from inferiorly in the mediastinum, histopathologic evaluation of the excised mediastinal specimen demonstrated a 5.3 cm by 2 cm thyroid tissue mass with large oedematous loose areas and foci of calcification and fibrosis (figure 5). the excised right thyroid lobe had a nodular architecture composed of follicles of varying sizes with focal lymphoid aggregates.thyroid function tests at 3 months were normal. further investigation with whole body radioiodine i123 scintigraphy demonstrated expected residual uptake in the remaining left thyroid lobe and surgical bed but no uptake suggestive of other ectopic thyroid tissue or metastatic disease. ultrasound surveillance of a 9 mm nodule in the remaining left lobe demonstrated no change in size over 6 months. in humans, the thyroid gland begins to develop at the 24th day of gestation. the thyroid is the first endocrine gland to develop and originates from between the first and second branchial arches. an invagination of endodermal epithelial cells begins at the midline of the developing pharyngeal floor forming a diverticulum. this site, known as the foramen caecum, lies between the tuberculum impar (median tongue bud) and the hypobranchial eminence (copula). the foramen caecum can be observed in adults as a small pit at the base of the tongue where the tongue is divided into an oral anterior two-thirds and pharyngeal posterior third by the sulcus terminalis. the initial path of descent of the bilobed thyroid diverticulum is anterior to the pharyngeal gut, the hyoid bone, and the laryngeal cartilages. at the same time embryological studies have shown that the strap muscles pull downward on the hyoid bone during development causing a forward tilt. this tilt pulls the tract posteriorly and causes it to be hooked up behind the hyoid bone. this explains the importance of excision of the median portion of the hyoid bone in the sistrunk procedure to excise a thyroglossal duct cyst. the gland reaches its final location anterior to the trachea by the 7th week gestation. by this stage, it has acquired a median isthmus and two lateral lobes. as it descends, it is joined by the ultimobranchial bodies which form the parafollicular c cells. thyroid function begins at around the end of the 3rd month when the follicular cells commence production of colloid and follicles appear. during migration, from the 5th week gestation, the thyroid is still connected to the tongue by the thyroglossal duct. in normal development, this tubular structure subsequently obliterates entirely at approximately the 8 to 10th week. in some individuals, however, abnormalities in the embryologic development and migration of the thyroid gland can result in ectopic thyroid tissue. these remnants can appear at any point along the migratory path and are always located at or near the midline. the majority of remnants are found at, or just inferior to, the body of the hyoid bone as a thyroglossal cyst. remnants can also be found in the tongue base as a lingual thyroid or close to the thyroid cartilage. the inferior end of the thyroglossal duct may fail to obliterate, and in at least half of individuals, a pyramidal lobe of the thyroid can be seen to persist. this pyramidal lobe itself may be attached to the hyoid bone or may be incorporated into a thyroglossal duct cyst. ectopic thyroid tissue in other locations is rare and generally the subject of single case reports. the most important diagnosis to exclude is metastatic lymph node deposits of well-differentiated thyroid carcinoma. indeed, it is generally accepted that early reports of lateral aberrant thyroid tissue may represent papillary thyroid carcinoma metastases. another hypothesis is that a thyroid nodule may become detached from the gland. in this case, the vascular supply should come from branches of the superior or inferior thyroid artery. there are reports of thyroid deposits in the soft tissues of the neck representing surgical implantation of thyroid neoplasms. in one case, infiltrating thyroid tissue in muscle and fibrous tissue presented 3 years after major blunt trauma to the neck. the tissue resembled that in a disrupted thyroid nodule present in the gland itself and was regarded as traumatically implanted. there are, however, a number of ectopic thyroid masses reported which do not have features consistent with these theories and are considered truly developmental. animal studies suggest that ectopic location of thyroid tissue may be related to vascular development. the normal arterial supply of the thyroid gland consists of paired superior and inferior thyroid arteries. the superior thyroid artery is generally considered to be present in 100% of cases, and its absence has only been reported once. an unusually large superior thyroid artery may replace the contralateral vessels or the inferior thyroid artery. the inferior thyroid artery when unusually small or absent may be replaced or supplemented by a thyroidea i m a artery. published reports place the incidence of a thyroidea i m a artery between 2% and 12%. in the case the ectopic mass was separate from the thyroid gland but in the same anatomical plane. careful histological examination of the mass and the excised ipsilateral thyroid lobe demonstrated no evidence of neoplasia, and exhaustive investigations showed no evidence of thyroid malignancy elsewhere. the authors therefore conclude that this represents developmentally ectopic sequestered thyroid tissue in the anterior mediastinum. despite this, the authors suggest that abnormally situated thyroid tissue, other than that in the central neck along the path of embryological descent of the thyroid gland, should be excised for careful histological analysis in order to exclude metastatic disease.

an 80-year-old female presented with an incidental finding of a retrosternal mass on magnetic resonance imaging. ultrasound demonstrated a mediastinal lesion adjacent to but separate from the inferior pole of the right thyroid lobe. fine needle aspiration cytology demonstrated colloid and follicular cells. at surgery, the right thyroid lobe was found to be normal. a discrete 5 cm nodule was found in the anterior mediastinum separate from the thyroid and just anterior and to the right of the trachea and thymus. the nodule had a vascular pedicle arising from the mediastinum. the differential diagnosis included metastatic thyroid carcinoma. histology was consistent with a benign ectopic sequestered thyroid nodule. extensive investigations demonstrated no sign of a thyroid malignancy.

PMC3197173

pubmed-1257

pregnant women and neonates are at greater risk for influenza-related complications than the general population [1, 2]. most institutions and organizations recommend that all pregnant women receive the trivalent inactivated influenza virus vaccine [38]. such endorsements rely on the immunogenic response of the mothers, the lack of teratogenicity, and the contrandication of immunization in children younger than six months [7, 911]. despite the broad recommendation to vaccinate pregnant women against influenza, coverage is still limited. a survey held by the centers for disease control and prevention involving women who were pregnant from october 2011 to january 2012 showed that only half of the respondents had been vaccinated and fewer than 10% had received the vaccine before giving birth. similar patterns were found in previous studies. although there is clear evidence of the efficacy of the influenza vaccine for the general population, to our knowledge, a systematic approach with regard to the evidence of the therapeutic effects of influenza vaccination in pregnant women is lacking. our objective is to review the effects of influenza vaccination in preventing influenza-related outcomes in pregnant women and their infants. we selected randomized controlled trials or cohort studies that assessed the effects of inactivated influenza vaccine in preventing influenza-related outcomes in pregnant women and their offspring compared with placebo, other vaccines, or no vaccines. we excluded studies that assessed monovalent vaccines, such as the h1n1 influenza vaccine, because they are used for specific epidemic situations. we searched for eligible studies in the following databases (from inception to september 2013): medline, embase, scopus, centre for reviews and dissemination (crd), cochrane central register of controlled trials (central), metaregister of current controlled trials (mcrt), latin american and caribbean center on health sciences information (lilacs), and scientific electronic library online (scielo). references to relevant publications in the field were also screened to identify potentially eligible studies. there were no restrictions on language, length of followup, publication date, or publication status. we used the following search terms to search medline (via pubmed) and adapted the strategy for the other databases: (influenza, human[mesh] or influenza[tiab] or human flu[tiab] or influenza[tiab] or influenzas[tiab] or grippe[tiab] or flu[tiab] or cold[tiab ]) and (influenza vaccines[mesh] or influenza vaccines[tiab] or vaccine[tiab] or vaccine[tiab] or vaccines[tiab ]) and (mothers[mesh] or mothers[tiab] or pregnant women[tiab] or pregnant[tiab ]) and (infant[mesh] or infant[tiab] or infants[tiab] or infant, newborn[mesh] or newborns[tiab] or fetus[mesh] or fetus[tiab] or foetus[tiab] or fetal[tiab] or pregnancy). two independent reviewers (eb and labl) selected the studies by assessing titles and abstracts and extracted the data. the extracted data consisted of the following: year, country, study design, gestational age, type of vaccine, posology, comparators, sample size, followup, and outcomes. when necessary, we contacted the corresponding authors for additional information. to assess the risk of bias of randomized controlled trials (rct), we used the cochrane collaboration tool, which includes judgments about the sequence generation, allocation sequence concealment, blinding, incomplete outcome data, selective outcome reporting, and other sources of bias. for observational studies, we evaluated the following: eligibility criteria, measurements of exposures and outcomes, control of confounding, and followup. we assessed the quality of evidence for each relevant outcome with the grading of recommendations assessment, development, and evaluation (grade) [17, 18]. for this evaluation, we separated the bodies of evidence into experimental and observational centered on rct and cohort studies, respectively. following the grade approach, then, we assessed the evidence against five items that could decrease its quality: limitations (risk of bias), inconsistency, indirectness, imprecision, and publication bias. after assessing these items, the resulting quality of evidence could be rated as high, moderate, low, or very low. when distinct levels of quality were available for the same outcome, we considered the experimental design (rct) evidence in rating the quality. the final judgments regarding the risk of bias and evidence quality were achieved by consensus. the primary outcome was the incidence of influenza-like illness, which was defined as fever and either cough or sore throat. for infants ' outcomes, we defined small for gestational age as a weight below the 10th percentile and prematurity as birth before a gestational age of 37 complete weeks. we extracted the estimates along with 95% confidence intervals (95% ci) according to the data available in the original studies (relative risk (rr), odds ratio (or), or hazard ratio (hr)). if reported, we only considered the adjusted estimates and did not perform further calculation. we attempted to perform meta-analyses using random effects models, if numerical data from studies allowed a summarization. twenty-three records were selected for full-text assessment, and nine were included in the review. were related to eight unique studies that enrolled 182,820 pregnant women and 182,246 neonates [3442]. we identified one rct conducted in bangladesh and seven cohort studies performed in the united states. the trivalent inactivated vaccine was the most common intervention and was assessed in seven studies [3541]. newborns were not vaccinated. only the rct had an active control group (pneumococcal vaccine). nearly the entire sample of pregnant women came from three retrospective cohorts [35, 37, 41]. the length of followup of each outcome varied among studies and lasted up to 36 weeks. some studies adjusted their results for confounding factors, such as the women's age, week of delivery, infant's gender, and gestational age. observational studies that compared baseline characteristics of the groups showed that most variables did not differ between the groups. some studies showed that vaccinated women had a worse profile than unvaccinated women, which were of higher risk for complications, were older, and had higher body mass index, higher parity, and more multiple gestation. we could not perform meta-analysis because the studies used different measures of association for the same outcome; the estimates are presented as available in the studies. mothers who received the influenza vaccine had a lower incidence of influenza-like illness, as did their infants (high-quality evidence), but there was no difference found for influenza-like illness with fever higher than 38c (moderate-quality evidence). a lower incidence of influenza in infants, as confirmed by laboratory tests, was also observed (moderate-quality evidence). very-low-quality evidence showed no difference between comparisons with regard to the incidence of upper respiratory infection, hospitalization, and medical visits for influenza-like illness in mothers and infants. two studies found no difference in the incidence of hospitalization for influenza-like illness in infants [35, 37]; in one study, the reduction in the rate of hospital admission was significant. with regard to medical visits for influenza-like illness in infants, the observational studies showed no significant differences [35, 37, 40], and the rct showed a reduction in such rate (moderate-quality evidence). for the outcomes prematurity and small for gestational age, conflicting results were found across the studies. one single cohort indicated significant reduction in stillbirth and neonatal death among the influenza-vaccinated group (moderate-quality evidence). we did not assess the incidence of adverse reactions because the included studies did not systematically evaluate this outcome. in general, influenza vaccination had no association with local or minor systemic effects, fever, apgar score at one minute, hyperbilirubinemia, or major malformations. the influenza vaccine was found to reduce the risk of influenza-like illness in mothers and infants as well as the risk of laboratory-confirmed influenza in infants. adverse reactions were not systematically assessed across the studies, but there was no evidence of increase in clinically relevant risk related to influenza vaccination during pregnancy. a big cohort study that focused on the safety of trivalent inactivated influenza vaccine, however, did not find any increased risk of adverse events and adverse obstetric events in the vaccinated mothers, when compared to unvaccinated pregnant women [43, 44]. other factors can prevent influenza in infants such as the effect of breast-feeding and immunization of all the infant's close contacts, also known as cocooning. to avoid confounding and enable comparison, groups should be set by randomization. in the present review, however, only one rct was identified and included. although most observational studies performed multivariate analyses, residual confounding may remain even after adjustment because this statistical procedure can not control for all biological variabilities. women receiving influenza vaccine would have more medical attention and be healthier than unvaccinated pregnant women; thus, the result found would be attributed to the health profile of vaccinated women rather than to the vaccine itself. however, some studies reported that vaccinated women were at high risk during gestation, and this difference was also statistically controlled [37, 42]. studies consistently reported that the patients and the clinicians made the decision about taking influenza vaccine or not. surveys about attitudes and beliefs of these actors regarding influenza vaccination in pregnancy show that the proportion of people who do not believe vaccine is safe is still high [48, 49]. another limitation of our review is the absence of the systematic reporting of adverse reactions. although the incidence of adverse reaction was not shown to be a concern in the included studies, this lack of evidence may inadvertently lead to the conclusion that this risk is minimal or nonexistent. individuals with egg allergy, for example, require caution when receiving trivalent inactivated influenza vaccine. previous narrative reviews concluded that influenza vaccination is safe, that there has been no evidence of teratogenicity, and that many countries recommend influenza vaccination among women with both healthy and high-risk pregnancies [9, 5156]. the role of education of patients and doctors in increasing adherence to maternal vaccination was also emphasized [5759]. one systematic review assessed the benefits and dangers of the influenza vaccine in special populations pregnant women included but limited the eligible studies to rcts only. apprehensions about the use of thimerosal-containing influenza vaccines, based on theoretical risk of harm to the fetal brain, were widely spread in scientific and lay communities during the past years [55, 62]. subsequent research proved that no causal relation existed between immunization with vaccine containing thimerosal preservative including exposure during pregnancy and neuropsychological outcomes [63, 64]. the most recent report of the global advisory committee on vaccine safety of the world health organization considered that available evidence strongly supports the safety of the use of thimerosal as a preservative for inactivated vaccines. it is expected that with the availability of higher-quality evidence, such concerns can be demystified. attending to claims for more evidence [66, 67], several rcts assessing influenza vaccination in pregnancy are planned and some are ongoing [6877]. such rcts focus on different populations, such as hiv-positive mothers, and factors that interfere with immunization coverage. we expect that, following the publication of these trials, the availability and quality of the evidence will radically improve. additionally, the issue about the comparability between the vaccinated and unvaccinated groups will be more properly addressed. maternal immunization for influenza significantly reduced the incidence of influenza-like illness in women and infants. for clinical practice, further studies should address this lack of evidence and enhance the overall quality of the outcomes.

objective. to assess the effects of the inactivated influenza virus vaccine on influenza outcomes in pregnant women and their infants. methods. we performed a systematic review of the literature. we searched for randomized controlled trials and cohort studies in the medline, embase, and other relevant databases (inception to september 2013). two researchers selected studies and extracted the data independently. we used the grading of recommendations assessment, development, and evaluation (grade) approach to assess the quality of the evidence. results. we included eight studies out of 1,967 retrieved records. influenza vaccination in pregnant women significantly reduced the incidence of influenza-like illness in mothers and their infants when compared with control groups (high-quality evidence) and reduced the incidence of laboratory-confirmed influenza in infants (moderate-quality evidence). no difference was found with regard to influenza-like illness with fever higher than 38c (moderate-quality evidence) or upper respiratory infection (very-low-quality evidence) in mothers and infants. conclusions. maternal vaccination against influenza was shown to prevent influenza-like illness in women and infants; no differences were found for other outcomes. as the quality of evidence was not high overall, further research is needed to increase confidence and could possibly change these estimates.

PMC4045453

pubmed-1258

since the first practical implementation of electrospray ionization (esi) with mass spectrometry reported by yamashita and fenn in 1984, a number of different atmospheric pressure ionization (api) techniques have been developed. many api techniques require extensive sample preparation as well as costly consumables to obtain mass spectra. one of the most cost effective and widely available media for chemical separation and analysis is paper. it has been shown that an electro-osmotic flow similar to that of static electrospray can be utilized to interface a triangle of wetted paper to a mass spectrometer (ms); this form of analysis is called paper spray (ps) [49]. the use of a chromatographic medium such as paper allows for the analysis of complex mixtures since separations will occur as the analyte is being driven along the paper medium. separating analytes on a paper medium provides the benefits of reduced probability of ion suppression and rapid analysis. the paper medium allows for analysis of complex samples such as biological tissue and dried blood spots which are not suitable for traditional infusion techniques due to their complex matrices [4, 7]. one limitation to ps, like many ms techniques that utilize incomplete chromatographic separations is the detector being utilized to perform the analysis. when dealing with complex samples or unknown samples, there is a need for high-quality data in order to determine what is being analyzed. fourier transform ion cyclotron resonance (ft-icr) mass spectrometers are powerful tools for analysis of complex mixtures due to their unsurpassed capability to obtain broadband high-resolution mass spectra with high mass accuracy. utilizing ps with a fourier transform ion cyclotron resonance (ft-icr) mass spectrometer as a detector allows for rapid analysis of both unknowns and/or complex mixtures. in this present work, we show the suitability of a 12 tesla ft-icr ms to obtain high-resolution broadband mass spectra utilizing a coffee filter paper spray medium. high-resolution mass spectra were first obtained for polypropylene glycol (ppg) and polyethylene glycol (peg) standards to test the source. second, urine and calf lung surfactant extract (clse) were analyzed to test the ps source for analysis of complex mixtures not suitable for direct infusion. all solvents used were hplc grade obtained from sigma aldrich (st. louis, mo, usa). ppg was obtained in the form of a tuning standard from applied biosystems (bedford, ma, usa). peg standard was made by dissolving 1 mg of peg 2000 obtained from sigma aldrich (st. louis, mo, usa) in 1 ml of hplc grade water. coffee filter, wegmans brand (rochester, ny, usa), was cut by hand into an isosceles triangle with dimensions of approximately 5 mm (base) by 10 mm (height). samples for ppg and peg ps analysis were prepared by wetting the coffee filter triangle with 10 l of the standard solutions. the wet filter paper samples were allowed to dry under ambient conditions before ps analysis was performed. mass spectra were then obtained by wetting the dried paper triangle with 10 l of 50: 50 methanol: water and using the method described in section 2.5. (amherst, ny, usa). prior to analysis, the sample was stored in chloroform in the refrigerator. samples for clse analysis were produced by wetting a coffee paper triangle with 2 l of clse and allowing the sample to dry under ambient conditions before ps analysis was performed. mass spectra were then obtained by wetting the paper with 10 l of 50: 50 methanol: water using the instrument method described in section 2.5. urine samples were collected from two healthy adult males into sterile polypropylene centrifuge tubes (vwr scientific) and immediately placed in the freezer. the samples were collected in accordance with the health sciences institutional review board at the university at buffalo (hsirb no. prior to analysis, the samples were thawed to room temperature and 1 ml was pipetted into 2 ml sample vials (agilent, santa clara, ca, usa) for use. ten l was injected directly onto a triangular coffee filter and allowed to dry under ambient conditions. once dry, the filter paper was attached to the mass spectrometer via a copper clip and rewetted using 50: 50 methanol: water for ps analysis using the instrument method described in section 2.5. high-resolution spectra were obtained on a 12 t bruker solarix ft-icr ms (bruker daltonics inc., the positive potential was obtained by grounding the paper triangle using a copper clip and applying a negative voltage at the inlet to the mass spectrometer (figure 1 shows an image of the source). mass spectra were obtained at various mass to charge ranges with a pre-ft-icr-trap hexapole accumulation time of 0.400 seconds and an ft-icr trap accumulation time of 0.025 seconds. all mass spectra were obtained in the positive mode at a spray voltage of 2 kv. polymeric standards were utilized to determine the suitability of ps with high-resolution ft-icr ms. in ft-icr ms the resolution of the mass spectra is relative to m/z as well as the number of data points that can be obtained during the detection process. high-resolution spectra require a time domain signal stable for longer than several hundred milliseconds. polyethylene glycol and polypropylene glycol produce a broad m/z range of ions ideal for testing the suitability of ps with high-resolution mass spectrometry. high-resolution mass spectra of peg and ppg are displayed in figures 2 and 3, respectively. identifies of the oligomer ion detected and their associated errors is given in tables 1 and 2, respectively. signal-to-noise obtained for peg and ppg after tuning spectra was in excess of 100/1 with absolute intensities that varied shot-to-shot by less than 20%. this allowed ample time for adjustment of the filter paper towards the inlet of the mass spectrometer so as to optimize instrument setup and analysis. utilizing polymers of known m n values (provided by the manufacturers; polydispersity not given) such as peg 2000 and ppg 2000 provides a detection window several hundred m/z wide for broad mass accuracy calibrations for ft-icr ms. calf lung surfactant extract (clse), which is commercially available in drug form as infasurf, is used to treat respiratory distress syndrome. clse is obtained by doing a total lipid extraction on the lavage fluid from a slaughtered calf lung [11, 12]. phosphatidylcholine (pc) makes 79% of the molar distribution of lipids and consists of a choline head group attached to the phospholipid with varying degrees of unsaturation in the fatty acid chains. based on the optimized setup determined from previously analyzing the standards, clse was then analyzed. ps mass spectra of clse are given in figures 4 and 5, and the identities with associated error provided in table 3. due to the complex nature of urine, many small molecules and metabolites were detected at varying intensities using ps with ft-icr. ps mass spectra, showing numerous lipids and other metabolites obtained from the analyzed urine specimens of two healthy adult male subjects, are shown in figure 6; an inset of one of these ps mass spectra is shown in figure 7 while the other is shown in figure 8. a few of the metabolites identified in the 27-year-old volunteer using the exact mass capability of ft-icr are provided in table 4. metabolites identified in the 44-year-old volunteer using the exact mass capability of ft-icr are given in table 5. of note, the 44-year-old volunteer, who was taking prescribed vitamin d supplements, showed indications of the vitamin d metabolite 25-hydroxyvitamin d2-25-glucuronide in the urine. interestingly, both adult male subjects had indications of one or more glucuronides in urine. this may signal a particularly strong propensity for glucuronides to ionize via the ps ionization mechanism; this capability of ps should be further explored. we developed a ps source for bruker ft-icr instrumentation that can be utilized for a broad range of chemical species. the ps source was tested on a range of samples and determined the source was suitable for both polymer and biological samples. the ps source provided an api medium for the analysis of complex mixtures while mitigating the possibility of cross contamination due to the disposable nature of coffee filter paper.

paper spray ionization is an atmospheric pressure ionization technique that utilizes an offline electro-osmotic flow to generate ions off a paper medium. this technique can be performed on a bruker solarix fourier transform ion cyclotron resonance mass spectrometer by modifying the existing nanospray source. high-resolution paper spray spectra were obtained for both organic and biological samples to demonstrate the benefit of linking the technique with a high-resolution mass analyzer. error values in the range 0.23 to 2.14 ppm were obtained for calf lung surfactant extract with broadband mass resolving power (m/m50%) above 60,000 utilizing an external calibration standard.

PMC3347743

pubmed-1259

streptococcus agalactiae (s. agalactiae) is also known as group b streptococcus (gbs), and causes invasive disease in neonates and pregnant women. however, the incidence of invasive gbs disease among non-pregnant adults has recently increased, especially among elderly persons. nevertheless, invasive gbs disease is rare among non-pregnant immunocompetent adults, and most patients have a chronic underlying disease, such as diabetes mellitus, neurological disease, renal failure, malignancy, or liver disease,, ,. s. agalactiae is also a rare cause of infective endocarditis (ie), and most cases require early surgery because of serious complications, such as major emboli or heart failure with rapid heart valve destruction,, ,. identification of the causative pathogen is essential for the diagnosis and appropriate treatment of ie, although blood culture-negative endocarditis accounts for 2.531% of all cases of endocarditis. one of the most common causes of blood culture-negative endocarditis is the administration of antimicrobial agents before performing the blood cultures. in this report, we describe our experience with a case of rapid progressive culture-negative ie in an immunocompetent man, which was caused by s. agalactiae, based on the results from 16s ribosomal rna (16s rrna) gene sequencing using the resected valve. a 43-year-old previously healthy japanese man presented with a 3-week history of a high-grade fever (> 39 c), rigor, and lumbago. he was treated for a common cold using cefditoren pivoxil, although his family physician did not perform blood cultures. the patient was subsequently referred to our hospital because his symptoms did not resolve. on the first day, chest computed tomography revealed bilateral lower lobe consolidation with pleural effusion. therefore, we performed two sets of blood cultures and started treatment using ceftriaxone (2 g once daily), based on a preliminary diagnosis of community-acquired pneumonia. one week after his first visit, he was admitted to our hospital after he developed dyspnea with or without effort (body temperature: 36.5 c, heart rate: 117 beats/min, blood pressure: 98/55 mmhg, respiratory rate: 20 breaths/min, and oxygen saturation: 95% in room air). chest radiography revealed a butterfly shadow with cardiomegaly and bilateral pleural effusion, which was consistent with congestive heart failure. transthoracic echocardiography revealed a 12 10-mm vegetation on the aortic valve and severe aortic regurgitation (fig. 1). a detailed medical interview revealed that he had undergone dental care without tooth extraction at 1 month before his presentation to our hospital. at the admission, we started treatment using ampicillin/sulbactam (12 g daily in four doses) and gentamicin (180 mg daily in three doses) instead of ceftriaxone for native valve endocarditis, and the patient immediately underwent emergency aortic valve replacement. the resected aortic valve was bicuspid (fusion of the right and left coronary cusps), and both leaflets of the bicuspid aortic valve (bav) were ruptured by the vegetation (fig. no organisms were detected from a total of four sets of blood cultures that were performed before the surgery.. therefore, we performed 16s rrna gene sequencing using the resected valve, which revealed bacterial dna that was 99% identical to the sequence of s. agalactiae. magnetic resonance imaging on day 8 also revealed spondylodiscitis in the l5 and s1 vertebrae and in the l5s1 disc space (fig. 3). therefore, we diagnosed the patient as having ie that was caused by s. agalactiae and complicated by lumbar spondylodiscitis. on day 29, the antimicrobial treatment was changed to ampicillin monotherapy (12 g daily in six doses). on day 43 after the surgery, the intravenous ampicillin was changed to oral amoxicillin (1500 mg daily in three doses) to treat the spondylodiscitis. the patient was discharged from our hospital on day 51, and subsequently completed the 106-day regimen of antimicrobial treatment. first, s. agalactiae should be considered a potentially causative pathogen in cases of rapid progressive ie, even among non-pregnant immunocompetent adults. although gbs is a rare cause of ie, gbs endocarditis is associated with a poor prognosis (a mortality rate of 3556% because of major emboli or heart failure), compared to ie that is caused by other streptococci (e.g., the viridans group); therefore, early surgery is recommended for gbs endocarditis,, ,. in the present case, the man did not exhibit the typical risk factors for gbs disease, although we discovered that he had undiagnosed congenital bav. similarly, most cases of bav are not detected until the onset of infection or calcification, despite bav being the most common congenital cardiac abnormality (found in 12% of the population). the second important clinical issue from the present case is that antimicrobial stewardship should be considered, as the administration of antimicrobial agents before performing blood cultures is a major cause of culture-negative ie. given that blood culture-negative ie accounts for 2.531% of all ie cases, it remains challenging to diagnose and appropriately treat. up to 61% of culture-negative ie resulted from administration antimicrobial agents before performing blood cultures. thus, genetic testing (e.g., 16s rrna gene sequencing) can be useful for identifying the causative pathogen in patients with culture-negative ie. furthermore, 16s rrna gene sequencing using the resected heart valve specimen is an important tool for complementing the diagnosis and therapeutic management of culture-negative ie ,. one reported case of culture-negative ie was caused by s. agalactiae identified using 16s rrna gene sequencing, although the patient died from progressive heart failure despite receiving antimicrobial treatment without surgery. to the best of our knowledge, the present case is the first successfully treated case of ie that was caused by s. agalactiae identified using 16s rrna gene sequencing in a non-pregnant immunocompetent adult. although gbs has been considered completely susceptible to penicillin, an increasing number of japanese isolates exhibit gbs with reduced penicillin susceptibility (prgbs). the minimum inhibitory concentration (mic) of penicillin g for prgbs is 0.251.0 g/ml ,. however, the clinical significance of isolated prgbs remains unclear, and most isolates are from elderly individuals respiratory tissue specimens. in the present case, the patient was successfully treated using antimicrobial therapy (ampicillin/sulbactam and gentamicin) and surgery. therefore, although we could not perform antimicrobial susceptibility testing, we selected ampicillin to replace the ampicillin/sulbactam as definitive therapy for the ie, after we had identified the s. agalactiae using 16s rrna gene sequencing. s. agalactiae can causes rapid progressive ie, even among non-pregnant immunocompetent adults. furthermore, antimicrobial stewardship is important, as the administration of antimicrobial agents before performing blood cultures is a major cause of culture-negative ie. therefore, although s. agalactiae is a rare cause of rapid progressive ie, it should be considered as a potentially causative pathogen, even among non-pregnant immunocompetent adults. the increasing incidence of prgbs with elevated -lactam mics may make it increasingly difficult to select an appropriate antimicrobial treatment for gbs endocarditis.

streptococcus agalactiae (s. agalactiae) is a major cause of invasive disease in neonates and pregnant women, but has also recently been observed among non-pregnant adults, especially elderly persons or persons with underlying chronic disease. s. agalactiae is also a rare cause of infective endocarditis, and most cases require early surgery. we report the case of a 43-year-old previously healthy man who experienced rapid progressive culture-negative infective endocarditis with aortic valve vegetation and severe aortic regurgitation, which was complicated by lumbar spondylodiscitis. emergency aortic valve replacement was performed on the day of his admission, which revealed a congenital bicuspid aortic valve was ruptured by the vegetation. the resected aortic valve specimen was submitted for 16s ribosomal rna gene sequencing, which revealed that the pathogen was s. agalactiae. therefore, s. agalactiae should be considered a potentially causative pathogen in cases of rapid progressive infective endocarditis, even if it occurs in a non-pregnant immunocompetent adult.

PMC5107641

pubmed-1260

after their biosynthesis at the endoplasmic reticulum, secreted proteins are transported to the golgi complex, where they are post-translationally modified and sorted for secretion, plasma membrane delivery, or delivery to prelysosomes. the golgi contains multiple subcompartments, termed cis (early), medial (middle), and trans (late) cisternae; each of these subcompartments houses different sets of glycosyltransferases and other enzymes. proteins enter the golgi at the cis compartment and exit at the trans compartment, but how they move from one cisterna to the next is still being determined. two possible models are widely discussed [1-3]. according to the cisternal maturation (or progression) model, cargo remains in a given compartment and different enzymes arrive there to convert a cis cisterna into a medial one or a medial cisterna into a trans cisterna. alternatively, cargo moves from one golgi compartment to the next, encountering different enzymes in each subsequent compartment until it reaches the trans cisterna, where it is then sorted into carriers bound for post-golgi destinations. this second class of model could use vesicles to transport cargo from one compartment to the next or compartment-connecting tubules through which cargo could pass. conversion of one golgi compartment into another by high-resolution, live cell video microscopy. a limitation of those studies is that one of the compartment markers that was monitored is a peripheral membrane protein that is likely to reversibly bind to and release from the golgi surface. the situation may be more complex in mammalian cells, where golgi cisternae are stacked tightly together, unlike yeast; it is hard to imagine a single cisterna moving from one side of the well-stacked structure to the other. nevertheless, large procollagen cargo traverses the golgi without ever leaving a cisterna, in support of a maturation model. to complicate matters, membrane tubules have been detected between golgi cisternae under conditions of active secretion; this scenario would permit cargo movement from one side of the stack to the next without maturation or vesicle transfer. important new clues to how golgi compartments might mature come from a study of golgi-localized, ras-related, rab family gtpases in yeast. rab gtpases are localized to different membrane compartments and catalyze the formation of functionally distinct, membrane microdomains that are important for transport vesicle formation, vesicle motility, and vesicle (or compartment) docking and fusion. rab gtpases help early endosomes mature into later endosomes by a process called rab conversion. the early endosomal rab5 protein recruits a specific guanine nucleotide exchange factor (gef) that activates rab7. rab7 then recruits rab7-specific effectors to that compartment, thereby converting an early endosome into a late endosome. this type of rab cascade (figure 1b) was first described for a yeast golgi rab, ypt32p, recruiting the gef for the subsequent acting sec4p rab. (a) endoplasmic reticulum, golgi, and endosome membranes are capable of homotypic fusion and fission. (b) rab cascades occur when sequentially acting rabs recruit guanine nucleotide exchange factors (gefs) and gtpase-activating proteins (gaps) to membranes. raba recruits a gef that will convert the subsequent acting rab to its active form. rabb can then recruit a gap that will inactive the previous acting rab, thereby removing it from the newly formed, second compartment. rivera-molina and novick have now used live cell video microscopy to detect rab conversion at the yeast golgi: they see compartments containing the early golgi rab, ypt1p, convert into a compartment containing the late golgi rab, ypt32p. (although the light microscopy method employed could not resolve structures smaller than about 200 nm, the images were nevertheless highly compelling.) the process involves the recruitment of ypt32p by the gtpase-activating protein (gap) that inactivates ypt1p: gyp1p. upon inactivation, ypt1p becomes a substrate for removal from membranes by another protein, gdi (gdp-dissociation inhibitor). the removal of rabs from the membranes makes this work subject to one of the same limitations of the previous studies; nevertheless, these markers permitted the authors to detect an important molecular transformation. the data provide a direct molecular mechanism for compartment inter-conversion at the golgi, reminiscent of maturation in the endocytic pathway. golgi compartments were seen to be dynamic, undergoing a certain amount of fission and fusion. in some cases (30%), a ypt32p compartment appeared to fuse to a ypt1p compartment to yield a mixed compartment or a mixed compartment appeared to undergo segregation and fission to yield separate ypt1p and ypt32p compartments. what this means is that, yes, yeast golgi compartments undergo apparent maturation by rab conversion, and at the same time, cargo may get the fast track from one compartment to the next by intermittent cisternal fusion and fission events (figure 2a, b). importantly, although a compartment will seem to mature, it is actually forming from a stable predecessor. early golgi has the capacity to fuse with other early golgi cisternae and so on. fission is favored in the absence of microtubules or when golgin proteins are depleted from cells. glycosyltransferases are localized to cis (red), medial (yellow), and trans (orange) golgi compartments but rarely have a perfectly sharp distribution. thus, for example, cis-golgi homotypic fusion events can occur with another cis compartment (step 1) or perhaps a medial (yellow) compartment (or both). similarly, a medial cisterna may be able to fuse with a trans cisterna (orange). in this manner, a large cargo may be able to encounter all golgi-processing enzymes without entering a transport vesicle, indicated by fusion steps (arrows) 1, 2, and then 3. the ability of golgi cisternae to undergo fission and fusion has been known since the 1970s: simple, nocodazole-triggered depolymerization of microtubules causes the mammalian golgi to fragment into mini-stacks that disperse throughout the cytoplasm, and drug washout leads to rapid stack reassembly (figure 2a). this indicates that the golgi is capable of fission as soon as microtubules are lost and of fusion with itself as soon as microtubules repolymerize. compartment collisions likely enhance fusion, and cytoskeletal motor proteins that decorate the golgi and connect it to both microtubules and actin cables are sure to contribute to both fusion and fission events, as is true in the endocytic pathway (figure 1a, c). are intercisternal fusion/fission connections required for membrane traffic? transport is only partially blocked in nocodazole-treated cells and this condition favors stack fission. but homotypic fission and fusion are likely much more prevalent than previously anticipated because cellular depletion of any one of many different golgi proteins (golgins) generates mini-stacks that are clustered near the microtubule-organizing center. such transient fusion and fission could yield the tubules that have been detected in electron micrographs of mammalian cell golgi complexes. fission and fusion would make it possible to accommodate extra-large cargoes, such as collagen, that are too big to fit into conventional transport vesicles. as is well established for the endocytic pathway, rab gtpases would define specific subdomains and retain specific golgi enzyme subsets there. compartments would be defined by their distinct rab gtpases, and adjacent cisternae might fuse at some frequency that allows cargoes to encounter sequentially acting, processing enzymes. in a mixed compartment, rab gefs and gaps would segregate individual rabs into separate regions that would be segregated upon the simple action of a membrane-associated, cytoskeletal motor protein to drive fission. order within the stack would be maintained by the relationship between specific rabs and their cognate activators (gefs) and inactivators (gaps), which are designed to permit rabs to function in a sequential cascade. indeed, the proteins that stack the cisternae may use a rab cascade to achieve their position in the stack. at the trans golgi, rabs would also initiate the process by which specific cargoes are collected into distinct transport carriers and delivered to their final destinations. validation and clarification of this model will require defining which rabs build which specific golgi enzyme microdomains and determining the specific molecular interactions that permit fission, fusion, and enzyme organization. vesicles are likely to be involved in golgi transport: we know that cop-i-coated vesicles collect kdel receptors for delivery back to the endoplasmic reticulum; in this case, we can postulate that such vesicles bud from a rab gtpase-organized, functional membrane microdomain. the same proteins that drive vesicle targeting and fusion may also participate in cisternal docking and fusion to permit protein transport across this central cellular compartment. the mechanism by which depletion of any one of at least 10 different golgin proteins leads to mini-stack formation will likely tell us much about how proteins move through the mammalian golgi stack.

the golgi complex is a central processing station for proteins traversing the secretory pathway, yet we are still learning how this compartment is constructed and how cargo moves through it. recent experiments suggest a key role for ras-like rab gtpases and provide important new ideas for how the golgi may function.

PMC2897732

pubmed-1261

major depressive disorder, especially in later life, has heterogeneous clinical characteristics and treatment responses. classification of depression according to age of onset may be a useful approach to understanding its intricate and complex properties. several studies of late-life depression have focused on age of onset between 40 and 60 years.1,2) however, although many studies have been conducted regarding differences between early-onset depression (eod) and late-onset depression (lod), only a few consistent differences between these disorders have been identified. research has shown that eod is associated with more chronic features3) and a higher tendency toward suicidal behavior.4) in patients with eod, there are more commonly feeling of worthlessness, suicidal ideation and anxiety, whereas psychomotor retardation, lack of energy, and apathy tend to be more common in patients with lod.1,5) the person with lod have more problems in cognitive function, neurological abnormalities and increased medical comorbidity. despite recent advances in psychopharmacologic treatments, 20% to 30% of patients with mood disorders experience inadequate responses to medication, often resulting in a trial of electroconvulsive therapy (ect).6) a large body of evidence suggests ect as an effective treatment especially for severe and treatment-refractory depression,7) and studies indicate that up to 90% of people with severe treatment-refractory depression improve dramatically with ect.8) with f-fluorodeoxy-d-glucose positron emission tomography-computed tomography (f-fdg pet/ct) we can obtain the status of brain metabolism of patients with neuropsychiatric disorders and the metabolic changes due to ect. several studies have described the relationship between depressive disorder and brain metabolism. in this report, we described a case of 55-year-old female patient who suffered treatment refractory depression and cognitive deficit. the changes of brain metabolism before and after ect were presented by f-fdg pet/ct. a 55-year-old female patient who had suicidal attempt with organophosphate was treated in the department of nephrology for a while and transferred to the department of psychiatry. she had experienced no earlier episode of mood change, but two months prior to admission she started feeling depressed mood and feelings of guilt towards her family members. she had paranoid delusion that her husband had another partner and wanted to murder her for her insurance. the patient had feelings of hopelessness, helplessness, worthlessness, and poor self-esteem. a complete biochemical workup was negative, including cbc, thyroid hormones, hiv, and syphilis blood tests. she underwent full psychometric evaluation including wechsler test, cognition and memory function test, rey-kim memory test, minnesota multiphasic personality inventory-ii (mmpi-ii), sentence completion test, symptom check list-90-revised (scl-90-r), draw-a-person test, mini-mental state examination (mmse), clinical dementia rating (cdr), rorschach test, positive and negative syndrome scale (panss), and hamilton rating scale for depression (ham-d). her ham-d score was 43, panss score was 113 and mmse was 20. the differential diagnosis comprised initially psychotic mood disorder, neurodegenerative disease and iatrogenic delirium secondary to medication. the patient presented as medication-resistant during the course of duloxetine, mirtazapine, trazodone, aripiprazole, and quetiapine. medications for 4 weeks showed no significant improvement of symptoms. to rule out organic brain damages, the patient underwent brain magnetic resonance imaging but there were no significant findings. for evaluating her cerebral function, f-fdg pet/ct scans were obtained with a biographmct 128 scanner (siemens healthcare, knoxville, tn, usa). she was intravenously injected with 185 mbq of f-fdg approximately 60 minutes before the imaging. the blood glucose level was<150.0 mg/dl before f-fdg injection. the patient was stable for 30 minutes prior to f-fdg injection and in the subsequent uptake phase. each pet/ct scan after ct scanning, a pet scan was performed in the three-dimensional mode. a depressive episode was still suspected clinically and, because medication had little effect, it was progressively reduced. modified bilateral ect was applied and the patient underwent 3 weeks of an index course ect (9 times). thereafter, there was impressive improvement in the patient s global functioning with complete resolution of symptoms. her ham-d score was 4, panss score was 34, and mmse was 29. the final diagnosis was major depressive disorder of high severity with mood congruent psychotic features. after the course of ect, the patient was discharged on minimal dosage of mirtazapine, aripiprazole, and quetiapine. after discharge, she showed no signs of residual psychosis or mood disorder and greater emotional expressiveness and had no psychomotor retardation or cognitive deficit. seven weeks after the last ect, the patient underwent f-fdg cerebral pet (fig. acquired pre/post pet images were co-registered and the relative standard uptake value changes were estimated by image subtraction algorithm. the image was compared with the f-fdg cerebral pet that was obtained before ect procedure and showed recovery of cerebral metabolism without any evidence of a neurodegenerative process (fig. this case report presented the change of brain metabolism before and after ect procedure in a patient with treatment-refractory lod by pet/ct imaging. there was increased metabolic activity in cerebral cortex and basal ganglia during the course. in other studies, the patients with lod showed decreased cerebral glucose metabolism than the healthy group in multiple areas of brain, which might be the cause of cognitive impairment that commonly accompanies lod.9) the use of pet as a probe of the cerebral metabolic rate of glucose (cmr) in depressive patients appears especially promising, because depression is reportedly associated with changes in cmr in different cerebral regions and can be used to display the resting state metabolism of the brain.10) repeated pet provides an opportunity to explore changes in cmr associated with the use of ect in vivo. thus, persistent changes in glucose metabolism patterns are expected to provide a reliable estimate of neuroanatomic metabolism.11) drevets et al.12) reported reduced cerebral blood flow (cbf) and metabolism in the anterior cingulate cortex (acc) ventral to the genu of the corpus callosum in patients with depression. and bell et al.13) reported decreased cbf in dorsomedial/dorsoanterolateral prefrontal cortex in patients with depression. reduced glucose metabolism in bilateral anterior and posterior frontal areas represented the most consistent finding of previous studies, despite considerable methodological heterogeneities. the study with 6 depressive patients treated ect found that significant increases of cmr which were presented by pet in basal ganglia, brainstem and occipital lobe.14) some researchers found that significant decreased metabolism in left subgenual acc and hippocampal area in patients with depression; and ect led to increased metabolism in these areas.15) however, there are different results that the ect-induced seizures change from the site of initiation to other specific brain regions and decrease cbf in cingulate and left dorsolateral frontal cortex 30 minutes after induction of seizures.16) and nobler et al.17) have reported reduced regional cerebral metabolic rate for glucose in 10 patients with depression in post-ect state. suwa et al.18) presented the synthetic view of these difference in metabolic change after ect. in those studies, they have shown that ect changed cbf and cerebral glucose metabolism which were decreased in depression patients. in our case, increases in glucose metabolism in the frontal, parietal, and occipital regions may have represented the antidepressant effect of ect. in this case, we described a patient who did not have any other history of mental disorder before and presented severe cognitive deficit and treatment-resistance. the patient presented a severe and diffuse cerebral glucose hypo-metabolism on f-fdg. awata et al.19) have reported similar recovered regional cerebral blood flow patterns were shown at 2 weeks and 12 weeks after ect in depressive patients, because of that the post ect image was taken 7 weeks later after the last ect to minimize the effect of psychotropic agent and to evaluate the long-term treatment result. this case illustrates the potential of f-fdg brain pet to demonstrate the normalization of brain metabolism in major depressive episode in patients benefiting from ect. it also illustrates the efficacy of ect in the treatment of major depression and suggests that its clinical mechanism is related with changes in metabolic function. the multifactorial effect of numerous psychoactive drugs might have increased the severity of the deficits.20) we presented the cerebral metabolic changes in the patient with severe lod and cognitive disturbance. there were several studies reported cerebral metabolic changes during the ect in depressive patients, but the little of those researches concern with both lod and cognition. this case report is not only the evidence of classical view of ect in treatment resistant depression but also the treatment choice of lod with cognitive deficit. first, we described one female patient and were unable to compare with other patient(s) with the same conditions. second, our patient was unipolar depression and it is not certain that the ect-induced brain metabolic change is replicable in bipolar depressions. third, the patient had lod, therefore findings might differ in eod. despite these limitations, fdg-pet represents a promising marker of neuronal cell functions and reflects an epiphenomenon of a complex and dynamic interaction of different neurobiochemical processes. regardless of these methodological difficulties, research into this area highlights the potential of functional neuroimaging. changes in metabolic patterns have been correlated with changes in behavioral variables reflecting the severity of the underlying disease; in addition, a link between changes in frontal activity and improvements in clinical symptoms was identified.17) further study should include more patients who have depressive episode, and focus on objectifying clinical improvement of depression after ect to allow a correlation with possible changes in cmr. follow up pet scans after six or twelve months should be performed to assess possible long-term effects.21) clearly, a separation of patients with unipolar depression from patients with bipolar depression should be considered and larger samples of patients are needed to increase statistical power and account for heterogeneity in study cohorts.

major depressive disorder, especially in later life, has heterogeneous clinical characteristics and treatment responses. symptomatically, psychomotor retardation, lack of energy, and apathy tends to be more common in people with late-onset depression (lod). despite recent advances in psychopharmacologic treatments, 20% to 30% of patients with mood disorders experience inadequate responses to medication, often resulting in a trial of electroconvulsive therapy (ect). however, the therapeutic mechanism of ect is still unclear. by using 18f-fluorodeoxy-d-glucose positron emission tomography-computed tomography (18f-fdg pet/ct), we can obtain the status of brain metabolism in patients with neuropsychiatric disorders and changes during psychiatric treatment course. the object of this case report is evaluating the effect of ect on brain metabolism in treatment-refractory lod by pet/ct and understanding the mode of action of ect. in this case report, we presented a 55-year-old female patient who suffered psychotic depression that was resistant to pharmacological treatment. several antidepressants and atypical anti-psychotics were applied but there was no improvement in her symptoms. the patient presented not only depressed mood and behaviors but also deficit in cognitive functions. we found decreased diffuse cerebral metabolism in her brain 18f-fdg pet/ct image. ect resulted in amelioration of the patients symptoms and another brain pet imaging 7 weeks after the last ect course showed that her brain metabolism was normalized.

PMC5290710

pubmed-1262

while the recent strategies of lung-protective ventilation and proper fluid restriction therapy have reduced mortality in patients with acute lung injury (ali) and acute respiratory distress syndrome (ards), the mortality rate remains high. although many pharmacological therapies have been attempted to treat ali/ards, no specific therapy has demonstrated a clear effect so far. accordingly, mesenchymal stem cells (mscs) have been introduced as a possible therapy. mscs are multipotent adult stem cells that have the ability to differentiate into many different cell lineages and the capacity for self-renewal (1). in early research on the use of mscs to treat ali/ards, the main mechanism of action of these cells seemed likely to involve the replacement of injured lung epithelium. however, subsequent studies have suggested that the most important therapeutic effect probably comes from the paracrine properties of mscs (2, 3). thus far, the therapeutic potentials of mscs have primarily been supported by preclinical evidence and the need for clinical trials has been suggested (4). as there is a lack of clinical data, we here introduce a patient who was treated with mscs in the course of ards and subsequent pulmonary fibrosis. a 59-yr-old man with a history of pulmonary tuberculosis (tb) and no significant family history was diagnosed with idiopathic thrombocytopenia in june 2008., he developed a cough, sputum, and rhinorrhea and five days later displayed fever and dyspnea. his chest radiography and computed tomography (ct) scan showed multifocal patchy ground-glass opacities (ggos) in both lungs with underlying emphysema with large bullae in the left lower lobe and focal irregular nodular lesions in the right upper lobe that were presumed to be tb sequelae. after five days, he was transferred to our hospital, a tertiary referral center, and admitted to the medical intensive care unit. at admission, he had tachypnea with a fever of 39. he had progressive bilateral diffuse infiltrations on his chest radiography. his initial sao2 level was 75% and his pao2/fio2 (p/f) ratio was 166 mmhg. we started a course of methylprednisolone at a dose of 40 mg twice a day to treat a possible pneumocystis jiroveci pneumonia in conjunction with a continuing regimen of empirical antibiotics and an antiviral agent. however, no specific pathogen was identified in any specimen from this patient, including bronchoalveolar lavage, blood or sputum. then, his chest radiography was stationary and his p/f ratio began to gradually improve. on hospital day (hd) 7, he underwent tracheostomy to enable early weaning from mv. however, he then developed hospital-acquired pneumonia and hydropneumothorax due to a ruptured bullae with bronchopulmonary fistula (bpf). he was conscious during this time but he could not communicate with the medical team or even his family members. on hd 87, he had a follow-up high-resolution ct, which showed aggravation of diffuse ggos and interlobular septal thickening in both lungs. this indicated the progression of pulmonary fibrosis as a post-ards sequelae. after discussing the possibility of administering mscs with his family, we commenced with a trial of this therapy. the umbilical cord blood (ucb)-derived mscs (ucb-mscs) were prepared as follows. ucb-mscs were produced at the good manufacturing practice facility of medipost co., ltd. quality control and quality assurance for the production of these cells were performed according to the standards of the korea food and drug administration. flow cytometry analysis of expressed surface antigens showed that these cells were uniformly positive for cd29, cd44, cd73, cd105, and cd166 and negative for the hematopoietic lineage markers cd34, cd45, cd14, and hla-dr. the final ucb-mscs preparations used in the infusion were harvested from cell culture passage 6 and suspended at a final density of 7.510/1.5 ml in normal saline. on hd 114, the patient underwent the intratracheal administration of ucb-mscs at a target dose of 110/kg. before the procedure, the ventilator was set to the pressure-controlled ventilation (pcv) mode of 26 cmh2o of inspiratory pressure (ip) and 0.45 of fio2. there were no peri-procedural complications. the next morning (16 hr later), he was able to communicate effectively with our medical team, which had not been possible in the previous few months. after 24 hr, we changed the pcv to a pressure support mode with 18 cmh2o of support pressure with 0.4 of fio2. after 48 hr, pcv was applied again with 22 cmh2o with 0.35 of fio2. the dynamic compliance of his lung improved from a pre-procedure value of 22.7 to 26.5, 27.3, and 27.9 ml/cmh2o after 24, 48, and 72 hr, respectively. his p/f ratio subsequently increased from pre-procedural 191 to 328 mmhg on day 1 after the procedure and to 334 mmhg on day 3 (fig. 1). a follow-up chest radiography showed a slight decrease in bilateral lung infiltrates (fig. 2). on day 3, however, he suddenly experienced six generalized tonic-clonic seizures during the course of the day with a fever near to 39. he had no focal lateralizing signs. a brain ct was performed and showed no hemorrhage. we discussed the case with neurological specialists and determined that the current use of carbapenem was the most probable cause of his seizure. the weaning trial was restarted for our patient and the ip was reduced to 12 cmh2o with 0.3 of fio2 during the following week. however, he could not be weaned because his repeated infection by multidrug-resistant pathogens was not controlled. eventually, he suffered septic shock due to empyema and died on hd 231 or day 118 after msc administration. our current case involved a patient who developed ards due to pneumonia. he subsequently had post-ards pulmonary fibrosis. weaning from mv was difficult as he did not improve for a long-period (four months). although he failed to survive, his immediate improvement after cell infusion is worthy of mention. his mental status, his lung compliance, p/f ratio and his chest radiography all showed improvement over the course of at least three days. we speculate that these clinical, physiological and radiological improvements might be related to the paracrine properties of mscs, with immunomodulation, alveolar fluid clearance and regulation of lung protein permeability known as potential mechanisms (2). the main reasons for our failure to help this patient survive by mscs therapy are likely to be the following. first, his pulmonary fibrosis was so advanced that it could not respond to the stem cell therapy. its effectiveness may depend mainly on paracrine activity rather than regeneration by stem cell engraftment. thus, a long-term effect would not be expected due to his advanced stage of fibrosis. second, his bpf-related repeat infections may have caused the lack of improvement in his lung compliance. since the pathogens that were responsible for the infection were multidrug-resistant to current antibiotics, a proper antimicrobial treatment could not be performed in advance of the stem cell therapy. a third possible reason concerns the dose and the number of stem cells administered. we intratracheally infused 110/kg stem cells on one occasion but no study regarding the proper number of cells for intrapulmonary administration in humans has thus far been reported. accordingly, our dose was based on that used intravenously in a trial in acute graft-versus-host-disease after allogeneic hematologic stem cell transplantation (5). we decided to infuse using a local route as we expected more intrapulmonary action and less systemic adverse effects than for an intravenous injection. however, our dose or the one-time-only administration may have been insufficient to produce a significant effect. this report has a number of notable limitations that hinder the interpretation of the definite effect of mscs in post-ards pulmonary fibrosis. first, we did not check the level of cytokines or other soluble factors that have been shown to be associated with the functions of mscs in preclinical studies. in addition, our interpretation in this case study relied on clinical parameters and radiological images only. thus, it is difficult to precisely link the patient's short-term clinical improvement to the definite action of the mscs. finally, even though the clinical studies of msc therapy for acute lung injury are currently underway, there is no definite evidence so far. thus, our decision on the msc therapy may have some ethical problems although we had no other way as a salvage therapy for his fibrotic stage of ards. our current findings suggest the possibility of using msc therapy in an ards patient and it is the first clinical case of ucb-mscs therapy ever reported. for a clear verification of msc therapy in ali ,

umbilical cord blood (ucb)-derived mesenchymal stem cells (mscs) have been introduced as a possible therapy in acute lung injury and acute respiratory distress syndrome (ards). this case history is reported of a 59-yr-old man who was treated with mscs in the course of ards and subsequent pulmonary fibrosis. he received a long period of mechanical ventilation and weaning proved difficult. on hospital day 114, he underwent the intratracheal administration of ucb-derived mscs at a dose of 1 106/kg. after cell infusion, an immediate improvement was shown in his mental status, his lung compliance (from 22.7 ml/cmh2o to 27.9 ml/cmh2o), pao2/fio2 ratio (from 191 mmhg to 334 mmhg) and his chest radiography over the course of three days. even though he finally died of repeated pulmonary infection, our current findings suggest the possibility of using mscs therapy in an ards patient. it is the first clinical case of ucb-derived mscs therapy ever reported.

PMC3945142

pubmed-1263

today, pharmaceutical business is among greatest businesses of the world. by spending a lot in order to produce new drug, in fact, while pharmaceutical industry is a sensitive one, it is an advantageous industry as well (1). studies of drug status and recent anticipations indicate a progressive trend in world drug market, so that the rate of drug sale around world (world market) has reached 900 billion dollars in 2011 from 309 billion dollars in 1998 (around 2.9 times more) (2). it is expected that the drug world sale would go beyond 1 trillion (1043.4 billion) dollars in 2013, and reach 1.3 trillion dollars until 2020 (3). the drug world market during 1998 to 2008 had enjoyed an average growth of about 12%, while the rate is much lower in other industries. naturally, the world market of pharmaceutical industry could be divided into four areas: united states, japan, europe, and the rest of the world (4). iran s pharmaceutical industry share is around 2 billion of the 750 billion dollars, while turkey as one neighbor of iran has a share of 6 billion dollars (1). drug expense share is averagely 30% of the whole health expenses among different world countries. drug expenses of iran in 2011 have indicated an increase from 276 million to 829 million dollars for imported drugs. generally, the ratio of rial sale of imported drugs to the drugs produced inside the country has changed from 0.28 to 0.71 in recent years (5). since most world countries have joined world trade organization, iran as a developing country with no significant role and presence of non-oil economy in world economy should not be in isolation from world progress and evolutions (6). therefore, iran s principal problem in this regard will be summarized in persistent and concentrated effort to make its membership possible with the least expense and the most profit. hence precise recognition of effects and outcomes of membership in the organization will considerably help passing the membership path successfully (3). therefore, studying effects of decreasing import tariff on major variants of drug field is a very important issue. finally, we aimed to evaluate the impacts of trade openness on pharmaceutical industry using cge model. model details associated with activities, production factor, and institutes are summarized in table 1. activities include agriculture, pharmaceutical industry, industry and mine sector, construction sector, and finally service sector which use two factors of labor and capital for their products. the model utilized include product associated trades, household and government consumption, saving, investment, and foreign business. it is assumed in this model that economic sectors use labor and capital as primary institutes for production. in order to realize the model, beside primary institutes, it is assumed that sectors use mediator institutes for production as well. for more convenience, it is assumed that in lower stage, additional value (composite primary factor) is obtained from composition of labor and capital with cobb-douglas type production function (equation 1) (7). vaj=bj fdhjhjj as sectors index, h as index of primary production factors (labor and capital), vaj as additional value of jth sector, fdhj as demand for h th production factor by j sector, bj as efficiency parameter in production function, and bhj as share parameter in production function or production elasticity of jth sector to hth institutes ratio so that hj 1 and 1 hj are considered in this equation. using leontief type production function in this stage, gross output is produced by combining additional value and mediator institutes (8). according to these two stages with regard to production, each sector maximizes its profit function. yj=min (xijaxij, vajayj)in this equation, i as sectors index, yj as gross output of jth sector, axij as coefficient of minimum demand for mediator institutes of jth in order to produce a gross output unit of jth sector (technical coefficients of input-output), ayi as coefficient of minimum need for additional value in order to produce one unit of gross output, xij as ith sector production that is consumed as jth sector mediator are considered. according to these two stages xij=axij.yj j vaj=ayj.yj fdhj=hj.pnjwh.vaj h psj=ayj. pnj+axij.pqi j pnj as additional value price of jth sector, wh as production factor wage, psj as supply price of jth sector, and pqi as goods price of jth sector are considered. it is assumed in this article that production factors (labor and capital) are in a state of equilibrium, and the supply of factors is constant (7). hence change in tariffs does not change the whole demand of labor and capital, while just transmission of production factors from one sector to another takes place. in order to calculate private sector consumption (households), it is assumed that consumption basket is optimally selected by consumers (9). their revenue is obtained from supply location of production factors (labor and capital) added by transmission payments of government to households and pure external moneys received. the favorability function is a cobb-douglas function which is maximized due to a budget equal to pure household revenue (household revenue minus direct tax amount and saving). ci.pqi=ci (wh.fsh-taxdir-savhoh) in this equation, ci is considered as ith object household consumption, ci as share parameter in favorability function or each object share in household consumption basket so that 0 ci 1 and ci 1, fsh the amount of primary hth factor supply (exogenous variable) is dir revenue derived tax of the households. according to public sector consumption, it is assumed that government earns through implementation of sales tax, direct household income tax, and oil import and export tax. taxind, j=txj.psj.yj taxdir=td.wh.fsh tariffj=tmj.pmj.mj gi.pqi=gi (taxdir+taxind, j+tariffj+eoil-savg in these equations, taxind, j is considered as indirect tax (sales tax), txj as sales tax rate, td as direct tax rate, tarifj as import tariff, tmj as import tariff rate, pmj as import domestic price, mj as import amount, gi as government expenditures in jth sector, savg as government saving, gi as share parameter of government expenditure in each sector, and eoil as oil export. investment in every sector depends on total investment which is equal to total saving, and is obtained from private, public, and foreign savings together. savhoh=shohwh.fsh savg=sg(taxind, i+tariffi+taxdir+eoil saving=(savh+savgov+exr.savf) saving=invest idi.pqi=i.invest in these equations, shoh is considered as mean tendency of private sector saving, sg as mean tendency of government saving, savf as foreign saving, saving as total saving, invest as total investment, idi as investment demand of ith sector, i as share parameter of ith sector investment, so that 0 i 1i and i 1. in external world and foreign trade, it is assumed that the country is small i.e. the country has no effect on world prices and international market. pei=pwei.exr pmi=pwmi.exr in these equations, pei is considered as domestic export price, pwei as world export price (exogenous variable), exr as foreign exchange rate. when the model is considered for an open economy, it is necessary to consider some precautions about alternating imported, exported and domestically supplied goods (12). in general equilibrium models, there is difference between imported and exported goods as well as goods produced for export and goods produced for domestic sale. it is assumed that imported and domestically supplied goods make composite good (argminton good). it is assumed that institutes are an incomplete alternate to domestic productions (13). the relation between imports and domestic production is indicated as a constant elasticity of substitution (ces) function (14). qi=i(mi.mimi+di.dimi)1mi in this equation, qi is considered as composite good, di as domestically produced good, i as efficiency parameter in production function of composite good, mi as share parameters in argminton function, so that mi+di 0, mi as argminton function power or the parameter associated with substitution elasticity, so that mi=(i-1)i and i 1, ias argminton function elasticity is i =- d(mi-di)mi/di/d(pmipdi)pmi/pdi. regarding the aim of problem maximization, demand functions for import and domestic production would be as equations number 20 and 21. mi=(imi.mi.pqi(1+tmi).pmi)11-mi.qi i di=(imi.di.pqipdi)11-emi.qi in this equation, pdi would be the price of domestically produced good. equation number 22 indicates the relation between export and domestic production which is stated on a constant elasticity of transmission (cet) as well. yi=(i(ei.eiei+di.diei)1ei i in this equation, ei is considered as the amount of export, qi as efficiency parameter of transmission function, ei and di as share parameters in transmission function, so that di+ei 0, ei, as transmission function power, or parameter associated with transmission elasticity, so that ei=(i+1)/i, transmission elasticity as i=d(eidi)eidi/d(peipdi)peipdi regarding maximization problem, export supply and domestic good functions would be the equations number 23 and 24 respectively. ei=(qiei.ei(txi+psi)pei)11-ei.yi di=(qiei.di(txi+psi)pdi)11-ei.yi associated prices are the modifying factors for supply and demand equality in each market in order to develop balance in four markets of labor, capital, composite good, and foreign exchange. profit in labor market, composite good price in composite good market, and foreign exchange rate in foreign exchange market are mediating factors. since there are uncountable solutions with similar relative prices, price normalizing equation is used in order to assure there is just one solution which is equilibrium solution. price index is constant in this equation and other price changes are measured in proportion to this price which is shown in equation number 28(16). pindex=wipqi in order to solve applied general equilibrium models, a complete set of statistics and data is needed. since the aim of research is to study the effect of decrease in tariff on key variables of pharmaceutical products, the decrease has been studied gradually in two general scenario formats. each scenario has been studied as 10, 30, 50, and 100% gradual decrease of custom tariff rate: 10, 30, 50, and 100% decrease in tariff rate of pharmaceutical products import on key variables of drug field10, 30, 50, and 100% decrease in tariff rate of other sectors (except pharmaceutical products) import on key variables of drug field 10, 30, 50, and 100% decrease in tariff rate of pharmaceutical products import on key variables of drug field 10, 30, 50, and 100% decrease in tariff rate of other sectors (except pharmaceutical products) import on key variables of drug field the results of scenarios simulation are summarized in table 2. this table shows the impacts of tariff s cut for key variables during trade liberalization; decrease in import tariffs affects good import and services. normally, increase in import of capital goods and mediator materials increases production, while good import and consumed services which decreases people demand for good and internal services decreases production. according to results of table 2, when tariff decreases in pharmaceutical products, each sector import will increase as a function of tariff rate in that sector according to equation number 16. in fact, when tariff rate decreases, imported good price will decrease in the country which leads to increase in demand for export. therefore, 10, 30, 50, and 100% decrease in tariff rate of pharmaceutical products in the first scenario will increase pharmaceutical product import by 0.2% on average, while decrease in tariff rate of agriculture sector (scenario 2) will decrease pharmaceutical product import by about 0.96% on average, and this variable will decrease by about 2.81% due to tariff rate decrease in industry and mine sector. also, export variable has increased with decrease in tariff rate of pharmaceutical products in scenario 1. this result is completely logical since according to its direct relation with supply (equation 22), increase in pharmaceutical products supply will undoubtedly increase drug export rate. since most items of drug import are raw materials in iran, it is natural that increase in pharmaceutical raw materials would increase manufactured drugs, and become the main factor in drug export. in various studies performed in iran, trade liberalization has increased import variable of associated product while has decreased the product s import on the other side. it is worth noting that change percent and drug export growth is lower than drug import due to gradual decrease in tariff rate. pharmaceutical product export indicates a 0.64% decrease due to decrease in tariff rate of agriculture sector, and 1.91% decrease due to decrease in custom tariff rate of industry and mine sector. according to results, the supply rate of pharmaceutical products has gradually increased by decrease in tariffs which is a logical result; since the rate of goods supplied in markets will increase by decrease in pharmaceutical product tariff and increase in drug import. drug supply rate has decreased by 0.19% due to decrease in tariff rate of agriculture sector, and by 0.69% due to decrease in tariff rate of industry and mine sector. since labor market has been considered in equilibrium state and labor supply in entire economy has been assumed to be constant in the current study, any change in tariffs would not change in total employment, while will change employment in the considered sectors. therefore, if employment decreases in a sector, it means that labor has been transmitted to other sectors. various studies indicate that one negative effect of globalization in developing countries is employment reduction in drug field; since due to competitiveness, these countries have to use new equipments and technology on one hand which reduces labor rate, and production of some pharmaceutical products will face problems due to lack of competitive advantage on the other hand which might lead to stopping drug production or unskillful labor unemployment. results of the current study indicated that in the first scenario, gradual reduction of tariff rate will not make any change in labor employment in drug field due to model conditions; while in the second scenario, decrease in tariff rate of agriculture sector will reduce labor employment in drug field by 0.14% as compared to the first scenario. in the second scenario, decrease in tariff rate of industry and mine sector has led to a mean 0.35% decrease in labor employment of this field. however, according to capital factor, there is a mean 0.12% increase in capital factor employment in the first scenario, and about a mean 0.82% decrease due to decrease in tariff rate of agriculture sector, and about a mean 0.13% decrease in capital factor in drug field due to decrease in tariff rate of industry and mine sector. investment in each sector directly depends on total investment and reversely depends on composite good price in each sector. in the first scenario, decrease in custom tariff of pharmaceutical products will increase the capital rate by a mean of about 0.1%, and in the second scenario, decrease in custom tariff rate of agriculture sector will increase capital rate by about 0.05%, and the same effect in industry and mine sector will lead to 0.13% increase. therefore, trade liberalization will increase capital rate in all scenarios. the model results indicated that decrease in custom tariff rate in the first scenario shows a mean 0.13 increase which is a thoroughly logical result, since household drug consumption will increase due to decrease in tariffs of pharmaceutical products and import increase, while in the second scenario, drug consumption rate will decrease by a mean of 0.52% of household consumption of pharmaceutical products due to decrease in tariffs of agriculture sector, and household drug consumption will 1.57% decrease due to decrease in tariffs of industry and mine sector. since the aimed function of this study model has been maximization in favorability function of society people, and this function indicates total society welfare, in the first scenario, total society welfare shows 0.63%, 0.64%, 0.96%, and 0.16% increase respectively with regard to the basic amount. however, according to effect of tariff decrease in agriculture sector, total society welfare has 0.83%, 0.60%, and 0.38% decreased respectively, and about 0.15% increased due to complete elimination of tariffs of agriculture sector. however, according to effect of tariff decrease in industry and mine sector, total welfare has 2.23%, 1.69%, and 1.15% decreased respectively, and 0.16% increased due to complete elimination (100%) of tariff in this sector. therefore, total society welfare will increase with regard to basic year due to complete tariff elimination in other sectors (except pharmaceutical products), while society welfare will increase by an average of 0.09% due to gradual decrease in tariffs of pharmaceutical products. sensitivity of results as compared to argminton elasticity on import variable of different economic sectors has been surveyed in this study. argminton function in cge model indicates supply for composite good which is in fact a function with constant elasticity of transmission of demand for import and internal productions. since this parameter evaluation has been selected exogenously from other studies, in order to model correctitude and define sensibility of results as compared with difference in this elasticity, it is assumed that the scenario of 50% decrease in tariff has taken place in all economic sectors, and argminton elasticity changes from 50 to 150% of primary elasticity is in 25% intervals. results demonstrate that import amount has increased in elasticity s lower than basis, while it has decreased in elasticities higher than basis. table 3 shows the elasticity of substitution, elasticity of transformation that is so important for sensitivity analysis. values of behavioral elasticity s are usually estimated outside the model or taken from other studies. considering the elasticity s drug in this research is critical and that is difference, we used it for sensitivity analysis. although, cge models are standard in the world and shows usually a multi-sector model based on real world data (e.g. social accounting matrix table or i-o table) (17) of one or several national economies to model the interactions of individual households and other agents on interdependent markets but cge model maybe give different results when we reduced tariff (14) .in iran and brazil increased export and import aftershock of tariff cut but the quantity of increasing depends on structure of pharmaceutical sector. in iran, the most of drug s import are material raw that with reduction of tariff, volume of imports increased that this is natural. so, all of results in cge models depends on real data in the countries. generally in iran, however, there are many studies related to different economic sectors some of which would be mentioned. according to precise searches of researchers up to mid 2012, four studies in the framework of general equilibrium in health field has been performed out of iran which has been in england (nottingham university) brazil, japan, and ghana. the study in england has been performed by dr martin rotten (2009) in form of phd thesis named economic effect of providing health care: evaluation of a cge model for england in 2005.in this thesis, nine economic sectors including agriculture, mine, pharmaceutical products, medical equipment, financial sector, defense sector, health sector, and other services has been considered, and production factors have been divided into two groups of work force (skilful and unskillful) and capital (10). households are also divided into five groups including employed households who have children, unemployed households who have children, unemployed households with children, employed households with children, and finally those who are retired. the data of general equilibrium model is obtained from british sam, 2005, and the alternative and transmission elasticity is considered to be 2. four scenarios have been considered in the thesis including: a) effects of 10% increase in government expenditure on health care, b) 20% increase in prices for economic sectors, c) 10% increase in expert forces of medical fields from other countries, d) 10% increase in efficiency of production factors in health field. results indicated about 8%, 1%, 7%, and eventually 5% improvement in health status by the first to fourth shock respectively. the scenarios have significantly affected on patient expectation list as well, so that there will be 15%, 3%10% and 15% change in patient expectation list respectively. the other study associated with trade liberalization has been performed by francisco (2003) on brazil labor using computable general equilibrium approach (18). 48 goods and two production factors, labor and capital have been used in this study. the first scenario is to impose custom tariff rate of 1990 for brazil economy structure in 1996. results of this study in different scenarios demonstrate that pharmaceutical products export would decrease 2.408% as well as 0.575 for drug pharmaceutical import in performing the model for the first scenario. the second shock will cause 15.7% increase in pharmaceutical export and 0.26% decrease in pharmaceutical import. finally, pharmaceutical export will increase 9.66% and this would be 5.24% for pharmaceutical import in the third scenario. according to results of this study, the general society welfare increases by decrease in tariffs. the third study named tax and subsidy policies of medical services and pharmaceutical industry has been performed in the international university of japan by kato in 2003 using computable general equilibrium analysis. decrease in import tax (custom tariff) of drugs will increase import and export and general society welfare. it is also worth to note that implementing the policy of decreasing custom tariff will cause import growth to be higher than export (19). the fourth study named government expenditure effect on health, economic growth in wealthy country using computable equilibrium model by ernest indicated that government expenditure has significant effect on health followed by economic growth. results indicate that generally, tariff decrease in globalization process will increase imports of this sector in the present conditions of economic structure and pharmaceutical sector of the country. it is obvious that in each economic sector, those products which have production stability will have competitiveness and relative competitive advantage. however, since our pharmaceutical sector is strongly dependant on raw material import, competitiveness in this sector is reduced. therefore, one significant way to increase competitiveness of pharmaceutical sector in the country is to decrease dependability on raw materials so that to decrease final price of such products by increasing efficiency and adopting suitable supports in the framework of one applied policy in pharmaceutical sector. finally, the most important efforts that should be made by iran s pharmaceutical industry to increase exports include: promotion of drugs produced in accordance with gmp and dmf preparation guidelines and regulations on the pharmaceuticalobtain international confirmations and verifications such as fda, who and etc.develop skills and international expertise in the field of marketing for increasing of drug s exportestablish mechanisms and institutions needed for driving and reducing the risk of export services, such as transportation and insurance claims services exporters, especially in export markets in the region, central asia, africa and so on.strategic planning for export markets promotion of drugs produced in accordance with gmp and dmf preparation guidelines and regulations on the pharmaceutical obtain international confirmations and verifications such as fda, who and etc. develop skills and international expertise in the field of marketing for increasing of drug s export establish mechanisms and institutions needed for driving and reducing the risk of export services, such as transportation and insurance claims services exporters, especially in export markets in the region, central asia, africa and so on. strategic planning for export markets finally, pharmaceutical factories should compare their product with the global reference product, using qualitative studies before it s supplied. also, iranian, pharmaceutical factories should attempt to offer various documents in the form of drug master file (dmf) international protocols to the ministry of health of target countries in order to provide proof of quality and register our pharmaceutical products. ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors .

backgroundcomputable general equilibrium models are known as a powerful instrument in economic analyses and widely have been used in order to evaluate trade liberalization effects. the purpose of this study was to provide the impacts of trade openness on pharmaceutical industry using cge model. methods:using a computable general equilibrium model in this study, the effects of decrease in tariffs as a symbol of trade liberalization on key variables of iranian pharmaceutical products were studied. simulation was performed via two scenarios in this study. the first scenario was the effect of decrease in tariffs of pharmaceutical products as 10, 30, 50, and 100 on key drug variables, and the second was the effect of decrease in other sectors except pharmaceutical products on vital and economic variables of pharmaceutical products. the required data were obtained and the model parameters were calibrated according to the social accounting matrix of iran in 2006. results:the results associated with simulation demonstrated that the first scenario has increased import, export, drug supply to markets and household consumption, while import, export, supply of product to market, and household consumption of pharmaceutical products would averagely decrease in the second scenario. ultimately, society welfare would improve in all scenarios. conclusion:we presents and synthesizes the cge model which could be used to analyze trade liberalization policy issue in developing countries (like iran), and thus provides information that policymakers can use to improve the pharmacy economics.

PMC3640784

pubmed-1264

allergic rhinitis (ar) is a recurrent or chronic allergen specific, ige-mediated hypersensitivity disorder affecting the nasal lining and characterized by nasal congestion, rhinorrhea, sneezing, nasal itchiness, and/or postnasal drip. limited studies on ar exist and epidemiological studies based on the allergic rhinitis and its impact on asthma (aria) criteria are lacking. according to aria guidelines, allergic rhinitis is defined if two or more symptoms of rhinorrhea, nasal itching, nasal blockage, or sneezing are present in a patient for at least one hour per day for 4 days or more a week and also for 4 or more weeks a year. based on duration, symptoms are intermittent (< 4 days/week or<4 weeks/year) or persistent (> 4 days/week or>4 weeks/year). severity grading is either mild or moderate-severe based on the absence or presence of sleep disturbance and impairment in daily activities, school, and work, respectively. skin prick test (spt) is a standardized test widely used in the diagnosis of suspected cases of ige-mediated allergy. generally accepted indications for spt include allergic rhinitis, asthma, atopic dermatitis, suspected food allergies, latex allergy, and conditions in which specific ige is suggested to play a role in the pathogenesis. it provides information about the presence of specific ige to protein and peptide antigens (allergens). identification of common aeroallergens in an area is necessary, in order to educate the patient on what allergens to avoid and also help find the best formulation of allergen immunotherapy for effective ar treatment. to date, there has been no information regarding the common allergen from the federal capital territory (fct) of nigeria, abuja. the aim of this study is to identify the clinical profile of ar patients according to aria guidelines and investigate the common allergens in abuja, nigeria. this cross-sectional study was conducted for a period of 18 months (march 2014 to september 2015). sample population was based on the ar patients referred to the allergy clinic, affiliated to medicaid radiodiagnostic center, wuse 2, abuja. patients were consecutively recruited (convenient sampling method) and fall within the age range of 5 to 65 years. they were patients with a positive history of nasal inflammation (at least 2 or more of the following symptoms: rhinorrhea, sneezing, nasal blockage, nasal itchiness, and postnasal drip) for at least one-year duration. patients ' symptoms were categorized as sneezers-runners and blockers based on the predominant complaint. patients whose chief complaints include sneezing, rhinorrhea, and itchy eyes and nose were classified as sneezers-runners, while those with nasal blockage, postnasal drip, and difficulty with breathing were classified as blockers. spt was performed on patients who have stopped taking antihistamines at least 5 days before the test, while patients with severe dermatographism were excluded from the study. a total of 22 allergens were used in this study; these allergens make up the subtropical prick test batch of alk-abello, denmark. these include tree pollen (oak, pecan, black willow, pine, cypress, red cedar, and box elder), weed pollen (pigweed, ragweed, and plantain), grass pollen (bermuda, bahia, johnson grass, and grass mix (meadow, orchard, timothy, june, rye, and redtop)), house dust mites (dermatophagoides pteronyssinus and dermatophagoides farinae mix), molds (alternaria tenuis, c. cladosporioides, penicillium mixed, and aspergillus mixed), animal dander (cat hair and dog epithelium), and cockroach extracts. of the pollen allergen used in this study, about 80% of the plants are present in abuja environment. spt was performed according to international guidelines as a one-time test done on two forearms with lancets and allergens (alk-abello skin prick test kit, berge alle, 2970 hrsholm., histamine hydrochloride (1%) and normal saline (0.9%) were used as positive and negative controls, respectively. the patients ' data was classified according to the aria guidelines and spt results were analysed in allergen-clusters of tree pollen, weed pollen, grass pollen, house dust mites (hdm), molds, animal dander, and cockroach extracts. spss 16 software (chicago illinois) was used in the analysis and p value of less than 0.05 was considered significant. a total of 96 new patients with suspected allergic rhinitis presented at the allergic clinic within the study period. only 74 patients from these had a positive spt result and were enrolled into the study. twenty-one respondents (28.4%) were categorized as children (5 to 17 years of age) with a male: female ratio of 1.3: 1. the majority of the study population resided in abuja municipal area council (amac) territory which is basically the city center. the prevalence of asthma, urticaria, and conjunctivitis as comorbidity was lower when compared with comorbidity such as hypertension. positive family history of atopy was seen in 56.8% of patients and 20.2% of the subjects had animal contact within their environment. according to predominant symptoms, the proportion of sneezers-runners was higher than blockers sneezers-runners tend to have persistent ar, while blockers symptoms were more intermittent (p=0.007). based on aria guidelines, most patients (67.2%) had moderate-severe ar (intermittent and persistent) and this was significantly related to animal exposure (p=0.035) and not to age, gender, or family history of atopy. there was a significant association between ar severity and the predominant complaints (table 2) by the patients (p=0.005). moderate-severe persistent ar was more common among sneezers, while moderate-severe intermittent ar was common with the blockers. there was no significant association between ar severity and the presence of asthma (p=0.26) or family history of atopy (p=0.19). house dust mites allergen yielded the highest number of positive responses (22.6%) followed by tree pollen (16.8%). weed pollen allergen yielded the least (7.4%), while animal dander and fungi allergen both came to 13.1% each (figure 1). also, house dust mites was significantly related to a positive family history of atopy (p=0.035). there was no significant difference between the positive skin tests and gender as well as history of asthma. furthermore, no relationship was observed between the allergens tested and duration of ar (intermittent and persistent), as shown in table 3. most of the patients with a positive spt were in the persistent ar category (66.2%). the number of allergens which produced a positive skin response from each patient was closely distributed. the highest was reaction to 3 or more allergens (35.1%) followed by reaction to 2 allergens (33.8%) and then to one allergen (31.1%), as shown in table 4. statistical analysis did not reveal a relationship between ar severity and skin prick test reactivity. the prevalence of ar is increasing worldwide, yet it remains underdiagnosed and undertreated especially in developing countries. a self-reported survey of ar among adult nigerians observed a prevalence of 29.6% and a mean age of 29.3 years which was close to the mean age observed in this study. the number of patients in this study with concomitant asthma is lower than other nigerian studies [7, 8] and this could be due to small sample size or poor awareness of asthma symptoms by the respondents.. observed a low level of awareness of asthma by patients in nigeria, such that most people with asthma symptoms do not present to the physician but prefer unorthodox means of medical care. about a third of the children (33%) in this study reported persistence or recurrence of rhinitis symptoms after adenoidectomy. the slight female predominance observed in our adult ar patients is in consonance with the findings in malaysia and india [3, 5]. we also corroborated the study that showed a higher male predominance of ar among children. we recorded a higher proportion of sneezers-runners to blockers similar to the findings by lee et al. and shah and pawankar but different from study by deb et al. sneezers having persistent ar and more blockers having intermittent ar (p=0.007). we could not establish any relationship between these predominant symptoms and the aeroallergens used in this study. blockers were more sensitized to hdm, house dust, and fungi, while this study observed that a majority of ar patients were categorized as moderate-severe persistent ar, according to aria classification, while the least were mild intermittent ar. this has been observed by most studies [3, 5, 12] with warm climate like nigeria. a constantly high environmental temperature and humidity could lead to a persistently high concentration of indoor and outdoor allergens all year round. there could also be a selection bias, since patients would more likely present for treatment when their condition is severe and persistent. no relationship was established between the type of ar and the allergen to which the patients were sensitized. this was similarly observed in a national, cross-sectional study of ar patients in mexico. the most common aeroallergen was house dust mites, followed by tree pollen as was seen in other spt studies in nigeria [8, 1416]. however, there are no ar studies in relation to tree pollen sensitivity to compare with. this study highlights the importance of pollen allergens among ar patients living in nigeria, a tropical country with high humidity. this supports an earlier observation of increased tree pollen sensitization in tropical environment and emphasizes the need for increased research in this aspect. further studies are needed to record the season of pollination of the different pollens found in abuja and to correlate these findings with the timing of symptoms in sensitized patients. thus a patient with positive sensitization to house dust mites could also be sensitized against pollen, insect, or fungi allergens. this supports the argument that time of exposure (seasonal or perennial) does not properly define ar patients and also creates difficulty with regard to immune therapy via hyposensitization. in addition, there is need for the use of spt that incorporates wide variety of allergens within a specific environment in order to avoid skipping some of the sensitive allergens attributed to each individual., most ar patients presenting for treatment in abuja, nigeria, have moderate-severe persistent ar and show similar spt sensitization pattern with other countries having similar climatic conditions. sensitization patterns are not related to aria classification or any predominant ar symptoms but rather may rely on the environmental condition of study area and genetic makeup of the study population.

allergic rhinitis (ar) is prevalent in nigeria, though little information exists on the allergen. we assessed the clinical features of ar patients in our environment based on the allergic rhinitis impact on asthma (aria) classification. only patients with positive skin prick test (spt) were recruited. seventy-four patients participated in the study. ar and asthma comorbidity were observed in 13.5%. the proportion of sneezers-runners was higher than blockers with significantly more sneezers-runners having persistent ar (p=0.007). no relationship was established between these predominant symptoms and the aeroallergens used in this study. intermittent mild and moderate/severe ar were evident in 13.5% and 31.1%, while persistent mild and moderate/severe were seen in 20.3% and 35.1%, respectively. house dust mites allergen yielded the highest number of positive responses (22.6%) followed by tree pollen (16.8%). no relationship was observed between the allergens tested and ar severity. majority of patients were oligosensitive (33.8%) and polysensitive (35.1%) and were not significantly associated with ar severity (p=0.07). most ar patients presenting for treatment in abuja, nigeria, had moderate-severe persistent ar and showed similar spt sensitization pattern with countries having similar climatic conditions. sensitization patterns were not related to aria classification or predominant ar symptoms.

PMC4877477

pubmed-1265

most cases were reported in patients with chronic liver disease and cancer, and cases of s. algae infection in end-stage renal disease (esrd) patients are few. the problems related to clinical practice are that little is known about the etiology of s. algae infection in esrd patients and that this bacterium is often confused with shewanella putrefaciens, although these two species may have different pathogenicities in humans. we herein report a case of s. algae bacteremia in a patient with esrd and review previous case reports on shewanella infection in esrd patients. a 71-year-old man was admitted to our hospital in mid-july because of a 5-day history of nausea and abdominal pain. he had been diagnosed with chronic kidney disease (ckd) due to chronic glomerulonephritis and had had an arteriovenous fistula in his left arm in preparation for renal replacement therapy since two month prior to the onset. he had been given an erythropoiesis-stimulating agent (esa; darbepoetin alpha 180 g/month) for renal anemia; he was not on any iron supplements. the laboratory data 1 month before the onset of symptoms showed hemoglobin of 6.9 g/dl, fe of 141 g/dl, transferrin saturation (tsat) of 76.6%, and ferritin of 704 ng/ml, findings that were indicative of dysregulated iron metabolism. he had eaten sliced raw fish or sashimi of mackerel and squid three days before the manifestations of nausea and abdominal pain, for which he visited the outpatient unit, where he was diagnosed with infectious enterocolitis and was given oral fosfomycin. however, his symptoms worsened, and he was admitted to our hospital five days after the onset of symptoms. a physical examination showed that his blood pressure was 160/70 mmhg, heart rate 83 beats/min, and body temperature 39.5. he had abdominal tenderness without rebound tenderness. laboratory tests on admission showed a white blood cell count of 3,700 /l with 83% segmented neutrophils, hemoglobin of 7.6 g/dl, mean corpuscular volume (mcv) of 93.4 fl, mean corpuscular hemoglobin (mch) of 30.2 pg, mean corpuscular hemoglobin concentration (mchc) of 32.5 g/dl, and platelet count of 156,000 /l. serum chemistries showed serum sodium 134 meq/l, potassium 5.0 meq/l, chloride 91 meq/l, calcium 5.0 mg/dl, phosphorus 11.7 mg/dl, magnesium 1.6 mg/dl, uric acid 11.8 mg/dl, blood urea nitrogen 166.9 mg/dl, creatinine 23.0 mg/dl, total protein 6.8 g/dl, albumin 2.8 g/dl, aspartate aminotransferase 27 u/l, alanine transaminase 19 u/l, lactate dehydrogenase 882 u/l, alkaline phosphatase 210 u/l, -glutamyl transpeptidase 19 u/l, creatine kinase 1,809 u/l, and c-reactive protein 11.7 mg/dl. abdominal computed tomography (ct) revealed intestinal swelling and fluid collection, compatible with infectious enterocolitis (figure). a, b: abdominal computed tomography on admission revealed intestinal swelling and fluid collection, compatible with infectious enterocolitis. two sets of blood cultures were collected before cefmetazole (1 g/day) was started. on the second hospital day, he went into cardiac arrest; cardiopulmonary resuscitation resulted in the return of spontaneous circulation. antimicrobial therapy was changed to meropenem (2 g/day). on the third hospital day, gram-negative bacteria appeared in both sets of the blood culture obtained on admission and were identified as s. putrefaciens 4 days later. ceftazidime (2 g/day) was given based on the susceptibility test (table 1). mic: minimum inhibitory concentration his condition improved; therefore, continuous hemodiafiltration was switched to hemodialysis 3 times per week. on the 13th hospital day, the antimicrobial agent was changed to levofloxacin and continued for 1 week. he was discharged on the 26th hospital day without any complications and continued maintenance hemodialysis. the bacterium recovered from the blood culture was further analyzed because the patient's condition had deteriorated. a 16s rrna gene sequence analysis revealed that the bacterium was s. algae s. algae is a rare causative pathogen of bacteremia, skin and soft tissue infection, or hepatobiliary infection. we reported a case of s. algae bacteremia in an esrd patient and demonstrated the probable link between raw seafood consumption and shewanella infection. this patient had iron overload that was related to a dysregulated iron metabolism caused by esrd. raw seafood consumption by an esrd patient may be a risk factor for shewanella infection. shewanella species are gram-negative bacteria that are mainly isolated from the marine environment. this species was once named pseudomonas putrefaciens; it was originally classified in the family vibrioneceae until the 1990s, when it was reclassified as genus shewanella (1). most case reports of shewanella infection have described an association with exposure to seawater; such contact can cause ear infection, skin or soft tissue infection, and bacteremia (1-3). two species, s. putrefaciens and s. algae, have been reported to be pathogenic in humans (1). it is often difficult to distinguish between these two species, because automated identification systems only include the database of s. putrefaciens, but not s. algae (2). therefore, some authors have suggested that the previously reported s. putrefaciens infections may have actually been caused by s. algae, which is thought to be more virulent (3). the accurate distinction between these two species requires a 16s rrna gene sequence analysis or phenotypic tests; s. algae can only grow at 42 and in 6% nacl (1). in the present case, an automated identification system (microscan walkaway 96 plus, siemens healthcare diagnostics, tokyo, japan) initially determined the bacterium to be s. putrefaciens; however, a 16s rrna gene sequence analysis confirmed the bacterium to be s. algae. shewanella infection can be fulminant and fatal and is often misdiagnosed as vibrio vulnificus infection, because of the similarities in their clinical manifestations (2,4). patients with underlying diseases, such as hepatobiliary disease or malignancy, were reported to be predisposed to shewanella infection, as well as to v. vulnificus infection (3); the mortality rate of shewanella bacteremia was increased in these patients (5). shewanella species belong to the microflora of the marine environment, and exposure to seawater was reported to be one of the risk factors for shewanella infection, especially in temperature regions (3). therefore, more attention should be paid to the pathogenicity of this bacterium, especially in patients with underlying diseases who are exposed to these environments. there have been seven previously published case reports on shewanella infection in esrd patients (4,6-11) (table 2). two cases were reported to be caused by s. algae and four cases by s. putrefaciens; however, only two cases were further analyzed by a biochemical procedure. the most common route of infection was skin ulcer (3 cases), followed by catheter-related infection (2 cases); no case was related to oral intake of seafood. in the present case, the onset of symptoms was related to seafood consumption, and abdominal ct scan showed signs of infectious enterocolitis. therefore, we concluded that the shewanella infection in this patient was probably caused by dietary intake of raw fish or sashimi. this was the first case of s. algae infection that might have been transmitted orally in an esrd patient. esrd: end-stage renal disease, flux: flucloxacillin, gm: gentamicin, pipc: piperacillin, cpfx: ciprofloxacin, cez: cefazolin, lvfx: levofloxacin, mino: minocycline, cfpm: cefepime, caz: ceftazidime, mepm: meropenem, vcm: vancomycin, doxy: doxycycline, drpm: doripenem, amk: amikacin although the reason why the esrd patient was predisposed to shewanella infection is still unclear, dysregulated iron metabolism may be a risk factor for this patient. iron is not freely available, since it binds to transferrin in the blood (12). pseudomonas and vibrio species have the ability to produce siderophore, which plays an important role in supplying iron to bacteria (12,13). siderophore has a high affinity to iron and is able to displace iron from transferrin. s. algae was reported to produce siderophore and was capable of absorbing iron into its body (14). patients with hepatobiliary diseases who are susceptible to s. algae often had the complication of iron overload. the association between shewanella infection and hepatobiliary diseases may be due to iron overload (5). in the present case, the patient did not have hepatic dysfunction but did have esrd with a dysregulated iron metabolism; his anemia was esa-resistant, and there seemed to be excessive iron levels, although he was not taking any iron supplement. it was suspected that uremia of the patient contributed to the esa-resistant anemia, because the patient was just starting renal replacement therapy, and the serum creatinine level on admission was extremely high. recent studies have demonstrated that hepcidin, which reduces iron release from reticuloendothelial and hepatocyte stores, plays an important role in the disordered iron metabolism of uremia including esa resistance (15,16). the serum hepcidin level is related to the residual renal function and elevates in ckd and in esrd patients (15,17). serum hepcidin can be removed by renal replacement therapy (18), and insufficient hemodialysis is known to cause esa-resistant anemia (19). therefore, we speculated that the esrd, especially the accompanying iron overload, might have been a risk factor for shewanella infection in this patient. to our knowledge, no previous report has described the status of iron metabolism in the esrd patients with shewanella infection. the limitation of this report was that the data for serum ferritin and tsat on admission were not available. however, we measured both of these markers at one month before the onset, when the patient was in a stable condition without acute inflammation or infection. iron status should be evaluated based on ferritin and tsat in ckd and esrd patients (19,20). the ferritin level and tsat of the patient in the present study were compatible with iron overload (21,22). in addition, we were unable to recover s. algae from a stool culture on the eighth hospital day, when the patient had already received courses of an antimicrobial agent to which the bacterium was susceptible. we could not collect a stool sample at an earlier time, such as at admission, because of the patient's critically ill condition. in conclusion, we reported a case of s. algae bacteremia that was probably caused by oral intake of raw fish in an esrd patient. in addition, a dysregulated iron metabolism in esrd may be a risk factor for shewanella infection.

a 71-year-old man was admitted because of nausea and abdominal pain. he was receiving an erythropoiesis-stimulating agent for anemia and dysregulated iron metabolism due to stage g5 chronic kidney disease. he had a history of raw fish intake and was diagnosed with infectious enterocolitis, which worsened and led to septic shock. shewanella putrefaciens grew in the blood culture, but shewanella algae was identified in a 16s rrna gene sequence analysis. we herein report a case of s. algae bacteremia believed to have been transmitted orally. we also reviewed previous case reports on shewanella infection in end-stage renal disease patients.

PMC5410489

pubmed-1266

educational evaluation (ee) is a formal process performed to evaluate the quality of effectiveness and/or value of a program, process, goal or curriculum.12 it deals with data collection and assessment of the progress of academic programs.34 by considering some principles related to educational measurements and data collection, ee may result in a better understanding of such programs.57 during the past thirty years, theorists have presented numerous methods of evaluation. worthen and sanders2 mentioned that more than 50 different evaluation approaches has been developed in recent decades. among these, methods based on internal criteria are known as the ones that can interpret the scientific, educational, and therapeutic authenticity of different educational groups.48 this is greatly welcomed by the academic community and is widely spread to all universities in the world. that is because this method provided a scientific, appropriate, precise, timely, and valid basis regarding the interpretation of decision making system quality and programming for its promotion and development.3 such a method was successfully carried out in four medical education groups at supervisory and expansion of medical education council secretariat of ministry of health, treatment and medical education of iran in 1995.8 ee has its most effect, value, and results when it can provide needed information to individuals which are directly related, as well as those who may be benefited from its results.346 educating dental professionals consists of theoretical and practical (clinical, paraclinical, and laboratory) courses, differing in duration, and educational curriculum among different countries. it might vary from 4 years (e.g., in india, turkey, and russia) to 6 years or more (e.g., in iran consisting of 2 years of only basic medical sciences and 4+years of dentistry courses). due to numerous practical educational units in dentistry education and with regard to expensive but very critical protocols for infection control, a great deal of resources is consumed in governmental universities of iran over training every general practitioner with a degree of doctorate of dental surgery. on the other hand, the quality of dental services plays an important role in public health. considering these issues, dentistry education needs to be cost-effective in terms of optimizing its quality. to date, the pace of advancements in dentistry necessitates a continuous revision of educational programs by officials to achieve new expectations of educational system and determine or update the policies.9 proper evaluation and research in education are accounted as scientific instruments for moving along with these developments in order to achieve improvements in education, health, and treatment qualities.911 such advancements may depend on education of faculty members and panels and their cooperation, elimi-nation of shortcomings, and approximation of components and educational instruments to standard indices.9 aim and mission of this educational groups are training and tutoring students of undergraduate and residency courses in order to gain complete ability for diagnose and treatment of patients needing this kind of treatments, so that residents can provide treatment and disease prevention services with a desirable quality after learning these courses.10 this study was conducted in year 2010 to assess the efficacy of educational programs provided at dental school of tehran university of medical sciences to estimate and address the limitations and strengths. apart from educational groups dedicated to deliver basic medical sciences to dental students, dentistry school of tehran university of medical sciences includes twelve only-dentistry educational groups (table 1). the objective of this descriptive cross-sectional study was to assess the quality of education provided at this school, compared with the standards. the following aims would be fulfilled: 1) determining the educational and research needs of group directors; 2) determining those of other faculty members; 3) defining such needs of under- and post-graduate students; 4) surveying attitudes of graduated doctors; 5) surveying regarding research and educational facilities as well as residency resources; 6) determining educational procedures; 7) surveying patients regarding their satisfaction from received dental cares; 8) distinguishing human resources; and 9) determining poor fields, needing to be pushed. dentistry school of tehran university of medical sciences educational groups in 2010 through this descriptive cross-sectional study, the efficacy of provided education by mentioned 12 departments were assessed in 13 fields. research society, educational groups including group director and sub-societies which include faculty teachers, students, alumni, human, and support recourses were completely involved in this survey. these included aims and missions of groups, management and organization, scientific board, students, human resources and support, educational, research, health and treatment spaces, educational, diagnostic, research, and laboratory instruments, educational, research, health, and treatment programs, process of teaching and learning, evaluation and assessment, and satisfaction of alumni and patients. each field was evaluated through the following steps: 1) establishment of standards; 2) data collection; 3) determining the importance of components; and 4) analyzing the collected data. inspection, interview, and checklists were to evaluate educational, research, health, and treatment spaces of dentistry sections and educational, research, laboratory and diagnostic instruments. first questionnaire was used to gather the feedback of panel director and faculty scientific members to establish the coefficient of desired factors. another questionnaire was utilized to evaluate 11 fields by multiple-choice questions based on a 5-point likert scale. measurement instruments, evaluated factors and data collection sources we tried to establish study variables in line with objectives and research questions in providing data collection instruments. for this reason, before preparation of data collecting instrument, a table was created that precisely identified each research question's variables and based on that the instruments were generated. afterward, in order to increase the validity, questionnaires were reviewed by experts and the straight and vague questions were addressed.12 considering the reliability of data collecting instrument, after preparation of questionnaires according to arranged subjects, confusions about some questions were identified and removed, taking the use of a pilot study in a 15-individual group and interviews with academic board members in educational groups. eventually, the final data collecting instrument was designed. in order to determine the coefficient of each of the 13 criteria, feedback forms were utilized. besides, through interviews and delphi technique the academic board members opinions were recorded and the importance of each criterion was determined. results indicated that all academic board members have given equal coefficient values to all questions. after determination of evaluated factors and sources of gathering related data, criteria for each factor were designated (e.g., an evaluated factor was management and organization of group, and a criterion related to this factor was group director). a marker was created for each of these criteria. in order to perform this, a) specificities of desirable condition were described; b) a marker of desirable condition was set, c) for assessment of goal achievement, the criteria condition was compared to the desirable condition marker. in order to analyze likert multiple-choice questions, scores 1 to 5 were respectively assigned to highest and lowest scores. utility rate was determined by the percentage of the related index. to facilitate the assessment of components and evaluated factors, the desirability level of each factor was classified based on the score percentage: desirable, more than 75%; relatively desirable, 50-75%; and not desirable, less than 50% (table 3). average educational evaluation results of educational groups in dentistry school of tehran university of medical sciences in 2010 descriptive statistics were calculated using spss10. ee results were analyzed based on swots (strengths, weaknesses, opportunity, and threats) method in evaluated educational groups. educational evaluation committee accepted fluency and clarity of the missions and aims of educational groups in undergraduate and graduate courses. revision of aims was suggested in three areas of knowledge, attitude, and practice. the quality of departments educational systems were relatively desirable (55.98) in all 13 fields. for management fields, average results were relatively desirable (52.9%) based on specificities of desirable condition. in this field, 72% of group members believed that group managers have acceptable scientific and educational background, 55% were aware of group manager selection criteria and 90% of academic field members were satisfied with the organization and management abilities of the group manager. in evaluation on academic board, mean age of educational group members was 43.9 3.2 years old and mean teaching background was 14.4 3 .9 years. most of academic board members were men and all of them were assistant professors and officially occupied. 80% of academic board members were involved in research projects; more than 80% were mentoring specialty course thesis; and 50% were satisfied with workspace condition. an important specificity of this group was that they were involved in programming of theoretical and applied education of students in undergraduate and residency courses. mean ee result in academic board field was relatively desirable (56.92%). in the field of learners, all residents were asked about their association, correlation with academic board, study duration, and educational, research activities, and student projects. desirability in the field of human resources and support in sections educational, treatment, research, audiovisual, library, diagnostic laboratory, radiology, and facilities was 54.58%, considered relatively desirable. and in both fields of educational, research, health, and treatment spaces and educational, research, diagnostic, and laboratory facilities ee results were relatively desirable (50.36% and 51.55% respectively). in the field of educational, research, teaching process and learning courses, nearly all academic board members (91%) 82% of group members believed that aims and mission of educational courses in groups were of their interests. most of the group members (91%) believed that cultural revolution committee headings were applied in educational programming and 82% of them were involved in both basic and clinical teachings. these fields had the average of 58.09% and 60.16% respectively and considered relatively desirable. in the field of graduates, mean age of individuals which have entered the university in year 1997 and graduated in year 2003 was 26.3 and 80% were men. considering the service condition, most graduates (66.7%) were spending their duty project. regarding their satisfaction, this field was in a relatively desirable (58.72%) condition. in the field of patients, more than 63% of patients of general (undergraduate) ward and 73% of patient which referred to residency wards stated that their reason for selecting this university was that they trusted in the precise and effective treatment delivered by the students at this university. after comparison of results to the desirability criteria regarding the trust of service takers to precise treatment and a good referring size of the ward, this fields condition was considered relatively desirable (61.32%). the evaluated educational groups have given suggestions about quality improvement in education, research, health, and treatment in four levels (group, faculty, university, and ministry) considering their strength and weaknesses.10 the mean of educational evaluation in departments of educational school of dentistry tehran university of medical sciences, 2010. ee can study and assess the educational programs utilizing standards, predetermined aims, or educational quality236710 after such assessments, it is possible to address these shortages in educational system and arrange an efficient educational system.11314 ee of educational programs is an important and basic task of medical universities.1 rate of occupational capabilities and medical alumni performances in order to offer educational and research programs, health and treatment services with the aim of supplying and improving society members health is related to the rate of educational programs realization.1 if educational programs are not well designed and performed, it can impose irreparable damage and harmful social, economical, and cultural effects on individuals, society, alumni, and also faculty and finally university's credit.1 considering the importance of education and its major role in improving nearly all aspects of societies, educational programs should be carefully probed to elucidate shortcomings and advantages in order to improve programs as well, teaching methods such as workshops, seminars, presentations, interactive teaching methods which may involve the students and thus increase their learned topics and introducing outlines of the lessons in the first place.16 however, unfortunately most teachers are not trained regarding teaching techniques and are not completely ready to undertake educational responsibilities.1718 an effective way of improving this shortcoming is conducting courses in teaching techniques and skills.19 evaluating the draw backs, preferences, and priorities of teachers teaching skills, as well as other educational shortcomings may enable the program directors of such courses to canalize the materials to which is more needed by teachers and educational groups.20 universities may determine their position in national and international levels to further improve their programs.2122 for example, india, as a country with the highest number of medical universities and thus the most number of medical faculty members, has developed such programs called national teachers training centers (nttc). these programs train teachers to gain skills, and to find certain teachers as group directors and leader, in long-term. such programs are globally called faculty development.23 the overall ee findings in educational groups were relatively desirable (mean score=55.98%). based on the findings, activities of group managers, educational management, and academic board members in these groups were performed in order to improve the procedure of assessment. results of other national studies shows that school of medicine,13 school of nursing and midwifery,24 and school of rehabilitation25 had average 75.3%, 80.4% and 77.8%, respectively and the quality of education, research, and treatment were desirable. was started with implementation of a pilot ee study in six educational groups in 1996.26 results showed that ee in iranian culture would lead to improvement.26 30 national medical educational groups in medical universities of the country implemented the ee project.27 farzianpour and bazargan in 1999 revealed that ee is the best measurement index for evaluation of university hospitals.27 in 2004, fifteen basic sciences and clinical educational groups of tehran university of medical sciences have reported their ee results desirable.28 saberian et al29 from school of midwifery and nursing of semnan described results of ee in surgery ward desirable in international congress of educational evaluation in 2004 which was held in edinburgh. olyaei et al14 from rehabilitation school noted that the results of their ee was 76.2% and was desirable. farzianpour et al28 from tehran university of medical sciences, and harden et al30 from dundee university of england had positive attitudes towards ee and improvement of education and research in clinical fields. researches in world's educational system report that ee is an effective way to find out the strengths and weaknesses of an educational system.142734 universities might follow standards of education evaluation so that the result could be better comparable. however, diverse techniques have been described for educational evaluation.35 these included expertise-oriented, management-oriented, and objectives-oriented.35 kirkpatrick36 has introduced a 4-step assessment. the application of theoretical knowledge of learners in their practice would be assessed. finally the impact of program on the institution and community would be evaluated. until conducting a unified method, international comparisons would be difficult to perform. quality of patient care and students learning were the best fields (respectively 61% and 60%). educational aims and objectives, and research and educational spaces had the poorest results (respectively 49% and 50%) .

background: educational evaluation is a process which deals with data collection and assessment of academic activities progress. in this research, educational evaluation of dentistry school of tehran university of medical sciences, which trains students in undergraduate and residency courses, was studied. methods:this descriptive study was done with a model of educational evaluation in ten steps and 13 fields including purposes and mission objectives, management and organization, academic board members, students, human resources and support, educational, research, health and treatment spaces, educational, diagnostic, research and laboratory tools, educational, research, health and treatment programs and courses, process of teaching and learning, evaluation and assessment, alumni, and patients satisfaction. data were collected using observation, interviews, questionnaires, and checklists. results:results of the study were mainly qualitative and in some cases quantitative, based on defined optimal situation. the total mean of qualitative results of educational evaluation of dentistry school in all 13 fields was 55.98% which is relatively desirable. in the case of quantitative ones, results of some fields such as treatment quality of patients and education and learning of the students were relatively desirable (61.32% and 60.16% respectively). conclusion: according to the results, educational goals and missions, educational and research facilities and spaces which were identified as the weakest areas need to be considered and paid more serious attention.

PMC3177397

pubmed-1267

the incidence of cerebrospinal fluid (csf)-related complications (i.e., csf leak, pseudomeningocele, and meningitis) after intradural spinal tumor surgery (ist) remains high following resection of intradural extramedullary spinal tumors (iest) and intradural intramedullary spinal tumors (iist) (1-17). treating these complications is a vexing problem and often requiring prolonged postoperative bed rest, operative re-exploration, the placement of an external lumbar drain, and a prolonged use of antibiotics (18-30). furthermore, these complications may lead to additional complications, and even the death of the patient. despite mayfield s (18) and black s (5) recommendations many years ago to use a fat graft to prevent potentially life-threatening csf complications in patients undergoing posterior fossa and spinal surgery, the practice did not gain routine use for those undergoing surgery for ists. we were not able to find any reports of the use of intraoperative fat grafting in the ist literature or reports of any other efficient way that ensures preventing this vexing postoperative complication problem while csf-related complications of surgical ist series have been regularly reported in up to 18% of patients (1, 2, 4, 6, 7, 9, 11-17, 19-21, 25, 28-30). in our earlier practice, csf-related complications were similar to those reported by other authors (1-3, 6, 7, 9, 11-14, 17-19, 22, 23, 25, 26). as a consequence, this study was approved by the appropriate institutional review board and was a retrospective analysis of all patients treated operatively by the senior author for ists over 13-year period (september 2003 through september 2016). medical records were reviewed for pre and postoperative exams, pre and postoperative magnetic resonance imaging (mri), and follow-up visits. in june 2005 was noted because this was when a patient with a sacral dumbbell schwannoma developed a large pseudomeningocele postoperatively, prompted us for the prospective application of autograft fat during dural closure for all other patients. the patients demographics, the tumors histology, the degree of tumor resection, and neurological outcomes were evaluated. all patients underwent postoperative mri scans with and without contrast in the hospital 2 months after surgery, yearly for 2 years, and then every 5 years during follow-up. fat harvest and application technique after endotracheal intubation and line placement in the supine patient, the left paraumbilical area is prepared and draped (figure 1). the harvested fat is placed in antibiotic saline in a sterile cup until needed for closure. once the tumor was resected, the dura was closed with 5-0 prolen stitches in a running fashion. if the incision was in the midline, the valsalva maneuver was done at 30 cm h20 for 510 seconds to ensure that it was watertight. if any area in the suture line leaked csf, an additional suture was placed there and a piece of fat tissue was cut and positioned inside the stitch, which was then tightened. if the dural incision was t-shaped (i.e., for dumbbell, intra/extradural, foraminal tumors), or y-shaped (i.e., for sacral canal/foraminal tumors), the dural incision in the midline and its t or y extension over the nerve root were closed in running fashion with 5-0 prolen. multiple pieces of fat tissue were then incorporated into single additional dural sutures to achieve watertight closure and reinforce the dural suture. when suturing was complete, a layer of fat tissue 68 mm thick was placed over the entire exposed dura to obliterate the dead space that remained after a laminectomy or facetectomy. the fat graft also created small pressure onto the dural suture line, lessening the chance of csf seepage and pseudomenigocele. in other words, the graft prevented a potential low-pressure space into which csf may migrate and form a pseudomeningocele, later producing a csf leak. the muscle, fascia, subcutaneous tissue, and skin layers are then closed in the usual fashion (figures 24). note the fat graft overlying the dura dorsally and enforcing the patch graft/dural suture line. the patient regained his full strength postoperatively and his balance problems resolved. a woman in her early 30s with an iist (hemangioblastoma) at c1. b) postoperative post-contrast t1-weighted mri showing the tumor resection and collapse of the cyst. note the fat graft overlying the dura dorsally. the patient s balance and swallowing problems resolved postoperatively. b) axial t2-weighted mri showing the left-sided tumor, which involved the c1c2 intervertebral foramen and compressed the spinal cord. over the course of 13 years (september 2003september 2016), 40 patients with an ist were operated on by the senior author. of these, 34 were iest, comprising 13 meningiomas, 14 schwannomas, 6 myxopapillary ependymomas, and 1 breast cancer metastasis. six patients had iists: 3 astrocytomas (one high grade), 2 ependymoma, and 1 hemangioblastoma. there were 11 men (28%) and 29 women (72%) with ages ranging from 2489 years (mean 56 years). thirteen patients (33%), all with iest, were older than 65 years. the length of hospital stay ranged from 2 to 8 days (mean, 4 days), and follow-up ranged from 1 to 36 months (mean, 45 months). abbreviations: iest=intradural extramedullary spinal tumors; iist=intradural intramedullary spinal tumors; ist=intradural spinal tumors except for 5 patients with schwannomas who presented with pain and numbness in the involved nerve areas, all other patients presented with motor, sensory, sphincter, and neurological deficits in various combinations. six patients (15%) had dumbbell tumors that extended into the corresponding intervertebral neural foramen; 5 of these had schwannomas and 1 had a meningioma. one patient, a woman in her mid 40s with a grade iii conus/cauda astrocytoma, died 3.5 years later as a result of disease progression and csf seeding throughout her neuraxis despite postoperative irradiation and chemotherapy. the third patient in the series, a man in his late 40s with perineal numbness and tingling, impotence, and severe low back pain harbored a dumbbell schwannoma at s1s2 and underwent resection. ten days after surgery, he presented with a large subcutaneous swelling in the sacral area, consistent with a pseudomeningocele, which was verified with computed tomography. he underwent a second surgery, during which the dural closure was revised with a fat autograft along with external lumbar drainage for 5 days bed rest. all patients after this patient received a fat autograft at the time of dural closure, and no evidence of a csf leak or a pseudomeningocele was noted on physical exam or neuroradiologic follow-up in these other patients. no evidence of complications related to the fat harvest site was noted. on the second postoperative mri, 2 months after surgery, the fat tissue routinely showed signs of significant resorption, and the 1-year follow-up mri scans in all patients showed that the fat was totally reabsorbed. efficacy of the technique in their classic work, yasargil and colleagues (34) reported the successful radical removal of ists using microsurgical techniques with favorable neurological outcomes. since then, numerous other reports of series of ists with good results followed (1-9, 11-31, 35). nonetheless, the incidence of csf-related complications (i.e., csf leak, pseudomeningocele, and meningitis) after ist tumor surgery remains high and in reports from major high volume centers ranges between 5% up to 18% (1-4, 6, 7, 9, 11, 13-17, 19-22, 25, 28-30). treating these complications may often require prolonged postoperative bed rest, external lumbar drainage, operative re-exploration, and the prolonged use of antibiotics. these complications may frequently lead to additional complications, such as deep vein thrombosis, pulmonary embolism, pneumonia, urinary tract infection, skin breakdown, subdural or cerebellar hematomas, and even death. they also significantly increase medical expenses and the length of the hospital stay, which in turn may worsen the outcome scores of both the hospital and individual surgeons, as well as patient satisfaction surveys. weber et al. (32) reported a 50% increase in mean hospital cost per case with csf-related postoperative complications after elective spinal surgery. the effect of csf-related complications on outcomes is particularly true as patients undergoing this surgery may be older (> 65 years) and have medical co-morbidities, as we found in one-third of the patients in our series. a higher number of elderly patients with ists who require surgical treatment has also been reported by sacko and colleagues (25). while such patients can have a successful surgery with radical tumor resection and frequently no neurological consequences, they may not tolerate the prolonged bed rest required to repair the csf-related postoperative complication. the additional complications mentioned earlier can also jeopardize an otherwise successful surgery and rehabilitation process. two recent studies (10, 33) evaluated the efficacy of polyethylene glycol sealants in an attempt to decrease the rate of csf-related complications after intradural spinal surgery. goodwin and colleagues (10) reported that csf leaks occurred in 5% of patients and meningitis occurred in 1%. similarly, wright and associates (33) compared the use of polyethylene glycol sealant to the standard of care dural closure. csf-related complications that required re-operation in the sealant treated group was 7% compared with 13% in the standard dural closure cohort. in addition, each cohort had additional 4% rate of pseudomeningocele formation that did not require re-operation. thirty-five and 15 years ago, respectively, both mayfield (18) and black (5) described and recommended the use of a fat graft to prevent potentially life-threatening csf complications after posterior fossa and spinal surgery. however, we were not able to find any reports of the use of intraoperative fat grafting in the literature or reports of any other efficient way that ensures preventing this vexing postoperative complication problem. our own experience in the surgical series of ists treated at our institution mirrored the experience of others. as a consequence, we prospectively adopted the intraoperative use of fat grafting after the third patient in our series developed a large pseudomeningocele, was readmitted to the hospital, and underwent a second surgery with revision of the dural closure and placement of an autologous fat graft and external lumbar drainage for 5 days. since then, we have operated on an additional 37 patients with ists prospectively using this graft at dural closure and have not seen any csf-related complications in these patients. technical considerations the spinal dura appears to be thickest dorsally in the midline and thins laterally, particularly along the dural sleeves of the spinal nerves. we use the running dural midline suture to achieve watertight closure after the tumor is removed and then use the valsalva maneuver to ensure watertight closure. still, there is no guarantee that the suture line wo nt weaken or that a leak wo nt occur after the patient is mobilized postoperatively, when csf fully replenishes and patient engages in full, physiologic csf pressure challenges of the dural suture line. csf may still seep into the low pressure dead space created after a laminectomy, leading to a pseudomeningocele or csf leak. we found that reinforcing the dural suture line with autologous fat is useful particularly in cases when the valsalva maneuver revealed csf seepage. in addition, the fat graft obliterates the dead space created by a laminectomy and muscle dissection, and creates gentle pressure to the dural suture line that may prevent the formation of a pseudomeningocele and a csf leak. in some cases, it may be impossible to achieve watertight dural closure, and these cases are particularly at risk for csf leak and therefore clearly benefit from this concept and technique: 1) an ist in a sacral location (one of our cases); 2) a craniospinal ist requiring a y-shaped dural incision and patch grafting (figures 2 and 3, 27% of cases in our series); 3) when the tumor invades the dura (e.g., meningioma) and necessitates dural excision to achieve radical resection and subsequent dural patching; and 4) if the tumor is a dumbbell iest extending into the intervertebral foramen and beyond (figures 4 and 5). eighteen percent of patients in our series had dumbbell tumors (5 schwannomas and 1 meningioma) and after radical tumor resection, it was not possible to achieve a watertight dural closure without incorporating a fat graft into the sutures. fat tissue was harvested at the beginning of surgery via a horizontal incision of approximately 2 cm while the patient was supine. it was routinely done on the left side to prevent confusion in the case of a future appendectomy or other abdominal surgery on the right. fat deposits are abundant in the abdominal area even in patients with a low bmi. in our experience, the tissue harvest did not add any significant time or expense to surgery, nor did it lead to infection, hematoma, or cosmetic problems for our patients. in addition, fat autograft carries neither the risk of hypersensitivity reaction nor risk of infectious disease transmission. finally, we favored a separate incision for the tissue removal, rather than harvesting fat from the subcutaneous tissue in the area of the primary incision. this approach prevents the development of additional tissue pouches in the primary surgical area that may favor a pseudomeningocele or hematoma formation and jeopardize wound healing. the reported incidence of csf related complications after surgery for ist remains high in published series. the prospective use of autologous fat grafting ensures watertight dural closure and obliterates the dead space created during surgical exposure, muscle dissection, and bone removal. this technique appears to significantly reduce, if not completely eliminate, postoperative csf-related complications in patients with ists, without adding any significant operative time, expense or complications.

object: the incidence of cerebrospinal fluid (csf)-related complications after intradural spinal tumor (ist) surgery is high and reported in up to 18% of patients. however, no efficient way to prevent those complications has been reported so far. treating these complications may require prolonged bed rest, re-exploration, external lumbar drain, use of antibiotics, and possible precipitation of other complications. to alleviate the risk of csf-related complications, we prospectively adopted the intraoperative use of autologous fat grafting after ist surgery. methods:this is a perspective analysis of 37 cases (out of 40 cases series) that a prospective use of abdominal fat autograft was applied during dural closure. after the tumor was resected and the dura closed, we used the valsalva maneuver to ensure watertight closure. csf leak was prevented with the enforcement of suture with a fat autograft as necessary. in addition a thin layer of fat tissue was then placed over the dura to obliterate any dead space. fibrin glue was then applied over the graft. filling the dead space with the fat graft prevented a low-pressure space in which csf could pool and form a pseudomeningocele. results:after adopting the fat autograft technique, we did not observe any post-surgery csf-related complications in any of these patients. conclusions:the prospective use of autologous fat grafting can ensure watertight dural closure and obliterate the dead space created during surgical exposure and bone removal. this technique significantly reduces, and may completely eliminate, postoperative csf-related complications in patients with ists.

PMC5292229

pubmed-1268

pyomyositis affects the massive skeletal muscles and is most frequently caused by staphylococcus bacteria (1). necrotizing fasciitis is a localized spread of infection that follows events such as trauma or surgery (2), and which is commonly affected by group a streptococcus. rhabdomyolysis comprehensively affects the skeletal muscle and, like pyomyositis and necrotizing faciitis, shows serum creatinine kinase elevation; however, rhabdomyolysis occurs as a systemic disease and is not associated with the focal manifestation of abscesses (3). the pathogenesis of infectious rhabdomyolysis is thought to be associated with the systemic or local metabolic changes related to a systemic or local infection (4); however, the mechanisms underlying its pathogenesis have not been established. in addition, gram-negative bacillus rhabdomyolysis is rare, and its microbiological pathology remains to be elucidated. we diagnosed the patient, a 64-year-old japanese man with multiple myeloma, with bacteremia caused by morganella morganii. he was admitted to our institute due to septic shock at the sudden onset of a febrile episode. his vital signs at admission were as follows: blood pressure, 82/60 mmhg; heart rate, 78/min; body temperature, 39.1; respiration rate, 20/min; and spo2, 95% (room air). the laboratory findings at the time of his diagnosis showed massive myolysis and a significantly elevated level of creatinine kinase (ck; 3,582 u/l); thereafter, an isotype analysis of the patient's ck confirmed that the elevated ck was derived from the patient's skeletal muscle (98.8%). the status of the patient's multiple myeloma, which had been diagnosed 5 years earlier, was stage iiia, igg type. he had undergone treatment with a variety of chemotherapies: melphalan (mp; 12 mg/day for 4 days+prednisolone 60 mg/day for 6 days, repeated triweekly), vad (vincristine 0.4 mg/m for 4 days as a continuous infusion+doxorubicin 10 mg/m for 4 days as a continuous infusion+dexamethasone 40 mg/day, days 1-4, 9-12, and 17-20, repeated monthly), bd (bortezomib 1.3 mg/m on days 1, 4, 8, and 11+dexamethasone 40 mg/day for 4 days, repeated monthly), and thalidomide (100 mg/day). his last treatments were lenalidomide 20 mg/day, and igg gradually increased 6 months before the current infectious episode. after the patient's admission for sepsis caused by m. morganii, rehydration was immediately performed via central venous catheterization and a catecholamine infusion (dopamine 3 mg/kg/h) was administered; at the same time, antimicrobial treatment with meropenem (0.5 g three times a day) was initiated. cervical to pelvis computed tomography (ct) was performed on the day of admission, but there were no clear findings explaining the patient's massive myolysis. on day 2 of the patient's hospitalization, the patient's vital sign were as follows: blood pressure, 61/45 mmhg; heart rate, 87/min; and body temperature, 36.8. we started hemodialysis combined with endotoxin absorption the same day. however, respiratory failure occurred, necessitating mechanical ventilation, on the night of day 2. the patient died of multi-organ failure two days after undergoing intubation. before his death, rhabdomyolysis developed day-by-day and was reflected in the remarkable elevation of the patient's ck level. the final ck value was 19,790 u/l (normal range: 40-200 u/l) (figure). we found that the m. morganii was sensitive to all of the broad spectrum beta-lactams, including carbapenems and cephalosporins, but only resistant to cefotiam, minomycin, and ciprofloxacin. his muscle damage manifested as with elevated levels of ck, aspartate aminotransferase (ast), alanine aminotransferase (alt), and lactate dehydrogenase (ldh) after the onset of the disease (day 1), with increasing leukocytes and c-reactive protein (crp). the days in the x-axis indicate the days after the patient s admission. the patient visited our outpatient clinic for a routine follow-up examination 6 days before his admission. infectious rhabdomyolysis is caused by various pathogens including viruses, bacteria and fungi (3). the clinical entity of infectious rhabdomyolysis was established in 1982 (5), and a review (3) identified the predisposing risk factors for rhabdomyolysis: alcohol ingestion, compression injury, and generalized seizures. regardless of the pathogen that causes infectious rhabdomyolysis, the underlying mechanism of the disease is metabolic. the most frequent differential diagnosis is necrotizing fasciitis, which occurs when the infection spreads into the deep fascial layers (2). necrotizing fasciitis is generally caused by infection, and the initial entry site is sometimes trivial. in contrast, rhabdomyolysis is caused by the following two critical conditions: (1) physical distress (for example, distress due to or involving excessive exercise, seizure, muscle compression, hyperthermia, or hypothermia); and (2) systemic metabolic disorders (for example disorders due to hypoxia, acidemia, drug abuse, infection, or inflammation). the localization of a soft-tissue infection can be distinguished by systemic imaging, especially by sensitive mri (6). in the reported cases of rhabdomyolysis, the local radiological findings are faint. in the present case, our evaluation of the present case raised an important question: what is the microbiological mechanism underlying the development of rhabdomyolysis accompanied by systemic infection (3,7)? thus, rhabdomyolysis is relatively common among patients with miscellaneous bacterial infections including, but not limited to, legionella (4), pneumococcal (8), and salmonella (7). we underscore that patients these types of infectious rhabdomyolysis share the following common findings: 1) the patients were immunocompromised due to malignancy or a severe wound; 2) precedent or concomitant acute renal failure were observed in the patient's clinical course; and 3) metabolic abnormalities such as dehydration or acidemia due to single/multi-organ failure existed as the underlying pathogenesis. thus, ischemia or hypoxia caused by systemic infection are considered to be involved in the mechanism underlying the development of rhabdomyolysis. m. morganii is rarely pathogenic in humans, but it sometimes develops into an opportunistic infection in a compromised host. although there are no reported cases of infectious rhabdomyolysis caused by m. morganii, arranz-caso et al. the authors noted that gram-negative bacterial pyomyositis can occur in an immunocompromised case followed by enteric organism translocation. gram-negative bacterial infection does not favor pyomyositis, which is usually raised by gram-positive bacteria. our case suggests that infectious rhabdomyolysis may be underdiagnosed as a comorbidity of gram-negative bacterial infections, even if the causative organism is an opportunistic pathogen such as m. morganii. regarding myolysis/rhabdomyolysis complicated with a systemic bacterial infection and the accumulation of muscle damage the non-bacteremic mechanism includes alterations of systemic metabolism (i.e., hypoxia, acidosis) and an intracellular metabolism in situ (e.g., disturbances of glycolysis and oxidation in muscle cells) (7). the non-bacteremic mechanism of rhabdomyolysis accompanied by systemic infection is similar, regardless of the pathogen that is involved (3,7). the common mechanisms underlying muscle damage arise from a combination of hypoxia, dehydration, and acidemia in the tissue (4). in general, rhabdomyolysis develops during the course of systemic infection in up to 5% of bacterial and viral cases (7). rhabdomyolysis is a specific term describing the excess leakage of enzymes derived from muscular tissue, which can result in multi-organ failure. in our patient, the apparent infection focus was not identified by repeated systemic ct. this negative finding caused a delay in our differentiation of the cause of the patient's rhabdomyolysis. we treated the patient with meropenem, to which the causative pathogen, m. morganii, is sensitive; however, an adequate antimicrobial therapy can not always rescue a patient from advanced multiple-organ deterioration. rhabdomyolysis should thus be recognized as a more aggressive clinical situation than local myolysis, and one that requires intensive care. furthermore, non-specific myolysis/rhabdomyolysis can occur due to any pathogen (7); thus, it may be underdiagnosed during the infection. myalgia and scant laboratory signs of myolysis/rhabdomyolysis (usually appearing as liver damage in laboratory data) can be misdiagnosed in patients with the overlapping leakage of transaminases due to various etiologies. clinicians should keep in mind that abnormal liver function test results may be linked to a muscular problem. clinicians should be aware of the possibility of systemic infection-associated myolysis/rhabdomyolysis and closely observe immunocompromised patients. this is the first report of lethal rhabdomyolysis caused by m. morganii as a complication in a non-hiv patient. short description: fatal rhabdomyolysis caused by m. morganii can occur in a multiple myeloma patient without hiv.

a 64-year-old japanese man with multiple myeloma was admitted to our institute due to fever and hypotension. he had received multiple courses of chemotherapy just before his febrile episode. blood culturing detected morganella morganii. at the time of the diagnosis, his laboratory findings revealed massive rhabdomyolysis with a significantly increased creatinine kinase level (ck; 3,582 u/l); 98.8% of which corresponded to the ck-mb isotype. we diagnosed the patient with sepsis caused by m. morganii, complicated with severe rhabdomyolysis. he died of multi-organ failure 2 days later. clinicians should closely observe patients with possible systemic infection-associated rhabdomyolysis.

PMC5348465

pubmed-1269

only a minority of individuals living in the obesigenic environment currently encountered in developed nations are able to maintain a healthy body weight. for example, in the usa, evidence from the latest national health and nutrition examination survey (collected in 20072008) indicates that 68% of adults are overweight or obese. given the health risks associated with obesity (heart disease, diabetes, hypertension, cancer), this has important implications for global health, and in light of the additional health-care costs associated with obesity, the global economy. genome-wide association studies have identified numerous loci associated with obesity; however, their contribution to variation in body mass index and weight between individuals is estimated to be less than 2%, suggesting that environmental influences, such as increased availability of energy-dense food and an increasingly sedentary lifestyle, play a crucial role. accumulating evidence from epidemiologic studies and animal models indicate that maternal health and nutritional state during pregnancy and lactation play a critical role in programming the neural circuitry that regulates energy balance and behavior in offspring, having a sustained influence on their physiology and behavior. metabolic imprinting refers to the programming of an offspring's future metabolic responses by a stimulus occurring during a critical developmental period. as the type and amount of available nutrition are crucial determinants of survival and reproductive success, the evolutionary purpose of metabolic imprinting is to enhance offspring survival by programming energy balance regulation so that available metabolic fuels are most efficiently utilized. mammals are exposed to two environments during development, the intrauterine and early postnatal environments, both of which are heavily impacted by maternal diet and adiposity. perinatal nutrition has enduring effects on many aspects of physiology and behavior including the regulation of energy balance, susceptibility to metabolic disorders, programming of body weight set point, stress response and mental health-related behaviors. in addition to perinatal nutrition, a number of factors associated with maternal consumption of a high-fat diet (hfd), including maternal adiposity, hyperlipidemia, lipotoxicity, glucose levels and insulin resistance, also have a long-term impact on the developing offspring and are associated with increased risk of obesity, metabolic disorders, and mental health disorders. as maternal obesity is associated with consumption of a hfd in humans and a hfd is used to promote maternal obesity in most animal models, a major challenge faced by the field is the ability to disassociate the effects of the hfd from the maternal metabolic phenotype. the most common perturbation of maternal nutrition is nutritional excess and maternal obesity. in 2008, over 64% of women of child-bearing age in the usa were overweight or obese and the majority consume an excess amount of calories and fat. epidemiological studies clearly indicate that maternal obesity is associated with increased incidence of obesity and metabolic syndrome in children. although this review focuses on the impact of maternal hfd consumption, it is important to note that maternal undernutrition during gestation also increases the incidence of offspring obesity; thus, the relationship between birth weight and adult adiposity is thought of as a u-shaped curve with both a very high and a very low birth weight increasing the risk of obesity in adulthood. the relationship between maternal obesity and the development of offspring obesity appears to be independent of gestational diabetes as women who are obese, but able to maintain normal glycemia, also have heavier offspring with increased adiposity. although epidemiological studies implicate the intrauterine environment, including maternal diet and energy status, in programming offspring obesity, in these studies it is not possible to directly link maternal diet and energy status with the offspring's metabolic profile, as several other factors could contribute to the association between maternal and offspring obesity, including genetics and shared environment factors such as access to energy-dense foods and a sedentary lifestyle. also, there is relatively limited information on normal brain development in humans, making it challenging to examine the impact of maternal diet on offspring's brain development. furthermore, it is difficult to accurately monitor and potentially unethical to manipulate the diet of pregnant women. thus, it is critical to use animal models to directly examine the effects of maternal overnutrition on subsequent generations and to develop effective therapeutic intervention strategies. providing animals with a hfd during pregnancy and lactation is a common method of inducing maternal obesity. however, the duration of exposure to the diet (acute vs. chronic), the percent of calories from fat, and diet composition are variable across studies, hindering comparisons. the diets commonly used to promote maternal obesity are either a purified hfd with fat in place of carbohydrates as an energy source or a cafeteria diet in which animals are provided with a selection of palatable food items that have a high fat content along with their regular diet. the cafeteria diet is most effective in promoting obesity; however, as several food items are provided, it is difficult to calculate the amount and composition of the diet consumed by each animal and the diet consumed by animals in the same group is variable. several studies have used purified diets with different sources of fat to compare the impact of maternal consumption of saturated fat, polyunsaturated fat 3 or 6 fatty acids on offspring physiology and brain development. it is clear from these studies that the source of fat in the hfd matters. for example, a study in rodents determined that while a maternal diet high in saturated fat programs hyperphagia in offspring, exposure to a diet of equivalent percent of calories from fat as fish oil does not. thus, it is essential that future studies determine which sources of fat in a mother's diet are beneficial and which are detrimental to the developing offspring in order for physicians and nutritionists to make appropriate recommendations to expecting mothers. most rodent studies report that offspring exposed to a hfd during gestation and lactation have increased body weight and adiposity at weaning. however, several studies report that maternal hfd consumption results in lighter offspring which they speculate is due to hfd/obesity-induced impairments in the initiation and production of milk by obese mothers. the differences in offspring's body weight phenotypes across studies are likely due to differences in the duration of hfd consumption and fatty acid composition of the diets. using nonhuman primates, our group is examining the impact of chronic maternal hfd consumption on offspring body weight regulation. briefly, infant offspring from hfd mothers are underweight at birth and display rapid catch-up growth, so that by 6 months of age they are heavier and have increased adiposity. this offspring phenotype appears to be independent of whether the hfd-consuming mother is obese with insulin resistance or lean with normal sensitivity to insulin. these studies indicate that in primates, as in rodents, maternal hfd consumption, independent of maternal weight and metabolic status, predisposes offspring to increased risk of developing obesity and metabolic disorders early in life. rodents that experience an early environment in which they are exposed to maternal hfd consumption or overnutrition are consistently reported to be hyperphagic as adults. a number of studies have found that maternal hfd consumption plays a critical role in programming hypothalamic pathways that regulate feeding. early postnatal overfeeding increases the orexigenic peptides neuropeptide y and agouti-related peptide (agrp) in the arcuate nucleus of the hypothalamus (arh) of juvenile rats. offspring of rat dams fed a hfd during the perinatal period also display a long-term upregulation in the expression of orexigenic peptides including galanin, enkephalin, and dynorphin in the paraventricular nucleus of the hypothalamus (pvh), and orexin and melanin-concentrating hormone in the perifornical lateral hypothalamus. exposure to hfd during gestation stimulates the proliferation of neuronal and neuroepithelial cells of the embryonic third ventricle of the hypothalamus and increases their migration to hypothalamic regions resulting in an increase in the proportion of neurons expressing orexigenic peptides. also, offspring from hfd mothers have reduced sensitivity to the anorectic effects of leptin. thus, in rodents it is postulated that perinatal exposure to overnutrition or maternal hfd consumption results in disruption of the homeostatic feedback regulation and nutrient sensing capabilities of the hypothalamic feeding circuits leading to hyperphagia. though rodent models have significant advantages, such as a relatively short period of gestation and the ability to manipulate genetics, the critical periods for the development of energy balance regulatory systems differ between rodents and humans. in rodents, the neural pathways regulating energy balance are immature at birth and are not completely developed until the third postnatal week (mice). in contrast, in humans, nonhuman primates, pigs, and sheep, the hypothalamic circuitry that regulates feeding develops primarily prenatally. thus, models of maternal overnutrition in which the development of energy balance regulation occurs prenatally are particularly relevant. in the nonhuman primate model of maternal hfd-induced obesity, our group has determined that fetal offspring also display alterations in the development of the hypothalamic melanocortin system that may contribute to disrupted homeostatic signaling. in addition to hyperphagia, there is evidence that feeding behavior and food choice are also programmed by perinatal nutrition. overweight children are reported to have increased preference for high-fat foods which is associated with increased parental adiposity. also, children with one or two overweight parents consume a larger percentage of energy from fat than children who have two normal-weight parents. in these studies it is unclear if the children's increased fat preference is due to programming as a result of perinatal hfd exposure, genetics or increased availability of high-fat food during childhood. animal models in which offspring are exposed to a hfd during the perinatal period provide further evidence for an influence of perinatal hfd exposure on food choice. for example, adult rat offspring exposed to junk food during either gestation or lactation displayed increased preference for fatty, sugary and salty foods. the source of fat in the perinatal diet influences the programming of food preference, as rat pups from mothers that consumed a hfd with lard as the main source of fat displayed increased preference for the hfd, whereas offspring exposed to maternal consumption of hfd with fish oil as the fat source do not. in rodents, there is evidence that maternal hfd consumption causes perturbation in the dopamine system of adult animals in areas associated with the rewarding value of food such as the nucleus accumbens and ventral tegmental area. preliminary findings from our studies examining the impact of exposure to maternal hfd consumption on the food preference of nonhuman primate offspring also indicate that offspring display increased preference for diets with a high sugar and fat content [sullivan and grove, unpubl. together, these studies provide compelling evidence that perinatal nutrition has a long-term influence on dietary preference and feeding behavior and may be an important contributing factor to the development of obesity. as differences in food preference and diet consumption will have a long-term impact on stress response and depressive behaviors of adult offspring, future studies examining the impact of perinatal hfd exposure on food preference should include the examination of stress response and behavioral disorders. although many studies have examined the impact of perinatal nutrition on food intake and food intake regulation, the number of studies examining the other component of energy balance, energy expenditure, are limited. offspring from mothers undernourished during gestation offspring exposed to maternal overnutrition have also been reported to be hypoactive or to have no difference in physical activity as compared to offspring exposed to a control diet. the effect of maternal diet on physical activity level depends on the type of fat in the diet. rat pups from dams fed a diet rich in polyunsaturated fat displayed increased locomotor activity when compared to offspring from dams fed a saturated fat or standard laboratory diet. this study also reported increased locomotor response to stimulants in offspring from dams that consumed a saturated fat diet. currently, the impact of early exposure to hfd on metabolic rate has not been examined. thus, a major gap in the knowledge of the field of metabolic programming is the impact of perinatal hfd exposure on the regulation of energy expenditure. future studies are needed which examine not only the effects of perinatal exposure to hfd on average basal energy expenditure, but its impact on compensatory changes in energy expenditure in response to metabolic challenges, such as fasting, dieting and chronic consumption of a hfd. in addition to being associated with metabolic disorders, epidemiological data indicate that obesity is associated with increased risk of behavioral/mental health disorders, such as depression, anxiety, and attention deficit hyperactivity disorder. moreover, anxiety and depression influence the feeding behavior, food preference and physical activity level. depression and anxiety are associated with increased craving for palatable food items and decreased physical activity level. variations in mood also alter food choice, with increased preference for high-fat/high-sugar foods being reported during negative emotions. maternal nutrition has long-term implications for the offspring's risk of developing mental health disorders. perinatal exposure to a hfd may contribute to this association by altering the development of key pathways implicated in regulating mood and behavior such as the serotonin system. recently, maternal hfd consumption has been associated with increased anxiety in rodent and nonhuman primate offspring. in a rat model, male adult offspring from mothers exposed to either a diet high in saturated or trans fat during gestation and lactation displayed increased anxiety and deficits in spatial learning. using a nonhuman primate model, our group recently demonstrated that perinatal exposure to a hfd causes a decrease in serotonergic tone perturbations in the serotonin system, which predisposes female offspring to increased anxiety. the finding that female nonhuman primate offspring exposed to maternal hfd consumption are more sensitive to developing anxiety than male offspring is consistent with evidence in humans which suggests that females are more prone to anxiety than males and that the association between obesity and anxiety is stronger in women than men. thus, nonhuman primates appear to be an ideal model in which to examine the impact of maternal hfd consumption on the development of mental health disorders such as anxiety and depression. although there is clear evidence from studies in both rodents and nonhuman primates that maternal hfd consumption leads to increased risk of obesity and metabolic diseases, the mechanisms responsible are largely unknown. we have very limited information on the impact of maternal hfd consumption on the brain and the complex neural circuitry that regulates physiology and behavior. recent evidence indicates that circulating factors such as hormones (leptin, insulin), nutrients (fatty acids, triglycerides and glucose) and inflammatory cytokines play important roles (fig. 1). maternal obesity and diabetes result in maternal hyperglycemia, and as glucose can readily pass through the blood-placenta barrier, it is transferred to the fetus. however, the elevated insulin levels associated with maternal obesity do not cross the placenta; thus, the fetal pancreas must secrete increased levels of insulin to respond to the maternal hyperglycemia. this fetal hyperinsulinemia is postulated to be involved in the programming of obesity and diabetes in the developing offspring. administering insulin to rats during the last term of gestation produces obesity in the offspring and administering insulin to the hypothalamus of rat pups during the time that projects from the arh to the pvh results in elevations in body weight, insulin level, impaired glucose tolerance, and increased vulnerability to diabetes. insulin is an important growth factor in the central nervous system; thus, it is postulated that early exposure to hyperinsulinemia alters the development of the brain circuitry regulating energy balance and behavior. the hyperleptinemia that offspring from obese mothers experience during development is also implicated in metabolic imprinting. there is substantial evidence in rodents that postnatal leptin is a critical factor in the development of neural pathways in the hypothalamus. human studies report that leptin is elevated in obese and diabetic mothers and lower in infants that experienced intrauterine growth restriction at term. however, in human and nonhuman primate gestation, circulating leptin levels do not rise until after hypothalamic development is mostly complete. though critical for brain development in rodents, there is limited evidence for leptin's role in the development of primate brains. increased adiposity is associated with elevations in peripheral markers of inflammation, such as c-reactive protein, interleukin-6, interleukin-1, and tumor necrosis factor-. these inflammatory markers are associated with increased risk of cardiovascular disease, heart disease, insulin resistance, type 2 diabetes mellitus and hypertension. the association between obesity and increased inflammatory cytokines has been confirmed in pregnant women such that obese pregnant women have increased levels of inflammatory cytokines which lead to endothelial dysfunction. exposure of the developing fetus to increased circulating cytokines has been proposed as a potential mechanism by which maternal hfd consumption impacts brain development. the development of many neural systems that are critical in regulating energy balance, such as the melanocortinergic system, the serotonergic system, and the dopaminergic system, is sensitive to circulating cytokine levels. also, rodent studies report that agrp and pro-opiomelanocortin neurons in the arh are directly impacted by cytokines. a recent study by bilbo and tsang reported that offspring from mothers consuming a saturated fat diet had increased microglial activation in the hippocampus at birth that persisted into adulthood. we have recently observed that fetuses from nonhuman primates consuming a hfd have increased circulating and hypothalamic cytokines. we postulate that exposure to increased inflammatory cytokines leads to the perturbations in the melanocortin and serotonin system observed in fetal offspring. given the large number of neurotransmitter systems that are influenced by inflammation, future research is needed to examine the impact of maternal hfd-induced inflammation on each critical regulator of physiology and behavior. in summary, many common brain regions and neurotransmitters regulate energy balance, stress response and mental health disorders including the melanocortinergic system, serotoninergic system and dopaminergic system (fig. thus, it is not surprising that maternal hfd consumption has a long-term impact on metabolic and behavioral regulation. as maternal hfd consumption and obesity are commonplace and rapidly increasing, we speculate that future generations will be at increased risk for both metabolic and mental health disorders. given the prevalence of maternal obesity, future studies need to identify therapeutic strategies that are effective at preventing maternal hfd-induced malprogramming.

the perinatal environment plays an important role in programming many aspects of physiology and behavior including metabolism, body weight set point, energy balance regulation and predisposition to mental health-related disorders such as anxiety, depression and attention deficit hyperactivity disorder. maternal health and nutritional status heavily influence the early environment and have a long-term impact on critical central pathways, including the melanocortinergic, serotonergic system and dopaminergic systems. evidence from a variety of animal models including rodents and nonhuman primates indicates that exposure to maternal high-fat diet (hfd) consumption programs offspring for increased risk of adult obesity. hyperphagia and increased preference for fatty and sugary foods are implicated as mechanisms for the increased obesity risk. the effects of maternal hfd consumption on energy expenditure are unclear, and future studies need to address the impact of perinatal hfd exposure on this important component of energy balance regulation. recent evidence from animal models also indicates that maternal hfd consumption increases the risk of offspring developing mental health-related disorders such as anxiety. potential mechanisms for perinatal hfd programming of neural pathways include circulating factors, such as hormones (leptin, insulin), nutrients (fatty acids, triglycerides and glucose) and inflammatory cytokines. as maternal hfd consumption and obesity are common and rapidly increasing, we speculate that future generations will be at increased risk for both metabolic and mental health disorders. thus, it is critical that future studies identify therapeutic strategies that are effective at preventing maternal hfd-induced malprogramming.

PMC3700139

pubmed-1270

dermatophytosis, one of the most common infectious diseases in the world, can be caused by dermatophyte fungal species of the genera trichophyton, microsporum and epidermophyton. onychomycosis is a fungal infection of the nails, which may be caused by many types of fungi, other dermatophytes or yeast species (1). treatment of onychomycosis is a challenge and infections are typically more severe and difficult to treat in toenails than in fingernails (1). treatment requires long-term therapy with oral antifungal medication (with the cure rates ranging from 48% to 76%) which may have side effects, and/or topical agents such as ciclopirox (34% cure rate) (2, 3). oral therapies (terbinafine, itraconazole) provide access to the nail bed and matrix and are also used to treat concomitant skin infections such as tinea pedis. to prevent side effects prior to and periodically during oral therapy, liver function the newer imidazoles may lead to a higher cure rate (4), but these drugs are considerably more expensive than routine treatments. topical therapies are associated with low rates of adverse events and mild localized reactions at the application site, but due to inadequate penetration, especially when the nails are thick, their efficacy is limited. uv radiation induces dna damage leading to inactivation of the pathogenic fungus commonly measured by viability count. uv-a with long wavelength (315-400 nm) rays has a slow effect on the skin and is poorly absorbed by the ozone layer. this ray is 1,000 times more common than uv-b with the middle-range of 290-320 nm that causes visible damage, commonly observed as redness and blistering and are a major cause of sunburn and skin cancer (5). uv-c with wavelengths less than 290 nm (100-280 nm) is the shortest and has the highest uv energy. it is filtered by the ozone layer; these wavelengths do not reach the earth surface and do not penetrate the atmosphere. thus, people are exposed to large doses of uv-a throughout their life, but uv-b rays do not penetrate glass (5). high doses of uv-a and uv-b radiation have significant inhibitory effects on dermatophytes (6). the antibacterial activity of uv-c radiation reduces the number of bacteria on environmental surfaces and vegetative bacteria including mycobacterium tuberculosis, viruses and fungi (7, 8). the current study aimed to investigate the fungicidal effect of uv radiation on the growth of dermatophytes isolated from nails, considering the low efficacy of current treatment options and ease of exposing nails to uv light. the sample comprised all the patients referred to the dermatology clinic (shahid faghihi hospital, shiraz university of medical sciences, iran) with suspected onychomycosis. the inclusion criteria were clinical manifestations such as distal or proximal sub ungula onychomycosis, white superficial onychomycosis, pitting, paronychia, onycholysis and nail plate discoloration. all diagnoses were confirmed by the mycology laboratory of shahid faghihi hospital, shiraz university of medical sciences, iran. since this procedure was part of the patients usual treatment protocol, informed consent for research purposes was not specifically sought. fragments of nail plate and nail bed scrapes were inoculated onto sabouraud dextrose agar (sda) (merck, germany) with the antimicrobials cycloheximide (sigma-aldrich, germany) and chloramphenicol (merck, germany) (9), and incubated at 30c for 14 days. isolated species were identified by macroscopic examination of colony morphology (size, color of the colonies from the top and bottom of the plate, and growth rate). microscopic examinations were performed to identify dermatophyte species using lacto phenol-cotton blue and diagnostic methods such as growth on trichophyton agar media (quelab, uk) and rice, and hair perforation test were performed to identify (9). to determine the influence of different doses of uv light on the fungicidal effect, colony count and size, and growth rate of the isolated fungi were evaluated under laboratory conditions. two strains of trichophyton mentagrophytes and two strains of t. rubrum were isolated from the patients and used in the current study. trichophyton rubrum type 1 was white and had fluffy colony with pink to burgundy on reverse. type 2 was granular with powdery-velvet, radially folded and pale brown in color and the reverse was dark tan. t. mentagrophyte type 1 was downy, floccose and white in color with the fade yellow on reverse. finally, type 2 was coarsely granular and light buff in color with the buff-tan color on reverse. as in the skin samples from patients, only mycelium can be observed, in the current study mycelial colonies were harvested, suspended in sabouraud dextrose broth (sdb) (merck, germany) and dispersed to small part suspensions using a sonic dismembrator (dr. hielscher gmbh, stahnsdorf, germany). cell densities of the suspensions were counted in a neubauer hemocytometer (hausser scientific, horsham, pa, usa), and reached 10 pieces/ml. fifty l of suspension was inoculated onto sda to observe macroscopic and microscopic colony characteristics. in each test, a 10 ml aliquot of the dermatophyte suspension was inoculated onto a petri dish. uv-a and uv-b lights were supplied by a waldmann uv801kl apparatus (waldmann, villingen-schwenningen, germany) at 470 w, and uv-c light was supplied by an f30t8/gl apparatus (young deungpo-gu, seoul, south korea) at 30 w. five different doses of uv light were used: uv-a (315-400) 3, 6, 9, 12, 15 j/cm, uv-b (280-315) 0.5, 1, 1.5, 2, 2.5 j/cm and uv-c (100-280) 0.3, 0.6, 0.9, 1.2, 1.5 j/cm. one positive control without exposure to uv light was cultured in each round with the test samples. this distance is widely used in dental and cosmetic fields to sterilize tools (10). in the current study, for uv-a and uv-b radiation, the lamps were utilized in the centers for dermatological diseases in order to obtain some practical implications. since radiation reduces the volume and raises the count because of evaporation, which may lead to errors, the specimen volumes were made up to the primary volume (10 ml) after irradiation, with sdb. for each irradiated specimen, the fungicidal effect of uv was evaluated by preparing five cultures to incubate at 30c under laboratory conditions; therefore, 75 cultures for each fungus were prepared. colony size, morphology and count, and growth rate of all isolates were checked in the plates every two days and mean values were calculated. since most colony growth occurs during the first 14 days, the results were graphed after this period. all statistical analyses were conducted using microsoft excel software.the current research conformed to the helsinki declaration and local legislation, and was approved by the local ethics committee (91-5) in clinical microbiology research center, shiraz university of medical sciences. in response to the uv treatments, compared with suspension control without irradiation, colony morphology for each dermatophyte remained unchanged, but colony count and size changed depending on the species and type of isolate. of the two t. rubrum isolates (figure 1), type 1 was less sensitive to uv-a and uv-c, and more sensitive to uv-b. continuous irradiation with the uv-a and uv-b doses used in the study did not completely suppress the growth of the isolates, but higher intensities of uv-c light inhibited the growth of t. rubrum. mean colony counts at different intensities uv-a (315-400 nm), uv-b (290-320 nm) and uv-c (100-280 nm). of the two t. mentagrophytes isolates (figure 2), colony count in type 1 decreased as the dose of all three types of uv radiation increased. however, colony count in t. mentagrophytes type 2 showed different patterns as the dose of uv radiation increased. type 2 strain was slightly responsive to uv-abut compared to control cultures; there was no significant effect on colony count. neither uv-b nor uv-c decreased the type 2 colony counts at low doses, but higher doses led to decreases in colony counts. mean colony counts at different intensities of uv-a (315-400 nm), uv-b (290-320 nm) and uv-c (100-280 nm). according to table 1, uv-a irradiation led to a slight increase in colony size in the four strains except t. mentagrophytes type 1, in which colony size increased with the lowest dose of irradiation and significantly decreased with increasing the doses. irradiation with uv-b increased colony size in t. mentagrophytes type 2, but in type 1 uv ray increased colony size at the lowest dose and decreased colony size with increasing doses. uv-b irradiation decreased colony size in t. rubrum type 2 but had no significant effect on type 1 or either of the two strains of t. mentagrophytes. irradiation with uv-c decreased colony size except for t. mentagrophytes type 2, in which higher doses increased colony size. values are presented as mean sd. values are presented as j/cm. according to obtained data, uv light can affect colony count and size of t. rubrum and t. mentagrophytes in the culture, the two fungal species frequently isolated from patients with onychomycosis. the estimated prevalence of onychomycosis caused by these species ranges from 10 to 21.3% in the general population in australia, the united kingdom and iran (11, 12). this prevalence rate may increase to 30% in specific populations such as people over 70 years old (13-15). it is challenging to determine the optimal treatment and circumventing drug resistance in pathogenic fungi, including dermatophytes. since the nails and skin are being exposed to sunlight, many photodynamic treatments are developed in dermatologic studies. for example, uv light therapy is investigated in diseases such as scleroderma (16) and onychomycosis (17). the mechanism of uv radiation is the primary photochemical reaction, which damages dna and affects survival and rate of germination (18). this molecular change makes the dna unstable for essential biological processes such as transcription and replication. as a rule, the simpler the microorganism is in anatomical terms, the more easily it is inactivated by uv radiation. this is why viruses and prokaryotic cells such as bacteria are more easily destroyed than a complex of microorganisms such as eukaryotic cells, yeasts and vegetative fungi. in particular, fungal spores in which the dna is protected by a concentrated cytoplasm and pigmented cell wall some data suggest that solar uv radiation can inhibit the growth of fungi on the soil surface in the antarctic terrestrial environment (19), and has disinfectant effects that can vary depending on the type and dosage of irradiation, condition of the surface and exposure time. the effective exposure time was reported to be 45 minutes for yeast and 75 minutes for mold (20). different doses of uv-b light show different effects on the growth of candida albicans. increasing doses of uv-b strongly reduced hyphal growth in this fungus and triggered enhanced blastospore formation (21). the mechanism of resistance to uv radiation is not completely known, but intra hyphal growth and recolonization of old cells by new ones were observed in all the strains investigated by gorbushina et al. uv-c radiation affected t. rubrum type 1: as the dose increased, colony count decreased. irradiation with uv-a and uv-b also decreased the colony count of some strains tested in the current study which was proportional to the dose. this finding may be due to the synthesis of new hyphae through the old hyphae not exposed to irradiation, or may be related to the age of the exposed hyphae (i.e. recolonization of older hyphae) in the suspension. trichophyton rubrum is one of the most frequent causal agents in nail infections, and recurrence of the infection and resistance to therapy are the most important limitations to treatment success for this anthropophilic dermatophyte (22). this species was unaffected by infrared irradiation, responded with increased pigmentation to uv-a at13.5j/cm twice daily, and was inhibited by a single dose of uv-b at 15.1 j/cm (6). growth rates, surface colony color and the texture of colony morphology are different in each strain. the current study compared two strains of t. mentagrophytes. according to figure 2, uv-a, uv-b and uv-c had similar effects on t. mentagrophytes type 1, but affecting type 2 strains differently. increasing pigmentation in response to uv-a (13.5 j/cm twice daily) was observed in t. rubrum (6). electron microscopy studies showed a marked thickening and blurred contours of the cell walls grown with a brightener effect related to interference by optical brighteners with the formation of normal chitin fibrils and thickening of the cell walls (23). in the present study, increasing doses of uv-a, uv-b and uv-c irradiation increased colony size in t. mentagrophytes type 2. uv-b irradiation of the skin is particularly well studied, and is accepted as the main cause of skin cancer. the depth of penetration of uv-c radiation into the human skin is very low; therefore, the risk of skin cancer associated with this type of treatment is low. there are studies which report the treatment of localized infections with multidrug-resistant microorganisms (24) and subungual onychomycosis (25) with uv-c light irradiation and favorable responses. as mentioned in these studies, the cases were limited and side effects should be taken into account more carefully. when uv irradiation is used to treat onychomycosis, it is important to minimize exposure by carefully masking the area with adhesive material. in addition, the dose of radiation must be minimized by careful dosimetery calculated with reference to the individual patient s nails, the etiologic agent, and optical properties. if there were enough samples, by using different types and doses of irradiation such as uv-a uv-b and uv-c for every single nail sample separately, the current study could directly examine the effects of radiation on them, but since the volume of samples was low, they were just cultured after the isolation of fungal strains and it was possible to evaluate different types and doses of irradiation in the isolated strains. uv-a, uv-b and uv-c seem to be effective in decreasing colony growth in some prevalent fungi, which caused onychomycosis in the patients. further studies are needed to determine the efficacy of this therapy, identify possible side effects and establish appropriate dosages for the antifungal action of uv radiation therapy.

background: the treatment of onychomycosis is a challenge and infections are typically more severe and difficult to treat in toenails than in fingernails. objectives:the current study aimed to investigate the fungicidal effect of ultraviolet radiation on the growth of dermatophytes isolated from nails. patients and methods: samples from patients with clinical manifestations of onychomycosis were inoculated onto sabouraud dextrose agar and incubated at 30c for 14 days. isolated species were identified by specific laboratory examinations; uv-a, uv-b, and uv-c light were used to irradiate two strains of trichophyton mentagrophytes and t. rubrum. colony count, size and growth rate of the isolated fungi were evaluated under laboratory conditions. results:trichophyton rubrum type 1 was less sensitive to uv-a and uv-c, and more sensitive to uv-b than type 2. t. mentagrophytes type 2 was slightly responsive to uv-a therapy, although no decrease in colony count was observed. increased doses of uv-b and uv-c irradiation decreased the counts. the effect of radiation on colony size was dependent on the dose and type of irradiation. conclusions:uv-a, uv-b, and uv-c light seem to be effective in decreasing colony growth of the most prevalent fungi, which caused onychomycosis in the current study samples. further studies are needed to determine the efficacy of ultraviolet light therapy, identify possible side effects, and establish appropriate dosages for the antifungal effect of this therapy.

PMC4548401

pubmed-1271

a 39 year old female who was seven weeks pregnant presented to a community hospital emergency department with a first episode of chest pain. she had a twenty pack per year smoking history and a significant family history of coronary artery disease (cad) with her father developing cad in his thirties. she described the pain as a pressure sensation, retrosternal in location, with radiation down both arms. it was associated with intense nausea and vomiting, and she had some relief with aspirin. in the emergency department, she denied recreational drug use, and did not have any constitutional symptoms. clinically there was no evidence of deep vein thrombosis (dvt), pulmonary embolism (pe), or pericarditis. on physical examination she was afebrile and hemodynamically stable, with a heart rate of 69 and regular, equal blood pressures in both arms of 95/65 mmhg, and an oxygen saturation of 97% on room air. there were no signs of congestive heart failure with no pedal edema, clear lungs, and no jugular venous distension. the precordial exam was normal with no heaves, thrills, normal s1 and s2 with normal physiological splitting and no extra heart sounds, rubs or murmurs. a 12-lead electrocardiogram (ecg) was completed that showed normal sinus rhythm at a rate of 60 bpm, normal axis, normal intervals with no evidence of chamber enlargement with 1 mm st segment depression in lead v4 and<1 mm depression in v5 (fig. 1). initial blood work showed a significant elevation of the cardiac markers with a troponin t of 0.96 g/l and a creatinine kinase (ck) level of 718 u/l, all of which decreased on serial measurements. an echocardiogram demonstrated severe inferolateral wall hypokinesis with a preserved left ventricular systolic function and ejection fraction of 60% with no other abnormalities identified. her past obstetrical history included three pregnancies with one full term delivery, one preterm delivery, and one therapeutic abortion. the patient was transferred from a community hospital to our tertiary center for further management. pulmonary, gastrointestinal, psychiatric, neuromuskuloskeletal, along with non-ischemic cardiac causes of chest pain must be considered in these patients. the incidence of myocardial infarction in pregnancy has been estimated to be 6.2 per 100 000 deliveries with a mortality rate of 5.1%11% in recent reviews.1,2 prior estimates of mortality have reported it as substantially higher at 37% and estimates of incidence substantially lower at 2.8 per 100 000 deliveries.1,3 this incidence is approximately 34 times higher than the estimated age associated risk for non-pregnant women.2 the decreased mortality and increased incidence in the recent literature is likely due to use of more sensitive and specific serum cardiac markers, such as troponins, identifying more cases of subendocardial myocardial injury as well as the increasing cardiovascular risks, such as advancing maternal age.1 in a nationwide us population based study of acute myocardial infarction (ami) during pregnancy performed between 2000 and 2002, the anterior coronary circulation was found to be more commonly involved with 20% of reported infarctions occurring in this territory.1 although not elaborated on within the original articles, the preponderance of anterior circulation culprit vessels in ami may be due to the greater clinical presentation of these patients, while missing the smaller myocardial infarctions in the other vascular territories. ladner et al found that in pregnant women with ami, 38% occurred in the antepartum, 21% occurred in the intrapartum, and 41% occurred in the 6-week postpartum period.4 within pregnancy, badui and colleagues identified that women in the third trimester had the highest risk of ami.5 the increased stroke volume and heart rate during pregnancy causes an increased myocardial oxygen demand, while the decreased diastolic blood pressure and related physiologic anemia result in decreased myocardial perfusion that may contribute to the ischemia when coronary blood flow is compromised. with labour, myocardial ischemia may be precipitated by a further increase in myocardial oxygen demand driven by pain, uterine contraction, and anxiety. after delivery, caval compression is relieved and blood flow is shifted from the uterus back to the systemic circulation resulting in further stress on the myocardium and likely contributing to the increased incidence of myocardial infarction in the puerperium. with a compromise in the coronary blood flow, the high demand physiological state of normal pregnancy would precipitate myocardial ischemia and potentially infarction.2 james et al reviewed the coronary anatomy through angiography and autopsy of pregnant women diagnosed with ami and found that 40% had evidence of atherosclerosis with or without thrombosis, 8% had thrombosis without atherosclerosis, 27% had coronary artery dissections and 13% had normal coronaries.1 in the general population, nearly all cases of acute myocardial infarction are due to coronary atherosclerotic disease and acute plaque rupture resulting in coronary artery occlusion. rarely, vasculitic syndromes, hypercoagulable states, coronary artery spasm, increased myocardial demand, coronary emboli, congenital coronary anomalies, trauma and aneurysm may cause ami.6 the increased events associated with thrombosis without atherosclerosis, vasospasm and coronary artery dissection may be related to the physiological alterations associated with pregnancy. pregnancy is a known hypercoagulable state.7 the association of thrombophilia with mi in pregnancy may be due to the increased testing for this in this particular population.1 in regards to vasospasm, the pregnant woman has more reactive vessels to norepinephrine and angiotensin ii, has associated endothelial dysfunction and has an increased renin secretion and angiotensin activity associated with uterine malperfusion with the supine position and the use of ergot derivatives to control post-partum hemorrhage all may contribute.8 this vasospasm may also be the precipitating mechanism for thrombosis in coronary vessels that have no evidence of atherosclerosis, with the spasm impeding blood flow and the physiologic hypercoagulable state resulting in a thrombosis.8 the risk factors for ami are also commonly seen in pregnancy, including diabetes mellitus, smoking, advanced maternal age, dyslipidemia, significant family history and hypertension.1,2 in addition novel risk factors such as black race, pre-eclamplsia, eclampsia, anemia, migraine headaches and thrombophilia have been identified.1,4 the associated risk with migraines may be due to overall disorder of the woman or due to heightened awareness of the physician for possible acs events in these patients.1 the association of pre-eclampsia and eclamplsia may be due to endothelial dysfunction that has been shown to persist up to one year post partum.9 the increased incidence of coronary dissection is thought to be due to the changes in progesterone resulting in several structural and biochemical changes within the vessel wall; however, other theories include changes in eosinophil activity and decreased prostacyclin activity have been postulated. it is these systemic changes in conjunction with the physiological changes of increased blood volume and cardiac output that likely result in increased shear forces that result in dissection occurring not only in single vessels, but frequently in multiple coronary arteries.2 the treatment of acs has been well established for the non-pregnant patient, but many uncertainties remain in the management of pregnant patient which may delay treatment. a classification scheme has been established to identify the associated risks with certain medications in pregnancy (table 1).6 nitroglycerine (class b) is widely used for ischemic pain, however there are concerns about maternal hypotension and uterine malperfusion.1,2 studies are required to fully elucidate the effect of nitrates in pregnancy.10 heparin (unfractionated heparin class c, low-molecular weight heparin lmwh class b) has been proven to be safe in pregnancy in numerous studies, however it is recommended to stop heparin prior to delivery and monitoring anti-xa levels if lmwh is used due to the pregnancy associated pharmacokinetic changes.11 beta-blockers (metoprolol class b, atenolol class c) have been used successfully; however there are anecdotal reports of fetal bradycardia, hypoglycemia, hyperbilirubinemia, and apnea.2 a cochrane review looking at oral beta blockers for use in treatment of mild to moderate hypertension in pregnant women found that there was a trend toward small for gestational age infants, but the results were skewed by a small outlier trial. there was insufficient data to comment on perinatal mortality or preterm delivery.12 atenolol has been linked to a possible increase in fetal growth restriction, especially when used in the first trimester.13 asa (class c) is debateable for use during pregnancy because animal studies have shown increased incidence of fissure of spine and skull, facial and eye defects, and malformations of the central nervous system (cns), viscera, and skeleton.2,10 chronic use of high dose asa during pregnancy should be avoided because of increased fetal hemorrhage, increased perinatal mortality, intrauterine growth restriction and teratogenic effects.2,6 a meta-analysis looking at antiplatelet agents found that low dose asa is safe in pregnancy.14 clopidogrel (class b) has very limited data for its use in pregnancy. it is recommended that clopidogrel be stopped 1 week prior to any regional anesthesia procedures.2 glycoprotein iib/iiia inhibitors (eptifibatide and tirofiban class b, abciximab class c) have not been studied in pregnant patients as all randomized trials of these agents excluded pregnant patients. these drugs can not be recommended in pregnant patients, however if they are used a c-section delivery is recommended to decrease the potential for fetal intracranial hemorrhage. 2 angiotensin converting enzyme (ace) inhibitors and angiotensin receptor blockers (arbs) are contraindicated in pregnancy due to teratogenic side effects. many animal and human studies have found that ace inhibitors and arbs cause multiple birth defects including renal dysgenesis, oligohydramnios, iugr, prematurity, bone malformations, limb contractures, death and multiple others.6,15 a recent retrospective analysis of fetuses exposed to ace inhibitors in the first trimester identified ace inhibitors as an independent risk factor for developing malformations of the cardiovascular and cns.16 statins (class x) are not recommended in pregnancy as information on use in pregnancy is limited. although laboratory models show potential placental growth disruption and animal studies have shown skeletal abnormalities and increases in mortality, a recent systematic review found that most data of human teratogenicity were only case reports and that the overall risk is likely minimal. the authors stated that statin exposure did not warrant termination of pregnancy as a sole reason.2,17,18 a prospective cohort of 134 women inadvertently exposed to lovastatin and simvastatin found no difference in the incidence of adverse pregnancy outcomes.19 the use of invasive catheter procedures for management of ami in pregnancy is also not clearly identified. numerous case studies have been published that describe results of both invasive and conservative management. in one report a patient was managed conservatively with asa and beta-blockers, while waiting for the post-partum period to undergo cardiac catheterization. other reports have described treating pregnant women with early percutaneous coronary intervention (pci) and stent placement. both reported favorable fetal and maternal outcomes.2022 bare metal stents have been used with success in the literature; however, there is limited data for the use of drug eluting stents and its necessary long-term clopidogrel treatment.2 the teratogenic effects of radiation were first reported in 1929 when goldstein and murphy observed a high rate of micorcephaly and reduced cranial circumference in women who had undergone radiation treatment for uterine cancer during pregnancy. while many studies have shown that a fetal dose of 5 rads is not related to teratogenicity at any period of gestation, the most vulnerable time for the fetus is 815 weeks of gestation.23 coronary angiography exposes patients to 2.55.0 msv (equivalent to 125250 chest x-rays), and percutaneous coronary intervention exposes patients to 5.015.0 msv (equivalent to 1151000 chest x-rays),24 which are both below the threshold for teratogenicity at any gestational age. the amount of radiation that reaches the fetus is a percentage of the total amount delivered to the patient and depends on the body parts being irradiated and the type of protection used. no necessary radiodiagnostic examination that is clinically justifiable should be avoided due to pregnancy, and protective measures for the mother and fetus should be taken. other diagnostic procedures that are equally as effective but not as dangerous to the fetus should be preferentially used. a number of therapies ranging from multiple drugs to pci are available for the pregnant patient presenting with acs. it is important to weigh the risks and benefits of each potential therapy and tailor the management according to the clinical presentation. the patient was started on asa, beta-blockers, and intravenous unfractionated heparin. a quantitative -hcg and pelvic ultrasound were arranged to verify the viability of the pregnancy prior to starting medications with known teratogenic side effects and unclear risk profiles. pci was discussed however not pursued as she had a normal left ventricular ejection fraction, no recurrent chest pain, no electrical or mechanical complications. a pelvic ultrasound showed an intrauterine pregnancy with no fetal heartbeat, consistent with fetal demise. expectant management of the fetus was chosen and follow-up ultrasound was arranged with the obstetrics team. the cardiology team arranged follow-up regarding long term medications and planning for further risk stratification. although acs in pregnancy has been historically uncommon, the increasing prevalence of atherosclerotic risk factors in women of child bearing age combined with the normal physiological changes of pregnancy will cause the incidence of this presentation to increase in clinical practice. it is important that physicians are familiar with the clinical presentation, risk factors, potential management options and their interactions with both the pregnant female and the fetus.

acute coronary syndrome (acs) in pregnancy has traditionally been considered to be a rare event, but the combination of normal physiological changes of pregnancy and more prevalent cardiovascular risk factors are increasing its incidence in this population. the present report describes a 39 year-old woman that is seven weeks pregnant presenting with a non st elevation myocardial infarction. the incidence, risk factors, pathophysiology and management of acs in pregnancy are discussed.

PMC2872582

pubmed-1272

clavicle fractures occur frequently, with the reported rates ranging between 8% and 15% of all paediatric fractures [13]. the vast majority of these injuries can be treated nonoperatively with excellent results [4, 5]. reported indications for operative management include markedly displaced fractures with compromised skin integrity, open fractures, concomitant vascular injury requiring repair, and compromise of the brachial plexus [610]. more recently, there has been some support for operative management of middle third fractures with marked displacement or shortening [1113]. some of these studies have specifically recommended fixation in children and adolescents [14, 15]. the aim of this study was to review the outcome of clavicle fractures in paediatric patients at our institute and to determine the number of such fractures that require operative management. we retrospectively reviewed all clavicle fractures in children treated at our institute over a two-year period. we used the agfa impax web1000 system to identify all radiographs of the shoulder region performed in children aged up to and including 15 years old. these radiographs were then reviewed to identify all clavicle fractures in the patient cohort group. medical records and theatre records were then reviewed to establish the classification of each fracture, the treatment method used, the duration of radiographic followup, the duration of clinical followup, and the clinical outcome. exclusion criteria were any patient aged 16 years or older, and any fracture as a result of birth trauma. we identified 487 clavicle fractures in 483 patients treated in our institute during the two-year period. ten neonates were excluded because their injuries were related to birth trauma, and 283 patients were excluded because they were 16 years old or older. all fractures were classified using the system described by robinson [16, table 1]. one hundred and twenty-four fractures were undisplaced (65%) and 66 were displaced (35%). immobilisation in the sling was continued until the patient was comfortable enough to mobilise without support. further radiographs were only taken if the patient continued to have pain or limitation of function when reviewed. initial review was within one week of injury, and second review was at three weeks after initial review. further review appointments were arranged at the discretion of the clinician and were determined by clinical and/or radiographic assessment. the mean radiographic follow-up of the group as a whole was 35 days (5 weeks), and the mean clinical follow-up was 44 days (6.3 weeks). the mean radiological and clinical follow-up of the group when subdivided by age can be seen in figures 2 and 3. forty-four of 190 fractures (23%) had radiographic confirmation of fracture healing. the remaining 77% had radiographic examinations discontinued when clinical symptoms of pain and limitation of function had resolved. all fractures in this study had healed clinically when the child was discharged from follow-up. clavicle fractures are common injuries in general, and, in this study, 39% of all fractures treated over a two-year period involved children of 15 years or younger. despite this, there are surprisingly few published studies that specifically discuss paediatric clavicle fractures. traditionally, clavicle fractures have been treated nonoperatively, particularly in children. this is largely due to the relatively low incidence of complications following non-operative management. indications for operative management in the acute setting have included markedly displaced fractures with compromised skin integrity, open fractures, concomitant vascular injury requiring repair, and compromise of the brachial plexus [610]. howard and shafer described fourteen clavicle fractures with associated neurovascular complications, but only one case occurred in a child, a ten-year old with a depressed clavicle fracture compressing the subclavian vein. mital and aufranc also described a venous occlusion following a greenstick fracture of the clavicle. keating and von ungern-sternberg recently published a case report entitled compression of the common carotid artery following clavicle fracture in a twelve-year-old but the report actually describes a clavicle dislocation. fixation of a clavicle fracture associated with a dislocation [19, 20] and fixation of a fracture associated with a sternoclavicular physeal fracture have also been described in children. operative management for nonunion of a clavicle fracture in a child has also been described. reports of complications of clavicle fractures and operative management of clavicle fractures in paediatric patients are few. the examples cited above demonstrate that complications do occur, but these are extremely rare. in our group of 190 patients, none had significant associated injuries and none required operative management. displaced midshaft fractures of the clavicle have received some attention recently, with some authors recommending operative management. it has been demonstrated that a periosteal hinge is important for fracture stability. in childhood, the periosteal sleeve is thick and protects the cortex, and the bone is softer and more pliable than in adults. in displaced fractures this periosteal sleeve and hinge has been mostly or completely disrupted (using robinson's classification, displaced fractures are those that are translated by 100% or more). sixty-six patients (35%) in our group sustained displaced fractures of the midclavicle, and all of these healed clinically with non-operative management. this included five fractures that were comminuted segmental (robinson type 2b2), thereby having almost or complete disruption of the periosteal sleeve in at least one part of the bone. as figure 4 shows, displaced fractures occur more commonly in children as they get older. this can be explained by the more adult type bone and periosteum as the child grows and develops. indeed, figures 3 and 4 demonstrate that the mean period of radiographic and clinical follow-up increased with increasing age of the children. this can in part be attributed to the larger percentage of displaced fractures being encountered with increasing age. in our institute, radiographic evaluation of clavicle fractures is discontinued when symptoms resolve. some authors recommend that torus/buckle fractures do not require any radiographic review whatsoever, as the incidence of complications is so small [24, 25]. in our experience, clavicle fractures in children, whether displaced or undisplaced, heal clinically, as demonstrated by the absence of pain and the return of full function. this is achieved at a mean duration of six weeks for all fractures. for all age groups, clinical followup continued for at least as long as radiographic follow-up, and in all cases radiographs were only requested when clinically indicated. some authors have recommended that paediatric patients with clavicle fractures require no follow-up at all. this is based on the fact that most paediatric clavicle fractures heal and is justified by detailed written instructions given to parents informing them of symptoms to be aware of and when to seek further review. the vast majority of patients reviewed in this study lived locally, and all patients are advised to seek further review if they develop symptoms after discharge. patient records were reviewed at a mean of nineteen months after injury. had any of these patients developed late complications within this time period, this would have been documented within their records in the form of referral back to the orthopaedic clinic by their family doctor or the local emergency department. we accept that we still may have overlooked complications in the small number of patients who did not live locally or those whose symptoms were not felt severe enough to warrant further orthopaedic consultation. however, radiographs take time, cost money, and expose patients to radiation [2729]. we firmly believe that radiographs should only be performed when they are likely to alter the management of the patient. our radiographic follow-up in this study was a mean of five weeks, but we do recognise that only 23% of our patients had fracture union confirmed by radiographs. some studies have suggested that radiographs are not required at all in the assessment of clavicle fractures [31, 32], but we feel that an initial radiograph to confirm and classify the injury is appropriate even where the fracture is obvious clinically. there is evidence in the literature that highlights the aetiology and risk factors for some of these complications. most published articles report these complications in adult patients, and there is a relative paucity of the literature available that is specifically reporting upon clavicle fractures in children. despite this, there are a small number of reports of complications in this group. in our experience, all paediatric clavicle fractures can be treated with simple immobilisation and analgesia, without development of complications. radiographic review of paediatric clavicle fractures is unnecessary in the absence of clinical findings suggestive of delayed union.

paediatric clavicle fractures have traditionally been treated nonoperatively. recent studies have recommended operative management for displaced midshaft fractures. we conducted a retrospective review of all clavicle fractures in children aged one to sixteen over a two-year period. we classified fractures and evaluated followup and clinical outcome. we identified 190 fractures. there were 135 boys and 55 girls. 65% of fractures were displaced and 35% undisplaced. mean radiographic and clinical followup was 35 days and 44 days, respectively. clavicle fractures in children heal with nonoperative management. radiographs of clavicle fractures in children are unnecessary in the absence of clinical symptoms.

PMC3236468

pubmed-1273

the objective of this study is to quantitatively estimate the effect of medicare supplementation on use of health services by medicare beneficiaries. however, the extent of supplementary health insurance coverage is only one of many factors influencing the use of health services by medicare beneficiaries. previous studies of health services utilization have found such factors as age, sex, race, income, and health status to be capable of exerting independent influences on the use of health services (leopold and langwell, 1978). in order to isolate the influence of supplementary health insurance coverage, it is necessary to control for the variation in utilization arising from these other factors. accordingly, our empirical results are based upon a multivariate model that relates an individual's use of either physician or hospital services to a variety of underlying determinants. in particular, the empirical specification for the model is: where the subscript i denotes the ith medicare beneficiary; ui measures the person's annual use of health services (two alternative models are estimated: one for annual number of physician visits and one for annual number of hospital days); privi is a binary variable indicating whether the ith beneficiary supplements medicare with some type of private health insurance; caidi is a binary variable indicating whether the ith beneficiary received medicaid benefits during the past 12 months; x3i through xni compose a set of variables to control for other factors influencing the ith person's utilization; and ei is a stochastic error term, assumed to have a truncated-normal distribution. the set of control variables in this model is designed to capture the influence of family income, race, sex, region of residence, education, family size, health status, age, marital status, labor-force status, veteran status, and the availability of medical resources. the primary data source is the 1976 health interview survey (usdhew, 1977). observations were excluded for persons for whom family income or education was unknown or not reported, reducing the sample for the analysis of hospital utilization to 8,325. the elimination of observations pertaining to persons who failed to report the number of physician visits during the preceding 12 months further reduced the sample for analyzing physician utilization to 8,239. for each of the dependent variables about 30 percent of the sample reported no use of physician services during the preceding 12 months, and approximately 80 percent did not use inpatient hospital services. accordingly, the tobit estimation technique (tobin, 1958) was used for this study. covariance analyses revealed that our investigation of physician utilization should be conducted with data stratified according to the presence or absence of chronic health problems. thus, results from the analysis of physician utilization are reported separately for (1) elderly medicare beneficiaries who have no chronic health problems and (2) those who have one or more such conditions. in contrast, covariance tests for hospital utilization indicated that it was appropriate to pool over all health conditions. these tables highlight the influence of supplementary coverage on the utilization of health services by elderly medicare beneficiaries. table 1 reports the average utilization of health services among medicare beneficiaries by type of supplementation, both in absolute terms and relative to the average utilization by medicare beneficiaries who do not supplement (shown in parentheses in the table). these average rates were tabulated directly from the health interview survey (his) data, and thus are unadjusted for other determinants of utilization. the differentials between these averages should not be interpreted as reliable indicators of the influence of supplementation on utilization. since we shall compare these unadjusted averages with utilization rates adjusted for other determinants, a brief examination of the apparent implications of the results in table 1 is merited. these unadjusted averages reveal that beneficiaries who supplement medicare with private health insurance generally make greater use of health services than those who do not supplement. the largest differential between those with private supplementation and those with only medicare arises in connection with physician visits by beneficiaries with no chronic health problems: those who have private supplementation in this group report 39 percent more visits to physicians than their counterparts who rely solely on medicare. private supplementation is also associated with somewhat greater use of hospital services (2.79 days per year), relative to the average utilization among those with no supplementation (2.51 days). in contrast, those with one or more chronic health problems and private supplementation tend to have fewer physician visits compared to those with no supplementary coverage. while the differential is small (6.23 visits versus 6.72 visits), it is nevertheless paradoxical. public supplementation of medicare is associated with relatively high rates of utilization of both physician and hospital services. for example, medicare beneficiaries with medicaid supplementation spent an average of 76 percent more days in the hospital than those with no supplementation and 58 percent more than those with private supplementation. based upon this type of evidence, one might be tempted to conclude that public supplementation of medicare has greatly stimulated the use of health services. however, while such a conclusion may be correct, it does not necessarily follow from the evidence in table 1. the high utilization rates in the medicaid category may simply reflect the relatively poor health status of persons in this group. table 2 contains the key findings of this study, namely, the predicted utilization rates by type of supplementation, adjusted for other determinants. these estimates are derived from a multivariate model that controls for a variety of determinants of utilization and thus isolates the influence of supplementation on utilization from the influence of other factors such as health status. the values reported in table 2 represent predicted utilization rates for typical medicare beneficiaries; that is, they are derived under the assumption that all of the determinants of utilization (except the supplementation variables) equal their mean values. what do these predicted utilization rates imply about the influence of supplementation on the use of health services by elderly medicare beneficiaries? first, other things equal, supplementation always stimulates use of health services, generally by statistically significant amounts. in particular, the paradox of relatively low demand for ambulatory care by those chronically ill beneficiaries with private supplementation disappears once the utilization rates are adjusted for other determinants. second, public supplementation always stimulates more utilization of health services than does private supplementation. however, in no instance is the differential in utilization rates between those with private supplementation and those qualifying for medicaid statistically significant. we are justified in concluding only that, other things equal, medicaid supplementation of medicare permits elderly beneficiaries to use physician and hospital services at least as often as those who purchase private supplementary health insurance. this conclusion contrasts sharply with the one implied by the unadjusted average utilization rates in table 1: those estimates suggest that the influence of public supplementation on the demand for health services far outweighs the influence of private supplementation. a final important implication of the estimates reported in table 2 pertains to the differential effectiveness of supplementary coverage at stimulating demand for health services. supplementation greatly increases the use of both hospital services and physician services among elderly persons with no chronic health problems. however, among those elderly beneficiaries with one or more chronic health problems (about 78 percent of the beneficiary population), persons with some type of supplementation have only slightly more physician visits than those with no additional coverage. apparently the deductibles and coinsurance provisions of medicare's part b medical insurance do not represent an important barrier to ambulatory medical care for those beneficiaries who suffer from chronic health problems. for these individuals, supplementation particularly through medicaid serves mainly to redistribute income to the chronically ill medicare beneficiaries. conclusion that public supplementation was very effective at stimulating demand for ambulatory care among those with chronic illnesses. the estimates presented in table 3 provide an additional perspective on supplementation's role in determining utilization rates among elderly medicare beneficiaries. mean utilization for a group, u, can be defined as: where p is the probability that a person with mean characteristics will use a particular health service and u is the mean utilization rate among those who actually use the health service. thus, any change in a group's utilization rate (u) can be divided along the following lines: this relationship can be restated in percentage terms as table 3 partitions the percentage increase in utilization arising from supplementation into the three components identified in equation 3: the percentage increase in the probability of using a particular health service, the percentage increase in the utilization rate among those that make use of the health service, and the interaction between these two factors. the estimates in table 3 reveal that supplementation raises a group's mean utilization rate (adjusted for other determinants) largely by increasing the proportion of the group that uses medical services. when compared to no supplementation for example, medicaid supplementation raises the use of hospital services by 47 percent. most of this gain (29 percent out of the 47 percent) arises from the hospitalization of persons who, in the absence of medicaid supplementation, would not have received treatment on an inpatient basis. private supplementation has a similar influence on the utilization of hospital services: most of the gain comes from more people being admitted to hospitals rather than from an increase in average length of stay. these results suggest that the part a deductible (approximately equal to the average charge for an inpatient hospital day) represents a significant barrier to the utilization of hospital services by the elderly. supplementation increases utilization by persons in this group mainly by permitting a larger fraction of the group to visit a physician than would otherwise be the case. the part b deductible ($ 60) thus appears to also serve as an important barrier to the use of ambulatory medical services by those with no chronic health problems. in addition, the part b coinsurance rate (20 percent of covered services) apparently serves as an important deterrent to physician utilization by those elderly persons with no chronic conditions. this conclusion follows from the relatively large positive effect of supplementation on utilization among those who, in the absence of supplementary coverage, would have still made some use of physician services. to illustrate, private supplementation leads to a 42 percent increase in physician visits by persons in this health-status group. of this increase, 16 percent is due directly to greater utilization among those who would have seen a physician even without supplementary coverage. as we noted earlier, supplementation stimulates physician utilization only to a small degree among those elderly with chronic health problems. table 3 reveals the two underlying components of mean utilization to be about equally responsible for this small gain in the use of physician services. apparently neither the deductible nor the coinsurance provisions of part b represent important barriers to physician utilization among those elderly beneficiaries with chronic health problems. our principal finding is that beneficiaries ' utilization of health care services rises when medicare is supplemented by private insurance coverage or by medicaid, though in varying amounts depending on individuals ' health status. to turn the conclusion around, we find that medicare cost-sharing (when not vitiated by private or medicaid supplementation) leads to significantly lower levels of hospital and physician utilization than would have prevailed in the absence of the program's deductibles and coinsurance. the estimates we report allow for an illustrative calculation of the magnitude of the cost savings owing to medicare cost-sharing. cost-sharing under parts a and b is an effective economic incentive for the 29 percent of the 24 million elderly beneficiaries (about 7 million) who have neither private nor public supplementary health insurance. the predicted hospital utilization rates in table 2 imply a utilization reduction of between 630 and 890 days per thousand beneficiaries per year when cost-sharing is effective (compared to private supplementation and medicaid, respectively). therefore, the part a cost-sharing provisions result in 4.4 million to 6.2 million fewer days of hospital care for the elderly. assuming a cost per day of $160 (the part a hospital inpatient deductible for calendar year 1979), medicare cost-sharing results in a reduction in total hospital expenditures by the elderly of between $700 million and $1 billion. of course, only part of these savings accrue to the medicare program since some of the reduced utilization would have been subject to the part a deductible. however, cost control is not the sole goal of the medicare program. it has been argued that medicare cost-sharing is particularly perverse because it largely reduces the health services utilization of certain disadvantaged groups the medicaid-ineligible low income and nonwhite populations for whom medicare was designed to equalize access to medical care (gornick, 1976; davis and schoen, 1978). thus it is conceivable that the advantage of cost-sharing in controlling health expenditures might be outweighed by the disproportionate reduction in utilization among certain population groups. to test the hypothesis that the availability of private health insurance supplementation leads to inequitable utilization advantages for whites and higher income groups, we re-estimated the above-described model after omitting the private insurance variable. examination of the changes in the coefficients on the race and income variables, respectively, will reveal unequal access afforded by the private market availability of supplementary insurance, if any. in all three regressions physician visits for chronics and non-chronics and hospital days the coefficients for southern and non-southern blacks changed insignificantly. similarly, as reported in link, long, and settle (1980), the variables representing income classes did not change significantly. whatever inequitable access to services exists for medicare beneficiaries is apparently caused by factors other than the availability of private supplementary insurance. in summary, we conclude that medicare cost-sharing, in the absence of private or public supplementation, reduces medical care utilization and, therefore, costs to the program. moreover, it appears that the burden of this cost-sharing is not so concentrated among race and income groups as to cause uniformly inequitable access to medical care. however, fine-tuning among these competing objectives remains in the province of policymakers, not researchers.

this paper investigates the extent to which private supplementary insurance and medicaid, which vitiate the effect of medicare cost-sharing, encourage elderly beneficiaries to seek additional medical care. a multivariate model of health services utilization is estimated with the tobit technique, using the 1976 health interview survey. we find that either private or public supplementation induces greater use of hospital and physician services, though in amounts that vary considerably according to health status. the paper closes with observations on cost savings brought about by medicare cost-sharing and some implications for equity among beneficiaries.

PMC4191144

pubmed-1274

parkinson s disease (pd) is one of the most common neurodegenerative disorders, with an estimated prevalence of approximately 1 to 2% among people 65 years of age or older. clinically, pd patients show motor (e.g., tremor and rigidity) and nonmotor (e.g., dementia and depression) symptoms. it is believed that motor symptoms are largely attributable to the loss of dopaminergic neurons in the substantia nigra, whereas nonmotor symptoms involve many other brain regions. effective treatment of pd has been hampered partially due to the fact that even newly diagnosed patients are already at relatively advanced stages pathologically. the other difficulty is lack of objective assessment of disease progression, which has led to extensive research on biomarkers that can be employed for early diagnoses of pd or assessing its progression. as of today, the best pd protein biomarkers are those found in cerebrospinal fluid (csf), the body fluid that is in direct contact with brain and spinal cord. discovery of peripheral biomarkers reflecting motor components of pd is highly desirable because csf-based tests depend on the comparatively more invasive process of lumbar puncture that requires more specialized training to collect samples and are therefore not acceptable by some subjects, particularly those without apparent clinical symptoms. however, peripheral biomarker discovery has been largely unsuccessful, due primarily to factors intrinsic to blood; specifically, the plasma or serum proteome is quite complex, proteins are present across a wide dynamic range, and target proteins are often of extraordinarily low abundance. thus, accurately detecting and quantifying brain-derived or disease-specific proteins in blood is challenging for current technologies. that said, a few blood biomarkers discovered recently, for example, egf and apoa1, appear to be related to cognitive impairment or disease onset in pd. additionally, it has been suggested recently that -synuclein pathology, a key component of pd pathogenesis, exists in peripheral nerves, leading to a report demonstrating abnormal autonomic nerve staining in skin biopsies in pd. to facilitate peripheral biomarker discovery, in the current investigation, we began with proteins identified in our previous proteomic investigations using human brain tissues obtained at autopsy and csf obtained from living patients, with a focus on glycosylated proteins, which are highly enriched in body fluids, including plasma. we hypothesized that some of the brain or csf -derived proteins will reach plasma via mechanisms yet to be defined and that a subset of these proteins or peptides will be detectable in plasma using sufficiently sensitive measurements. to study the presumably low-abundance brain-derived proteins in plasma, we turned to a current quantitative mass spectrometry (ms) technique, selected reaction monitoring (srm), which has emerged as an alternative to immunoaffinity-based measurements of defined protein sets. srm has the benefit of fast and cost-efficient assay development, and protein quantification by srm in complex samples using predefined assay coordinates is reproducible across different laboratories and instrument platforms. however, the main advantage of srm is the capacity to quantify multiple proteins in parallel at a low limit of detection and high accuracy. it has been reported that srm has the ability to detect plasma proteins at g/ml levels without any sample enrichment or fractionation, which suggests that a further enrichment would be necessary to detect central nervous system (cns)-derived proteins at lower levels. in recent years, a multitude of enrichment approaches have been developed. among them, immune affinity depletion can be used to remove the most abundant proteins, improving detection of low-abundance proteins. however, one risk associated with this kind of predepletion is the proteins of interest might also be partially removed. in contrast, specific peptides of interest can be enriched after digestion (immuno-srm), dramatically increasing their relative concentration; however, such techniques are limited by the need for highly specific and high-affinity antibodies, which are either not available for all targets or expensive to generate. we chose to use the n-glycocapture technique, which is an antibody-free, hydrazide-based approach to selectively enrich n-glycopeptides. additionally, glycosylation is known to be important in pd, and glycoproteins, which are prevalent in extracellular surface proteins and secreted proteins, are ideal sources of biomarkers. therefore, to test our hypothesis in this study, n-glycoproteins were captured from plasma of a larger cohort of pd patients, along with healthy and diseased controls, by a hydrazide-based solid-phase capturing approach, followed by quantification of peptides that are uniquely associated with pd diagnosis or severity based on our previous experimentations. a total of 282 subjects, including patients with pd or alzheimer s disease (ad) and age-matched controls, recruited at the veterans affairs puget sound health care system/university of washington school of medicine, the oregon health and science university, and the university of california at san diego, were included in the investigation. the subjects consisted of two subcohorts: those collected prior to 2011 (75 pd, 15 ad, and 30 controls) and those collected more recently (98 pd, 15 ad, and 49 controls). all plasma samples were obtained after informed consent from patients, and all patients underwent medical history evaluation, physical, and neurological examinations, laboratory tests, and neuropsychological assessments. all pd subjects met the uk pd society brain bank clinical diagnostic criteria for pd, while ad cases were diagnosed according to nia criteria. pd patient samples were further categorized based on updrs part iii on-state motor scores to approximate disease stage. patients with updrs scores<15 were defined as early-stage pd, those with scores ranging from 1530 were classified as midstage pd, while those with scores>30 were classified as late-stage pd patients. control subjects were community volunteers in good health and had no signs or symptoms of cognitive impairment or neurological disease; all control subjects had a mini mental status examination (mmse) score between 28 and 30, a clinical dementia rating (cdr) score of 0, and new york university paragraph recall scores (immediate and delayed) of>6. all samples were collected and processed following standard clinical protocols and quality-control procedures at all participating sites, as defined previously. isolation of n-linked glycopeptides (n-glycopeptides) from plasma was performed as previously described. in brief, starting with aliquots of 25 l individual or pooled plasma, samples were diluted 10-fold with ammonium bicarbonate (100 mm), then denatured with 50% tfe (2,2,2-trifluoroethanol, j.t. baker, philipsburg, nj) and digested with mass-spectrometry-grade trypsin (promega, madison, wi). the peptides were desalted using c18 cartridges (waters, milford, ma) and were then oxidized with 10 mm naio4. the resulting oxidized glycopeptides were coupled to hydrazide resin (affi-prep, bio-rad, hercules, ca) by incubation in coupling buffer (100 mm sodium acetate and 1.5 m sodium chloride, ph 4.5) overnight at room temperature with bottom-over-head rotation. the unbound nonglycosylated peptides were removed by several washes of sodium chloride (1.5 m), 80% acn, and ammonium bicarbonate (100 mm), respectively. n-glycopeptides were finally eluted from the resin by the addition of pngase f in 50 mm ammonium bicarbonate, ph 7.5, and incubation overnight at 37 c. an mcx (mixed-mode cation exchange) desalting step using an oasis elusion plate (waters, milford, ma) was performed before lc glycoproteomes of brain tissue and csf samples from pd and ad patients and age-matched controls were previously investigated. all n-glycopeptides with quantitative alterations in brain or csf were selected in the initial candidate library. to evaluate the utility of these glycoproteins as diagnostic markers for pd, we tested the 133 srm assay feasible glycopeptides (derived from 73 n-linked glycoproteins) in the initial pilot study using a few pooled plasma samples from pd and controls. peptide selection criteria include: (1) length of 820 amino acid residues; (2) no chemically unstable residues (e.g., ng, dg, qg, n-terminal n, and n-terminal q); (3) fully tryptic; (4) avoiding cysteine residue if possible; and (5) sequence specific for the target protein (i.e., proteotypic peptides). all peptides used in this study were evaluated using blat (http://genome.ucsc.edu) and protein blast (http://blast.ncbi.nlm.nih.gov/blast.cgi) searches to ensure uniqueness of the target proteins at both proteomic and genomic levels. finally, an srm theoretical collision calculator tool (http://proteomicsresource.washington.edu/cgi-bin/srmcalc.cgi) was applied to confirm the uniqueness of every q1/q3 pair for the target peptides. all srm analyses were performed on a tsq vantage triple quadrupole (qqq) mass spectrometer (thermo scientific) at the university of washington proteomics resource. the mass spectrometer was coupled to a nanoelectrospray ionization source and a waters nanoacquity uplc system. glycopeptides from 1 l of original plasma (1 g peptides) were loaded onto a c18 trap column (20 mm long, 75 m i d) and separated by a 150 mm c18 column (75 m i d) over a 60 min 235% linear acetonitrile gradient. spray voltage was set at 1700 v. scheduled srm was performed with 5 min retention time windows for most of the peptides and an instrument cycle time of 2000 500 ms. dwell times were varied depending on the number of concurrent transitions; in all cases they were at least 10 ms. both unpurified (thermo-fisher scientific, germany) and purified (aqua-grade, sigma-aldrich, usa) peptide standards that correspond to natural counterparts (light peptides) were synthesized with heavy isotopic lysine (c6n2) or arginine (c6n4) at the c-termini (heavy peptides). collision energies (ces) were determined using the default formula from thermo (0.034 precursor mass m/z+3.3140) and then optimized with four additional ce steps (5 v, 10 v). the top four abundant transition (q1/q3) pairs, including 4 light and 4 heavy determine the limits of detection (lod) and quantification (loq) for each target glycopeptide, we titrated heavy peptides at seven concentration points in a reference glycocaptured plasma matrix. a linear regression algorithm was used for fitting the seven serial dilution data points for each curve. the endogenous peptides were also monitored in these assays to help determine the amount of each peptide standard to spike-in. all raw srm data were processed using the skyline targeted proteomics environment (v1.3) (mccoss lab, university of washington) software developed for srm data sets. settings including 0.055 th match tolerance m/z, default peak integration, and savitzky peptides with signal-to-noise ratio of at least 3 were considered detectable. information including peak area and area ratio of light/heavy peptide pair were output from skyline to a text-delimited format worksheet. it should also be noted that the glycopeptide capturing method used in the current study has been well-optimized and widely employed, with cvs typically controlled in 1520% range. to further control this variable, in this study, we included one to two reference plasma samples in every batch of sample preparations as inter- and intrabatch controls. these samples showed an average of 16.5 and 14% for preanalytical (capture) and analytical (srm) variation (cv), respectively; that is, the variations associated with the capture and srm stages were reasonable in our investigation. the key challenge for the present analysis is a high dimension of the feature vector (large number of potentially predictive proteins) versus size of samples within the data set. missing data were handled using k-nearest neighbor imputation algorithms (k=10). repeated (duplicate) measurements for the same protein all other analyses were generated in prism 6.0 (graphpad software, la jolla, ca) or spss 18.0 (ibm, chicago, il). one-way analysis of variance (anova), followed by the tukey s hsd post hoc test, was used to compare differences between groups. receiver operating characteristic (roc) curves were used to calculate the relationship between sensitivity and specificity for pd versus the healthy control group and hence to evaluate the diagnostic performance of the analytes, either individually or in combinations. logistic regression was used to determine the best linear combination of peptide analytes for predicting disease status (versus healthy controls), followed by roc analysis on the linear combination. cutoff value from a roc curve is determined when the sum of sensitivity and specificity is maximal. additionally, relationships between the analytes and the unified parkinson s disease rating scale (updrs) were analyzed with bivariate correlation using pearson s correlation coefficients. stepwise multiple linear regression analysis was used to screen for the best predictors (linear combination of peptide analytes) that correlate the disease severity (updrs) to enrich secreted proteins in body fluids, in the past few years, we profiled n-linked glycoproteins in the brain and csf of patients with pd as compared with age-matched healthy and diseased controls (patients with ad). a subset of proteins has been previously published. drawing from our previous work, we identified candidate peptides with quantitative alterations (defined by 50% over control cases; full list not shown) and selected a total of 133 n-glycopeptides, representing 73 n-linked glycoproteins based on peptide characteristics, especially amino acid sequence, suitable for srm assays, as defined in the methods section. with the aid of synthetic heavy-isotopic labeled peptides, srm conditions including q1/q3 transitions, collision energy (ce), retention time (rt), and the spiked-in amount of heavy peptide, were first optimized with a set of pooled samples obtained from healthy controls and pd patients. on the basis of the detectability of target peptides in the pooled samples (either control or pd), 50 formerly n-glycosylated peptides (deamidation happens during glycocapture, the n-glycopeptides monitored in srm are not glycosylated anymore) derived from 40 glycoproteins (supplemental table 1 in the supporting information, along with reported biological functions) were selected as srm targets for further analysis, with spiked synthetic corresponding heavy peptides in the initial stage of the validation to be discussed later. brief workflow outlining the pipeline for screening plasma pd glycoprotein biomarkers by srm. in the pilot study, a primary synthetic peptide library containing 133 unpurified, deamidated n-glycopeptides was used to generate srm assays and test the detectability of these formerly n-glycosylated peptides in glycocaptured plasma. refined srm assays with optimized settings including retention time (rt) were applied in next stages in the pipeline. in initial validation, 50 n-glycopeptides that could be detected in the pilot study were measured in a cohort including 15 pd, ad, and control samples pooled from 75 pd, 15 ad, and 30 control individuals, respectively. the final srm assay library containing 12 purified, deamidated n-glycopeptides was then used to detect the candidate biomarkers in an independent validation cohort for roc calculation and assessment of disease severity association (n=162). having identified peptides that are reliably detectable in plasma, we then aimed to narrow down the panel of potential biomarker candidates to those showing the largest alterations under the disease conditions. a total of 120 cases from pd, ad, and age-matched controls were included in this study, with the samples pooled based on disease status. to allow approximation of disease stage correlations, we further split plasma from pd subjects into three subgroups according to updrs-defined disease severity. to reduce the within-pool heterogeneity and facilitate statistical analysis, we combined samples in each group into three small pools (control: n=3, early pd [updrs<15]: n=3, intermediate pd [updrs 1530]: n=3, late pd [updrs>30]: n=3, and ad: n=3). each pooled sample consisted of 10 individuals with age and gender evenly distributed, except the ad and early pd groups, where plasma from only five individuals was used because of limited samples in these subcohorts (table 1). quantitative assessment of 15 pooled samples revealed clear differences among different diagnostic groups or between different stages of the disease (supplemental table 2 in the supporting information). figure 2 shows the hierarchical clustering analysis, demonstrating a clear separation of pd with updrs>15 (right side, pd2_*and pd3_*columns) from the three controls (left side columns). interestingly, this phenomenon is missing in the three ad pools as well, suggesting that these elevations are unique in pd with updrs>15. the three updrs<15 samples (pd1_1, pd1_2, and pd1_3) did not present obvious changes compared with the controls, indicating that the current set of glycoproteins may lack the ability to distinguish pd in the early stage from controls, at least in these pooled samples. overall, a total of 12 n-glycopeptides derived from 11 glycoproteins were detectable, with more than 50% difference between pd versus controls or between the stages of disease. consequently, these 12 n-glycopeptides were selected to be the targets in the next stage of validation experiments. heat map of hierarchical clustering analysis of the 50 n-glycopeptides that were detected in the initial validation cohort containing 15 pooled control, ad, and pd plasma samples. the analysis was based on srm measured peptide relative abundance against average of three pooled controls and conducted using average linkage and euclidean distance. pd1, pd2, and pd3 represent pd at early, middle, and late stages of the disease, respectively, based on updrs. (also see table 1.) pooled samples, even with multiple pools as performed in our initial validation previously discussed, do not provide precise sensitivity and specificity information. accordingly, in the final validation stage (figure 1), a total of 162 individual plasma samples from an independent cohort of subjects recruited from the same medical center were investigated. additionally, to achieve higher-level accuracy, we used aqua level purified heavy isotopic-labeled peptides, with srm running conditions further optimized for the new peptide standards (supplemental table 3 in the supporting information). peptide sequences, estimated plasma concentrations, and limits of detection and quantification of these 12 peptides are presented in table 2. of note, calibration curves were generated in a reference glycocaptured plasma matrix to fully characterize the performances of srm assays. calibration curves for each aqua peptide are plotted on linear scales in supplemental figure 1 in the supporting information. representative srm chromatograms of the 12 glycopeptides (both endogenous and spiked-in standard) are shown in supplemental figure 2 in the supporting information. with the exception of peptide sema4d-aadytsslnlpdk, the remaining 11 peptides were detected in more than 50% of the individuals (supplemental table 4 in the supporting information). p values and posthoc comparisons between each diagnostic group and age-matched controls were calculated for each of the 12 srm peptides using one-way anova, followed by tukey s hsd post hoc tests (table 3). three of the 12 n-glycopeptides, derived from glycoproteins prnp (prion protein), golm1 (golgi membrane protein 1), and ncam1 (neural cell adhesion molecule 1), were significantly altered between pd versus age-matched controls (age 50; pd, n=96; control, n=34; p<0.05). among them, the plasma concentration of peptide ncam1 was also significantly increased in ad patients compared with age-matched controls (p<0.05), suggesting its change may be related to neurodegeneration but not specific to pd. furthermore, levels of 5 n-glycopeptides (chl1_iip, chl1_isg, golm1, hspg2, and tnc) were lower in younger controls (age<50), indicating that the blood level of these glycoproteins was likely associated with aging processes. notes: n, deamidated asparagine residues. the limit of detection (lod) of each peptide was obtained from the average of lowest concentration point at which all three or four transitions were confidently detected. the limit of quantification (loq) was obtained from the average of lowest concentration point at which the intensity of the most abundant transition is on the linear scale along with the calibration curves. settings in tukey s hsd post hoc tests: alpha=0.05; n=average sample numbers in the two comparison groups. proteins and peptides in bold are those with statistical alterations unique to pd (vs age-matched controls and ad). significance level: p<0.01. significance level: p<0.05. on the basis of the performance of each individual peptide on the group difference, a logistic regression-based multivariate analysis was performed to evaluate the sensitivity and specificity of the three pd unique peptides, alone or in combination with the rest of the 12 peptides for discriminating of pd from healthy controls (age 50) (table 4). as shown in supplemental figure 3 in the supporting information, the best individual performing peptide was prnp (area under curve (auc): 0.648; sensitivity: 76.8%; and specificity: 47.1%). the performance of roc became progressively better when more peptides were added to the panel; a combination of four peptides (prnp, hspg2, megf8, and ncam1) improved auc to 0.753, sensitivity to 90.4%, and specificity to 50.0% (figure 3a). notably, however, adding more peptides only enhanced roc performance slightly; for example, a panel of 10 peptides (prnp, hspg2, megf8, ncam1, icam1, tnc, golm1, chl1_iip, chl1_isg, and lsamp) achieved the following values: auc, 0.840; sensitivity, 71.7%; and specificity, 83.3%. also, as expected, there were more cases having missing values (i.e., peptides not detectable) when more peptides were included; specifically, the number of missing values for prnp, 4-peptide combination, and 10-peptide combination were 1, 4, and 53, respectively. logistic regression of a panel of four n-glycopeptides and correlation with pd progression. (a) receiver operating characteristic (roc) curve and corresponding area under the curve (auc) of the classifier in distinguishing pd from controls are presented. this classifier includes four formerly n-linked glycopeptides (prnp, hspg2, megf8, and ncam1). (b) combination of two n-glycopeptides (icam 1 and megf8) correlates with pd progression.. significant differences among pd subgroups at different disease stages (updrs<15, updrs 1530, updrs>30) and age-matched controls were also observed (table 3). next, the correlation of pd severity (as determined by updrs) with plasma levels of single peptide or a combination of peptides was evaluated by pearson s correlation. a stepwise multiple linear regression analysis was used to screen for the best predictors (linear combination of peptide analytes) for disease severity. although none of the single peptides significantly correlated with updrs, when peptides were considered together, a combination of megf8 and icam1 was identified to correlate significantly with updrs (figure 3b; r=0.293, p=0.004). it has been exceptionally challenging to detect peripheral markers unique to cns diseases, including pd, largely because human blood is extremely complex, with proteins contributed from many organ systems, and cns-derived proteins are exceedingly low in concentration. in this study, we took a targeted approach, using previously identified cns-related proteins with changes specific to pd as the starting point. several objectives are achieved in this study, including (1) detection of 11 cns-related glycoproteins (related to pd diagnosis or severity in previous proteomics profiling) in plasma, a much less invasive sample source (than brain, csf, or skin biopsy), (2) a combination of several peptides, that is, prnp, hspg2, megf8, and ncam1, provided good diagnostic sensitivity (90.4%) and specificity (50.0%) between pd and controls, and (3) a combination of two peptides, megf8 and icam1, significantly correlated with pd severity, as measured by updrs. all 12 of the n-glycopeptides studied in the final validation study are either relatively specific to the cns or have functions potentially important to pd pathogenesis or cns diseases in general. however, those that provided good sensitivity and specificity, alone and in combination, warrant further discussion. prnp (prion protein or prp) is most predominantly expressed in the nervous system but occurs in many other tissues throughout the body. brain barrier (bbb) and, when aggregated, becomes a major contributor to a variety of cognitive deficiencies and neurodegenerative diseases, especially creutzfeldt jakob disease. prp has been implicated as a receptor and binding partner for a oligomers in ad, and its discovery as a protein altered in pd suggests interesting potential for a role in pd pathogenesis as well. neural cell adhesion molecule 1 (ncam1) plays important roles in the inflammatory mechanisms associated with neurodegeneration and participates in the neuroprotective response in neurodegeneration. its up-regulation in pd cases could be a compensatory mechanism during the disease process. similarly, heparan sulfate proteoglycan 2 (hspg2) is an extracellular matrix protein that is primarily synthesized by vascular endothelial and smooth muscle cells, with several proposed functions, including maintenance of the integrity of the bbb, which has been reported to be compromised in pd. finally, very little is known about megf8 (multiple epidermal growth factor-like domains 8) and its role in either pd or any other neurodegenerative disorder. interestingly, a recent study has suggested that this protein could potentially be involved in phagocytic function of astrocytes. it should be stressed that while the four-peptide panel probably does not reach sufficient (80%) sensitivity/specificity to be used as a sole diagnostic criterion, it performed similarly to the best biomarkers discovered thus far. for example, in a previous study of -synuclein and dj-1 in csf of pd patients, sensitivity and specificity for distinguishing control from pd were 94 and 50% for dj-1 and 93 and 39% for -synuclein, respectively. this result is especially notable when considering that the previous results were obtained in csf, a biological fluid in direct contact with the extracellular space in the brain, while the current study used a peripheral fluid. thus, while the current test performed similarly, it provides at least a promising lead to use a less invasive sample by sensitive detection of cns-related proteins in blood samples. that said, even a test without ideal sensitivity or specificity could be useful in clinical and research settings (e.g., selecting patients for clinical trials or screening for preclinical/premotor patients), where they may be useful for screening subjects or patients whose diagnosis would then be confirmed by more expensive or more invasive tests. of note is also the observation that another glycoprotein, golm1 (golgi membrane protein, also known as golgi phosphoprotein 2 or golgi membrane protein gp73), was significantly changed between pd and controls but did not perform well in logistic regression screening for roc performance. however, this does not necessarily mean that it is not important to pd pathogenesis or development. indeed, the golgi complex, including gp73, plays a key role in the sorting and modification of proteins exported from the endoplasmic reticulum, which has been demonstrated to be dysfunctional in pd or pd models. to date, biomarkers that could robustly correlate with pd severity or progression (assessed in longitudinal collected samples) are quite rare, if there are any. in fact, we are unaware of any such pd candidate markers (or ad for that matter) that have been validated by independent studies. to this end, our candidate markers, although vetted through discovery and validation phases, require yet another round of validation by independent groups, particularly for the identified models/combinations. that said, two candidate peptides, megf8 and icam1, were associated with pd severity in this study. as previously indicated, there is no clear evidence that megf8 is involved in cns disease at this point, and its potential role should be investigated, particularly if these findings are validated, as it may be a novel pd-related protein. intercellular adhesion molecule 1 (icam1) is a cell-surface glycoprotein that is typically expressed on endothelial cells and cells of the immune system. one of the proposed cns functions of icam1 is involvement in vasoconstriction associated with subarachnoid hemorrhage, indicating its role in endothelial dysfunction. in addition to regulating endothelial functions, icam1 is also a mediator of cellular inflammation and reported to be regulated by dj-1 and -synuclein, two proteins intimately involved in pd, during neurodegeneration. the increase in circulating icam1 as a function of increasing pd severity is in line with the argument that there is persistent inflammation during pd development and progression. it remains to be determined, though, whether plasma icam1 is originated from the cns or the other way around. the two-peptide panel that correlated with disease severity may also be useful in developing an assay to track the progression of pd. however, as in all biomarker studies, the biomarkers studied here must be further validated in future studies. moreover, because of the unbiased nature of the discovery stage of this study, some of the peptides discovered here (including the pair correlating with updrs) have not previously been associated with pd and are therefore also new putative candidates for future studies of pd pathogenesis. interestingly, most of these peptides showed a pattern of greatest increase in the midstage pd group, followed by a decrease in the late pd group. the reasons for this pattern are not yet clear, but it should be emphasized that updrs does not reflect a linear pattern of degeneration in specific brain regions but rather a combination of the neurodegenerative and compensatory processes occurring throughout the brain. moreover, pd processes are highly heterogeneous, and individual disease courses vary substantially. thus, biomarker patterns may similarly be nonlinear and require interpretation depending on disease stage (e.g., screening tests at early stages vs tests for following progression at more advanced stages). it is also notable that although several peptides showed significant differences between control and pd subjects no single peptide correlated with pd severity. this result resembles those observed for -synuclein, which is reduced in the csf of pd patients but does not correlate with disease severity. for example, some biomarkers show floor/ceiling effects, in which the maximal change occurs early in the disease and therefore no correlation is observed with further progression. furthermore, proteins involved in the disease process may undergo nonlinear changes, such as early compensatory up-regulation, followed by decreases accompanying more severe neurodegeneration. moreover, it must be considered that the current results were obtained in plasma, while the proteins measured may have originated in the cns. therefore, alterations in peripheral clearance or mechanisms of transfer across the bbb may change during the course of the disease as well, so plasma levels reflect a complex combination of disease processes. in the original selection of candidate peptides, we included peptides that changed 50% compared with controls. while this criterion is lenient, the low threshold could be justified in the preliminary steps, where the goal was choosing a pool of candidates for further analysis. because further elimination of candidates would occur in following steps, to narrow a large number of detectable peptides to a feasible number of promising candidates for srm, failing to identify a candidate was more problematic than including a peptide that may not really change. an additional limitation was the pooling of subjects used for the first test set. in the preliminary validation step, the primary goal was to identify which of the candidate peptides were most altered in the disease conditions. we followed our previous strategy of combining samples into small pools by disease status. this represents a compromise, limiting costs by decreasing the samples to be processed and analyzed but also allowing statistical analyses. this strategy does have statistical costs, most notably dramatic reduction of the power to detect subtle changes (due to the reduction from the number of subjects to the number of pools) and masking the true variability of individual subjects. however, this limitation can be justified in the context of a preliminary study, in which the goal was to rank peptide candidates by the magnitude of their changes. that is, while we may not have detected subtle differences between groups in some peptides, these differences may not be the changes of the greatest relevance. finally, an important aspect of the current study is the increase in efficiency of the use of large proteomics data sets. in biomarker research, unbiased studies can produce very large pools of candidate biomarkers, but validation of these candidates is hampered by limitations in assay development for individual proteins. thus, this strategy of prioritizing peptides that change in the cns under the disease condition, while moving to peripheral fluids and using sensitive, relatively high-throughput assays such as srm, may allow improved use of these data in the future. in conclusion, using a relatively large cohort totaling 282 subjects, several cns-related protein markers, implicating potential novel mechanisms in pd pathogenesis were readily detected in human plasma. several of them provided good diagnostic sensitivity/specificity for pd or correlated with pd severity. these results, if validated in independent investigations, could potentially help with establishing cns specific markers in blood for early disease diagnosis, monitoring disease progression or assessment of treatment effects.

despite extensive research, an unmet need remains for protein biomarkers of parkinson s disease (pd) in peripheral body fluids, especially blood, which is easily accessible clinically. the discovery of such biomarkers is challenging, however, due to the enormous complexity and huge dynamic range of human blood proteins, which are derived from nearly all organ systems, with those originating specifically from the central nervous system (cns) being exceptionally low in abundance. in this investigation of a relatively large cohort (300 subjects), selected reaction monitoring (srm) assays (a targeted approach) were used to probe plasma peptides derived from glycoproteins previously found to be altered in the cns based on pd diagnosis or severity. next, the detected peptides were interrogated for their diagnostic sensitivity and specificity as well as the correlation with pd severity, as determined by the unified parkinson s disease rating scale (updrs). the results revealed that 12 of the 50 candidate glycopeptides were reliably and consistently identified in plasma samples, with three of them displaying significant differences among diagnostic groups. a combination of four peptides (derived from prnp, hspg2, megf8, and ncam1) provided an overall area under curve (auc) of 0.753 (sensitivity: 90.4%; specificity: 50.0%). additionally, combining two peptides (derived from megf8 and icam1) yielded significant correlation with pd severity, that is, updrs (r=0.293, p=0.004). the significance of these results is at least two-fold: (1) it is possible to use a targeted approach to identify otherwise very difficult to detect cns related biomarkers in peripheral blood and (2) the novel biomarkers, if validated in independent cohorts, can be employed to assist with clinical diagnosis of pd as well as monitoring disease progression.

PMC4224986

pubmed-1275

langerhans ' cell histiocytosis (lch) is a proliferative disease characterized by monoclonal proliferation and the infiltration of organs with langerhans ' cells. several organ systems may be involved in lch including the lungs, bone, skin, pituitary gland, liver, lymph nodes, and thyroid. localized forms of lch in bone have been referred to as eosinophilic granuloma since lichtenstein first described them in 1940. the term " pulmonary langerhans ' cell histiocytosis (plch) " was first coined by farinacci in 1951 and refers to disease in adults that affects the lungs, either in isolation or in addition to other organ systems. multi-systemic variants of this disease are known by a variety of names, including systemic histiocytosis x, letterer-siwe disease, and hand-schuller-christian disease. to avoid confusion, the histiocyte society has established a simplified classification system. according to this system, plch is a disease in adults that affects the lungs, either in isolation or in addition to other organ systems. the most common findings on high-resolution computed tomography (hrct) of the chest are multiple nodular and cystic changes, which occur predominantly in the middle and upper lobes. nodular lesions are predominant in the early stage of plch and progress to cystic lesions in later stages of the disease. in korea, multiple cystic lesions are the main radiological findings of plch, and no cases with multiple nodular lesions without cysts have been reported. here, we report a case of plch with multiple nodules without cysts throughout the lungs. a 31-year-old male was admitted to our hospital for a cough and exertional dyspnea, which had been present for 2 months. he had no hypertension, tuberculosis, or diabetes, and no history of surgery, medication, or travel. his vital signs were blood pressure 120/80 mmhg, pulse 84/minites, respiration rate 20/minites, and body temperature 37.2. physical examination of the head and neck revealed no palpable cervical lymph nodes or masses and no neck vein engorgement. auscultation of the chest revealed a regular heart beat with no murmurs and clear breath sounds with no crackles or wheezing. laboratory examination indicated a white blood count of 10,310/mm, hemoglobin concentration of 16.0 g/dl, and platelet count of 202,000/l, and the chemistry was unremarkable. arterial blood gas analysis revealed a ph of 7.455, pao2 99.1 mmhg, paco2 38.2 mmhg, hco3 27 mmol/l, and 98% o2 saturation. the results of the pulmonary function test were forced vital capacity (fvc) 5.46 l/minites (105% of predicted), forced expiratory volume in 1 second (fev1) 4.59 l/minites (110% of predicted), and fev1/fvc 84%. we performed fiber optic bronchoscopy for bronchoalveolar lavage and observed no abnormal findings in the gram stain or culture, acid-fast bacillus stain or culture, tuberculosis-pcr, or cytology. the chest ct revealed variable-sized nodules with peribronchiolar or centrilobular distribution, some of which revealed thick-walled cavitary change (fig. the differential diagnosis based on the chest ct findings was that the pulmonary nodules represented hematogenous metastasis. histologically, multiple nodules showed an infiltration of eosinophils, lymphocytes, and langerhans ' cells (fig. treatment consisted only of smoking cessation, and a chest radiograph 4 months later indicated that the nodules had decreased in size. after 15 months, we observed that the symptoms had improved further, and a follow-up chest radiograph and hrct indicated that the multiple nodules had disappeared (fig. this case involved a young male who currently smoked and had multiple pulmonary nodules, which were suspicious of metastasis. plch is a rare disorder found in only 5% of biopsy-confirmed interstitial lung disease. reviewed 102 cases with histopathologically confirmed plch drawn from a group with a mean age of 40.3 years and a 1: 1.5 ratio of males to females and found that 95% of the cases involved smokers. the only consistent epidemiologic association is cigarette smoking, which is involved in the overwhelming majority (> 90%) of cases. several hypotheses have been proposed to explain the association between cigarette smoking and plch. according to one, cigarette smoke induces the secretion of bombesin-like peptides from neuroendocrine cells in the lungs. these peptides may have an important role in mediating lung injury and consequently induce lung fibrosis. other components of cigarette smoke, such as tobacco glycoprotein, have also been implicated in the pathogenesis of plch. nevertheless, plch occurs in a very small percentage of smokers, so genetic or environmental factors likely contribute to the development of this disease. a young male who currently smoked complained of a cough and exertional dyspnea, common clinical features of plch; however, the radiological findings did not support a diagnosis of plch. hrct of the chest is a useful, sensitive tool in the diagnosis of plch. the combination of diffuse, irregularly shaped cystic spaces with small peribronchiolar nodular opacities, predominantly in the middle and upper lobes, is highly suggestive of plch [6-8,13]. when these features are present on hrct, they allow the clinician to make a diagnosis of plch without a lung biopsy. the lung cysts of plch are often less than 20 mm in diameter and typically have thin walls (< 1 mm) [6-8]. the frequency of cystic change reflects the timing of imaging during the course of the disease [1,6-8]. in the early stages, the most common finding is nodular change, whereas in the later stages, cystic change and fibrosis predominate [6-8]. based on the radiological staging, this case represents an early stage of plch comprised of nodular lesions only without cystic change. this finding is rare; only one other case of plch with multiple cavitating pulmonary nodules has been reported, although several cases of plch presenting as a solitary pulmonary nodule have been reported worldwide. none of these cases progressed to a more severe stage, such as cystic change or reduced pulmonary function. brauner et al. reported two cases of plch in which only nodules were present: one progressed to cystic change and the nodular lesion disappeared in the other. it is thought that predominantly nodular lesions in plch appear early in the reversible state of the disease. in korea, a number of cases of plch have been reported based on variable radiological findings, including multiple cysts and a combination of nodules and cysts. however, no case involving only nodular lesions has been reported. atypical radiological findings as in this case need to be followed to determine whether sarcoidosis, pneumoconiosis, military tuberculosis, hypersensitivity pneumonitis, or metastatic lung nodules are present. the confirmative diagnostic tool is a pathological examination of a lung biopsy. in this case, an open lung biopsy was performed, which resulted in the diagnosis of plch based on positive staining for s-100 protein. the only treatment was smoking cessation, and clinical and radiological improvement was observed 15 months later. here, we report a case of early stage plch in a young male that had atypical radiological features, which consisted of variable-sized nodules only without cystic changes.

a 31-year-old man presented with a dry cough and exertional dyspnea. the chest x-ray showed multiple nodular opacities throughout the entire lung field. chest computed tomography (ct) revealed variable-sized nodules with a peribronchiolar or centrilobular distribution, some of which revealed thick-walled cavitary change. based on the chest ct findings, it was initially assumed that metastatic lung nodules with hematogenous spread were present; therefore, we performed an open lung biopsy. on microscopic examination, several compact cellular interstitial infiltrates composed of langerhans ' cells, eosinophils, and lymphocytes were observed. immunochemically, the langerhans ' cells showed strong cytoplasmic staining for s-100 protein. based on these findings, the patient was diagnosed with langerhans ' cell histiocytosis of the lung. high-resolution ct of the chest is a useful, sensitive tool in the diagnosis of pulmonary langerhans ' cell histiocytosis (plch). a typical radiologic finding of plch is irregularly shaped cysts. the radiological finding in this case of nodular opacities throughout the lung fields only without cysts is rare in plch. we report a case of plch with atypical multiple nodules mimicking hematogenous metastatic lung nodules.

PMC2784986

pubmed-1276

in high-income countries, growing numbers of men as well as women are choosing to postpone parenthood. on average, if sustained, this demographic shift may have manifold implications for the health of successive generations. we discuss here the evidence for a beneficial effect of increasing paternal age at conception (pac) on the health of descendants. basic genetics offers a strong foundation for hypothesizing that increased pac creates an increased risk for a host of disorders in offspring. in utero, the female germ cells undergo an estimated 22 cell divisions before meiosis and two divisions during meiosis. however, as only one chromosome replication takes place during meiosis, the female germ cells undergo a total of 23 chromosome replications. postnatally, the meiotic process is arrested at the first meiosis and this persists until puberty. thus, between the mother's birth and the conception of her offspring, her germ cells undergo no chromosome replication and only one cell division (regardless of her age at conception). for instance, the estimated cumulative numbers of germ-line stem cell (gsc) replications in men by the ages of 20 and 40 years are 150 and 610, respectively. it has long been recognised that rare conditions such as achondroplasia (the prevalence of which is one per twenty thousand) and marfan syndrome (which has a prevalence of one per five thousand) may arise from mutations in the male gscs. however, in the past decade, a large number of studies have linked increased pac to severe conditions that are not so rare in offspring, including autism, schizophrenia, and other neurodevelopmental disorders. it is often assumed, but not proven, that this is also due to mutations in the male germ line. perhaps because of the gravity of these diseases, studies now suggest, however, that older pac may also confer benefits on the health of offspring. if increased pac may have both adverse and beneficial effects, understanding the balance of its risks and benefits will require the consideration of a broad scope of relationships between increased pac and offspring health. yet we have only just begun to explore the potential benefits of older pac, and the evidence for them is still not widely understood. we therefore integrated work done across disciplines to articulate the case for a potentially major benefit of older pac on the basis of an intriguing finding in recent studies that leukocyte telomere length (ltl) is on average longer in the offspring of older fathers. this association of pac with offspring ltl has no threshold, as it has been observed for increasing pac from the age of 20 up to 60 years. as a longer ltl predicts reduced atherosclerotic risk and longer survival in the elderly, it is possible that older fathers, by endowing their offspring with a longer ltl, may also confer on them resistance to atherosclerosis and an advantage for increased longevity. that older pac is related to longer offspring ltl, it might even seem counter-intuitive, given the widespread awareness of genetic abnormalities related to both increased maternal age at conception (e.g., chromosomal aneuploidy) and pac (e.g., de novo mutations). the pac effect on offspring ltl is also perplexing on a deeper level, however, because of what it implies about age-related changes in the male germ line and how they are transmitted to offspring. yet the outcome of these age-related changes, namely, longer telomere length (tl), is probably transmitted in mendelian fashion. we discuss below how this enigma could be resolved. for understanding of the following discussion, it is important to recognize four major aspects of tl in general and ltl in particular. first, telomeres are the ttaggg tandem repeats at both ends of each of the mammalian chromosomes, and together with telomere-binding proteins they cap the chromosomes. this capping stabilizes the telomere and prevents the chromosomal ends from being recognized by the dna repair processes in cells as dna break points and potential sites of chromosomal fusions. second, as somatic cells replicate, their telomeres undergo progressive attrition because dna polymerase can not completely replicate the 3 ' end of linear duplex dna. once telomeres become very short, they often cause cells to exit from the replicative cycle and become senescent. third, ltl is a complex human genetic trait in that it is determined by many genes, and its dynamics (birth ltl and age-dependent telomere attrition thereafter) reflect telomere dynamics in hematopoietic stem cells. fourth, because the hematopoietic system is probably the most proliferative system among somatic tissues, ltl, and by inference tl in hematopoietic stem cells, can become critically short during the long human life course, thereby imposing a limit on the longevity of some individuals. the mechanisms underlying the association of pac with the ltl of offspring are not yet understood. genome-wide association studies (gwas) of ltl have deciphered a number of genes and genetic loci associated with ltl in the general population. however, it is very unlikely that the pac effect on offspring ltl is mediated through increased mutation load with age in the paternal germ line. such mutations are too rare to explain the pac effect on the offspring ltl, for the reason that the effect of older pac is manifested as a shift to a longer average ltl of the offspring in the population. because there is no corresponding increase in variance of the offspring ltl, this population shift does not merely reflect an increase in the small subset of persons with extremely long ltl. an important clue to the causal mechanism of pac on ltl is that while telomeres undergo age-dependent shortening in replicating somatic cells, tl is longer in sperm samples donated by older men than in those donated by young ones. thus, postulated mechanisms for the pac effect on ltl must ultimately explain the reason for why, on average, sperm cells of older men have longer tls than sperm cells of younger men. each sperm is a distinct genetic package, ensuring genetic diversity among a father s offspring. age might exert selection pressure at the level of the male gscs such that surviving gscs are those with relatively long telomeres. indeed, there is some evidence for the overrepresentation of sperm with longer telomeres in older men. another explanation for the effect of pac on ltl, not mutually exclusive with gsc selection, relates to the age-dependent elongation of telomeres in male gscs. this could be due to the difference in telomerase activity between somatic cells and male germ-line cells. telomerase activity is repressed after birth in most human somatic cells, including hematopoietic stem cells. by contrast, telomerase displays robust activity in embryonic stem cells and in the testes of humans and other mammals, presumably because the enzyme is active in male gscs. the activity of telomerase is usually fine-tuned to maintain the length of telomeres constant in telomerase-positive cells. it seems, however, that a small increase in tl (only a few base pairs) occurs with each replication of gsc in males, suggesting that in these cells telomerase overshoots its mark. because of the high number of replications of male gscs, a small elongation would result in considerable lengthening of sperm-cell tl over many replications. although sperm have a long tl, the tl of mammalian oocytes is relatively short and is evidently however, regardless of mechanisms that affect tl in the embryo and fetus, human tl is evidently inherited in an allele-specific manner. accordingly, ordinary mendelian principles would seem to suggest that since a child receives roughly half of its dna from its father, the slope of its ltl vs. pac should be approximately one half of the slope of tl in sperm vs. the ages of the sperm donors. the magnitude of the pac effect on offspring ltl is large, at 1520 base pairs of a longer ltl in the offspring for each year of pac. this is close to the average rate of age-dependent ltl attrition in adulthood, of 2030 base pairs per year. moreover, the pac effect on offspring ltl appears to be cumulative across successive generations. given the considerable magnitude of the pac effect on offspring ltl and its additive nature across successive generations, current demographic trends of an upward shift in paternal age might affect tl in future humans. from this standpoint, the pac effect on ltl is also directly relevant to the biological limit of human longevity. short human telomeres could, in theory, impose a limit on human longevity, but the pac effect suggests that human tl is malleable. it is therefore essential to factor in the pac effect on tl dynamics in the offspring when considering the question of whether life expectancy is approaching its ultimate ceiling in modern humans. the large magnitude of the pac effect on offspring ltl also suggests that it could be worthwhile to use this knowledge to explore possibilities for improving population health. before doing that, however, it is necessary to answer some basic questions about pac and ltl in life-course and cross-generational epidemiological studies. the main determinants of ltl at any age are ltl at birth and the magnitude of its attrition during growth. yet the hypothesis of mendelian transmission of a longer tl from the paternal germ line to the offspring predicts that offspring of older fathers would be conceived with a longer tl. by the time of conception, the tl of the paternal germ line would already have been lengthened by gsc selection or age-related elongation or both, and this longer tl would be inherited by the offspring. but this prediction of mendelian inheritance of epigenetic changes has not yet been tested, leaving open the question of whether there might be some more complex process by which pac exerts a latent effect on ltl that becomes evident in utero or during the first two decades of life. we have noted above that increased pac is associated with rare mutations and related diseases. if increased pac also has beneficial effects on offspring health via increased tl, then we need to pose the following question: might the pac effect be understood from the perspective of natural forces that shape human biology with respect not only to disease but also to evolutionary fitness? evolutionary theories suggest that delayed reproduction leads to increased longevity because of larger investment in maintenance and repair. this feature has been displayed in model organisms. for instance, selection based on delayed reproduction causes a pronounced increase in the lifespan of the fruit fly drosophila melanogaster. because increased pac reflects delayed reproduction in humans, and longer ltl might predict increased lifespan, the pac effect may be a manifestation in humans of the same phenomenon as seen in the fruit fly. moreover, in aging male fruit flies, the number of gscs is much smaller than that in younger male flies. such a phenomenon may be the result of a stochastic depletion of gscs or may reflect the ability of surviving gscs to withstand the accruing burden of aging-related stress, primarily in the form of oxidative stress. the variant genetic and more likely epigenetic constitution that distinguishes the surviving gscs from the non-surviving ones might provide a potential mechanism for transmitting increased fitness and longevity from fathers to offspring. studies across a variety of species suggest that tl and the expression of telomerase activity in somatic tissues have been fashioned by evolutionary forces. consider, for instance, the body sizes of terrestrial mammals (among which humans are viewed as moderately sized mammals) and their life spans. increased mammalian body size is associated with repression of telomerase, whereas increase in life span is associated with shorter telomeres. humans, the longest-living terrestrial mammals, have short telomeres and repressed telomerase activity in their somatic cells during extra-uterine life. in principle, tl might hence curtail lifespan in humans to a greater extent than in other mammals. a postulated evolutionary explanation for repressed telomerase activity with increased body size and diminished tl with increased life span is that these features protect against cancer through the reproductive phase of the life span. because somatic cells from relatively large and long-living mammals tend to undergo many more replications for growth and maintenance than do those of small, short-living mammals, they should be subject to a greater risk of cancer. thus far, however, there is no empirical evidence for associations of cancer risk with body size or longevity among mammals. in this context, little is known about the effect among humans of inter-individual variation in tl with respect to the cost of having relatively short telomeres (perhaps less cancer risk early in life and more cardiovascular disease risk later in life) or long telomeres (perhaps more cancer risk early in life and less cardiovascular disease risk later in life). the pac effect on the health of offspring and, more broadly, on public health, requires fundamental re-thinking and new directions in research. insight into this phenomenon and its links to human telomere biology will significantly advance the understanding of aging-related diseases in modern humans. overall, the evidence supports the view that in some contexts the male germ line might drive the evolution of human tl. if so, this would represent a substantial addition to haldane s conceptualization of the evolutionary force of male-biased mutations and the subsequent recognition that the evolution of dna sequences is largely driven through the numerous replications of the male germ line. because tl at birth and its rapid attrition during early life strongly influence tl throughout the human life course, an understanding of the pac effect on offspring tl requires going straight to the source, namely, tl at birth and its attrition during childhood. ongoing studies, exemplified by the avon longitudinal study of parents and children (alspac) in the uk and the norwegian mother and child cohort study (moba), might be used to achieve this goal in a cost-effective manner. lastly, it is clear that the enigma of the father s age and the health of his offspring are bound to engage telomere researchers, evolutionary biologists, epidemiologists, and demographers for quite some time.

what are the implications for population health of the demographic trend toward increasing paternal age at conception (pac) in modern societies? we propose that the effects of older pac are likely to be broad and harmful in some domains of health but beneficial in others. harmful effects of older pac have received the most attention. thus, for example, older pac is associated with an increased risk of offspring having rare conditions such as achondroplasia and marfan syndrome, as well as with neurodevelopmental disorders such as autism. however, newly emerging evidence in the telomere field suggests potentially beneficial effects, since older pac is associated with a longer leukocyte telomere length (ltl) in offspring, and a longer ltl is associated with a reduced risk of atherosclerosis and with increased survival in the elderly. thus, older pac may cumulatively increase resistance to atherosclerosis and lengthen lifespan in successive generations of modern humans. in this paper we: (i) introduce these novel findings; (ii) discuss potential explanations for the effect of older pac on offspring ltl; (iii) draw implications for population health and for life course; (iv) put forth an evolutionary perspective as a context for the multigenerational effects of pac; and (v) call for broad and intensive research to understand the mechanisms underlying the effects of pac. we draw together work across a range of disciplines to offer an integrated perspective of this issue.

PMC3619950

pubmed-1277

frequently, the introduced air is limited to the subcutaneous space and leads to local swelling and crepitus in the absence of any signs of inflammation such as erythema, edema, tenderness or lymphadenopathy [1, 2]. however, there is a potential risk that the air disperses along the facial planes to the periorbital and mediastinal spaces. in the literature, the most common causes of subcutaneous emphysema are the use of air turbine handpieces [2, 3] and air syringes [2, 3]. in addition, subcutaneous emphysema can be caused by coughing, blowing, smoking, vomiting and using straws after a dental treatment. gilles de la tourette syndrome (gts) is a disorder that is characterized by multiple motor tics, especially affecting cranial and upper limb muscles and vocal tics. although several gts patients can temporarily suppress their tics to some extent, tic emergence is rather random and intrusive. therefore, it has been suggested that tics in gts patients are associated with an insufficient inhibitor motor control [6, 7]. to the best of our knowledge, the development of a subcutaneous emphysema in the context of the presence of gts has not been described yet. in this case report, a patient with gts who developed a subcutaneous emphysema following surgical wisdom tooth extraction is presented. a 30-year-old man was referred to the department of oral and maxillofacial surgery because of a soft tissue swelling in the facial area. this diffuse facial swelling had developed after bilateral surgical wisdom tooth extraction in the mandible in local anesthesia on the same day in a private dental office. gts is characterized by repeated, quick movements or sounds, which can not be controlled by the patients. this patient permanently repeated valsalva maneuvers and pressed air against his closed lips. at admission, the patient was hemodynamically stable, afebrile and the function of the facial and trigeminal nerves were normal. in addition, no general malaise was existing, and the c-reactive protein (crp) level was within normal ranges. clinically, an extensive diffuse swelling in the whole facial area was visible, and the patient had difficulty in opening his eyes (figs 1 and 2). mouth opening was slightly limited (cutting edge inter-incisor distance: 36 mm). on palpation, there was a manifest crepitus of the periorbital region, the cheek and the supraclavicular fossa. the floor of the mouth and the pharyngeal region were unaffected, and the patient was able to swallow but showed commencing respiratory distress. figure 1:clinical appearance of the patient with distinct swelling in the periorbital, cheek and supraclavicular region after bilateral wisdom tooth removal. clinical appearance of the patient with distinct swelling in the periorbital, cheek and supraclavicular region after bilateral wisdom tooth removal. the patient had difficulty to open his eyes. computed tomography (ct) of the head and neck region revealed a bilaterally located subcutaneous air collection in the area of the periorbital, temporal, paramandibular and supraclavicular region (figs 35). there were no signs or symptoms of severe complications such as pneumothorax or pneumomediastinum. figure 3:ct scan (coronal section in the first premolar region) showing multiple air inclusions within the facial soft tissue. figure 4:the frontal ct scan of the head and neck region (section in the retromolar region) illustrates bilaterally trapped air into the soft tissue reaching from the temporal fascia through to the supraclavicular region. figure 5:three-dimensional reconstruction of the ct scan showing the trapped air in the head and neck region. ct scan (coronal section in the first premolar region) showing multiple air inclusions within the facial soft tissue. the frontal ct scan of the head and neck region (section in the retromolar region) illustrates bilaterally trapped air into the soft tissue reaching from the temporal fascia through to the supraclavicular region. three-dimensional reconstruction of the ct scan showing the trapped air in the head and neck region. because of the extensive emphysema, the patient was admitted for an inpatient monitoring. in order to avoid an infection, 2x/d for 2 days, pfizer ag, zrich, switzerland) were prescribed. in order to prevent the tic-induced powerful injection of air into the soft tissue, caused by the repetitive valsalva maneuvers, a 10-cm-long small tube (redon drainage tube cut in shape) was given to the patient, and the correct handling was instructed. by applying this tube, the building-up air pressure within the patient's oral cavity figure 6:the patient after 3 days with tube in place, which served as a valve to avoid air pressure within the oral cavity. the patient after 3 days with tube in place, which served as a valve to avoid air pressure within the oral cavity. after 3 days, the facial swelling was reduced approximately by half, and the subcutaneous crepitus was almost undetectable, and so the patient could leave the hospital. oral antibiotic prophylaxis was continued for 7 days (amoxicillin/clavulanic acid, 875/125 mg tid; augmentin). at that time this case report was submitted and approved by the ethics committee of northwest and central switzerland (eknz). subcutaneous emphysema following dental procedures, albeit rare, might cause severe complications including pneumomediastinum and mediastinitis. spreading air in subcutaneous tissue increases the risk of subsequent connective tissue infections. due to the communication of the facial spaces with the mediastinum, there is an increased risk for spreading of bacteria and the development of life-threatening infections of the retropharyngeal space and the mediastinum [2, 8, 9]. in principle, two factors are prerequisite for the formation of a subcutaneous emphysema: (i) air forced under pressure and (ii) a communication between the oral cavity and the subcutaneous tissue. in this case, the combination of bilateral surgical wisdom tooth extraction in the mandible and the presence of the motor tic to press air against the resistance of the closed lips caused by gts led to the distinct subcutaneous emphysema. the formation mechanism in this specific case is comparable with the first published case of a subcutaneous emphysema more than 100 years ago: a horn blower, who played his instrument immediately after a tooth extraction. as motor tics of gts patients can hardly be controlled, the usage of a redon drainage was instructed in order to prevent the patient from elevating pressure within the oral cavity. the diagnostic work-up of a patient with suspected subcutaneous emphysema includes a clinical examination, laboratory analysis (crp levels) and a ct of the affected region. following the exclusion of potentially life-threatening complications, antibiotic prophylaxis to avoid infections concluding, a sudden swelling occurring after tooth extraction might be due to a subcutaneous emphysema. a thorough clinical examination has to be performed, and the patient should be referred to a specialized maxillofacial clinic.

subcutaneous emphysema is a rare complication in oral surgery. in most cases, it resolves spontaneously. however, air might disperse into deeper facial spaces causing life-threatening complications such as compression of the tracheobronchial tree or the development of pneumomediastinum. moreover, microorganisms might spread from the oral cavity into deeper spaces. hence, rapid diagnosis of subcutaneous emphysema is important. characteristic signs are both a shiftable swelling and crepitation. in this case report, a 30-year-old man, suffering from the gilles de la tourette syndrome, with a distinct subcutaneous emphysema after bilateral surgical wisdom tooth extraction is presented. induced by a specific motor tic, air accumulated from the periorbital through to the parapharyngeal region. applying a 10-cm-long redon drainage tube as air valve, 10 days after wisdom teeth extraction, the patient was asymptomatic with complete resolution of the emphysema.

PMC4466419

pubmed-1278

traumatic cervical artery dissection (tcad) represents a rare but under certain circumstances life-threatening injury. due to improvements in the diagnostic algorithms in the emergency room, blunt the most common injury mechanisms are combined movements of distraction and extension, distraction and flexion, and lateral flexion. the formation of a thrombus at the site of the intimal lesion can lead to an ischemic neurologic deficit if cerebral blood flow is compromised or embolization to the brain occurs. even injuries of the vertebral arteries alone without any lesion of the carotid arteries can lead to symptomatic cerebral lesions. interestingly, there seems to be no correlation between the size of the occlusion and the neurologic outcome.1 recent studies have shown tcad in 1.0 to 3.7% of all blunt trauma admissions.2 3 in patients at risk for tcad, aggressive screening has revealed an incidence of 13 to 39% of those serious vascular injuries.2 4 neurologic deficits were present in more than a quarter of patients suffering from tcad.4 cases of traumatic quadruple lesions of the cervical arteries have exhibited pronounced neurologic deficits.5 6 the following case report describes tcad of all four cervical arteries after a high-energy horse-riding injury of a woman with no resulting neurologic deficit. a 45-year-old woman was admitted to the emergency department after falling off a galloping horse without wearing a helmet. the patient was treated according to advanced trauma life support guidelines. at a glasgow coma scale of 15 points, the patient initially complained of right-sided chest pain and exhibited progressive respiratory insufficiency. after successful treatment of hematothorax with a thoracic drain, the patient was intubated and put under controlled ventilation. diagnostics revealed right-sided pulmonary contusion, fracture of the sixth through tenth ribs, and fracture of the scapula as well as a compression fracture of the second lumbar vertebra, accompanied by extensive soft tissue contusions of the right side of the neck and shoulder. following extubation and neurologic examination, the patient demonstrated paresis of abduction and anteversion of the right arm that was clinically allocated to an upper brachial plexus injury. magnetic resonance imaging (mri) of the right shoulder revealed a diffuse accumulation of fluid around the upper right brachial plexus. a secondary finding was an elongated dissection of both vertebral arteries in the v2 segment (fig. immediate mri examination of head and neck showed no signs of thromboembolic lesions and cerebral ischemia. however, imaging did show stenosis due to intimal flaps of both carotid arteries between the carotid bifurcation and the base of the skull without signs of thrombosis (fig. the diagnosis of quadruple cervical arterial injury was confirmed by computed tomography angiography (cta; fig. after 1 week, anticoagulation with heparin was stopped and antiplatelet therapy was initiated. images from 1.5-t avanto (siemens, munich, germany) magnetic resonance imaging (mri). flow voids in both common carotid arteries (arrows), indicating arteries are perfused normally. thin flow voids in both vertebral arteries (va), indicating reduction of arterial lumen (open arrows). flow voids in both vas (open arrows) but only partial flow voids in both internal carotid arteries (arrows), indicating reduced perfusion. computed tomography angiography on 16-slice sensation (siemens, munich, germany). (a) normally perfused common carotid arteries (white arrows) but filiform-perfused vertebral arteries (va) (black arrows) at the level of the larynx. (b) filiform-perfused internal carotid arteries (ica) and normally perfused vas at the base of the skull. (c) regular perfusion of basilar artery and both icas at level of carotid canal. at the follow-up examination after 3 months mri after 3 months showed that all arterial lesions had regressed in size (fig. (a) both common carotid arteries and vertebral arteries (va) open with residual reduction of lumen of right va. (b) at the base of the skull, all four arteries are perfused; however, both internal carotid arteries still show reduced lumen. as far as we know, there has to date been no other case of an asymptomatic dissection of all four brain-supplying arteries originating from high-energy trauma to the neck. this case is highly interesting due to three facts: (1) asymptomatic tcad may be easily missed. (2) cta should be routinely performed in the emergency diagnostic procedures according to the patient's history. of all diagnostic modalities, digital subtraction angiography (dsa) reaches the highest level of sensitivity and specificity in the detection of vascular lesions and represents the gold standard. the diagnostic accuracy of cta varies between institutions depending on the quality of the ct as well as the quality of image interpretation by the radiologist.7 in a recent prospective study, only 68 of 112 patients with cta results suggestive of tcad were confirmed by dsa.8 meta-analysis of the diagnostic accuracy of cta and dsa in the detection of traumatic cervical vascular injuries showed that sensitivity of cta (66%) was inferior to dsa, yet cta specificity (97%) was high. it was suggested that patients with cervical artery injury detected by cta, who suffered accompanying injuries indicating high risk of tcad, may receive treatment without confirmation of diagnosis by dsa.7 in clinical practice, cta is faster, less prone to complications, and more useful for diagnosing secondary injuries.9 in the face of these advantages on top of the increasing sensitivity of modern ct instruments, cta has become more and more important for the diagnosis of traumatic arterial dissections.10 with regard to our trauma protocol in the emergency room, cta has been included into the secondary trauma survey in the presence of a patient history indicating high risk of tcad. in contrast to dsa, not only does mri angiography allow screening of tcad, but it is also capable of detecting thromboembolic cerebral lesions.11 in addition, many patients with multiple injuries do not tolerate further invasive and sometimes long-lasting diagnostic procedures such as dsa. because diagnosis of quadruple arterial dissection was positive in both cta and mri angiography, we did not perform dsa as a third diagnostic procedure after risk-benefit evaluation. in our case, surgical therapy of the vascular injuries was never considered due to the fact that neurologic deficit, signs of acute hemorrhage, or ischemia was not detected. in fact, tcad may be more benign than previously thought and therefore only requires aggressive surgical treatment in very rare cases.12 finally, any surgical procedure to be performed in a patient with multiple injuries has to be carefully evaluated. this latter point clearly makes the difference between a patient with tcad and a patient suffering from chronic stenosis of the carotid arteries. most authors agree that some form of antithrombotic therapy is necessary after diagnosis of those injuries, although the overall incidence of ischemic stroke among patients with tcad is low.8 however, the type of antithrombotic therapy is controversial. the use of heparin is more popular than antiplatelet therapy, but the risk of hemorrhagic complications is high.2 13 14 15 16 nevertheless, we decided to use heparin in our patient with multiple injuries because of its capability to maintain continuous and immediate control of anticoagulation. after 1 week, during which the patient remained asymptomatic, her vital functions had stabilized, and no surgical procedures had been necessary, the treatment was changed to antiplatelet therapy. the latter have been found to be equivalent or even superior to heparin therapy with regard to thromboembolic complications, but with a more favorable profile of potential side effects. 17 18 19 this is in agreement with a recent study by cothren and coworkers.17 the authors looked at the therapeutic options after blunt tcad and clearly demonstrated that the type of treatment, heparin versus antiplatelet agents, does not appear to significantly affect either the risk of stroke or the injury healing rates.17 we conclude from the experience with our case and the recent literature that anticoagulation in the presence of tcad is necessary but may be different from patients with chronic atherosclerosis. we recommend heparin at the beginning because of its excellent capability of dose and effect regulation.

study design case report and review of the literature. objectives case report of a traumatic dissection of all major brain-supplying arteries resulting from a horseback-riding accident. overview of the literature on diagnostic and therapeutic recommendations. methods case presentation. for the discussion, handpicked articles and pubmed database research with the keywords dissection, vertebral artery, spine trauma, computed tomography, magnetic resonance imaging, and angiography were used. results despite high-energy induced acute lesion of all four cervical arteries, this 45-year-old patient did not demonstrate signs of microemboli nor suffer from stroke. conclusion in case of high-energy trauma of the head and/or the neck, emergency physicians must consider traumatic cervical artery dissection (tcad). thus, emergency care algorithms should routinely include computed tomography angiography and magnetic resonance imaging. although the incidence of tcad-induced stroke is low, antiplatelet therapy is recommended in the presence of tcad.

PMC4111942

pubmed-1279

the apoptotic cell death pathway, in its basic tenents, is conserved among metazoans. during development, resistance to apoptosis is a hallmark of cancer, whereas excessive programmed cell death is associated with degenerative diseases. apoptosis is controlled and amplified through a variety of mechanisms, many of which converge on release of proapoptotic factors from mitochondria (danial and korsmeyer, 2004). (2011) now uncover how regulated trafficking of the proapoptotic protein bax off mitochondria and into the cytosol prevents mitochondrial permeabilization, reconciling discordant models for how antiapoptotic proteins on the mitochondrial surface inhibit proapoptotic proteins in the cytosol. the permeabilization of mitochondria is controlled by proteins belonging to the bcl-2 family, which is composed of anti- and proapoptotic members sharing homology in short stretches of amino acids called bcl-2 homology (bh) domains. the prosurvival members, including bcl-2, bcl-xl, and mcl-1, contain bh domains 14 and antagonize the prodeath members. the multidomain proteins, such as bax and bak, contain bh domains 13 and directly mediate mitochondrial permeabilization, whereas the bh3-only proteins (e.g., bid, bim, bad, etc.) are thought to act as sensors of cellular stress (danial and korsmeyer, 2004). despite clearly defined roles for the bcl-2 family members, two major questions have remained: how are proapoptotic members activated, and how do the antiapoptotic members inhibit them? the simplified rheostat model, first proposed in the early 1990s, suggests that pro- and antiapoptotic proteins directly counterbalance each other, with the more abundant protein dictating whether the cell dies or survives (danial and korsmeyer, 2004). complicating this model is the existence of two types of bh3-only proteins: the activators that directly activate the proapoptotic multidomains and the sensitizers that neutralize the antiapoptotic inhibition of the multidomain proteins (letai et al., 2002). a modified version of the rheostat model postulates that activator bh3-only proteins act as receptors for multidomain proapoptotic proteins on the mitochondrial outer membrane (om), whereas antiapoptotic proteins act like a sponge, soaking them off of the mitochondrial surface (lovell et al., a mutually exclusive model postulates that the antiapoptotic proteins directly bind and inhibit bax and bak, with bh3-only molecules acting as inhibitors of the inhibitors (youle and strasser, 2008). this latter model suffered from the lack of evidence that could explain the spatial paradox of this direct inhibition: under normal circumstances, bax is in the cytosol, whereas bcl-2 is on the surface of mitochondria. upon induction of apoptosis, bax accumulates on the mitochondrial om, undergoes conformational changes to expose a helical domain, and triggers the release of cytochrome c and other proapoptotic effectors. to gain insight into the regulation of bax, edlich et al., (2011) examine the localization of a mutant version of bax that is constrained in its cytosolic conformation by engineered disulfide bridges. surprisingly, this mutant protein, which does not interact with the mitochondrial antiapoptotic protein bcl-xl, localizes to mitochondria but does not induce apoptosis. following up on this observation, the authors ask whether bax normally translocates to the mitochondrial surface. using fluorescence loss in photobleaching, they monitor the localization of gfp-bax in cells and find that bax cycles on and off the mitochondria in healthy cells. the presence of antiapoptotic proteins in the mitochondrial om is required to constitutively retrotranslocate bax into the cytosol. notably, the rate of retrotranslocation is almost doubled in cells overexpressing bcl-xl and requires the physical interaction between bcl-xl and bax. once in the cytosol, bax quickly returns to its monomeric form, ready to cycle back to the mitochondrial surface. edlich and coworkers observe an acceleration of the retrotranslocation rate upon the coexpression of bcl-xl and bax. similarly, the bcl-2/bcl-xl inhibitor abt-737, as well as bh3-only proteins, disrupts the equilibrium between cytosolic and mitochondrial bax by reducing its retrotranslocation (figure 1). this new study provides a new paradigm for understanding bax regulation during apoptosis, but how the two categories of bh3-only proteins antagonize bax retrotranslocation by bcl-xl remains an open question. one possibility is that the activators (e.g., bid and bim) directly inhibit bax retrotranslocation by unmasking its 9 helix and anchoring bax in the mitochondrial om. conversely, the sensitizer (e.g., bad) could obstruct the binding of bax to bcl-2. the interplay between proapoptotic and antiapoptotic proteins is part of the normal regulation of the trafficking of bax in healthy cells. under nonapoptotic conditions, the equilibrium between the cytosolic and mitochondrial pools of bax is maintained by its bcl-xl-dependent retrotranslocation. during apoptosis, however, this balance may be tipped by bh3-only proteins, which increase the insertion of bax into the mitochondrial om (figure 1). thus, the retrotranslocation model is an important step forward, reconciling two opposing models of how bh3-only, multidomain, and antiapoptotic proteins cooperate to dictate whether a cell lives or dies (e.g., cheng et al., 2001; lovell et al., 2008 versus willis et al., in addition to this new model, edlich and colleagues also find that the weak interaction between bcl-xl and bax is sufficient to extract bcl-xl from the mitochondrial om and transiently localize it to the cytosol. this highlights an unappreciated conformational change that facilitates the removal of an antiapoptotic protein from the mitochondrial om. interestingly, the rate of bcl-xl retrotranslocation increases upon treatment with abt-737, raising the possibility that inactive bcl-xl might be predominantly cytosolic. it will be important to verify whether this is a feature common to extramitochondrial antiapoptotics, such as endoplasmic reticulum (er)-localized bcl-2/bcl-xl, and to understand the mechanism by which interaction with bax inactivates the transmembrane domain of bcl-xl. interestingly, a fraction of inactive bax is associated with intracellular membranes, including mitochondria and er in transformed cells, but not primary hepatocytes (scorrano et al., 2003). understanding how oncogenic transformation influences the on and off rates of bax translocation could open new perspectives to apoptosis in cancer cells. from a teleological perspective, one might wonder why a cell would maintain a continuous flux of bax cycling on to the mitochondria and then back to the cytosol. one reason could be to prime cells to rapidly execute death, positioning bax in a ready to attack mode such that minor changes in the kinetics of bax retrotranslocation can swiftly induce apoptosis. another possibility is that bax has additional functions that require its regulated presence on cellular membranes. interestingly, inactive bax controls transient membrane events such as mitochondrial fusion (hoppins et al., 2011) and, however, bax can not remain on the organelle without becoming active and promoting apoptosis (nutt et al. the retrotranslocation model not only provides a paradigm for understanding how apoptosis is regulated, but also provides a platform for integrating the diverse functions of bax and other bcl-2 family members.

antiapoptotic bcl-2 proteins on mitochondria inhibit prodeath proteins, such as bax, which are found primarily in the cytosol. in this issue, edlich et al., (2011) show that bax and bcl-xl interact on the mitochondrial surface and then retrotranslocate to the cytosol, effectively preventing bax-induced permeabilization of mitochondria.

PMC3072571

pubmed-1280

oxygen-sensing mechanisms enable the cell to adapt to low oxygen environments and are also critical for normal development and apoptosis. disruptions of oxygen-sensing pathways can lead to the development of certain forms of cancer [115]. oxygen sensing is mediated partly via the egln hydroxylases (egln1/egln2/egln3) that are dependent upon molecular oxygen, iron (ii) and -ketoglutarate to perform proline hydroxylation on their target [1620]. therefore these enzymes are considered oxygen and also metabolic sensors. the availability of molecular oxygen is absolutely required for the hydroxylation reaction, because it donates the oxygen atom to the hydroxyl group. but equally important for the hydroxylation reaction is the electron donor -ketoglutarate, a metabolite from the krebs cycle. since egln is dependent upon metabolites that take part in the krebs cycle, they are directly tied into the cellular metabolic network (fig. egln prolyl hydroxylases (also referred to as phd) belong to the 2-oxoglutarate-dependent oxygenase superfamily. the reaction proceeds via the incorporation of dioxygen into the prolyl residue forming hydroxyproline at the substrate. as electron donor for this reaction serves -ketoglutarate (= 2-oxoglutarate) which is oxidized to succinate and co2. the dependency of the metabolite -ketoglutarate for catalytic activity makes these enzymes sensitive to -ketoglutarate levels and ties them directly into the metabolic network. the first identified substrate for the egln prolyl hydroxylases is the transcription factor hypoxia inducible factor (hif-) [16, 17]. the identification of hif- as a direct hydroxylation substrate provided the first and direct link between tumour suppressor function and oxygen sensing. the tumour suppressor von hippel lindau (vhl) acts as an ubiquitin ligase by targeting hydroxylated hif- for degradation when oxygen is available, as where under low oxygen environments, hif- escapes hydroxylation and subsequently escapes vhl recognition [35, 16, 2227]. the escape from vhl recognition allows hif- to accumulate and to transactivate its target genes that are important for adaptation to low oxygen environment including energy metabolism and angiogenesis [2830]. both hif-dependent and hif-independent vhl functions contribute to vhl-defective tumorgenesis [3134]. the vhl disease is caused by inactivating germline mutations of the vhl gene and predisposes to a variety of tumours including haemangioblastoma of the retina and nervous system, clear cell renal carcinomas (rcc; the most common form of kidney cancer) and phaeochromocytomas/paragangliomas, tumours of the sympathoadrenal nervous system. hif- deregulation appears to have a causal role in vhl-defective clear cell rcc and in vhl-defective blood vessel tumours haemangioblastomas (hb). although hif- regulation has been a major focus of vhl research, the genotype phenotype correlation in the vhl disease gave insight into hif-independent vhl function (table 1). specific vhl germline mutations corresponding to a specific subset of tumour phenotypes have a different relationship to hif- deregulation (categorized in type i/iia/iib/iic disease) [3537]. the type i disease associates with rcc and hb, and their tumours reflect deregulated high hif- expression, whereas a very different clinical outcome is observed in the type 2c-vhl disease. more importantly, hif- is not deregulated and maintains in low levels in the type 2c tumours [31, 32]. these findings suggest that hif- deregulation is not necessary for phaeochromocytma development in vhl disease. phenotype correlation in the vhl disease interestingly, a second hydroxylation target of the prolyl hydroxylases egln has recently been identified. the 2-adrenergic receptor is a prototypic g protein-coupled receptor, which is hydroxylated by egln3 and also oxygen dependent degraded by pvhl. the discovery of another hydroxylation substrate by the egln enzymes not only broadens the functionality of prolyl hydroxylation, but also expands our understanding of cellular response to oxygen and its relationship to disease. therefore, the identification of other egln substrates and the clues from the genotype phenotype correlation that emerged in the vhl disease clearly indicates hif-independent vhl functions. of great insights are studies from egln3-mediated neuronal apoptosis during sympathetic neuronal development, which shed light onto the genesis of phaeochromocytoma by presumably hif-independent pathways. phaeochromocytomas are rare, with only five to eight cases diagnosed per million people a year. extra-adrenal phaeochromocytomas are sometimes referred to as paragangliomas. in short, these tumours are sympathetic nervous system tumours. the most frequent causes of phaeochromocytoma susceptibility are vhl missense mutations, activating mutations in c-ret, mutations in neurofibromatosis type 1 (nf1) and mutations in succinate dehydrogenase subunits of mitochondrial complex ii (sdh b/c/d). loss of nf1 has been reported to promote survival of embryonic sympathetic neurons in the absence of the nerve growth factor ngf. further, mutations in subunits of mitochondrial complex ii suggest impairment of mitochondria-mediated apoptosis and have led to the early hypothesis that failure of apoptosis in neuroendocrine precursor cells could result in the development of phaeochromocytoma and paraganglioma. given the evidence that nf1 promotes sympathetic neuronal survival and further that these neoplasias originate from the sympathetic nervous system, a closer look into the sympathetic neuronal development reveals important clues to the genesis of these tumours. since the discovery by rita levi-montalcini and viktor hamburger of the neurotrophic factor ngf, our understanding of developmental apoptosis in the sympathetic nervous system has greatly increased. during neuronal development competition for ngf is an important developmental process for matching the size of a neuronal population, especially in the peripheral nervous system. as much as 50% of neurons produced during embryogenesis die by apoptosis during neuronal development. abnormal ngf signalling has been linked to paediatric nervous system tumours such as neuroblastoma [44, 45] and disease-associated mutations such as nf1 have been shown to enhance signalling by ngf receptors and promote neuronal survival in the absence of ngf. in the last decades it became evident that jnk/c-jun signalling is required for apoptosis when ngf is limiting in the developing nervous system [4650]. egln3 is induced in sympathetic neurons deprived of ngf and further has pro-apoptotic activity when overexpressed. given that egln3 is known to hydroxylate hif- and has been implicated in developmental apoptosis in sympathetic neurons, the following questions arise: (i) does egln3-mediated apoptosis depend upon its hydroxylation activity? (ii) does it depend upon hif hydroxylation or does it involve hydroxylation of unknown substrates? (iii) is failure of egln3-mediated apoptosis implicated in the genesis of phaeochromocytomas and other tumours arising from the neural crest origin? the ability of egln3 to induce neuronal apoptosis appears to be unique among the egln family members. induction of apoptosis is dependent upon egln3 hydroxylation activity, because catalytic impaired egln3 fails to induce apoptosis [9, 52]. importantly, egln3-induced apoptosis is not diminished in the presence of stable hif1 or hif2 variants that can not be hydroxylated on their prolines. this suggests that hydroxylation targets of egln3 other than hif- are crucial for apoptosis function. egln2 and egln3 are induced by hypoxia and dampen the hif- response under chronic hypoxia [5357]. however, egln1 appears to be the primary hif prolyl hydroxylase under normal conditions. consistent with this, mice lacking egln2 or egln3 are viable and grossly normal whereas mice lacking egln1 are not viable. conditional inactivation of egln1 in mice leads to polycythemia due to hif- stabilization and increased transcription of hif target genes including erythropoietin [60, 61]. further, patients carrying egln1 mutations have been reported to develop polycythemia concluding that egln1 couples hif- stability in vivo[13, 62, 63]. consistent with the unique role of egln3 in neuronal apoptosis are studies obtained from the egln3-deficient mice that clearly demonstrates the requirement for egln3-mediated apoptosis during the sympathetic neuronal development. egln3 deficient sympathetic neurons are resistant to apoptosis after ngf withdrawal, as well as certain neurotoxins. consistently, egln3 mice have an increased number of cells in the super cervical ganglia and in the adrenal medulla and show abnormalities in the sympathoadrenal system including systemic hypotension. in fact, it appears that some of the adrenal abnormalities are caused through deregulation of the 2-adrenic receptor (2-ar). loss of egln3 results in 2-ar up-regulation and accordingly, hypoxia stabilizes the 2-ar. this is consistent with the observations from the type 2c vhl patients that develop phaeochromocytoma. type 2c patients often show excessive secretion of catecholamines (endogenous 2-ar ligands) and increased sympathonervous system activity. in summary, this points towards distinct and unique functions within the family of egln prolyl hydroxylases. identification of novel egln hydroxylation targets will open new oxygen sensing pathways independent of what we have learned from hif-. given the evidence that (i) phaeochromocytomas are sympathetic nervous system tumours and that (ii) egln3 is critical for apoptosis during sympathetic neural development, an attractive hypothesis emerged in which failure of egln3-mediated apoptosis during sympathetic development predisposes to the genesis of phaeochromocytomas and perhaps other neoplasia arising from neural crest origin. interestingly, apparently unlinked phaeochromocytoma lesions (vhl, nf1, c-ret and sdh) all appear to act on a single common pathway by decreasing egln3-mediated apoptosis at the time during development when levels of ngf become limiting (fig. egln3 appears to act downstream of the c-jun in the ngf signalling pathway. therefore, a single pathway was established in which the genetic phaeochromocytoma defects act either directly on egln3 or upstream of egln3 to impair apoptosis (fig. 2). for instance, the genetic defect in sdh acts directly on egln3-mediated apoptosis function. sdh inhibition results in accumulation of intracellular succinate, which in turn can product-inhibit the egln prolyl hydroxylases [9, 71]. this succinate-mediated inhibition not only results in hif stabilization, but also importantly inhibits egln3-mediated apoptosis. the succinate-mediated product-inhibition of egln3 was overcome by re-addition of -ketoglutarate restoring ngf-mediated apoptosis. this suggests that sdh inhibition acts on the prolyl hydroxylases via succinate and not as alternatively suggested through the generation of reactive oxygen species., ngf is required for neuronal survival but is also limiting. neurons that successfully compete for ngf survive whereas unsuccessful neurons undergo c-jun-dependent apoptosis. c-jun transcriptionally activates prolyl hydroxylase egln3. as ngf levels become limiting, familial phaeochromocytoma mutations (vhl, nf1, c-ret, sdh and kif1b) all decrease apoptosis mediated by egln3. in addition to the predisposing sdh genetic lesion, other phaeochromocytoma lesions have been implicated upstream of egln3 to promote neuronal survival in the ngf signalling pathway. in the case of vhl predisposing lesion, pvhl suppresses junb and all vhl mutants tested (including type iic vhl disease mutant) abrogate its ability to do so. junb acts as an antagonist of c-jun and increased junb levels attenuate the induction of c-jun-mediated apoptosis in sympathetic neurons deprived from ngf. finally, previous evidence indicated that ret (the receptor for glial-derived neurotrophic factor) and nf1 could act through this pathway by modulating the action of the ngf receptor trka. activation of c-ret, like loss of pvhl, leads to the induction of junb and attenuates apoptosis when ngf becomes limiting. thus, when mutated, all the genetic phaeochromocytoma lesions (sdh, vhl, c-ret and nf1) impair ngf-mediated apoptosis in neuroendocrine precursors during development. these findings provide an explanation as to why the mutations in tumour suppressors that are found in familial phaeochromocytoma are rare in sporadic phaeochromocytoma. this is because the pathway is no longer critical once development is completed. in summary, all the genetic lesions associated with phaeochromocytoma act on a single common pathway that impinges upon egln3 apoptotic activity during sympathetic neuronal development. mutations in sdh, vhl, c-ret and nf1 would allow sympathetic neuronal precursors to escape from developmental apoptosis and set the stage for their neoplastic transformation. it will be interesting to determine not only if egln3 is mutated in these neoplasias, but also, if failure of egln3 developmental apoptosis plays a broader role in paediatric cancers and other forms of hereditary cancer. an important challenge is to identify the link between this enzyme and apoptosis, which presumably involves hydroxylation of a protein other than hif-. early studies indicated that egln3 mrna and protein expression (at that time referred as sm-20) increases shortly after removal of ngf in primary sympathetic neurons and peaks between 10 and 15 hrs at a time when cells undergo apoptosis. overexpression of egln3 is sufficient to promote cell death in ngf-maintained sympathetic neurons [51, 73]. cell death is caspase dependent and accompanied by an increase of cytochrome c in cytosolic and mitochrondria-enriched subcellular fractions. the mechanism underlying egln3-induced cytochrome c is not known although it appears to involve an increase in cytochrome c mrna. other studies have identified egln3 as a growth factor inducible gene in vascular smooth muscle cells and later proposed, that it might function in growth arrest, differentiation and cell death during muscle differentiation [76, 77]. expression of egln3 was also reported in fibroblasts upon activation of a temperature-sensitive form of p53. however, egln3-induced apoptosis appears not to be impaired in cells lacking p53 or expressing a dominant negative p53 protein, indicating that p53 might not function downstream of egln3 to induce apoptosis. in addition, a recent study indicated that egln3 s ability to induce apoptosis correlates with the formation of aggresome-like structures. recently, an unbiased genome-wide approach has been undertaken to understand how egln3 causes neuronal cell death. this led to the identification of the kinesin kif1b as a downstream effector of egln3. kif1b, a member the kinesin 3 family, consists of two major splice variants and. kif1b and kif1b are motor proteins implicated in anterograde transport of mitochondria and synaptic vesicle precursors, respectively [79, 80]. the recent study, which identified kif1b as an egln3 downstream target showed how kif1b is both necessary and sufficient for neuronal apoptosis when ngf becomes limiting, but it remained unclear how egln3 regulates kif1b. interestingly, kif1b maps on to the chromosomal region 1p36.2, a region of the genome that is frequently deleted in neural crest-derived tumours including neuroblastomas. the existence of one or more human tumour suppressor genes on chromosome 1p has been suspected for decades [8284]. further suggestion that kif1b acts as a 1p36 tumour suppressor comes from the current model for phaeochromocytoma development. phaeochromocytomas develop when sympathetic neuronal precursors escape from egln3-mediated developmental apoptosis, suggesting that mutations in kif1b may be relevant to phaeochromocytoma and other tumours of neuronal origin. indeed, inherited loss-of-function kif1b missense mutations have been identified in phaeochromocytomas and neuroblastomas and an acquired loss-of function mutation in a medullablastoma, arguing that kif1b is a pathogenic target of these deletions. nonetheless, it has been further reported that the remaining kif1b allele in 1p deleted tumours and cell lines is often wild-type, contrary to the knudson twohit scenario [64, 8588]. also, the existence of multiple neuroblastoma and phaeochromocytoma suppressor genes on 1p has been suggested [89, 90]. additional studies are needed to address how often it is deregulated, epigenetically or genetically, in cancer. a spotlight is now on understanding the mechanistic basis of how egln3 regulates kif1b and how this translates into cell death. kif1b appears not be a direct egln3 hydroxylation target, but egln3 hydroxylation activity is required for kif1b regulation. therefore egln3 presumably involves hydroxylation of a protein that in turn might regulate kif1b to induce apoptosis. in 1924, otto warburg observed that cancer cells are highly glycolytic in the presence of oxygen and have reduced rates of oxidative phosphorylation. recent studies have argued that cancer cells might benefit from this persistence of high lactate production in the presence of oxygen, referred to as aerobic glycolysis or pseudo-hypoxia [92, 93]. from a bioenergetic perspective, it remains a conundrum how highly metabolic proliferative cancer cells undergo a pathway that results in 10 times less atp production compared to their normal counterparts that oxidize their glycolysis endproduct within the mitochondria via the krebs cycle (pyruvate conversion into acetyl-coa, fig. interestingly, three major enzymes of the krebs cycle (sdh, fh, idh) have been recently identified as bonafide tumour suppressors, but more importantly, inactivation in either of them directly affects egln activity [9, 10, 14, 71]. this is because the egln activity is not only dependent upon molecular oxygen to perform their hydroxylation reaction, but also require the krebs cycle metabolite -ketoglutarate as electron donor, which ties them directly into this metabolic network. therefore an attractive hypothesis arises: cancer cells favour aerobic glycolysis to inhibit egln activity in order to escape either certain oncogenic apoptotic signals and/or activate oncogenic hif-. about 70% or more of low-grade gliomas bear loss of function mutation in idh1 and idh2 [94, 95]. subsequently egln prolyl hydroxylases are inhibited in their hydroxylase activity with the consequences of hif- stabilization. presumably egln3-mediated apoptosis might be impaired in these settings as well, but this has not been investigated yet. further, these studies follow the discoveries that germline mutation in sdh are linked to phaeochromocytoma and that fh mutation lead to leiomyosarcoma and renal cell carcinoma, both of which affect also the egln prolyl hydroxylase activity [9, 10, 71]. this is because loss of function mutation in sdh and fh increases the accumulation of succinate and fumarate respectively. the excess of these metabolites inhibits the proline hydroxylases with the consequences in either accumulation of oncogenic hif- and/or blunting egln3-mediated apoptosis [9, 10, 71, 96]. the krebs cycle enzymes (sdh, fh and idh) are tumour suppressors and linked to egln activity. the egln prolyl hydroxylases are dependent upon the krebs cycle metabolite -ketoglutarate as electron donor for hydroxylation activity. loss of function mutation in idh decreases -ketoglutarate levels and subsequently impairs egln function. on the other hand, mutation in sdh or fh leads to accumulation of succinate or fumarate respectively, which in turn product-inhibits egln activity. loss of function of sdh, fh or idh would lead to impaired krebs cycle activity and impaired oxidative phosphorylation with the consequence of enhanced glycolysis (embden meyerhof pathway) to generate atp. the generated pyruvate during glycolysis is redirected away from the krebs cycle by conversion into lactate. cancer cells tend to convert most glucose to lactate regardless of whether oxygen is present (aerobic glycolysis), an observation that was first made by otto warburg in 1924. despite the striking difference in atp production, cancer cells might favour aerobic glycolysis to escape from egln hydroxylation, resulting in the accumulation of oncogenic hif. and/or resistant to egln3-mediated apoptosis. the recently identified mutations in the metabolic/mitochondrial enzymes (fh, sdhb/c/d, idh1/2) provide convincing evidence that alteration in cellular metabolism contributes to the pathogenesis of cancer. hence, 43 years later we are beginning to learn the depth of otto warburg s foresight that (in his words) cancer cells should be interpreted as a mitochondrial dysfunction and that the prime cause of cancer is the replacement of the respiration of oxygen in normal body cells by a fermentation of sugar[97, 98]. even if this is the prime cause for some cancers, the underlying mechanistic basis remains controversial. however, recently it has become more evident that the inactivation of the egln prolyl hydroxylases is a downstream effector of these mitochondrial alterations. the identification of sdh and fh mutations has already led to the development of cell permeable -ketoglutarate derivates with the potential to suppress the transforming effects of these mutations through reactivation of the eglns. the exciting part of identifying metabolic-enzyme mutations in specific cancers is that they are druggable. they might provide new opportunities as therapeutic targets that would be more susceptible and more effective than existing cancer therapies. future work is needed to further elucidate the direct impact of cancer metabolism in prolyl hydroxylase functioning.

introductionlessons from a rare diseasespecificity of function within the egln prolyl hydroxylasesfailure of egln3-mediated apoptosis in the genesis of phaeochromocytomaunderstanding the mechanistic basis of egln3 killingconnecting egln activity to the warburg conundrumfuture directionsoxygen-sensing mechanisms are often dysfunctional in tumours. oxygen sensing is mediated partly via prolyl hydroxylation. the egln prolyl hydroxylases are well characterized in regulating the hypoxia inducible factor (hif-) hypoxic response, but also are implicated in hif-independent processes. egln3 executes apoptosis in neural precursors during development and failure of egln3 developmental apoptosis can lead to certain forms of sympathetic nervous system tumours. mutations in metabolic/mitochondrial enzymes (sdh, fh, idh) impair egln activity and predisposes to certain cancers. this is because the eglns not only require molecular oxygen to execute hydroxylation, but also equally require the electron donor -ketoglutarate, a metabolite from the krebs cycle. therefore egln enzymes are considered oxygen, and also, metabolic sensors. -ketoglutarate is crucial for egln hydroxylation activity, whereas the metabolites succinate and fumarate are inhibitors of the egln enzymes. since egln activity is dependent upon metabolites that take part in the krebs cycle, these enzymes are directly tied into the cellular metabolic network. cancer cells tend to convert most glucose to lactate regardless of whether oxygen is present (aerobic glycolysis), an observation that was first made by otto warburg in 1924. despite the striking difference in atp production, cancer cells might favour aerobic glycolysis to escape from egln hydroxylation, resulting in the accumulation of oncogenic hif and/or resistance to egln3-mediated apoptosis.

PMC2847199

pubmed-1281

descriptive epidemiologic statistics assist public health planning and provide valuable information about the burden of illness to policy makers, funding agencies, resource planners, healthcare insurers, and manufacturers. information on malignancies that is compatible with their clinical classifications is of particular value to clinicians and public health professionals and increasing efforts are being made to collect data at this detailed level (e.g., the haemacare project). it is challenging, however, to assemble a database of descriptive epidemiologic statistics from the peer-reviewed literature alone. cancer registries are used worldwide to collect and analyze demographic, diagnostic, and survival data. some registries are fraught with poor quality and infrastructure; however, there is no standardized system for the collection and reporting of descriptive statistics worldwide [2, 3]. comprehensive reviews of descriptive epidemiologic statistics, such as this one, are warranted to better understand the totality of the currently available data and to optimize the utility of such data in the future. this paper uses a particular cancer subtype b-cell malignancies to evaluate a novel approach to assembling a database of worldwide, national-level, descriptive epidemiologic cancer statistics. this approach incorporates information from various sources, including the peer-reviewed literature, online reports, and query systems from cancer registries and health agencies, and direct contact with cancer registries, to provide a current, comprehensive database for a representative group of countries worldwide. the major b-cell malignancies were chosen as the cancer subtype to test this approach largely because their epidemiology has not been well characterized. further, some b-cell malignancy subtypes require detailed diagnostic evaluation, and this paper allowed us to broadly assess the extent to which detailed diagnoses are currently being reported to cancer registries. this is important because an understanding of these subtypes may inform the development of novel treatments. b-cell malignancies emerge in cells of the bone marrow, blood, or other tissues at various stages of b-lymphocyte differentiation and represent a rare (3% of all malignancies) and heterogeneous group of lymphohematopoietic malignancies (table 1). in 2001 and 2008, the world health organization (who) classified lymphoid hematologic neoplasms into 4 major categories based on the cell linage of the malignancy or the normal cell type that the tumor most resembles: b-cell malignancies, t-cell malignancies, natural killer (nk) cell malignancies, and hodgkin's disease. further divisions were based on cell maturity (e.g., precursor versus mature b-cell neoplasms), as well as morphologic, genotypic, genetic, immunohistochemical, and clinical criteria. the major b-cell lymphoid malignancies include diffuse large b-cell lymphomas (dlbcls), follicular lymphomas (fls), and plasma cell or multiple myeloma (mm). it is estimated that 85% to 90% of non-hodgkin's lymphomas (nhl) are of b-cell origin, including dlbcl and fl. certain leukemias also arise from the b cells and are categorized based on (1) whether the disease is acute or chronic and (2) what type of cell is infected. the leukemias associated with abnormalities of b-cell formation in the blood include b-cell acute lymphoblastic leukemia (all) and chronic lymphocytic leukemia (cll) or small lymphocytic lymphoma. all represents one of the most common forms of leukemia among children. in this paper, a novel, comprehensive approach to gathering descriptive, epidemiologic statistics was used to develop a current worldwide database of the major b-cell malignancies. the intent of this paper was to evaluate the feasibility of this novel approach for assembling a database of descriptive statistics and to broadly assess the level of detail collected by cancer registries worldwide. we also briefly describe our findings of the descriptive epidemiology of b-cell malignancies. we collected the most recent information available on the incidence, prevalence, and survival of the following b-cell malignancies: dlbcl, fl, cll, mm, adult b-cell all, and pediatric b-cell all. these data were collected for a representative selection of countries worldwide, categorized into the following groups: north and south america (brazil, canada, and the united states), the european union-5 (france, germany, italy, spain, and the united kingdom), asia (china, india, japan, and south korea), and australia. descriptive statistics for the countries constituting the united kingdom (england, scotland, wales, and ireland [including northern ireland and the republic of ireland ]) were also collected. we employed three general strategies: a structured literature review of the published, peer-reviewed literature in pubmed, a review of online documentation from cancer registries and relevant health agencies, and, finally, direct contact via email with personnel at cancer registries and key experts in the field. the first 2 strategies were employed in tandem; direct contact was used to address any remaining gaps and clarify the reasons behind missing data. various search strings (see table s1 in supplementary material available online at doi:10.5402/2012/129713) were used in the pubmed search to identify general articles describing the epidemiology of these malignancies in the countries of interest. the search was restricted to human studies published from january 1, 2000 to july 13, 2011. approximately 5600 citations were retrieved, and the titles and abstracts of these publications were reviewed. studies that appeared to report descriptive epidemiologic statistics (i.e., incidence, prevalence, or survival) for the b-cell malignancies of interest were included. articles reporting national-level data on the statistics, malignancies, and countries of interest were identified and the relevant information was extracted. in addition, case reports, case series, and letters to the editor were excluded. relevant full-text articles were retrieved and reviewed for inclusion. worldwide, national, and regional cancer registries and online documentation for these registries (including reports and online query systems) the websites of other relevant health agencies and government departments were also searched for reports and references to the data of interest. registries and relevant agencies were identified from previous experience, online research into the structure of public health in each country, and involvement in organizations such as the international association of cancer registries, globocan, and the european network of cancer registries. for some non-english language speaking countries (france, germany, spain, china, and japan), persons proficient in the language searched online data and translated some of the published literature; a translation of non-english language material (including online documentation and published studies) was not performed, however. the goal was to identify the most recent estimates of the incidence, prevalence, and survival associated with the relevant b-cell malignancies. if relevant data were reported by more than 1 source, the source with the most recent data and/or the most relevant data (e.g., data specific to b-cell all versus all) was used. once gaps in the availability of the data of interest were identified, cancer registry personnel and key experts in the field were contacted via e-mail to discuss the availability of missing statistics (these contacts are referred to as personal communication). table 2 describes the cancer registries and organizations that were contacted and the outcome of each respective communication. we did not collect data from globocan or the cancer incidence in five continents (ci5) series. these iarc programs provide a valuable resource for modeling estimates on the incidence and mortality of common cancers, including nhl and mm. however, since modeling estimates may not reflect the most recent national-level statistics, we did not rely on these databases for nhl and mm data. there are also standardized efforts in europe to report descriptive epidemiologic data: eurocare and the automated childhood cancer information system. similar to ci5; however, these standardized systems are not based on complete ascertainment from registries in the underlying countries, for example, only select regional registries participate in spain, italy, and france. as such, we only used data from these european programs in the absence of national-level data. the following information was extracted from each data source: source of the data, country or countries, malignancy type, including any available information on international classification of diseases (icd) codes, and data relevant to incidence, prevalence, and/or survival. we sought to assess the availability of recent statistics; therefore, only post-2000 diagnoses were assessed for incidence and survival, and only prevalence estimates for a date after 2000 were collected. incidence projections for future years were collected only in the absence of actual incidence estimates. limited-duration prevalence rates and prevalence proportions estimated on a date after 2000 were collected for all available time frames (e.g., 5 y, 10 y). relative survival rates were collected for all time periods (e.g., 5 y survival, 10 y survival) for diagnoses post-2000; it was noted whether survival rates were estimated using period methods. relative survival is defined as the ratio of the proportion of observed survivors in a cohort of cancer patients to the proportion of expected survivors in a comparable set of cancer-free individuals. in the absence of data on relative survival, data were collected by gender and methods for age standardization and any age restrictions were noted. data were reported from multiple citations for a particular malignancy and country if different information sources reported supplementary information. large gaps in the availability of national-level statistics on b-cell malignancies were observed at the outset of our review process and few descriptive statistics were available for the b-cell nhls (i.e., dlbcl and fl) and b-cell all. we, therefore, extended the review to the larger diagnostic categories of nhl and all. data on diffuse nhl, a diagnostic category of nhl that includes dlbcl, were available from numerous countries, and data were collected for this larger diagnostic category in the absence of data specific to dlbcl. as a result, we collected data on the larger diagnostic category lymphoid leukemia. finally, national-level statistics were lacking in several countries (e.g., brazil, india, and china) because of the status of cancer registration in these countries. to compensate for this deficiency, table 3 provides an overview of the availability of statistics by malignancy type and country; notations are provided where proxy data (as defined above) were used. supplementary tables 2 to 7 provide the most recent statistics by country and statistic type for nhl, dlbcl and fl, adult all, pediatric all, mm, and cll, respectively. cancer registries are well recognized as a valuable information source for public health planning and epidemiologic and therapeutic research because they provide essential epidemiologic data on the current burden of disease. nonetheless, we found that the implementation of cancer registries has been slow in some countries, and data were not made readily available or updated in a timely manner in others. in 2006, almost 80% of the world's population was not covered by a population-based cancer registry; most of this unrepresented population was from low- and middle-income countries. the uncoordinated nature of cancer registration worldwide has resulted in wide variation in the geographical areas covered, the methods used to estimate descriptive statistics, and the level of detail collected. this differential evolution of cancer registration worldwide yielded notable between-country variation in the availability of descriptive statistics on b-cell malignancies (table 3). the use of online resources and direct contact with cancer registries greatly expanded our database. the us surveillance, epidemiology, and end results (seer) registry provided the most complete data on b-cell malignancies. the seer registry provides incidence, prevalence, and survival data by icd-o-3 code using the seer*stat software. we were, therefore, able to calculate statistics for the b-cell malignancies dlbcl and b-cell all, which require icd-o-3 coding. nearly all countries in our paper had national-level incidence statistics for nhl and mm, although prevalence and survival data for these malignancies were less complete. data on dlbcl and fl were only available in the united states, the united kingdom, germany, and australia, but supplementary data were available on diffuse nhl from several countries (brazil, england, ireland, and germany). since the course of leukemia varies by its tissue of origin and whether it is acute or chronic, the reporting of data by specific leukemia subtypes is preferable. lymphoid leukemia incidence was often used as a proxy for all and cll, however, and prevalence and survival data were scarce for all categories of lymphoid leukemia. in many developed countries (e.g., canada, the united kingdom, and germany), only the prevalence of leukemia as a large diagnostic group was reported. france provided no national-level estimates of all incidence, except rates estimated using a statistical method based on unreported mortality data (belot a and mitchell m, personal communication with universit lyon, january 27, 2011). in germany, descriptive statistics were only available for leukemia as a large diagnostic group. regional statistics for lymphoid leukemia were found in the bavaria and bremen population-based cancer registries (i.e., registries that collected data on every person with incident cancer within a defined geographic region). italy, on the other hand, provided pooled data from its 28 regional cancer registries on adult and pediatric all and adult cll. among the asian countries, only japan had national-level data on all, whereas china and south korea had national-level data on lymphoid leukemia. the united states and the republic of ireland were the only countries with incidence data specific to adult b-cell all (1.0 and 0.22 (s. deady and m. wagner, personal communication, with national cancer registry of ireland, september 24, 2010), respectively, per 100,000 persons). in the united states, these data suggest that b-cell all comprises approximately 60% of all adult all. the united states was the only country with statistics on pediatric b-cell all, with an incidence of 1.77 (95% ci, 1.701.84) per one million children (representing only 5% of pediatric alls). of note, france, germany, and spain reported the incidence of mature b-cell lymphoid leukemias (international classification of childhood cancer [iccc] code ia2) among children 0 to 14 years of age (1.5, 1.0, and 0.6 per million children per year, resp.); these data demonstrate that the vast majority of lymphoid leukemias in children are of the precursor cell type [1921]. cancer registries in some countries indicated that the provision of descriptive data on specific nhl and leukemia subtypes was not possible because of small population sizes and the associated uncertainty with small numbers. the northern ireland cancer registry, for example, could not provide statistics by nhl or leukemia subtypes because of small numbers (d. donnelly and m. wagner, personal communication with northern ireland cancer registry, september 27, 2010). the national cancer registry of the republic of ireland, on the other hand, provided these data after e-mail contact was initiated with the registry (s. deady and m. wagner, personal communication with national cancer registry of ireland, other cancer registries could not provide the level of detail required for nhl and leukemia subtypes because the data were not coded appropriately to permit such analyses. for example, noted that it is not currently possible to calculate statistics for various hematologic subtypes in france because of issues with reliably classifying cases using a consistent coding scheme. similarly, the office of national statistics (which releases descriptive data for england) stated that they were only able to provide data by icd-10 codes because of variability in the coding information provided by regional registries and issues with mapping icd-o-3 (introduced in 2008) to icd-o-2 (n. jakomis and m. wagner, personal communication with national cancer intelligence center on descriptive cancer statistics, october 11, 2010). icd-10 codes do not allow for the calculation of statistics on b-cell all or dlbcl. we were able to capture statistics for fl with icd-10 code c82 and for diffuse lymphoma with icd-10 code c83, however. the icd-o-3 coding system provides the most relevant information for descriptive epidemiologic statistics because malignancies are coded by cell lineage and maturity, as well as morphologic, genotypic, genetic, immunohistochemical, and clinical behavior. icd-o-3 has been adopted by the us and most european countries, but the report of descriptive statistics by icd-o-3 coding is limited by the recent conversion from icd-o-2 in 2000 and the small numbers associated with these rare hematologic malignancies. one author noted that, even considering the whole of europe, some entities were too rare to calculate statistics by icd-o-3 coding. to address this issue, haemacare, a project funded by the european commission to improve the standardization of population-based data on hematologic malignancies, collected incidence data 20002002 from 48 european registries in 20 countries using a grouping system based on who recommendations and the icd-o-3 morphology codes. thus, categories represent cell lineage and prognosis, which are useful for epidemiologic and public health purposes. the haemacare coding system contains detailed information on mature b-cell malignancies, including dlbcl, fl, and cll. in their first publication of incidence data from the haemacare network, sant et al. reported european age-standardized incidence rates (per 100,000 persons) for dlbcl, fl, and cll of 3.81 (95% ci, 3.733.89), 2.18 (95% ci, 2.122.24), and 4.92 (95% ci, 4.835.01), respectively. compared to these haemacare european estimates, the incidence of dlbcl is considerably higher in the united states (6.83; see table s3) and the incidence of fl is higher in both the united states (3.7) and australia (4.1; see table s3). however, the haemacare european estimate of cll incidence is similar to rates observed in the united states (5.0), canada (5.0), and australia (4.9; see table s7). sant et al. noted geographic variability in the incidence of hematologic malignancies across europe, which they attributed to differences in diagnostic and registration criteria. this first publication from the haemacare network suggests that data on dlbcl, fl, and cll are increasingly available in certain registries across europe, but that cancer registries are reluctant to regularly publish and/or provide these statistics. another limitation of the diagnostic classification system used for reporting cancer statistics is the use of the iccc for childhood cancers. in the third version of the iccc, all is categorized along with other lymphatic leukemias in the category iccc-1a, which includes morphology codes 9820, 9823, 9826, 9827, 98319837, 9940, and 9948. although the vast majority of lymphatic leukemias in childhood are acute in nature, estimates using iccc-1a only provide a proxy for actual all rates. there was wide variation between countries in the methods used for estimating national-level descriptive statistics. some countries used direct estimates from national cancer registries, collecting information on all cancer diagnoses nationwide into 1 registry (i.e., wales, northern ireland, republic of ireland, scotland, and south korea) or a coordinated group of regional registries (i.e., england, canada, and australia). other countries calculated estimates of the national-level experience using statistical methods to extrapolate data from regional registries to the national level (i.e., united states, france, japan, and china). another group of european countries estimated the national-level cancer experience by pooling data from regional registries (i.e., italy, germany, and spain). this variability in the methods used to estimate national-level statistics limits the comparability of statistics between countries. one statistical method used to predict cancer incidence at the national-level is the use of mortality rates (which are often readily available nationwide) as a geographic correlate of incidence. observed incidence and mortality data are modeled in this approach using age-cohort techniques to provide estimated incidence rates. this method was recently used to estimate cancer incidence in france and china [22, 25] and is frequently used by globocan. while this method is known to be generally reliable, there are some questions about the representativeness of the data used. however, the reliability of this method to estimate the incidence cancers with high survival rates (e.g., pediatric all) is less clear. the vast majority of registries were population-based, but some countries had registries that collected data only on cancer patients seen at a particular hospital (i.e., hospital-based registries). hospital-based registries are limited compared with population-based cancer registries because the source population is not well-defined. brazil has several hospital-based cancer registries, and south korea has a nationwide, hospital-based cancer registry (the korea central cancer registry) that reportedly captures 90% of all cancer cases nationwide. the national registry of childhood tumors is a population-based cancer registries of malignancies and benign brain tumors diagnosed in children living in england, wales, or scotland. the french national registry of childhood hematopoietic malignancies has recorded all cases of hematologic malignancies, including lymphoma, since 1990; the french national registry of childhood solid tumors has recorded all cases of solid tumors, except lymphomas, since 2000. the german childhood cancer registry has collected data on cancer cases nationwide, with an estimated coverage of about 95%. finally, the australian pediatric cancer registry has provided full coverage of all australian states and territories since 1983. a substantial number of countries do not have a nationwide cancer registration program. rather, cancer registration consists of a group of regional cancer registries, collecting complete data on cancer cases in their respective geographic regions and coordinated by an overarching network. the level of standardization and coordination between these regional registries varies substantially by country. in the united states, seer currently collects and publishes cancer data in a standardized fashion from 17 population-based cancer registries covering approximately 28% of the us population there are 21 regional cancer registries in france coordinated under the association of french cancer registries (francim) that cover approximately 16% of the population. in italy, there are 28 registries that cover approximately 26% of the population coordinated through the italian association of cancer registries (airtum). in germany, there are regional registries in 13 states and 1 administrative district, coordinated through the robert koch institute. japan has approximately 30 population-based cancer registries that are coordinated through the japan cancer surveillance research group. some developed countries still lack coordination of their regional registries, which limits what can be done to predict the national-level experience of cancer. spain, for example, currently has 13 regional registries that cover approximately 27% of the population, but no network that coordinates and standardizes these registries; a network is currently under development, however. in less developed countries, where financial, resource, and logistical challenges dominate, coverage is typically limited to metropolitan areas, and differences between regional registries preclude the development of national estimates. substantial efforts are being made, however, to organize and standardize these regional registries into a coordinated network. in brazil, for example, efforts are being made to collect data from an expanding network of regional registries and to improve data quality to support nationwide cancer prevention and planning. epidemiology surveillance was formally organized and standardized in 1999 and now includes 22 population-based cancer registries, most of which include capital cities and surrounding areas. these population-based cancer registries vary in the source (hospital, laboratory, etc.) and quality of their data. because of this variation, pooled data can not be reported and statistics are provided only by individual population-based cancer registries for brazil. the age-adjusted incidence of nhl, for example, varied from 2.0 to14.1 per 100,000 among males in the ten population-based cancer registries with more than 2 years of data. the authors of the latest report cautioned that these results are not generalizable to brazil as a whole because the population-based cancer registries are still of heterogeneous quality, validity, and completeness. underreporting also remains a serious concern in brazil and other less developed countries. despite these limitations, some brazilian population-based cancer registries provided data on detailed diagnostic categories diffuse nhl, follicular lymphoma, and pediatric all. however, although incidence data were readily available from brazilian population-based cancer registries, more complex data (prevalence and survival) were not. similarly, india has 26 population-based cancer registries and 6 hospital-based cancer registries coordinated under the national cancer registry programme network and covering 7% of the population; statistics are only reported by population-based cancer registries. china also has no national-level coordination of its 50 population-based cancer registries, which cover 5.7% of the country. this survey represents the status of data that were available as of mid-july 2011 using comprehensive methods. it is clear from our paper, however, that the breadth and specificity of data reported by cancer registries is continually evolving, and this evolution will ultimately entail the reporting of data at the national level for detailed cancer subtypes. in france, for example, national level data were reported for the first time in 2003 for cancer incidence, in 2006 for cancer survival, and in 2008 for cancer prevalence. lacour et al. described the incidence of childhood cancers nationwide in france for the first time in 2010. population-based cancer registration in germany improved considerably after the federal cancer registry data act of 2009, which called on all federal states to report complete data to the german centre for cancer registry data at the robert koch institute, thus expanding the breadth of cancer statistics in germany from data reported only by the saarland cancer registry to pooled analyses of data from cancer registries meeting the minimum requirements for data completeness. current estimates of cancer incidence are based on data from 13 states and 1 administrative district, and the gekid cancer survival working group recently published their first comprehensive monitoring of cancer survival in germany based on a collaboration between 13 population-based cancer registries. some basic patterns in the descriptive epidemiology of b-cell malignancies emerged from reviewing the available data. specifically, the incidence rates of the b-cell neoplasms included in this paper were generally higher in the united states, australia, and the european union-5, compared with asia and brazil (possibly reflecting differences in the ascertainment of cases). further, the incidence of nhl was generally higher in men, and nhl survival rates were slightly higher in women (see table s2). the higher incidence of hematologic malignancies in men versus women may be explained by the traditionally higher exposures of men to occupational and environmental carcinogens. many cancer registries reported that the requested statistics were not readily available or were available at a cost (e.g., additional data were available at a cost from the office of national statistics, the welsh cancer intelligence service, and the australian institute of health and welfare). it is, therefore, possible that statistics beyond those reported in table 3 are available. some attempts to communicate with key experts and cancer registries failed, thus limiting our understanding of the true availability of certain data (table 2). for example, our attempts to contact the northern and yorkshire cancer registry and information service (nycris) and the cote d'or hematological malignancies registry (the lead registries for hematologic malignancies in england and france, resp.) yielded no responses. we were also unable to establish contact with cancer registries in italy, japan, and south korea. we limited the data we gathered to diagnoses post-2000 to allow for representation of the availability of recent descriptive statistics. some data were only available, however, for diagnoses that occurred in the late 1990s (e.g., survival data in ireland and france and pediatric all incidence in scotland). this paper was also limited to select countries, which precludes a full evaluation of the worldwide availability of data and an analysis of geographic differences. a comparison of statistics across countries is also limited by differences in the structure of cancer registries, age standardization methods, methods for estimating the statistics (e.g., period estimates for survival), and the year(s) of diagnosis. furthermore, the comparison of hematologic malignancies across time and place was significantly limited by regional differences in disease classification systems. this paper was also limited by reports of cancer data in non-english languages. translations of non-english language material were restricted to languages in which the research team had proficiency and were not carried out in a systematic manner. thus, it is possible that additional data is readily accessible in other languages. updates are published continuously by cancer registries, and investigators are frequently publishing manuscripts with additional, relevant epidemiologic data. we attempted to capture the most recent data available as of approximately mid-july 2011. at the time of the publication of this study, however, we were aware of numerous updates, namely the release of updated seer data. in addition, the nycris was planning to publish a report on hematologic malignancies for all english cancer networks in 2011. this paper used a novel approach to gathering descriptive epidemiologic statistics on cancer by combining information from the biomedical literature, internet-based query systems and reports, and direct contact with cancer registry representatives and key experts to provide the most up-to-date, comprehensive picture of a particular malignancy type. this paper highlights the importance of international coordination between cancer registries (e.g., haemacare) to promote standardization in data collection and reporting. we also recommend continued coordination and standardization between regional registries in countries such as brazil, spain, and india to promote the development of nationwide estimates. this novel approach provided a more comprehensive database of the current patterns of b-cell malignancies compared with a simple literature search. in addition, gaps were identified that highlight the need for more detailed reporting to cancer registries and the importance of developing assumptions to estimate the burden of specific b-cell malignancies in the interim. because research indicates that cellular lineage affects prognosis, treatment, and etiology, cancer registries should continue to work to improve reporting of detailed diagnostic information on malignancies.

the compilation of comprehensive, worldwide epidemiologic data can inform hypotheses on cancer etiology and guide future drug development. these statistics are reported by a multitude of sources using varying methods; thus, compiling a complete database of these statistics is a challenge. to this end, this paper examined the usefulness of a novel, multisource approach extracting data from the peer-reviewed literature, online reports, and query systems from cancer registries and health agencies and directly contacting cancer registry personnel for building a comprehensive, multinational epidemiologic cancer database. the major b-cell malignancies were chosen as the cancer subtype to test this approach largely because their epidemiology has not been well characterized in the peer-reviewed literature. we found that a multisource approach yields a more comprehensive epidemiologic database than what would have been possible with the use of literature searches alone. in addition, our paper revealed that cancer registries vary considerably in their methodology, comprehensiveness, and ability to gather information on specific b-cell malignancy subtypes. collectively, this paper demonstrates the feasibility and value of a multisource approach to gathering epidemiologic data.

PMC3549359

pubmed-1282

sd, a 45-year-old caucasian male, was referred for evaluation for liver transplantation because of end-stage liver disease caused by hepatitis b. he had been well until six months prior to his initial visit, when he started losing weight and developed severe fatigue. during the six-month period, he had lost 25 kilograms. his past medical history was significant for non-hodgkin's lymphoma that was diagnosed eleven years ago. he was treated with 8 cycles of chop chemotherapy and had been in remission since then. on examination, the patient appeared mildly icteric. the abdomen was soft with some tenderness in the right upper quadrant and ascites. complete blood count showed a leukocyte count of 2500 cells/mm, hemoglobin of 9.8 gm/dl, and platelet count of 29 000 cells/mm. his liver function tests were abnormal with a total bilirubin of 5 mg/dl (normal, 0.21.2 mg/dl), alt: 361 u/l (normal, 1763 u/l), ast: 750 iu/l (normal, 1540 u/l), total protein: 15.4 g/dl (normal, 6.17.9 g/dl), albumin: 2.9 g/dl (normal, 3.94.8 g/dl), and an inr of 1.9. serological tests for viral hepatitis were as follows: hepatitis b surface antigen positive (hbsag), hepatitis b surface antibody negative (antihbs), hbe antigen negative, hbe antibody positive, igm antibody to hepatitis b core antigen positive, igg antibody to hepatitis b core antigen positive, and hbv dna 4 150 000 iu/ml (by pcr). ct scan and ultrasound examination of the abdomen showed a small liver with moderate ascites, splenomegaly, patent portal, and hepatic veins. the striking elevation in his globulin fraction (12.5 g/dl) was further worked up. serum protein electrophoresis showed the presence of a large monoclonal protein spike (m protein) in the gamma region which on immunofixation electrophoresis (ife) was identified to be iga-kappa type (see figure 1). the iga levels were increased at 6730 mg/dl (reference range 70400 mg/dl) and the igg and igm levels were decreased at 466 mg/dl (reference range 7001600 mg/dl) and 25.6 mg/dl (reference range 40320 mg/dl), respectively. a bone marrow biopsy on two separate occasions showed normocellular bone marrow with less than 2 percent population of plasma cells. full body positron emission tomography (pet) scan and ct scan were performed, and no focus suspicious for lymphoma or myeloma or infection was identified. whole body mri was unremarkable. on retrospective review, the patient had an elevated total protein (12 g/dl) approximately six months prior to initial visit. within the next few weeks, the patient continued to deteriorate, and he developed worsening ascites with hyponatremia. it was clear that the patient had progressive liver disease from chronic hepatitis b with a relatively high meld score of 23, and he would benefit from a liver transplantation. our dilemma before proceeding with liver transplantation was whether he had an underlying hematological malignancy that was causing the monoclonal gammopathy. we were also concerned about potential vascular complications after liver transplant given his increased viscosity. however, due to his deterioration and absence of any clear-cut evidence of malignancy, it was decided to place the patient on the liver transplant wait list. anti-hbv therapy was initiated with a combination of entecavir (0.5 mg/day) and tenofovir (300 mg/day) to decrease his hbv dna to the lowest possible level before transplantation. however, he was on this antiviral therapy for less than a month as he received a deceased donor liver transplant ten days after listing. in the period between listing and liver transplantation, his bilirubin continued to trend up from 3.3 mg/dl to 4.6 mg/dl, and his total protein level fluctuated between 12.5 g/dl and 13.5 g/dl., he received two doses of rabbit antithymocyte globulin at 1.5 mg/kg during the time of transplant and on postoperative day 2. subsequently, the mycophenolate mofetil was withdrawn, and he is currently on tacrolimus monotherapy with trough levels between 35 mg/ml. he continues to receive hepatitis b immunoglobulin infusion every month along with entecavir (0.5 mg/day), and his hepatitis b antibody titre has been kept above 300 miu/ml. he is currently 9 months posttransplant, his total protein levels range 4-5 g/dl, and he has had no detectable m protein spike. the temporal relation of the liver transplant with disappearance of hepatitis b surface antigen and normalization of the monoclonal spike implicates hepatitis b as the possible cause of the m protein spike. there is very little data and uncertainty regarding the incidence and natural history of m proteins in a person with hepatitis b. it has been suggested that the immunological response elicited against hepatitis b virus in the host rather than the direct cytopathic effect of the virus may be the basis for the pathogenesis of hepatic and nonhepatic manifestations. hepatitis b virus seems particularly well suited to initiate a chronic immune disease because of its tendency to persist in spite of a good immune response. this may cause clonal expansion of the immunoglobulin secreting cells and may explain the above phenomenon. alternately, the m protein spike could represent increased serum immunoconglutinin titres. it has been previously suggested that elevated serum immunoconglutinin titers in patients with acute and chronic hepatitis b virus infection may represent a physiological autoimmune response to hbsag/anti-hbs immune complexes. although our patient did not have the anti-hbs antibody, it has been suggested that in the light of massive antigenemia present in chronic hbsag carriers, it is possible that anti-hbs exists in the form of hbsag/anti-hbs immune complexes. this would make it difficult to detect anti-hbs in serum samples by conventional serological methods. the primary issue was whether he was eligible for liver transplantation in the presence of an increasing m protein. the fact that he had lymphoma 10 years ago complicated this evaluation. additionally, an association between hepatitis b infection and non-hodgkin's lymphoma has been previously described. however, he had had regular follow-ups every six months after remission of lymphoma, and also his malignancy workup prior to transplant was negative. moreover, the m protein spike was noticed only 6 months prior to transplant and this coincided with the elevation of his liver function tests. surprisingly, his igm antibody to hepatitis b core antigen was positive, as was his total core antibody. he did have active hepatitis and this can be deduced by the fact that he had an alt level that was 10 times the upper limits of normal; his explanted liver showed significant inflammatory activity. it was felt that most likely he had a flare of his chronic hbv, which might have triggered the elevation in m protein. because of his elevated m protein and an urgent need for liver transplantation, we decided to initiate dual therapy with a nucleoside (entecavir) and nucleotide (tenofovir) analogue to decrease his hbv dna. however, he was on therapy less than a month before a suitable organ became available. we decided to accept the organ because of high meld score rather than to wait for hbv dna disappearance. since we were planning to continue therapy posttransplant along with hbig (target anti-hbs titers>500 iu/ml first three months and levels of>300 iu/ml after three months), we believed that the risk of recurrence of hbv was very small. unfortunately in the time period before the transplant and after initiation of antiviral therapy, we failed to follow his iga levels and hbv dna levels. however, his protein levels remained unchanged suggesting that there may have been very little change in his gammopathy, and this is not surprising given the fact that nucleos(t)ide therapy seldom leads to hbsag clearance in that time frame. after liver transplantation and with clearance of the hepatitis b surface antigen, the m protein levels became normal. one and the most likely reason is the removal of antigen source with native hepatectomy. his hepatitis b surface antigen, m protein, and total protein levels remain within normal limits 1 year after transplantation. the second possibility is the impact of thymocyte, given at the time of transplant, on plasma cells. antithymocyte globulin has been used in treatment of plasma cell dyscrasias due to its activity against several plasma cell antigens. this is less likely in our patient because thymocyte globulin was used only as induction therapy at the time of transplantation. the elimination half time of thymoglobulin is 2-3 days and is unlikely that its effect on plasma cell is sustained for a year. in conclusion, this case demonstrates that monoclonal gammopathy can be associated with chronic hepatitis b infection and can be eliminated after successful transplantation.

monoclonal gammopathy of undetermined significance (mgus) has been most commonly associated with diseases like multiple myeloma, waldenstrom's macroglobulinemia, primary systemic amyloidosis, hiv, and other lymphoproliferative disorders. there has been an isolated report of mgus in patients coinfected with hiv and hepatitis b, as the work by amara et al. in 2006. here, we report a case of iga-kappa light chain gammopathy secondary to hepatitis b infection, which resolved after liver transplantation. to our knowledge, this is the first reported case of m protein spike seen in the context of hepatitis b infection only.

PMC2809430

pubmed-1283

igg4-related diseases are systemic syndromes characterized by elevated serum levels of igg4 and igg4-positive lymphoplasmacytic infiltrative lesions in the body. it was first observed that mikulicz's disease correlated with igg4-related disease and later determined that igg4-related disease can occur in any ocular adnexal tissues [25]. here, we review the clinicopathological features, differential diagnosis, and treatments of orbital igg4-related disease on the basis of a meta-analysis of 42 patients including 3 case series studies. the median age of patients with orbital igg4-related disease is 59 years (range: 30 to 86 years) with a 1: 1 male-to-female ratio [35]. notably, there is a 1: 3 for bilateral lacrimal lesions similar to finding in mikulicz's disease. although orbital igg4-related disease can occur in men and women of any age, many patients have a history of allergic diseases such as asthma and allergic rhinitis. the signs and symptoms of orbital igg4-related disease are chronic lid swelling (figure 1) and proptosis (figure 2), but otherwise there are only mild signs, or no signs of inflammation or periocular pain. ocular motility is restricted mildly if at all, despite the presence of one or more enlargements of the large extraocular muscles (figure 1). there are generally no visual disturbances, although they may occur due to apical orbital lesions (figure 2). imaging studies show infiltrative lesions in ocular adnexal tissues such as the lacrimal glands (figure 1) [25], extraocular muscles (figure 1) [3, 4], infraorbital nerves (figure 2), optic nerve sheath, lacrimal sac, and even cavernous sinus (figure 2) or the intracranial extension. in cases of orbital igg4-related disease, 62% have bilateral lesions, 69% have lacrimal gland involvement, and 48% have bilateral lacrimal gland involvement [35]. patients with orbital igg4-related disease sometimes have systemic igg4-related lesions in their submandibular glands (29%), lymph nodes (14%), pancreas (5%), or bile ducts (5%) [35]. igg4-related lesions in the thyroid and pituitary may also be present [7, 8]. chronic rhinosinusitis with orbital igg4-related lesions has similar histology (figure 3), although nonspecific chronic rhinosinusitis can be also associated with igg4-positive plasma cells in the lesions. laboratory data of patients with orbital igg4-related disease show elevated serum levels of igg4 and ige, as well as hypergammaglobulinemia [25]. high igg4 levels are associated with elevated levels of the soluble interleukin (il-2) receptor. sclerosing pancreatitis and cholangitis, considered as igg4-related diseases, have lymphocytes that signal for il-4 and il-10 in situ. although an etiology for the igg4-related group of disease has been not determined yet, one plausible hypothesis for these serological and immunological abnormalities is that they result from in vivo activation of the immune system by activated th2 cells [3, 10]. the histology of orbital igg4-related disease includes different degrees of lymphoplasmacytic infiltration with dominant sclerosing lesions or reactive lymphoid follicle (reactive lymphoid hyperplasia, figure 3) [25]. rarely, orbital igg4-related disease may have an infiltration by lymphoplasmacytic cells and macrophages containing eosinophilic material. igg4-related diseases in the body are characterized histologically by obliterative phlebitis; however, it is rare in orbital igg4-related disease. immunohistochemical analysis shows igg4-positive plasma cells (figure 3), which differentiate igg4-related disease from other inflammatory conditions arising from the ocular adnexa [3, 4]. for optimal treatment and resolution, orbital igg4-related disease must be differentiated from the following: idiopathic orbital inflammation, idiopathic orbital myositis, marginal zone b-cell lymphoma, antineutrophil cytoplasmic antibody- (anca-) mediated systemic vasculitis (such as churg-strauss syndrome and wegener granulomatosis), and reactive lymphoid hyperplasia without igg4-positive plasma cells (figure 4). they are characterized by sudden onset of orbital inflammation, periocular pain, swelling and redness of the eyelids, proptosis, ptosis, and ocular motility restrictions. however, some cases of idiopathic orbital inflammation have atypical signs and symptoms, that is, they have lacked acute onset and inflammatory signs. in such cases, ocular adnexal marginal zone b-cell lymphomas make up the majority of lymphomas arising from the ocular adnexa. they are characterized histologically by the presence of reactive follicles in up to 64% of cases, sclerosis in up to 20% of cases, and plasma cells in up to 35% of cases. in addition, 9% of patients with ocular adnexal marginal zone b-cell lymphomas have infiltration of igg4-positive plasma cells and elevated serum level of igg4. evidence of light chain restrictions by in situ hybridization and immunoglobulin heavy chain gene rearrangements by southern blot analysis can differentiate between marginal zone b-cell lymphomas with igg4-positive plasma cells and igg4-related inflammatory disorder. ocular adnexal lymphomas are reported to arise in igg4-related sclerosing dacryoadenitis, indicating a possible link between the two conditions. the symptoms of patients with orbital lesions include periocular pain, which can differentiate these patients from those with orbital igg4-related disease thus, anca-related vasculitis may not only include nonspecific inflammatory lesions, but also have abundant igg4-positive plasma cells. finally, mikulicz's disease includes symmetrical bilateral lacrimal gland enlargements and frequently correlates with igg4-related disease. however, symmetrical bilateral lacrimal gland enlargements do not always indicate igg4-related lesions (figure 4). treatments for patients with orbital igg4-related diseases may include systemic steroids, radiotherapy, or rituximab [35]. studies of each of these treatments involved relatively few patients, making it difficult to evaluate the treatment outcomes via meta-analysis. orbital igg4-related diseases resolve after systemic steroid therapy, but relapse is often observed following therapy discontinuation. in mikulicz's disease, relapses of lesions are observed when steroids were discontinued. therefore, it may be best to continue prednisolone at 5 to 10 mg/day or to combine prednisolone with an immunosuppressant such as azathioprine. rituximab treatment leads to prompt clinical and serologic improvement in patients with refractory igg4-related diseases, although recurrence is also observed after rituximab treatment ends in some patients with orbital igg4-related disease. orbital igg4-related disease has several unique characteristics that distinguish it from other orbital inflammatory conditions. orbital igg4-related disease differs from other igg4-related diseases in the body in that it arises from nonglandular lesions and is not associated histologically with obliterative phlebitis.

orbital igg4-related disease, which can occur in adults of any age, is characterized by igg4-positive lymphoplasmacytic infiltrations in ocular adnexal tissues. the signs and symptoms include chronic noninflammatory lid swelling and proptosis. patients often have a history of allergic disease and elevated serum levels of igg4 and ige as well as hypergammaglobulinemia. orbital igg4-related disease must be differentiated from idiopathic orbital inflammation and ocular adnexal marginal zone b-cell lymphoma to ensure appropriate and effective treatment. systemic steroid therapy decreases the size of the lesions, but relapse often occurs when systemic steroid therapy is discontinued.

PMC3388428

pubmed-1284

the experimental models of myocardial infarction have largely contributed to a better understanding of the pathophysiology of myocardial infarction. studies on large animal models revealed many mechanisms involved in myocardial injury and repair [1, 2]. in the last few years, transgenic mice and gene targeting technologies allowed profound studies of the underlying pathomechanisms. for this purpose, we developed murine models of myocardial ischemia and reperfusion and used them to study the mechanisms involved in ischemic myocardial injury [3, 4]. we described pathological features of reperfused myocardial infarction in mice, that is, rapid formation of granulation tissue and subsequent development of a stable scar. our studies also revealed an important role for macrophages and their associated inflammatory and remodeling-related mediators [5, 6]. a profound understanding of these mechanisms is crucial for projects utilizing novel cellular therapies, since the local microenvironment seems to exerts a strong influence on the cells applied in our model of myocardial cryoinfarction [79]. this study compares the pathology and the sequence of cellular and molecular events in the two mechanistically different lesion models of myocardial cryoinfarction and reperfused infarction and reveals substantial differences in myocardial remodeling between them. both models show a transient inflammatory response associated with induction of chemokines, cytokines, and remodeling-related mediators. the cryolesioned heart showed a prolonged remodeling with postponed development of granulation tissue and scar formation, which was associated with persistent macrophage infiltration in the injured tissue when compared to reperfused infarction. wild-type c57/bl6-mice (charles river, sulzfeld, germany) of 18 to 25 g weight and 8 to 10 weeks old were used in our infarction models. group size in reperfused infarction model was n=8 mice and in cryoinfarction n=6 mice. all experiments were performed in accordance with an animal protocol approved by the local governmental authorities. in an initial surgery (2.5 g/g; merial, halbermoos, germany) as previously described. then, mice were intubated with a pe-90 tube (becton dickinson, sparks, md, usa) connected to a small animal respirator (rodent ventilator mod. millis, ma) and ventilated at a frequency of 110/min with tidal volume of 0.25 ml. pericardium was dissected and partially removed, and a prolene 8-0 suture (ethicon, norderstedt, germany) was placed around the left descending coronary artery (lad). suture ends were threaded through a sterile pe-10 tube (becton dickinson) of 3 mm of length, exteriorized through the thoracic wall, and then stored subcutaneously. the thorax was closed with interrupted prolene 6-0 stiches, and the skin was closed with a running prolene 6-0 suture. at the end, metamizol (100 mg/kg; novalgin) was given for analgesia in a mixture with cefuroxim as antibiotic prophylaxis i.p. (100 mg/kg, zinacef; bristol-myers squibb, munich, germany). the skin was reopened, and the ends of the lad-ligature were connected to heavy metal picks. pulling on the ligature ends induced lad-occlusion, and this ischemia was maintained for 60 minutes. after removal of the lad-ligature, the blood flow was restored in the reperfusion. ecg monitoring of einthoven lead ii during and after lad-occlusion confirmed successful ischemia and reperfusion. one-hour ischemia was followed by reperfusion for 6 hrs, 1, 3, 7, and 14 days. mice were euthanized using an overdose of pentobarbital i.p.; hearts were excised and fixated in zinc-paraformaldehyde (z-fix, 4%; anatech, battle creek, mi, usa) for histology or stored in rna-later solution (qiagen, hilden, germany) for mrna-studies. animals were sedated by brief exposure to narcotic gas containing 50% o2, 50% n2o, and 3 to 4 vol-% of isoflurane as previously described. thereafter, mice were positioned on a temperature-controlled plate (37c) in supine position, intubated, and ventilated using a small animal respirator (harvard apparatus) as described in the previous section. an anterolateral skin incision was performed 5 mm above the costal margin, the anterolateral thoracic muscles were transected, and the thorax was opened in the fifth intercostal space. the pericardium was opened, and the apex of the heart was exposed cryocoagulation was performed by placement of a copper probe (3 mm diameter, cooled in liquid nitrogen for 2 min) to the free left ventricular wall (3 times for 20 seconds) in order to achieve reproducible, large transmural myocardial lesion. after induction of the cryoinfarction, a 22-g chest drain (optiva, johnson&johnson, new brunswick, nj, usa) was inserted into the left pleural cavity to prevent pneumothorax. the thorax was closed with prolene 6-0 sutures, and air was drained using negative pressure on chest drain before extubation., the hearts were excised after the above-mentioned time periods and further processed for histological and molecular analysis. after excision, hearts were washed in cardioplegic solution containing 4 g nacl, 3.73 g kcl, 1 g nahco3, 2 g glucose (all from berlin chemie, berlin, germany), 3 g 2,3-butandion monoxime (sigma-aldrich, munich, germany), 3.8 g ethylenglycol tetra acetic acid (sigma), 0.2 mg nifedipine (sigma), and 10 ml heparin (1000 iu/ml; ratiopharm, ulm, germany), all of which were dissolved in 1 l of isotonic nacl (berlin chemie). hearts were fixated in zinc-paraformaldehyde for 24 hrs and further processed using standard paraffin embedding. hearts where cut from basis to apex, and at every 250 m, a set of ten 5 m sections were mounted on glass slides. myocardial sections below insertion of the papillary muscles were further used for histology and immunohistochemistry. photographic images were recorded on a computer system equipped with a digital camera (dp70, olympus, hamburg, germany), and planimetric evaluation was performed using analysis software (olympus). picrosirius red staining of total collagen was used to evaluate development of fibrosis in the myocardial scar. cell density was measured by cell count of immunohistochemically stained cells in infarcted myocardium and was expressed as number per mm. we used the following primary antibodies for immunohistochemistry: alpha smooth muscle actin mouse monoclonal antibody (clone 1a4; sigma, st. louis, mo, usa) for myofibroblasts, f4/80 rat monocyte/macrophage antibody (serotec, duesseldorf, germany) for macrophages and mca 771 g rat monoclonal antibody for neutrophils (serotec). furthermore, samples were stained for macrophage maturation marker osteopontin-1 using a goat polyclonal antibody (p-18, santa cruz, heidelberg, germany) and for macrophage elastase using a rabbit monoclonal anti-mmp12 antibody (abcam, cambridge, uk). immunohistochemical staining was performed using appropriate vectastain elite abc kits and diaminobenzidine (axxora, loerrach, germany). excised hearts were cleared of large vessels and atria and placed in rna-later solution (applied biosystems, foster city, ca, usa). hearts were minced using a tissue tearor (tissue tearor modell 398; biospec, bartlesville, ok, usa), and mrna was isolated using standard phenol/chloroform extraction method (trizol, applied biosystems). mrna was transcribed into cdna using high-capacity cdna transcription kit (applied biosystems) with random hexameric primers as described in the manufacturer's protocol. the mrna-expression was determined by taqman real-time quantitative pcr (rt-qpcr, applied biosystems). cdna was diluted 1/10 and then used for measurement of gene induction according to the manufacturer's instructions on an abi prism 7900ht sequence detection system using sds2.2 software (applied biosystems). all murine primers were commercially available and measured with fam tamra chemistry using the relative standard curve method. at the end of rt-qpcr cycle, dissociation curve analysis was performed to ascertain the amplification of a single pcr product. two-tailed, unpaired student's t-test was used to determine a significant difference between two groups. histopathological features of cryoinfarction and reperfused myocardial infarction in our model of cryoinfarction showed a transmural lesion with sharp infarction borders early after injury. the cryoinjury presented after 3 days with extensive cellular infiltration and partial granulation tissue formation starting at the periphery of infarction (figure 1(a)). the central part of the cryoinjury at the copper probe application site contained numerous dead cardiomyocytes and cellular debris. the formation of loose granulation tissue was almost finished after 7 days (figure 1(b)). the subsequent scar formation was largely completed after 14 days, but a substantial cellularity was still present in some parts of the cryoinjury whereas revascularization of large vessels was not observed (figure 1(c); arrow). in contrast, the reperfusion of myocardial infarction in our closed-chest model led to a nontransmural lesion. complete formation of granulation tissue was found after 3 days (figure 1(d)) and followed by a compacted scar formation with low cellular content after 7 days reperfusion (figure 1(e)), as we published before. the cryoinfarction showed large areas with loose collagen deposition after 7 days (figure 1(f)) in contrast to compacted collagen-rich scar in reperfused infarction at the same time point (figure 1(g)). this difference in scar formation was further underlined by a vast number of myofibroblasts in cryoinfarction after 7 days (figure 1(h)) whereas only few myofibroblasts were found in the reperfused infarction (figure 1(i)). the collagen was largely compacted in cryoinfarction after 14 days, but this was still associated with low myofibroblast persistence in the scar. the cryoinjury led to prolonged scar formation probably due to a longer time period needed for debris clearance, which could only progress from the periphery of the injury due to completely cryodamaged vasculature in the scar. in contrast, the reperfusion of myocardial infarction was associated with rapid scar formation due to an intact vascular network, which provided synchronous cell migration into the entire infarcted area. immunohistochemical staining of neutrophils revealed a strong myocardial infiltration early after the injury, suggesting a comparable myocardial debris clearance in both models. we found a significant difference in neutrophils density in infarcted area between the two models after 3 days (figures 2(a) and 2(b)) and slightly longer neutrophils persistence in cryoinfarctions after 7 days (figure 2(c)). we found no difference in cellular density of neutrophils in noninfarcted remote myocardium in both models (figure 2(d)). macrophage staining showed a persistent, strong infiltration of cryoinjured myocardium after 7 days whereas only few f4/80-positive cells were found after 7 days reperfusion of infarction, as previously published (figures 3(a) and 3(b)). we investigated macrophage activity using elastase staining after 7 days and found very strong signals in cryoinfarction, but no signals in reperfused infarction (figures 3(c) and 3(d)). interestingly, after 14 days, cryoinfarction showed much lower elastase activity, even though the total macrophage cell density was still high. the macrophage cell density data strongly support a short, rapid course of tissue remodeling after reperfusion of murine infarction, in contrast to a persistent macrophage infiltration even 14 days after cryoinfarction (figure 3(e)). to our surprise, macrophage density was also significantly higher in the remote, noncryoinjured myocardium, thus suggesting a dysfunctional development of the infarction border zone in this model (figure 3(f)). we further investigated the maturation of macrophages and found after 7 days a stronger infiltration of osteopontin-1-positive cells in cryoinfarcted hearts than in reperfused myocardial infarction (figures 4(a) and 4(b)). evaluation of osteopontin-1-positive cell density revealed a prolonged, but functional maturation of macrophages (figure 4(c)). the cell density of osteopontin-1-positive cells in the noninfarcted area was also comparable between the models (figure 4(d)). taken together, the course of cellular events involves a prolonged infiltration of cryoinfarction with inflammatory cells, thereby explaining the later development of a stable scar in this model. in contrast, a transient, short infiltration of reperfused myocardial infarction with inflammatory cells is associated with a rapid resolution of myocardial remodeling. the course of molecular events in cryoinfarction and reperfused infarction was assessed using mrna-expression measurements of specific inflammatory and remodeling-related mediators. the myocardial damage leads to a rapid production of reactive oxygen species and induction of several scavenger enzymes, for example, heme oxygenase 1, glutathione peroxidase, and so forth. the expression of heme oxygenase 1 in reperfused myocardial infarction accompanied the transient short inflammatory reaction with a peak induction after 24 h of reperfusion and a rapid downregulation thereafter (figure 5(a)). the cryoinfarction led to a significantly different expression pattern with prolonged induction of heme oxygenase 1. the cytokine tnf-, the next downstream mediator in the postischemic inflammatory cascade, presented with a significantly stronger mrna-induction early after reperfusion of infarction, which is probably triggering the rapid onset of inflammatory reaction (figure 5(b)). tnf--expression decreased thereafter in reperfused infarction, in contrast to its maximal upregulation 3 days after cryoinfarction. we measured the induction of the anti-inflammatory cytokine il-10 to investigate negative feedback regulation of proinflammatory response (figure 5(c)) and found a comparable time course in both models with a significantly higher induction of it in cryoinfarction after 3 days. based on our previous results regarding the role of chemokines in remodeling, we measured the expression of macrophage-related ccl2 and ccl4, as well as neutrophils-related ccl3. to our surprise, we found a significantly higher induction of ccl2 in reperfused infarction whereas cryoinfarction did not lead to its significant upregulation at a later time point (figure 5(d)). in contrast, the mrna-expression of ccl3 showed a significantly stronger early induction in cryoinfarction and a second maximum after 3 days when compared to reperfused infarction (figure 5(e)). the chemokine ccl4 showed a comparable expression pattern between the models, but also significant differences at an early and later time point (figure 5(f)). taken together, the expression of free radical scavenger enzymes, cytokines and chemokines, is directly influencing the course of cellular events in both models of infarction. still, the expression of chemokines shows a different time course, probably due to their additional role in collagen deposition during advanced stages of remodeling. since osteopontin-1 is not only related to macrophage maturation but also is involved in extracellular matrix formation and remodeling, we measured its mrna-expression and found an earlier peak after 24 hrs in reperfused infarction whereas in cryoinfarction its maximum was reached after 3 days (figure 6(a)). another marker of early remodeling, tenascin c, showed a significantly higher induction early after reperfusion of infarction, thus supporting the rapid granulation tissue formation (figure 6(b)). tenascin c-expression was significantly higher after 3 days in cryoinjured myocardium, thus mediating the prolonged granulation tissue formation. we also investigated the mrna-expression of tgf- and found a later induction of predominantly profibrotic acting tgf-1 and -2 isoforms (figures 6(c) and 6(d)). interestingly, we found a significantly higher induction of rather antifibrotic acting tgf-3 isoform 7 days after cryoinfarction, thus probably representing a strong signal for the resolution of myocardial remodeling (figure 6(e)). these data reveal specific mediators responsible for the prolonged granulation tissue formation and delayed remodeling process after cryoinfarction. our data also confirm our previously published findings on induction of tgf- isoforms in reperfused infarction and give us additional novel insights in osteopontin-1- and tenascin c-expression in the reperfused infarction model. studies using large experimental models of myocardial infarction brought a detailed analysis of pathology and some insights into mechanisms involved in myocardial ischemia. recent developments in transgenic mice and gene-targeting technologies provided tools for profound studies of the pathomechanisms and specific genes involved in cardiac repair. we developed a chronic, closed-chest murine model of myocardial ischemia followed by reperfusion, which leads to infarction with rapid scar formation. this closed-chest model has minimized the influence of the initial surgery trauma and is, therefore, particularly useful for studies of inflammatory response and early remodeling. we used this model in cellular therapy experiments and found a very poor engraftment of the i.v. injected whole bone marrow cells (unpublished observation) in contrast to a very good engraftment found in another model, that is, cryoinfarction. therefore, we assumed differences in myocardial remodeling and specifically in local microenvironment of injured myocardium. in order to investigate these differences, we compared the pathology and the course of cellular and molecular events during myocardial remodeling between the two models. in this study, we confirmed our previous findings on rapid development of granulation tissue and the time course of mediators involved in transient inflammatory reaction, as well as in subsequent remodeling [3, 4]. we found a transient, strong increase in proinflammatory cytokines and chemokines leading to a rapid tissue infiltration with neutrophils and macrophages, which was also described in large animal models. in addition, we observed a transient induction of heme oxygenase 1, which is caused by a massive production of reactive oxygen species during early reperfusion and precedes the cytokine release in the ischemic heart. development of granulation tissue was associated with anti-inflammatory action of il-10, with transient upregulation of early remodeling mediator tenascin c, and was followed by induction of macrophage maturation marker osteopontin-1 and, as previously published, upregulation of tgf- isoforms. these mediators lead to differentiation of myofibroblasts and subsequent collagen deposition, thus resulting in the stable scar formation 7 days after reperfusion of murine infarction. the timely resolution of myocardial remodeling is also confirmed by a lack of macrophage elastase and osteopontin-1 staining after 7 days reperfusion. the pathology of cryoinfarction showed a similar course of events, but a longer duration of this process until formation of a stable myocardial scar is completed. the basic histology revealed that cryoinfarction leads to an early, strong cellular infiltration of the damaged myocardium, which slowly decreased to a lower level until 14 days postinjury. we observed a permanent damage and occlusion of the large vessels in cryoinjured myocardium, which mechanically prevented an evenly distributed cellular infiltration of the injured myocardium and thus rapid myocardial remodeling, as it is observed in reperfused infarction. in cryoinfarction, macrophages and neutrophils persist up to 14 days in the last area near the center of the epicardial copper probe application site. this seems to be one of the main factors influencing the pace of remodeling in the cryoinfarction model. interestingly, we found a significantly prolonged macrophage infiltration of the noninjured area, which may be associated with a dysfunctional border zone formation and infarction limitation in this model. the concomitant appearance of elastase-producing macrophages and myofibroblasts 7 to 14 days postinjury represents an active myocardial remodeling process, which is further supported by not entirely compacted collagen in the scar. in consequence, this active myocardial remodeling is probably responsible for a good engraftment of the implanted cells in the cryoinfarction model. the mrna data provide novel insights into the time course of the expression of several mediators involved in remodeling of cryoinfarcted myocardium. the later induction of cytokines and remodeling-related mediators is leading to the prolonged remodeling in this model. in particular, the relatively high expression of heme oxygenase 1 after 3 days represents persistent reactive oxygen species production from cell debris, while the high ccl2 and ccl4 expression characterize the inflammatory response associated with strong macrophage activity. the concomitant upregulation of anti-inflammatory il-10 is acting towards resolution of the inflammatory reaction and formation of granulation tissue. the significantly higher expression of neutrophils-related chemokine ccl3 seems to reveal a strong migration stimulus into the damaged area, which is limited by the lack of intact vasculature as discussed above. chemokines, particularly the ccl2, have also been associated with myofibroblasts activity and collagen production, which may provide additional explanation for its upregulation after 3 days in cryoinfarction. using transgenic mice, we described a crucial role for the chemokine ccl2 and macrophages in timely resolution of myocardial remodeling and preservation of myocardial function. the later induction of tenascin c and osteopontin-1 as well as the tgf- isoforms represent the prolonged extracellular matrix production and collagen deposition in myocardial remodeling. this study shows substantial differences in local microenvironment and cellular and molecular events between the models of cryoinfarction and reperfused infarction. in a comparison between murine models of cryoinfarction and coronary ligation without reperfusion, a modest adverse remodeling was postulated for the cryoinfarction, and the cryoinfarction was suggested as a representative model for myocardial infarction encountered in clinical practice. we do not completely share this opinion, since the main goal in treatment of acute infarction is early revascularization with reperfusion. but for the experimental study purpose, the cryoinfarction indeed offers a very reproducible area at risk and infarct size whereas this is a major weakness of the reperfused infarction model due to the anatomical variability of coronary arteries. the prolonged myocardial remodeling seems to be beneficial in cell transplantation studies, since the cellular engraftment after direct application into injury is very good. our data provide novel insights into expression of cytokines and chemokines in cryoinfarction, where persistent proinflammatory milieu may be favorable for the cell engraftment. on the other side, the rapid resolution of inflammatory response in reperfused infarction may minimize the time span of suitable local environment for cell engraftment, but this concept still has to be further investigated. in respect to the ongoing clinical trials, the reperfused infarction model could be seen as a more relevant model, but it has not been widely used yet. first, the findings are mainly of experimental interest since the model of reperfused myocardial infarction is comparable to a clinical situation in patients presenting with acute infarction, but it seems to be less favorable for the cell engraftment studies. on the other hand, the cryoinfarction model provides good conditions for cell engraftment and it is very reproducible, but of a limited value to the clinical practice. second, we did not measure infarct size and one could argue that a difference in infarct size may explain the observed differences in pathological and molecular events between the two models. based on previously published work, the model of reperfused infarction [4, 5] affects mostly a larger portion of left ventricle than the cryoinfarction, but the reperfusion still leads to a faster resolution of inflammation and scar formation than the cryoinjury. therefore, the infarct size does not reflect the quality and pace of myocardial remodeling, and we did not found it to be necessary for explanation of the findings in this study. in conclusion, the cryoinfarction is associated with prolonged inflammatory response leading to a postponed granulation tissue formation and scar development, when compared to the reperfused myocardial infarction. several inflammatory mediators and remodeling factors are involved in this process, and they all contribute to a specific, dynamic local environment in ischemic myocardium. these substantial differences in remodeling may affect and even be favorable for cellular engraftment and should therefore be considered in cell therapy studies.

myocardial infarction is associated with inflammatory reaction leading to tissue remodeling. we compared tissue remodeling between cryoinfarction (cmi) and reperfused myocardial infarction (mi) in order to better understand the local environment where we apply cell therapies. models of closed-chest one-hour ischemia/reperfusion mi and cmi were used in c57/bl6-mice. the reperfused mi showed rapid development of granulation tissue and compacted scar formation after 7 days. in contrast, cmi hearts showed persistent cardiomyocyte debris and cellular infiltration after 7 days and partially compacted scar formation accompanied by persistent macrophages and myofibroblasts after 14 days. the mrna of proinflammatory mediators was transiently induced in mi and persistently upregulated in cmi. tenascin c and osteopontin-1 showed delayed induction in cmi. in conclusion, the cryoinfarction was associated with prolonged inflammation and active myocardial remodeling when compared to the reperfused mi. these substantial differences in remodeling may influence cellular engraftment and should be considered in cell therapy studies.

PMC2997608

pubmed-1285

laryngeal cancers represent about 30% of head and neck cancers with a high incidence for the glottic location (from 25 to 85%) [1, 2]. there is currently no recommendation from the uicc (international union against cancer) for pre-therapeutic assessment (endoscopy, computed tomography, and magnetic resonance imaging) specifying in particular the need to involve them. now the evaluation of the cartilaginous extension, especially thyroid, is an important element in the pre-therapeutic assessment of the endolaryngeal cancer. in case of infiltration the tumor is classified ct4a, and the patient is amenable to an aggressive therapy given the high risk of local recurrence and low radiosensitivity. the purpose of this study is to evaluate, by comparing, the existing correlation between preoperative radiological classification of endolaryngeal tumors involving the anterior commissure (endoscopy, ct scan) and postoperative classification (pt pathology). this is a single-center retrospective study (19982005) conducted at the croix-rousse hospital on 127 patients with endolarynx cancer involving the anterior commissure. all these patients were surgically treated and 32 of them with a total laryngectomy, the 95 other patients with a partial laryngectomy. squamous-cell carcinoma represented the main pathology (124 patients) with a case of adenosquamous carcinoma, a case of pseudosarcomatous carcinoma, and a last case of verrucous carcinoma. all patients underwent a nasofiberscopy (including a verification of chordal mobility) followed by panendoscopy using optical at 0 and 30. radiological examinations were performed in seven different centers in 2 mm maximum sections, in all cases after injections of contrast. the tumor extension should be clarified to the following areas: sub glottis, paraglottic area, preepiglottic area, and cartilages (thyroid, cricoid). the patients were 58 years old in average, 93% of them were smokers, and more than half had a regular alcohol consumption. table 1 summarizes the comparison of ct and pt stages for all tumors in the study. only 76% of tumors have been classified correctly with the endoscopy and ct scan combination. it is more likely to correctly classify tumors with a limited size since only 64% of t3 and t4 tumors were correctly classified preoperatively. 24.6% of ct2 and 33.3% of ct3 tumors are actually reclassified pt4 after histopathological examination. this shows that the cartilage invasion is frequently under-estimated by the radiological assessment with endoscopy and cervical ct scan. this underestimation of the cartilage infiltration is more common than on the assessment of the subglottis, paraglottic areasc and preepiglottic extension. only 2% of ct2 tumors were reclassified pt3, and 5% of the ct3 tumors were reclassified pt2 after pathological examination of the specimen. a decrease in the survival rates of laryngeal cancer in the united states, the terms of support, especially for the advanced stages, are currently controversial. if no conclusion of this study can be drawn in a formal and dogmatic manner, the proliferation of treatment options (radical surgery, partial surgery, transoral surgery, and laryngeal preservation protocols) requires more accurate and right pretreatment staging. one of the major means of all these studies, randomized or not, is the selection mean with the inclusion of patients with faulty pretreatment radiological staging. the underestimation of the thyroid cartilage invasion by cervical ct is found in many studies with variable rates. in a prospective study related to the cartilage invasion, realized on 40 patients, zbren et al. found a 67% sensitivity and a 87% specificity of ct. when studies involve all endolaryngeal tumors, it appears that the anterior commissure of the underestimation is greatest. thus, on nakayama and brandenburg's cases, 50% of t3 tumors were subclassified by breach of a micro-cartilage invasion. 90% of this situation involved tumors of the anterior commissure. the special case of the thyroid cartilage invasion via the anterior commissure was also clearly demonstrated. barbosa et al. then found a 25% overall underestimation of the ct stage of the anterior commissure tumors by the combination ct/endoscopy. however, reports show a stronger underestimation for smaller tumors classified as t1 and t2 (respectively 50% and 62% of correct estimation). showed that the redefinition of radiological criteria allowed the increase in accuracy of radiological conclusions in two successive audit cycles by increasing the accuracy rate of 45% to 71%. this also shows that our balance sheet allows us to achieve correct figures and that theses are not related to poor quality of the examinations. table 2 summarizes the various studies on the reclassification of endolaryngeal tumors after postoperative pathologic examination (initial radiological staging ct/endoscopy). some scan signs to look for can increase the relevance of this paper. for the thyroid cartilage, specificity (ability to correctly identify individuals who are not affected by the disease) would be about 93% for erosion or lysis the combination of these signs causes a high sensitivity (ability to detect cases of a disease) and reinforces the negative predictive value (probability of being healthy if negative). zbren et al. also studied retrospectively the reclassification of the endolaryngeal recurrent lesions. in these cases, it appears that the combination ct/endoscopy is even less accurate to correct a tnm stage assessment. they report indeed a 48% sensitivity on the study of the thyroid cartilage invasion and 47% for the cricoid invasion. our study did not use any mri as an examination to detect the cartilage infiltration. with a 8994% sensitivity and a 7488% specificity, its negative predictive value (npv) is around 9496%, but its positive predictive value (ppv) reaches only about 71% (probability of getting sick if positive). they are then many false positives with a significant risk of inadequate treatment by overestimation [1215]. this difficult pretreatment evaluation of the cartilaginous extension of endolaryngeal tumors leads us to suggest a surgical approach with resection of exposed cartilaginous portions as soon as the anterior commissure and the anterior portion of the subglottis are getting invaded. our study shows that radiological pretreatment classification (ct) of laryngeal cancer involving the anterior commissure is often inaccurate when compared with postoperative pathology (pt). the mri appears to offer a more effective accuracy but still below the pt. this finding should lead to a transdisciplinary consideration from the moment the treatment choice is not directed towards a first surgical resection.

background. the objective of this study is to assess the accuracy of pre- and posttherapeutic staging of endolaryngeal cancer involving anterior commissure. materials and methods. 127 patients were included in this retrospective study, and laryngectomy (partial or radical) was achieved in all of them. initial radioclinical evaluation (ct) was performed (endoscopy-ct scan) and compared with postoperative histopathological findings. results. 24,6% of ct2 and 33,3% of ct3 laryngeal tumors were reclassified pt4 after the histopathological examination. conclusion. pre-therapeutic staging (combining endoscopy-ct scan) of endolaryngeal cancer involving anterior commissure is inadequate and sometimes underestimates thyroid cartilaginous invasion. nethertheless, a precise diagnostic assessment by surgery with postoperative histological findings is possible. cartilage and/or paraglottic structures are involved, or not, on the laryngectomy specimen exam. so surgery should always be discussed in first line in transdisciplinary meeting for endolaryngeal cancer management.

PMC3671717

pubmed-1286

association of osteomalacia or rickets with neurofibromatosis has been documented only rarely. as a rule, osteomalacia in neurofibromatosis is characterized by later onset in adulthood, renal phosphate loss with hypophosphatemia, and multiple pseudofractures in the typical cases. the hypophosphatemic conditions that interfere in bone mineralization comprise of many hereditary or acquired diseases, all of them sharing the same pathophysiological mechanism-reduction in the phosphate reabsorption by the renal tubuli. this process leads to chronic hyperphosphaturia and hypophosphatemia, associated with inappropriately normal or low levels of calcitriol, causing rickets in children and osteomalacia in adults. a 43-year-old woman presented to the orthopedic department of our institute with progressive bone pain and difficulty in walking since two years. the patient was a known case of neurofibromatosis-1, with multiple cutaneous nodules, from her early childhood. she was otherwise healthy until the age of 40 years, when she started experiencing progressive bone pain affecting her thighs, pelvis, and left forearm. in recent times, she could walk only with the help of crutches. on examination, tenderness and deformity were present in the regions of her left forearm, bilateral thigh, left knee, and leg. multiple skin nodules were present all over her face, back, and abdomen [figure 1]. 43 yrs old, female known case of von recklinghausen neurofibromatosis with multiple skin nodules all over her face and legs her skeletal survey revealed generalized osteopenia, coarse trabeculations, and nodular shadows in the soft tissue. in addition, a radiograph of her left forearm with elbow revealed fractures of the upper shafts of the radius and ulna [figure 2]. a radiograph of her left knee revealed pseudofractures (looser's zone) in the distal femur, upper shafts of the tibia/fibula and mid shaft of the fibula [figure 3]. a radiograph of the pelvis with bilateral hips showed a deformed, triradiate pelvis, with bilateral symmetrical fractures of the upper shaft of the femur and pseudofractures of the inferior pubic rami [figure 4]. and lateral revealed diffuse osteopenia with fractures of upper shafts of radius and ulna (arrows) radiograph of left knee with leg a.p and lateral revealed generalized decrease in bony density with pseudo fractures (looser's zone) in the distal femur, upper shafts of tibia/fibula and mid shaft of fibula (white arrows). note nodular shadows in soft tissue (black arrows) radiograph of pelvis-a.p. view. triradiate pelvis with osteopenia and bilateral, symmetrical fracture of upper shafts of femur (white arrows) with bilateral, symmetrical pseudo fracture of inferior pubic ramii (black arrows) the laboratory data in our institute were as follows: serum calcium was 8.7 mg/dl (normal 8.5-10.5 mg/dl). serum phosphorus was 1.5 mg/dl (normal: 2.5-4.5 mg/dl). alkaline phosphatase was 650 lu/l (normal 44-147iu/l). the 24-hour urinary excretions of calcium and phosphorus were 98 mg/24 hours (normal: 0-300) and 440 nmol/24 hours (normal: 13-42), respectively. her serum parathyroid hormone (pth) and 25-(oh) vitamin d were within normal range. on the basis of the radiological and laboratory findings, a final diagnosis of hypophosphatemic osteomalacia in a patient of von recklinghausen disease was made. she has been prescribed a high dose of calcitriol and oral phosphate and in view of innumerable neurofibromas, surgical resection was not advised. her constitutional symptoms have improved, but the fractures have shown no radiological signs of healing in the last three months of follow-up. oncogenic hypophosphatemic osteomalacia (oho) is a rare endocrinological paraneoplastic syndrome, characterized by defective bone mineralization from renal phosphate loss. it is an unusual condition, but probably still is the most common cause of acquired hypophosphatemic osteomalacia in adult males. the affected age group range is between seven and seventy-seven years with a male: female ratio of 1.2:1. patients characteristically present with joint deformities, waddling gait, bone pain, muscle weakness, anorexia, fatigue, and occasionally long-bone fractures. the initial clinical presentation may be mistaken for rheumatoid arthritis, muscular dystrophy or primary neurological disorder in some cases. if the biochemical profile of the patient is that seen in hypophosphatemia, namely low phosphate in the serum and high phosphate excretion in the urine, and the patient is not responding adequately to oral calcium and vitamin d therapy, then the possibility of an underlying cause of either renal or oncogenic or hereditary x-linked hypophosphatasia must be entertained. the most common tumor described is hemangiopericytoma, but other tumor types described include fibrous dysplasia, osteosarcoma, chondroblastoma, chondromyxoid fibroma, malignant fibrous histiocytoma, giant-cell tumor, hemangioma, paraganglioma, prostate cancer, and oat-cell carcinoma of the lung. hypothesize that putative melatonin deficiency in cases of neurofibromatosis-1 may play a role in the pathogenesis of hyperphosphaturia, by decreasing the sodium-phosphate cotransport, increasing the level of cyclic adenosine monophosphate (camp) and the un-antagonized effect of dopamine on phosphate reabsorption, and increasing the glucocorticoid levels. parathyroid overactivity that may occur secondary to osteomalacia may have synergistic effects with dopamine and further exaggerate the phosphate loss in urine. on the other hand moreover, in the presence of hypophosphatemia, hypercortisolism may further inhibit melatonin secretion that may lead to progression of bony deformities in these cases. in recent times, it has been suggested that in the epidermal nevus syndrome and type-1 neurofibromatosis, hypophosphatemic rickets/osteomalacia is probably due to increased secretion of fibroblast growth factor 23 (fgf-23) by cells from the nevus or neurofibromas. x-linked hypophosphatemic rickets, autosomal dominant hypophosphatemic rickets, tumor-induced osteomalacia (tio), fibrous dysplasia, and the mccune albright syndrome share a common underlying pathophysiological condition: increased phosphorus renal loss secondary to augmented fgf-23 plasma levels and activity. (a gene on the x chromosome that codes for a zn-metaloendopeptidase proteolytic enzyme, which regulates the phosphate). fgf-23, the largest member of the fibroblast growth factor family (fgf), contains 251 amino acids and is encoded by a gene in 12p13. the metabolic abnormalities in oncogenic osteomalacia are hypophosphatemia, hyperphosphaturia, low or normal serum calcium, raised alkaline phosphatase, low concentrations of 1,25 dihydroxy vitamin d, decreased tubular resorption of phosphates, normal paratharmone levels, and normal urinary calcium. these are believed to be the result of mesodermal dysplasia, intrinsic to the disease and they appear early in life. they cause bone deformities, and are not associated with disturbances in calcium and phosphate metabolism. in contrast, osteomalacia of neurofibromatosis is very rare, presents in middle age, and is associated with marked disturbance of phosphate metabolism. the presence of multiple symmetrical pseudofractures (looser's zone), high alkaline phosphatase, low serum phosphate, and generalized demineralization of the bones pointed to the diagnosis of hypophosphatemic osteomalacia. if complete excision of the tumor is achieved, it can lead to improvement in the clinical course of the disease as well as the biochemical markers, and may be curative. in patients with neurofibromatosis and osteomalacia, although innumerable neurofibromas are present, it is probably the largest ones or those with recent growth that cause oho and their surgical removal should be tried, to achieve permanent cure. high doses of calcitriol and oral phosphate salts are indicated, similar to those used in the treatment of x-linked hypophosphatemia. osteomalacia in neurofibromatosis is a very rare entity and distinct from the more common dysplastic skeletal affections of this disease, as a rule, being characterized by later onset in adulthood, with renal phosphate loss, and with hypophosphatemia and multiple pseudofractures in typical cases. in patients with neurofibromatosis, although innumerable neurofibromas are present, it is probably the largest ones or those with recent growth that cause oho, and their surgical removal should be tried, to achieve permanent cure, along with high doses of calcitriol and oral phosphate.

osteomalacia in neurofibromatosis is a rare entity and distinct from more common dysplastic skeletal affections of this disease. as a rule, it is characterized by later onset in adulthood. there is renal phosphate loss with hypophosphatemia and multiple pseudofractures in the typical cases. the hypophosphatemic conditions that interfere in bone mineralization comprise many hereditary or acquired diseases, all of them sharing the same pathophysiological mechanism-reduction in phosphate reabsorption by the renal tubuli. this process leads to chronic hyperphosphaturia and hypophosphatemia, associated with inappropriately normal or low levels of calcitriol, causing rickets in children and osteomalacia in adults.

PMC4329691

pubmed-1287

chronic pain affects up to 20% of the population in developed nations.14 this represents a profound impact on individuals and their families alongside the sizeable burden on employers, health care systems, and society in general.3 when chronic pain occurs, it has the potential to become disease itself, and subsequently, chronic pain has emerged as a distinct phenomenon.5 management of chronic pain varies greatly between nations and even within nations. literature supports a multidisciplinary approach as the standard of care, although various health care systems may not always support this concept consistently.2 the current standard of care for chronic, noncancer pain typically includes many disciplines with the clinician developing an individualized treatment plan with the options of utilizing surgical interventions, pharmacology, and psychological and physical therapies. opioid analgesics are often prescribed, despite the lack of clinical evidence supporting their long-term use in the management of chronic pain.6 however, for many patients, this multidisciplinary approach is inadequate or ineffectual or is accompanied by the burden of side effects that are unacceptable and debilitating. only at this late stage, the field of neuromodulation for the treatment of pain has developed rapidly since the seminal paper on the electrical inhibition of pain by the stimulation of the dorsal column almost 50 years ago.7 the original term of dorsal column stimulation has evolved to become known as spinal cord stimulation (scs).8 scs has been particularly effective as an adjunct in treating mixed neuropathic/nociceptive and neuropathic/radicular pain conditions such as failed back surgery syndrome (fbss) and complex regional pain syndrome (crps). neuromodulation therapies offer a treatment option that has minimal side effects and that is relatively safe and potentially reversible.9 scs has been used to treat various pain conditions for many decades.8,1013 in traditional scs therapies, the objective has been to replace the pain sensation with paresthesia that requires mapping of stimulation to the region of pain.14 the anticipation is that the electrical current alters pain processing by masking the sensation of pain with a comfortable tingling or paresthesia. although patients mostly cope with paresthesia, a significant proportion report that the sensation is unpleasant, particularly with positional changes. the stimulation is provided either through electrodes that are placed percutaneously into the epidural space or through a surgical paddle lead that is delivered via a laminotomy.8 these devices are capable of delivering pulse frequencies in the range of 21,200 hz but are regularly utilized at 4060 hz. patients typically undergo a trial of neuromodulation with an externalized power source and if this trial proves to be positive and compelling, they subsequently have a subcutaneously implantable pulse generator for the long-term therapy. in recent years, the next phase in the evolution of neuromodulation has become available with the development of dorsal root ganglion (drg) scs and the emerging use of two novel advances in stimulation frequencies, being high-frequency scs (at 10,000 hz) and burst scs.1419 these recent advances have improved the efficacy and expanded the applicability of scs. drg scs is a highly targeted form of neuromodulation therapy.20 studies indicate that the drg plays a key role in both nociceptive and neuropathic pain.14,21 drg scs is particularly useful in treating focal areas of pain, in particular those that have been difficult to target with traditional scs systems such as groin and foot pain, by applying an innovative lead configuration and delivery system around the drg.8,9,15 high-frequency 10 scs (hf10) presents a significant development in the evolution of scs technologies.19 this involves application of a unique waveform at 10,000 hz at a subthreshold level and therefore provides pain relief without any paresthesia.18,22 the majority of patients have a clear preference for paresthesia-free stimulation, and hf10 has been approved for clinical use in australia and europe since 2011 and has received food and drug administration approval for the united states in 2015 for patients with chronic refractory pain of the trunk and/or limbs.22,23 burst scs offers another novel mode of stimulation whereby conventional frequency parameters are provided in bursts of five pulses. the burst frequency is 40 hz, and the pulse frequency is 500 hz. amplitude is reduced to try and achieve subthreshold stimulation, thereby providing pain relief with either reduced or no paresthesia.16,17,24 the most recent systematic and comprehensive review of the effectiveness of scs in treating chronic spinal pain demonstrated that there is a significant (level i ii) evidence for scs as a treatment for lumbar fbss, where conventional medical management has failed.23 furthermore, there is now level i evidence for high-frequency stimulation but only limited evidence for burst stimulation.23 in another recent and extensive review and meta-analysis of conventional scs, more than half of all patients experienced significant pain relief.25 the authors observed that this was maintained for a mean follow-up period of 24 months.25 these reviews demonstrate that traditional scs is an effective treatment option for a cohort that is notoriously difficult to treat. the existing scs literature has a large number of case series reports and only a limited number of high-quality, large prospective, consecutively recruited, randomized, or controlled comparative trials (table 1).8,23,25 furthermore, the literature, when viewed historically, must be tempered by the developments in skills, application, and technological advances.26 hence, the traditional scs papers have often reported successful pain relief as an undifferentiated generic pain that is not specific to the site of the primary or greatest pain (eg, back or leg).25 this observation is important because conventional scs therapy has historically been prescribed for limb pain and has had only limited success in managing back pain.10,2729 indeed, predominant back pain has been an exclusionary factor in many studies.25 recent studies that have included back pain as the primary source have involved hf10 therapy at 10,000 hz; this therapy has evolved to better capture significant back, leg, and radicular pain.10,22 tolerance to scs has been observed in patients where pulse amplitude needs to be increased to achieve the same analgesic benefit over time and/or efficacy has been lost.30,31 tolerance can not be predicted, and although the rate has not been widely reported in the literature, one study that researched over 10 years found it to be in the order of 29%.32 possible causes for stimulation tolerance include neuroplasticity of pain transmission pathways, cellular or fibrotic changes in the tissues around the electrodes, patients reframing their pain over time, and psychological or psychiatric affective disorders.30 however, data pertaining to hf10 scs have demonstrated no tolerance at this point.22 despite strict criteria for patient selection, a substantial number of patients fail to achieve optimal pain relief with scs.30,33 a number of factors have been identified as possible indicators for treatment failure including tobacco and drug use, age, and lengthy delay between times of original pain onset to scs implant.30,33 food and drug administration requirements for labeling the recent advanced iterations of scs systems have led, for the first time, to level i noninferiority comparative studies being undertaken to achieve labeling. drg scs has been demonstrated as effective in multiple etiologies, including fbss, crps, and chronic postsurgical pain.15 a recent study reported 1 year outcomes for drg with overall pain scores reducing from 77.6 to 33.6 (p<0.005). back pain reduced from 74.5 to 39.7 (p<0.05), and leg pain reduced from 74.6 to 28.7 (p<0.0005). the most compelling pain reduction happened for foot pain with scores reducing from 81.4 to 22.0 (p<0.05).15 approximately 60% of the drg scs patients reported>50% improvement in their pain, and the pain localized to the back, legs, and feet was reduced by 42%, 62%, and 80%, respectively.15 other outcome parameters including quality of life, mood, and satisfaction were improved and maintained throughout the 12 months.15 the accurate study is a us pivotal, noninferiority, randomized controlled trial (rct) between drg scs and traditional scs medtronic system (medtronic, inc., fridley, mn, usa). it is the largest rct in the history of crps and causalgia, running from 2013 with primary completion estimated for 2018. the sample size for the study is 152; with 76 randomized to drg scs and 76 to the control arm using medtronic traditional scs. the inclusion criterion was leg pain for more than 6 months duration with a visual analog scale (vas) score>6/10.34 in the intention-to-treat analysis, superiority was demonstrated in the drg scs group with 81% of patients achieving>50% pain reduction and meeting the primary endpoint at the 3-month mark, and 74% maintaining that primary endpoint at 12-month follow-up. the traditional scs arm demonstrated 56% of patients having>50% pain reduction at 3 months and 53% maintaining this through 12 months.34 in the subset analysis of the implant-only group, the data were even more impressive with 93% of the drg scs group meeting the primary endpoint at 3 months and 86% at 12 months. the traditional scs arm had 72% meeting the primary endpoint and 70% at 12 months.34 the statistical analysis demonstrated noninferiority and, beyond that, superiority in the intention-to-treat, modified intention-to-treat, and implant-only groups. furthermore, it was noted that 70% of patients achieved>80% pain reduction in the drg group versus 52% in the medtronic group. target specificity for stimulation and pain relief was achieved in 94.5% of the drg group and 61% of the medtronic group.34 the sunburst study is set to run from 2013, with primary completion in 2016. it is a prospective randomized, non-inferiority controlled trial with the st jude medical company (st jude medical, inc. patients with intractable pain were randomized for the order they would receive either traditional or burst scs. the study was performed with a one-to-one crossover at 12 weeks to the alternate therapy. the study was applied to an enriched cohort with patients who required to have pre-existing pain scores>6/10 and a>50% pain reduction in a traditional scs trial using tonic stimulation. the mean age of patients was 59 years, with a median duration of pain being 13 years.35 the trial demonstrated noninferiority, and further statistical analysis demonstrated superiority for burst stimulation over tonic stimulation (p=0.035).35 the mean difference in burst pain reduction compared with tonic stimulation was 6 mm vas points. this difference, while being statistically significant, does not meet the well-defined criteria for minimal clinical important difference.36 approximately 65% of the burst cohort experienced paresthesia-free stimulation and 69% of the cohort chose a preference for burst, with the majority of these having their preference related to no paresthesia, more so than better pain reduction.34 the senza rct is a level i study design run from 2012 with an estimated primary completion in 2015.22,37 this is the first-ever rct of two scs therapies with patients randomized to hf10 scs (senza system; nevro corp., redwood city, ca, usa) or traditional scs commercially available, precision plus, scs system (boston scientific corporation, marlborough, ma, usa). it is a noninferiority study with the statistical capability of demonstrating superiority supervised by the food and drug administration, and patients were monitored and programmed by the technicians associated with the respective devices. one hundred and ninety-eight patients were randomized with 101 to the hf10 scs group and 97 to traditional. of these, 90 hf10 scs patients and 81 traditional scs patients were subsequently implanted. the primary endpoint of>50% back pain reduction at 3 months was achieved in 80.9% of the hf10 scs group versus 42.5% of the traditional scs group.37 this met the criteria for noninferiority and statistical superiority (p<0.001). furthermore, at 12 months, this primary endpoint was met in 78.7% versus 51.3% of the patients. similarly, the primary endpoint for leg pain reduction was met in 80.0% of the hf10 scs group versus 49.4% of the traditional scs group.37 the responder rates for>50% leg pain reduction at 3 months was 83.1% in the hf10 scs group and 55.0% in the traditional scs group. the 12-month outcome data for the same groups were 78.7% versus 51.3% (superiority p value, p<0.001).37 this study demonstrated superiority of hf10 scs to traditional scs in all primary and secondary endpoints that has led to the labeling of hf10 therapy as superior to traditional low-frequency scs by the food and drug administration. in further analysis of pain etiology, it was demonstrated that for the conditions of fbss, radiculopathy, degenerative disc disease, and spondylosis, the relative ratio of patients meeting the primary endpoint with hf10 scs was approximately 2.0 times the traditional scs (eg, fbss 85.7% versus 41.4%).37 further subset analysis of patients who achieved vas pain scores of 2.5 showed that for those with back pain, relief was maintained for 12 months 68.5% of the time for hf10 scs, but only 35.8% of the time using traditional scs. whereas, for those with leg pain it was achieved 67.4% of the time for hf10 scs versus 42.5% of the time with traditional scs. superiority p-values for hf10 were significant (p<0.001).37 these data demonstrate compelling evidence for treating complex back pain that was previously unheralded in the literature. furthermore, paresthesia-free options allow the patient to keep stimulation on potentially 24 hours a day and hence sleep with the stimulator on and also perform activities such as driving. health care policy and funding decisions require evidence of clinical efficacy and information around cost-effectiveness of treatments.38,39 consequently, there have been a number of studies considering the economic factors associated with scs. most recently, a 2015 study investigating the cost-effectiveness of conventional medical management with or without scs in patients with fbss compared a summary of the total direct and indirect costs incurred in the 12 months prior and 24 months following scs.40 the costs were scaled to values of 2,009. the year of implant incurred a significant increase in costs of 20,902/patient-year, mainly attributed to the high cost of the scs devices. in the following 1214 months, scs implant had dropped to 5,430/patient-year.40 applying the current united kingdom national health service threshold (intervention considered not cost-effective if the incremental cost-effectiveness ratio is higher than 45,000/quality-adjusted life year [qaly]), scs with conventional medical management would be considered cost-effective around 40% of the time.40 however, if the willingness-to-pay threshold was shifted to 60,000/qaly, scs would be considered cost-effective by the national health service with an average of 80% of the time. in 2013, a study developed models to evaluate the cost-effectiveness of scs and conventional medical management together compared with conventional medical management alone for patients with fbss and crps.41 health effects were expressed as qalys and costs were expressed as canadian dollars (can$) scaled to 2012. the models were extrapolated over a 20 year-time period with 3.5% discounts annually (as per national institute of clinical excellence suggestion). the modeling data showed that scs with conventional medical management is cost-effective compared with conventional medical management alone for all presentations, with a cost-effectiveness ratio for scs of can$9293 for fbss and can$11,216 for crps per qaly gained.41 in a study in 2010, the cost-effectiveness of scs with conventional medical management was compared with conventional medical management alone in crps patients.42 this study models economic costs using a simulated model population, employing parameters and assumptions set from previously published randomized trials. here, scs was shown to be cost-effective in select crps patients, with a probability exceeding 80% that scs is cost-effective where the willingness to pay is set for a maximum of 30,000 per qaly.42 another cost-effectiveness study of scs was performed in 2010 using a fbss cohort.42 here, the authors compared scs versus conventional medical management versus reoperation. this study showed that in selected patients, scs is cost-effective both as an adjunct to conventional medical management and as an alternative to reoperation; that the likelihood scs would be cost-effective versus conventional medical management and versus reoperation exceeds 80%, where the willingness to pay is set for a maximum of 20,000 per qaly.42 in 2008, a systematic review for cost-effectiveness of three studies where fbss patients had been treated with scs demonstrated that scs is both more effective and less costly than conventional medical management alone in the long-term but there are high upfront implant costs associated with scs implantation and maintenance.43 in 2008, another study reported the generic health-related quality of life and costs of scs at 6 months follow-up (using data from the process trial) compared to the quality of life, resource consumption, and costs of conventional medical management alone in patients with fbss.39 the study found that the mean total health care costs for the scs group were significantly higher (12,653) than the conventional medical management group (2,594), when scaled for uk 20052006 national data.39 this result reflects the high upfront costs of scs over the limited 6-month follow-up period. the authors showed that 15% of the additional mean cost of scs was offset within 6 months by a reduced use of drug and nondrug therapies. they also demonstrated a gain in quality of life over the same period which was significantly greater for the scs group. the study concludes that, over the short term, scs treatment results in greater healthcare costs but also generates important health improvements for the patients over the same period.39 cost-efficacy studies show that despite significant initial costs, scs compared with other conventional treatments available to chronic pain patients results in long-term reductions in health care costs, which offset the high initial treatment costs over time.44 in the literature, scs is reported as a safe procedure due to its reversible and minimally invasive characteristics.30 although catastrophic complications are possible, they are very rare. however, the incidence of minor complications of scs has been reported at around 30%40%.13,30,31,4547 these minor complications tend to occur within 12 months of implantation and are readily reversible and generally resolved.13 the complications are divided into three main categories: mechanical, biological, and technique-related.32 complications of a mechanical origin are more common than those of biological origin.31,45 historically, hardware-related complications occurred at a rate of between 24%50%, whereas adverse biological events occurred in 7.5% of cases.30 mechanical complications include lead fracture or disconnection, which has a reported incidence of between 5% and 9%; lead migration has a reported incidence between 0% and 27%; implantable pulse generator failure occurred at a reported frequency of 1.7%.11,30 these complications can be minimized by using appropriate leads, anchoring, and suturing techniques. furthermore, minimizing patient movements in the first 3 months after surgery allows for postoperative scarring of leads into place.30 kapural et al22 demonstrated that lead migration of significance and requiring intervention in both the hf10 and traditional scs arms occurred<5%. this most likely reflects improvements in both lead design and the anchoring systems used (figures 13).14,22,26,48 biological complications include infection, allergic reaction, pain at implant site, implantable pulse generator seroma, epidural fibrosis, epidural hematoma, dural puncture, and, rarely, neurological injury.30,31,4952,5760 the most common biological complication is infection with a rate between 3% and 8%, and the majority of these are superficial.30 as is seen in the more recent literature,30 incidence of infection can be minimized via pre and postoperative antibiotic use and appropriate skin preparation. the occurrence of dural puncture is reported as between 0.3% and 2%.53 other adverse biological events such as epidural fibrosis, compressive phenomenon, or spinal cord injury, while serious, are rare. the neuromodulation appropriateness consensus committee has recommended a number of criteria and adaptations to practice to help reduce complications: physician training and mentoring, the appropriate and careful selection of patients, a continued focus on equipment development and innovation, as well as the dissemination and application of practice and research-based advances.30 as this field strives to provide high-quality, transparent, and independent empirical evidence for scs therapies, it is possible to overlook the primary rationale for scs treatment in the first place: patient benefit. improvements to training in pain management are needed, beginning at the undergraduate medical level. at present, the time allocated to pain management is generally inadequate; for instance, in the united kingdom, the median time spent on pain management by a medical student is 13 hours, and sometimes it is as little as 6 hours.2 indeed, when the undergraduate training of all healthcare professionals is analyzed, education about the identification, assessment, and treatment of pain represents less than 1% of university-based teaching yet pain is the most common reason for patients to consult their general practitioner.2 the undergraduate training of all healthcare professionals is analyzed, education about the identification, assessment, and treatment of pain represents less than 1% of university-based teaching yet pain is the most common reason for patients to consult their general practitioner.2 the ensuing repercussions may include inadequate diagnosis, inappropriate treatments, and extended periods of mismanagement. further complicating the treatment of this cohort has been the inclusion of neuromodulation therapies as a treatment of last resort.54 yet research has shown that shifting scs forward in the treatment algorithm of refractory chronic pain is associated with better patient outcomes.44,55 in recent years, many researchers and practitioners have included patient satisfaction (alongside empirical measures) as a clinically useful metric for assessing scs treatment success.23 when reported, overall patient satisfaction is high for the vast majority of scs patients, perhaps reflecting the efficacy of scs treatment, but may also reflect factors such as limitations of alternative treatments, safety, and tolerability of scs and the rapidity of onset and durability of scs treatment.56 moreover, under current arrangements, most patients access scs treatment through private health insurance schemes or through compensatory bodies such as workers compensation schemes.57 for patients who do not qualify for these funding systems, the immediate cost outlay would likely post a significant financial barrier to treatment.57 the scs specialty should aim to support the provision of equitable and accessible treatment for all. significant evidence exists for traditional scs as a safe, clinical, and cost-effective treatment for many chronic pain conditions. indeed, the field is rapidly evolving, and there is now level i evidence for newer techniques including hf10 scs and drg scs, which demonstrate dramatic improvements in overall efficacy in reducing pain in specific conditions, including failed back surgery, back pain, neuropathic leg pain, crps, and causalgia. furthermore, the field has increasingly met the challenge of not only having newer devices to achieve these outcomes but concurrently reducing the risks of complications and adverse events. the data supporting scs in its multiple forms are compelling and have reached a level that now demands that this therapy be considered earlier in the treatment continuum and to be no longer regarded as simply an end-stage salvage therapy.

spinal cord stimulation (scs) applications and technologies are fast advancing. new scs technologies are being used increasingly in the clinical environment, but often there is a lag period between the clinical application and the publishing of high-quality evidence on safety and efficacy. recent developments will undoubtedly expand the applicability of scs, allowing more effective and individualized treatment for patients, and may have the potential to salvage patients who have previously failed neuromodulation. already, high-level evidence exists for the safety, efficacy, and cost-effectiveness (level i ii) of traditional scs therapies in the treatment of chronic refractory low back with predominant limb pain (regardless of surgical history). more than half of all patients with chronic painful conditions experience sustained and significant levels of pain reduction following scs treatment. although only limited evidence exists for burst stimulation, there is now level i evidence for both dorsal root ganglion scs and high-frequency scs that demonstrates compelling results compared with traditional therapies. the body of evidence built on traditional scs research may be redundant, with newer iterations of scs therapies such as dorsal root ganglion scs, high-frequency scs, and burst scs. a number of variables have been identified that can affect scs efficacy: implanter experience, appropriate patient selection, etiologies of patient pain, existence of comorbidities, including psychiatric illness, smoking status, and delay to scs implant following pain onset. overall, scientific literature demonstrates scs to be a safe, effective, and drug-free treatment option for many chronic pain etiologies.

PMC4938148

pubmed-1288

testicular germ cell tumors comprise the most common group of solid malignancies in male population of 15 to 35 years old. these tumors account for approximately 95% of all testes cancers. the incidence of testicular tumors varies in different countries and ethnic groups with the highest reported incidence in scandinavia region (about 9 in 100,000 males). few reports are present about the incidence and epidemiologic features of testis cancer in the male population of asian and latin american countries, which indicate that testicular germ cell tumors are less common in these regions than most western countries. a recent study has shown that the incidence of testis cancer is 1.3 in 100,000 male population in iran. these include positive family or personal history of testis tumor, cryptorchidism and intratubular germ cell neoplasia (itgcn). earlier studies have claimed that concomitant itgcn is seen in 80%90% of testes affected by germ cell tumors using immunohistochemistry (ihc). thus, itgcn is also considered as a premalignant lesion and prompt treatment is essential if it is incidentally discovered in testis biopsy. in addition, in cases of testis sparing surgery, itgcn may be observed in the remnant tissue. in these situations,, we aimed to assess the prevalence of itgcn in the patients who have underwent radical orchiectomy due to testicular tumor, as well as its correlations with pathologic features of concomitant testis cancer and patients ' epidemiologic characteristics. to our knowledge, this is the first report on itgcn in association with testicular germ cell tumor in our region, also the first study which is looking for its prognostic role. this is a descriptive analytical and cross sectional study which was performed in our center. the study population was the patients who underwent radical orchiectomy due to testis tumor from 2003 to 2015. serum tumor markers had been measured before the surgery and consisted of alpha-fetoprotein (-fp), and beta-human chorionic gonadotropin (-hcg). cd-117 (c-kit) and placental-like alkaline phosphatase were two immuno-reactive agents that were used to identify itgcn via ihc. data about patients ' age at the time of surgery, predisposing factors (such as family history, cryptorchidism), testis atrophy, and serum tumor marker before surgery were recorded and gathered for further analysis. pathologic reports were categorized as pure seminoma, mixed germ cell tumors (mgcts; defined as containing at least two pathological subtypes of germ cell tumors), pure mature teratoma, embryonal carcinoma, yolk sac tumor, lymphoma and benign lesions (leydig or sertoli cell tumor). data was gathered and analyzed using ibm spss ver. 18.0 (ibm co., armonk, ny, usa). independent sample t-test was obtained to assess any relationship between itgcn and quantitative factors. for qualitative factors we used chi-square test and fisher exact test as needed. a p-value less than 0.05 was considered as significant. the institutional review board of iranian urology and nephrology research center approved the study design. mean age of the patients was 34.5 years old (range, 182 years). eighty-four point four percent (84.4%) of patients (n=151) were in the range of 20 to 50 years and 2.2% (n=4) were under 12 months old. history of testis atrophy (confirmed by ultrasound study) was observed in 8 cases (4.5%). right and left testicles were involved by tumor in 96 and 83 cases, respectively. calcification (diagnosed by ultrasound study) was observed in 87 patients (48.6%). pathologic reports showed that pure classic seminoma and mgcts were the most common histologic types. considering the pathologic components of all cases (including those with mgct), we found that seminoma was also the most prevalent component. pathologic features of the patients are demonstrated in table 1. while primary study of the specimens by light microscopy leaded to diagnosis of itgcn in only 21 patients (11.7%), further analysis by ihc staining increased this figure to 85 patients (47.5%). so, it seems that light microscopy missed about three-fourths (75%) of patients with itgcn. with exclusion of lymphoma and benign lesions, the prevalence of concomitant itgcn was determined as 49.4%. there was not a statistically significant difference in mean age, histologic type, histologic components, cryptorchidism, and lymphovascular invasion between the 2 groups (p=0.151, p=0.11, p=0.233, p=0.413, and p=0.14, respectively). however, calcif ication of testis parenchyma, as identif ied by ultrasonography, was lower in patients who had concurrent itgcn (35% vs. 59%, p=0.001). the prevalence of itgcn was significantly lower in the patients with stage t1 than those with higher stages (t2). among the 150 patients with stage t1, 62 (41%) had itgcn, while 18 out of 29 patients (62%) with stage t2 and t3 had itgcn (p=0.03). since patients with pure seminoma do not present with elevated serum tumor markers, any relationship between itgcn and tumor markers was assessed in 98 patients with nonseminoma histology. fifty-two patients with nonseminoma pathology had concomitant itgcn, of which 44 patients (84.6%) had elevated serum -fp before the surgery, while in the other group, only 24 patients (52%) had elevated serum -fp level. thus, elevated serum -fp level is much common in patients with itgcn (p<0.001). prevalence of elevated serum -hcg was 58% and 64% in patients with and without itgcn, respectively; which did not indicate a significant difference (p=0.6). there are several risk factors that contribute to testis cancer, including family or personal history, cryptorchidism, and itgcn. skakkebaek have noticed that almost all types of testicular germ cell tumors (except germ cell tumors in children and spermatocytic seminomas) originate from itgcn. the microscopic features of itgcn resemble that of seminoma and ihc is needed for exact diagnosis. the results of our study shows that light microscopy might underestimate itgcn in patients with testicular germ cell tumors. it is higher in western countries and scandinavia region, and lower in african americans and asian countries. the incidence of testicular cancer is about 5.3 cases per 100,000 in the united states, but the highest rate of testis cancer has been reported from scandinavia region as about 9 in 100,000. several studies have shown that the incidence of testicular germ cell tumors is increasing. a recent study by basiri et al. has shown that the incidence of testis cancer is lower in iran than western countries. in 2009, since itgcn is a major risk factor and precursor to most testicular cancers, we decided to evaluate the prevalence of itgcn and its correlations with other histologic factors in an iranian population who had testis tumor. earlier studies by skakkebaek and montironi indicated that 80%90% of patients who undergo radical orchiectomy have simultaneous itgcn in the adjacent tissue. another study by klein et al. have shown that 82% of seminomas and 75% of nonseminomas are associated with itgcn. although mean age of patients was lower in itgcn group (32.6 years old), it was not significantly different from the overall group.. the reason may be that both itgcn and seminomas both originate from primordial germ cells (gonocytes). current data in the literature supports the idea that microlithiasis is not an independent predisposing factor for testicular cancer, although it may accompany other possible premalignant features. a study by meissner et al. concluded that microlithiasis may be accompanied by itgcn, and thus, healthy patients with several risk factors who present with microlithiasis need to undergo routine testicular biopsy to rule out itgcn or testis tumor. this study and other previous surveys, evaluated the significance of microlithiasis and its correlation with itgcn in healthy patients who did not have testis cancer at the time. however, the results of our study indicate that in patients who suffer from testis cancer, microlithiasis does not accompany itgcn. in support of this idea, a recent literature review revealed that there are 5 conditions in relation to microlithiasis and testicular cancer: down syndrome, mccune-albright syndrome, cryptorchidism, infertility and familial disposition of testicular cancer. itgcn was much common in patients with higher tumor stage (i.e., t2, t3). this may indicate that itgcn is related with more aggressive tumors, which is a new finding. in addition, in patients with nonseminoma germ cell tumors, elevated serum -fp level was closely related to itgcn. more than 84% of patients with itgcn had high serum levels of -fp before orchiectomy. this study was the first to evaluate the associations of itgcn with epidemiologic and pathologic factors of testis cancer. high volume, multi center studies with larger sample size are needed to get more constant and reliable information about itgcn. the prevalence of concomitant itgcn in the iranian population who undergo radical orchiectomy is lower than most western countries. itgcn is more common in higher tumor stages and is accompanied with elevated serum -fp levels before the surgery. since the prevalence of itgcn is higher in the regions with high incidence of testis cancer and in high stages of germ cell tumors, presence of itgcn in adjacent tissue may suggest a negative cancer behavior.

purposeto assess the prevalence of intratubular germ cell neoplasia (itgcn) in patients with concurrent testis tumor and its correlation with histologic features and serum tumor markers. materials and methodsfrom 2003 to 2015, 179 patients underwent radical orchiectomy due to testicular mass. tissue specimens were evaluated by an expert uro-pathologist using immunohistochemistry (ihc) staining, in addition to light microscopy, to identify presence of itgcn. patients ' demographic characteristics, histologic subtypes, pathologic stage of tumor and serum tumor markers were gathered and analyzed. resultseighty-five out of 179 patients (47.5%) had concomitant itgcn according to ihc staining. there was not statistically significant difference in histologic type, histologic components, cryptorchidism, and lymphovascular invasion between the 2 groups (p=0.151, p=0.11, p=0.233, p=0.413, and p=0.14, respectively). the prevalence of itgcn was significantly higher in patients with stage t2 and t3 of tumor than those with stage t1. elevated serum alpha feto protein level is much common in patients with itgcn (p<0.001). conclusionsthe prevalence of concurrent itgcn in our region is lower than previous data from western countries. itgcn is more common in higher tumor stages and is accompanied with elevated serum alpha feto protein levels before surgery. presence of itgcn in adjacent tissue may suggest a negative cancer behavior.

PMC4869572

pubmed-1289

the nerves and their structures change and are subject to the law of neuroplasticity: they adapt to reflect the stimulus which is present (mechanical, biochemical, electrical, or metabolic).1 the peripheral nervous system is subjected to a daily mechanical tensile load, as when a joint moves, undergoing compression and stretching. compression happens when the surrounding tissues create a longitudinal force to a nerve, such as when the muscles are stretched, while stretching occurs when a force is parallel to the nerve, for example, when the elbow is flexed, the ulnar nerve is stretched.2 this mechanical stress can leave the physiological confines when there is direct trauma to the nerve or surrounding tissue.2 the physiological tensile load allows the nerve to regenerate, through autocrine and paracrine substances, generated by the same nervous structure. when the nerve is subjected to stress in elongation, the schwann cells can proliferate and synthesize many neurotrophic factors, which play roles in nerve repair and remodeling.3 the synthesized molecules are varied, with different functions depending on the site of production and the quantity and based on the combinations that occur; their direction and propulsion force also change.4,5 the synthesized molecules from nerve structure can travel along the nerve and stimulate the various receptors, activating or switching them off, so as to or not to interact with the molecule produced; the same molecules can behave in different modes based on the area where they were synthe-sized.4,5 in order for the nervous tissue to reshape correctly, it must have the ability to retain its elasticity. the correct sliding of the fascial structures that make up nerves and the sliding of nerves between the various tissues that cross and innervate it become fundamental, so that the mechanical stress can be communicated properly and has the ability to adapt and regenerate the nerves.6 an impediment to this slide will lead to dysfunction and pathology.7 the biochemical information carried by the nerve does not merely have the function of operating on the nervous system, but it is also able to assist trophism and tissue function that crosses the nerve and innervates it. nerves can carry multipotent cells for repair and remodeling of tissues and organs, which are innervated by the same nerves.8 the fascial structure of a nerve is divided into three layers: the endoneurium, perineurium, and epineurium. all the layers are innervated and have a subtle but potentially important plexus of nociceptors.9 the epineurium is composed mainly of type i and iii collagen, fibroblasts, mast cells, and fat cells. the inter-fascial epineurium is loosely attached to the perineurium, facilitating sliding of the various fascia. there is abundant epineurium in larger nerves, in order to disperse most of the compressive forces. the epineurium is attached to paraneural fascial components of the connective tissue that surrounds the nerve and is an extension of the dura mater. the vessels enter the epineurium in rolled-up form (to adapt better to the strain in elongation of the nerve) and periodically along its length, forming the vasa nervorum. therefore, the epineurium supports the blood vessels while keeping the microvascularity of the nerve constant and also supports the nervi nervorum (sensory nerve fibers) of the nerve itself. it contributes to the tensile strength of the nerve and to the sliding, but it does not provide a barrier function.10,11 the perineurium surrounds the axon filaments with a dense connective tissue and the same perinerium is formed by up to 15 layers of flat perineural cells. we can encounter collagen type i and ii, and other cells with different carriers modulating the tension recorded from the nerve, thereby regulating intraneural pressure. perineural cells synthesize several substances and are in close contact with laminin and collagen type iv, further acting as a shock absorber. another key task of the perineurium is to act as a blood barrier, ie, not allowing all the filtered substances to reach the endoneurium. arterioles that reach and penetrate the perineurium form an oblique angle; these vessels have developed little smooth muscle and therefore do not possess a great intrinsic ability to regulate blood flow. nevertheless, the perineurium performs important functions in the repair of nerve tissue by managing the traffic of molecules inside and out of the neural environment by active transcytotic transport.10,11 a study has shown the presence of many pinocytotic vesicles for this transcytosis, but more research is needed to draw conclusions.11 molecules can also be transported via specific membrane receptors.11 the endoneurium contains individual axons wrapped several times by schwann cells; this sheet is in turn wrapped by type iv collagen cells, fibronectin, laminin, and proteoglycans. inserted between the various filaments, collagen type i and ii with a longitudinal orientation, mast cells and macrophages, and endoneurial fluid (70% water) can be found. the capillaries that penetrate the endoneurium increase in size, allowing the blood flow to head in different directions. the arterioles, however, are tightly wrapped by the endoneurium cells to create an additional barrier to the blood. it is most likely that the blood flow comes into contact with the endoneurium by diffusion. venules carry the blood back to the venous system. the lymphatic system, however, is present only in the epineurium; there are no lymphatic drainage vessels into the nerves (figure 1).10,11 the nervi nervorum, or a nerve s nerves, can evoke local neuroinflammation when there is nonphysiological damage or mechanical stress to the nerve tissue, to assist in its repair.9 the fascial nerve system innervated by the nervi nervorum may become a source of local pain.9 another cause of pain by the fascial nerve system, connected to axonal and physiological dysfunctions and capable of generating dysesthetic or distant pain (for example, pain resulting from traction of the sciatic nerve, when the leg is stretched during tests), follows directly from the fascial structures of the axon. these fascial structures become more sensitive to mechanical stimulation and, after a few days of local inflammation, they are able to generate potential action similar to the mechanical stimulus that causes dysfunction. this mechanism is known as ectopic electrogen-esis.9 such nonphysiological situations may require from 1 week to 2 months to disappear and only involve the epineurium and perineurium.9 there is a strong hypothesis that impeded sliding of a nerve between its various layers and tissues that cross its fascial system will generate local and dysesthetic pain. when there is an impediment to the sliding of a nerve, the rigidity of its fascial structures during joint movement is increased, and this is even more true when the joint is moved swiftly, which often occurs with everyday gestures.10 an elongated nerve causes a reduction in its diameter, defined as transverse contraction, with an increase in the pressure of the endoneurial compartment.10 its return to size and length at rest happens due to the elasticity of the perineural tissue, while the endoneurial tissue has less elastic compliance.10 a decrease in the nerve s ability to stretch may damage the endoneurial integrity before a lesion to the epineurium is visible.10 repetitive elongation of a nerve with reduced fascial elasticity properties brings about further inability of the nerve to slide, decreasing blood flow and likely leading to ischemic problems.10 for example, use of a computer mouse for prolonged periods leads to a reduction of the flow capacity of the median nerve at the level of the carpal tunnel, with possible pathological consequences of nerve compression, including the formation of edema, inflammation, and the production of adhesions, along with pain and reduced axonal flow (figure 2).10 edema is found at the intraneural level and is a common response to trauma, compression, tension, or excessive vibration. a slight trauma may give rise to edema, more superficially, at the epineural level; this can be transformed into intraneural edema if compression continues, creating a mini-compartmental syndrome of the nerve, due also to the absence of lymphatic vessels in the endoneurium. all this will lead to fibrous adhesions, decreasing the sliding of the intrafascicular tissue. the fibrosis increases the compression both inside and outside the tissues with a thickening of the nerve. this is even more noticeable when the nerve passes through small areas.12 the situation described will still generate pain symptoms. many peripheral nerves can be palpated and directly moved, but at the same time, they are more susceptible to trauma than the central nervous system, which is protected by the cranium.9,13 many peripheral nerves can be palpated and directly moved for anatomical reasons.9 there are very few texts in the scientific literature on manual treatment of the peripheral nervous system. such therapy is used by manual operators with the intent of allowing adequate sliding of the peripheral nerve and surrounding tissue, using two methods in particular: the sliding technique or a technique that alters the tension of the nerve path.13 studies on the human model are scarce, and there are no precise data on the method of movement that should be performed on a patient s nerve path in order to optimize the results. specific work protocols are lacking, and a positive clinic outcome is not always observed.1419 within the field of osteopathy, there are no scientific articles that deal with palpation or manual techniques applied directly to the peripheral nerve, although there is a book that describes and shows how the osteopathic operator should palpate and treat emergencies superficial to the nerve path.20 the purpose of these therapies and treatments is to alter the mechanical properties of fascia, such as density, stiffness, and viscosity, so that the fascia can more readily adapt to physical stresses. in fact, some osteopathic physicians and manual therapists report local tissue release after the application of a slow manual force to tight fascial areas; these reports have been explained as a breaking of fascial cross-links, a transition from gel to sol state in the extracellular matrix, and other passive viscoelastic changes of fasciae.21 the fascial osteopathic technique is the application of a low-load, long-duration stretch into the myofascial complex that is intended to restore the optimal length of this complex.21 the operator places his or her hand on the fascial restriction identified previously by palpation, using different approaches, until the perceived resistance disappears or greatly decreases, inducing or stopping the preferential direction of the tissue.21,22 the time required for the technique varies according to the response of the patient.22 there are reports in the literature that improvement in the sliding of the different fascia layers, via manual manipulation, may decrease pain symptoms and reduce inflammation at the local level.21,23 palpation of the peripheral nerves is feasible and is also used to evaluate nerve function, because these nerves are very often superficial.24,25 our clinical experience encourages us to support osteopathic fascial treatment of the peripheral nerve, providing a strong incentive to launch new research aimed at understanding what happens to the nerve using fascial osteopathic techniques, in addition to quantifying the benefit derived by the patient. it is important to remember that evidence-based medicine involves not only scientific research highlighted in an article, but also the patient s experience of treatment and the clinical experience of the operator.26 to provide some examples of treatment of peripheral nerve fascia, a finger or fingers are placed on the nerve emergence, either at the elbow on the cubital tunnel, where it passes the ulnar nerve (figure 3), or in the middle third lateral of the humerus, where the radial nerve passes (figure 4).24 ulnar compression difficulties may be encountered at the cubital tunnel level, while the humeral area is subject to direct trauma that might damage the radial nerve.2729 the method of treating any emergence of the peripheral nerve is relatively simple and noninvasive. once the fingers are in contact with the emergence and the most superficial area of the nerve branches, a fascial osteopathic technique is used, during which the operator waits for the tissues of the fabric to release under the fingers.21 it is common for the area being treated to be characterized by tissue hardness, with layers that slide with difficulty under the fingers: the technique ends when the tissue in the area has been restored, as close as possible, to a softer, more flexible tissue. we do not know the exact scientific reasons for this fascial release, despite many studies showing that an osteopathic fascial treatment is useful in many clinical conditions.21 an in vitro study demonstrates how osteopathic techniques can influence the metabolic behavior of fibroblasts, such as proliferation and inflammatory response.21 another possible explanation is that improved sliding of the various fascial layers would allow resetting of the afferents of the free nerve endings, resulting in a physiologic response of the efferents.21 further studies are expected in order to facilitate choosing the best osteopathic approach to ensure the well-being of the patient. the peripheral nerves are composed of several layers of fascial tissue, which can become a source of pain if they fail in their ability to slide. it is only recently that fascial osteopathy research has been aimed at understanding what happens to the fascia following treatment and, as a result of the first studies, it has been possible to highlight some of the benefits, including a reduction in local pain and inflammation. the osteopathic approach to the fascial system of the peripheral nerve does not have a grounding in scientific research, being based only on the clinical experience of individual operators, despite the use of peripheral nerve palpation as a method for the evaluation and testing of the nerve s function. the authors wish to encourage the initiation of new research in the fields of academic and clinical osteopathy that is aimed at quantifying the possible benefits a patient may derive from osteopathic treatment of the peripheral nerve.

the peripheral nerve is composed of several layers of fascia tissue, which can become a source of pain if the way they slide is impeded. it is only recently that fascial osteopathy research has been aimed at understanding what happens to the fascia following treatment, and as a result of previous studies, we are able to highlight some of the benefits, including a reduction in local pain and inflammation. the osteopathic approach to the fascial system of the peripheral nerve does not have a grounding in scientific research, being based instead on the clinical experience of individual operators, despite peripheral nerve palpation being used as a method to evaluate and test its function. the authors wish to encourage the initiation of new research in the fields of academic and clinical osteopathy that is aimed at quantifying the possible benefits a patient may derive from osteopathic treatment of the peripheral nerve.

PMC4634830

pubmed-1290

the combination of methods from the behavioral decision-making literature such as risky decision-making tasks derived from the classic work of kahneman and tversky (1979), and methods of neuroscience such as functional magnetic resonance imaging (fmri) and lesion studies has led to breakthroughs in both fields. examples include how impairment in specific brain functions translate into disadvantageous decision-making inside and outside of the laboratory (bechara et al., 1994, 1996, 1997, 1999) and how common decision-making biases and heuristics can be understood at the neural level (sanfey et al., 2003; hsu et al., 2005; kuhnen and knutson, 2005; de martino et al., 2006; huettel et al., 2006; a major contribution of this work has been a better understanding of how emotion, in combination with cognition, guides our decisions, particularly in the realm of risky decision-making where conflicts often arise in balancing the lure of reward and the fear of loss. evidence is accumulating that emotional reactivity differs in response to risky gains and risky losses. logical questions are whether risk-taking for gains and risk-taking for losses can best be understood as separate psychological processes, and ultimately, whether they rely on different brain structures. in this paper, we integrate findings from our own work and that of others to come to conclusions that have some generality but also allow for differences between studies based on methodology. in order to frame this investigation, we start with a model put forth to support the findings from two studies we conducted with patients with lesions to areas of the brain known to be critical to risky decision-making, namely the ventromedial prefrontal cortex (vmpfc), the amygdala, and the insula (bechara et al., 1999; 2008). as summarized in figure 1 (from weller et al., 2007), we propose that risky decision-making is influenced by the opposing forces of lure of gain and fear of risk. we operationalize the lure of rewards as either the potential for a relatively large gain in the gain domain (in comparison to the small sure gain from a riskless choice) or the potential for avoiding a loss altogether in the loss domain, and the fear of risk as arising from risking a relatively large loss in the loss domain (in comparison to the small sure loss from a riskless choice) or not winning anything in the gain domain. we suggest that the vmpfc subregions, the amygdala, and the insula each contribute in different ways to the processing and utilization of these two critical pieces of emotional information. the mere presence of uncertainty induces a primary fear response elicited by the amygdala, which has been associated specifically with fear processing and avoidance behavior (ledoux, 2000; trepel et al. this fear response activates the vmpfc whose function it is to mediate decision-making and allows for more careful deliberative processes by linking together working memory and emotional systems (damasio, 1994). processing of primary inducers, mediated by the amygdala, triggers the ventromedial prefrontal cortex (vmpfc) system, which, in turn, conducts a more deliberative analysis of uncertainty. however, decisions involving potential losses may trigger redundant neural responding from structures such as the insula (anterior, posterior, or both) and the adjacent primary and secondary somatosensory cortices (si and sii), which are independent of the amygdala; these backup processes are represented here by dotted lines. while the amygdala has been studied extensively and shown to be a key substrate for triggering emotional responses, especially in connection with fear (ledoux, 2000), the fact remains that the triggering of emotional responses involves multiple neural regions, and not just the amygdala. thus, structures such as the insula, which are independent of the amygdala, are also likely to impact decision-making under uncertainty (kuhnen and knutson, 2005; clark et al., 2008; weller et al. in particular, we propose that the insula and the amygdala provide complementary systems for dealing with potential losses, which we attribute to the evolutionary significance of dealing with potential losses. our ancestors learned to avoid situations that risked the loss of things essential for survival and it is reasonable to assume that our brains have been primed for avoiding losses. this account parallels the proposed dual systems approach of system 1 (experiential) and system 2 (deliberative) for decision-making (kahneman, 2003). according to the somatic marker hypothesis (bechara and damasio, 2005; reimann and bechara, 2010), after the amygdala triggers an automatic emotional response (or primary induction), the vmpfc subsequently prompts a more careful deliberative analysis that triggers secondary emotional responses (secondary induction) that help guide advantageous decision-making. findings in support of the somatic marker hypothesis were key to new behavioral theories in which emotions play a pivotal role in decision-making (mellers et al. 2002). in the following sections of this paper, we review the evidence for our model based on studies involving the vmpfc, amygdala, and insula, but we also include studies involving other areas that have implications for addressing the basic question of whether there is evidence at the neural level of a distinction between risky decision-making in the gain and loss domains. we will provide evidence that separate psychological processes are involved in risk-taking for gains and losses in terms of both behavioral and neurological reactions that discriminate between risk-taking to achieve a gain and risk-taking to avoid a loss. we then address the more complex issue of whether distinct neural structures support these different reactions. in the case of fmri studies, we will see that results depend on when during the decision-making process the recordings are made. we start, however, with some more straightforward and well-known behavioral phenomena that motivate the search for neurological dissociations between risk-taking for gains and losses. it is typical to consider risk-taking as a unified behavioral concept when we talk about a person in terms such as she is a risk-taker or he likes to play it safe. however, it has been shown that risk-taking within the same individual varies across content domains such as monetary, health, and social risks (weber et al., 2002). within each of these domains, we may talk about an action as being risky because of the uncertainty of its outcome without differentiating between the potential for achieving benefits versus the potential for avoiding aversive consequences. kahneman and tversky (1979) demonstrated a fundamental principle that sparked decades of later research: individuals were more likely to take a risk to avoid a loss than to achieve a gain of the same magnitude. later work by the same authors revealed a fourfold pattern of risk-aversion for gains and risk-seeking for losses of high probability but risk-seeking for gains and risk-aversion for losses of low probability (tversky and kahneman, 1992). this was explained in terms of underweighting the likelihood of high probability but overweighting the likelihood of low probability events. this paper describes a relatively new component of this research: neuroscientific studies that provide additional sources of data that separate risk-taking to achieve a gain and risk-taking to avoid a loss. in presenting the most recent research in our laboratory, we focus on the cups task (levin et al., 2007), which we developed specifically to separate risky decision-making for actual gains and losses, both in terms of overall riskiness and sensitivity to expected value (ev) differences between choice options. gain trials involve some probability of an addition to the decision-maker s account while loss trials involve a possible reduction. decision makers choose between one array of cups in which the outcome is constant (the riskless choice) and one array of cups in which the outcomes vary (the risky choice). outcomes are displayed immediately after choices are made. by varying the number of cups and the amount to be won or lost, we create gain and loss trials with contingencies that either do or do not favor a risky choice (see figure 2). for example, a one-out-of-three chance of winning five coins is better in the long run than a sure gain of one coin but a one-out-of-three chance of losing five coins is worse in the long run than a sure loss of one coin. a key component of data analysis for the cups task is the extent to which an individual makes choices based on the consideration of relative ev between choice options, for both gain- and loss-related decisions. ev sensitivity represents an index of advantageous decision-making because consistently choosing the option with a more favorable ev will yield more positive outcomes in the long run. as will be described later, a somewhat simpler version of the task was adapted for use in scanner research. across many data sets, we demonstrated that kahneman and tversky s (1979) original finding of more risk-taking to avoid a loss than to achieve a gain of the same magnitude is reproduced in the cups task. beyond the initial demonstration of greater risk-taking for losses than for gains, our recent research with the cups task showed age-related differences in risk-taking as a function of decision domain (risk-taking to achieve a gain versus to avoid a loss). risk-taking in the domain of gains decreased monotonically from early childhood to older adulthood whereas overall risk-taking to avoid losses was remarkably constant across age groups (weller et al., 2011). within both domains, ev sensitivity increased from early childhood through adulthood with a slight decline for older adults note: in each case the riskless side is depicted on the left and the risky side is depicted on the right. in the experiments these were counterbalanced over trials. we turn to neuroscience for an exploration of brain functions that may help explain these gain/loss behavioral differences. our approach in this paper is to provide a body of evidence that is consistent with the proposition that risky decision-making is separable in the gain and loss domains rather than providing a single critical test. historically, the most fundamental functional division of the brain was thought to be the one that distinguished between approach and avoidance behaviors. however, many years of animal research failed to identify anatomically separate neural substrates neural systems underlying pain and pleasure seem to overlap considerably (e.g., craig, 2009). later human behavioral studies found equivocal support for a separation of neural systems whereby the left hemisphere is predominantly concerned with approach behaviors and the lure of reward, whereas the right hemisphere is critical for avoidance behaviors and the fear of uncertainty (davidson et al., more recently, neuropsychological research on the approach avoidance conflict evolved into studies of risky decision-making where the shift was to a more microscopic analysis of neural systems. neuroimaging data have been used to gain new insights concerning risky decision-making. in particular, fmri studies use changes in blood flow that accompany neural activity in different parts of the brain to associate these areas to particular behaviors. for instance, in a recent meta-analysis of fmri studies of risky decision-making using young, healthy adults, mohr et al. (2010) found evidence common to all studies that risk processing is associated with activation of specific emotional systems in the brain such as the anterior insula, especially when potential losses are involved. the dorsolateral prefrontal cortex and parietal cortex are also activated when making decisions involving risk. using fmri in conjunction with a paradigm in which individuals decided whether to accept or reject gambles offering a 50/50 chance of gaining or losing varying amounts of money, tom et al. (2007) found that activity in the ventral striatum and the vmpfc increased as potential gains increased but decreased as potential losses increased. also, in the anterior insula, activity was found more strongly associated with the anticipation of losses than with anticipation of gains (knutson et al., 2007). earlier research showed increased arousal following losses than following gains (bechara et al., 1999). such results motivated us to classify study results based on whether activation was measured before, during, or after a decision was made. in order to get a more complete picture using the keywords fmri, gains, losses, risk, and uncertainty, table 1 summarizes the results of a number of fmri studies in terms of which areas of the brain were studied and at what point in time, and whether the study provided support for distinct mechanisms involved in risky decision-making for gains and losses. while the results are mixed, a pattern emerges when the studies are separated based on whether brain activation was measured before, during, or after a risky choice was made. most noteworthy, while different regions were the focus of different studies, in 14 studies in which activation was assessed prior to a choice (i.e., anticipation), support for separate mechanisms was found in eight studies, four studies did not support separate structures, and two studies did not make claims about separate structures because they focused on a specific region only. for example, studies by kuhnen and knutson (2005) and knutson et al. (2008b) each found that the nucleus accumbens was activated in anticipation of a risky gain, whereas the insula was activated in anticipation of a risky loss. we think these results are particularly compelling because they suggest that different parts of the brain drive risky decision-making in anticipation of uncertain gains versus uncertain losses. whereas activation during or after a risky choice can influence subsequent risky choices, activation prior to a choice is unique in its potential to influence the current choice. this table is sorted by time of measurement (before, during, or after decision-making) and by result (supportive of separate structures or not). in each category, the table is sorted first in chronological order, then in alphabetical order. beside the dissociation at the pre-decision stage, recent evidence suggests that experienced gains and losses might also activate different regions, which then affect subsequent decisions making. in a recent study using the cups task, we found that at the feedback stage, experienced reward was associated with strong activation in the vmpfc and the ventral striatum, and the stronger reward-related responses in the vmpfc were positively associated with risk-taking (xue et al., 2009). in a follow up study, we explicitly examined how neural and behavioral responses to gains and losses were associated with subsequent decisions. we developed a modified version of the cups task in which a single array of cups was presented on a given trial where one coin would be lost for all but one randomly selected cup, but multiple coins would be won if the other cup was drawn (xue et al., 2011). the decision-maker indicated whether to take or not take the gamble. in one analysis, we focused on how an experienced gain versus an experienced loss could modulate subsequent risky decision-making, both behaviorally and neurally. we found that subjects took more risk after losing a gamble than after winning a gamble. at the neural level, we again found that at the feedback stage, win was associated with stronger activation than loss in the anterior cingulate cortex, the posterior cingulate cortex, the ventral striatum, and the insula. more importantly, decisions after loss were associated with stronger activation in the frontoparietal network, which was positively correlated with individuals increased tendency to take more risk. these results thus suggest that experienced gains and losses not only involve different brain regions, but also trigger differential neural responses and behaviors in subsequent decisions. despite this suggested anatomical separation, the fact remains that the same structure, for example, the insula, has sometimes been implicated in the processing of both painful and pleasurable stimuli (e.g., craig, 2009). indeed, when compared to a baseline of activation following trials on which the decision-maker decided not to take the gamble, both experienced gains and losses elicited strong insular activation, which then modulated subsequent decision-making (xue et al., 2010). this calls for caution when making absolute determination about the anatomical separation of these pleasure (gain)loss (pain) systems. in particular, a proper baseline should be included in this analysis since the same regions might show opposite modulation by gains and losses (tom et al., 2007). thus, the stronger activation for gains or losses in some regions might not necessarily reflect distinct neural structures for gains and losses. another reason for these difficulties in establishing absolute anatomical separations is that cellular physiological evidence of neurons responding to positive versus negative valence stimuli, at least within the amygdala, indicates separation, while anatomical evidence is highly inter-mixed (e.g., paton et al., 2006). this explains why the neural systems for risky gains versus losses can be functionally separate, but finding clear-cut separation viewed at the global anatomical level is more difficult, given the proximity and overlap of these two systems. next, we turn to lesion studies which are smaller in number in terms of addressing this issue but which should align with the anticipatory fmri studies because, of course, pre-existing brain damage would likewise serve to influence revealed choices. while neuroimaging studies argue whether a particular brain region is involved in a particular function, lesion studies test whether that brain region is necessary for that function, and thus form more direct tests of the model in figure 1 and our earlier reference to anatomically separate neural substrates. the logic here is that if a particular function is impaired in individuals with a localized lesion, then the affected neural region must play a crucial role in executing that function. lesion studies seem to reveal little dissociation between the domains of gains and losses within the prefrontal cortex region, but such dissociations are more likely to be revealed when one considers two other neural systems, the insula and amygdala, which feed information into the prefrontal cortex. indeed, within the prefrontal cortex, patients with damage to the vmpfc show deficits for both risky gains and risky losses (weller et al., 2007) compared to healthy controls, vmpfc patients showed increased levels of risk-taking and decreased sensitivity to ev differences in both gain and loss domains. in contrast, amygdala patients showed impaired decision-making and exaggerated levels of risk-taking to achieve gains. however, in the loss domain amygdala damage was not associated with significantly increased risk-taking or decreased ev sensitivity. given the abundance of literature suggesting that the amygdala is involved with avoidance of punishment, this finding suggests that other structures may act in concert with the amygdala to produce a signal that engages the vmpfc. when patients with insula damage were compared to controls, a different pattern emerged (weller et al., 2009). consistent with research suggesting that the insula is important for risk processing (preuschoff et al., 2008), insula lesion patients like vmpfc and amygdala patients showed decreased sensitivity to ev differences between choice options for both risky gains and risky losses. however, these individuals showed lower levels of risk-taking compared to healthy controls, especially on gain trials. thus the insula, with connections to the amygdala, ventral striatum, and the vmpfc, may serve the purpose of providing a gate to determine the effectiveness of excitatory and inhibitory motivational circuits, signaling approach or danger. subsequently, insula damage may result in a blunted response toward risk, and would lead to insensitivity to changes in environmental contingencies signaling the approach or avoidance of a risk, regardless of domain. because the amygdala and insula have long been implicated in the processing of negative emotions, evoked from stimuli that are particularly aversive and perhaps even a threat to survival (e.g., ledoux, 2000; paulus and stein, 2006; phelps, 2006), we argue that these emotional reactions may be processed by multiple neural structures and are thus more difficult to disrupt as a result of a focal lesion to the amygdala or the insula alone. specifically, a person with a damaged amygdala but an intact insula can still make reasoned decisions in the domain of losses even when they can not in the domain of gains. while a separation in processing gains and losses is achieved at the level of the amygdala versus insular cortex, the two neural systems may come closely together (and become more difficult to dissociate) by the time information reaches the prefrontal cortex, which responds similarly to risky gains and risky losses. nevertheless, when considering the evidence from both insula and amygdala lesions, support for separate processes for risky decision-making in the gain and loss domains seems to emerge. consistent with our model, the insula, in addition to its general role in processing risk, serves to especially aid in recruitment of the vmpfc to guide risky decisions in the more emotion-laden loss domain. taken individually, each of the neuroimaging and lesion studies reviewed here has its limitations. lesion studies are limited to the small sample of available participants who meet the criteria of damage to a targeted area. furthermore, the complexity and length of tasks that can be conducted in a scanner are limited. also, because different studies focus on different areas (see table 1), comparisons, and integration of findings can be difficult. finally, for present purposes, the tasks used in the different studies differed in their ability to separate the gain and loss domains nevertheless, we believe that we can provide a meaningful summary of the findings reviewed here. a study comparing different age groups suggests different developmental trajectories for risk-taking in the gain and loss domains. neuroimaging studies are sometimes inconclusive in mapping brain systems to differential reactions to risky gains and losses. for example, while there is evidence that a system such as the vmpfc or the striatum is involved in both risky gains and losses, different parts of the system may be differentially sensitive to gains and losses (xue et al., 2009). in such cases, the more general hypothesis of separate processes underlying risk-taking for gains and losses is still supported. with regard to the stricter hypothesis of separate structures, a breakdown of fmri studies in table 1 shows the strongest evidence for this hypothesis when recordings capture pre-decisional or anticipatory processes. although a more detailed meta-analysis is clearly warranted, table 1 shows that a wide variety of structures are involved in risky decision-making beyond those depicted in figure 1. nevertheless, we feel that the relatively simple depiction of the model represents a good start in capturing the different neurological underpinnings of risk-taking for gains and losses. the complementary roles of the vmpfc, amygdala, and insula depicted in the model are consistent with both the general hypothesis that separate processes underlie risk-taking for gains and losses, and the stricter hypothesis of separate neural structures coming together in different ways to guide risky decision-making in the gain and loss domains. in conclusion, we find that evidence of different neural responses underlying risk-taking for gains and losses favors the hypothesis that decision makers react differently to risky gains and losses, both in terms of overt risk-taking and neural activation. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

affective neuroscience has helped guide research and theory development in judgment and decision-making by revealing the role of emotional processes in choice behavior, especially when risk is involved. evidence is emerging that qualitatively and quantitatively different processes may be involved in risky decision-making for gains and losses. we start by reviewing behavioral work by kahneman and tversky (1979) and others, which shows that risk-taking differs for potential gains and potential losses. we then turn to the literature in decision neuroscience to support the gain versus loss distinction. relying in part on data from a new task that separates risky decision-making for gains and losses, we test a neural model that assigns unique mechanisms for risky decision-making involving potential losses. included are studies using patients with lesions to brain areas specified as important in the model and studies with healthy individuals whose brains are scanned to reveal activation in these and other areas during risky decision-making. in some cases, there is evidence that gains and losses are processed in different regions of the brain, while in other cases the same region appears to process risk in a different manner for gains and losses. at a more general level, we provide strong support for the notion that decisions involving risk-taking for gains and decisions involving risk-taking for losses represent different psychological processes. at a deeper level, we present mounting evidence that different neural structures play different roles in guiding risky choices in these different domains. some structures are differentially activated by risky gains and risky losses while others respond uniquely in one domain or the other. taken together, these studies support a clear functional dissociation between risk-taking for gains and risk-taking for losses, and further dissociation at the neural level.

PMC3273874

pubmed-1291

interstitial deletion of chromosome 3 is rare. to our knowledge, there are only 11 cases of 3p deletion reported in english literature. we report a girl with interstitial deletion of the short arm of chromosome 3 (46, xx, del 3 (p13 p21)) with features of joubert's syndrome (js). js is a rare brain malformation characterized by the absence or underdevelopment of the cerebellar vermis- an area of the brain that controls balance and coordination. this saudi female baby born to g4 p3 mother at 38 weeks gestation by emergency caesarian section due to fetal bradycardia. parents were non-consanguineous saudi couple and the mother is 34-year-old and father 40-year-old. family history was unremarkable. during antenatal period, there was no history of medication intake or exposure to radiation. birth weight was 1980 g (10 centile); length: 43 cm (10 centile); head circumference: 31.5 cm (10 centile). after birth, severe respiratory distress was encountered, which required intubation and mechanical ventilation. bilateral choanal atresia diagnosed and surgical repair of choanal atresia was performed at 1 week. poor and uncoordinated sucking was noticed, which necessitated tube feeding initially, and later on gastrostomy tube feeding. the subject had a broad fore head, low frontal hair line, hypertelorism, a short broad based nose with anteverted nares, bilateral microopththalmia, a short philtram, high and vaulted palate, small low set ears with hypoplasia of the upper part of helix. finger positioning showed camptodactyly of the second finger with the third and fourth finger overlapping the index finger. hands were positioned in an ulnar deviation and transverse crease was found in the palms. the magnetic resonance imaging showed molar tooth sign, absence of vermis with partial agenesis of corpus collosum, figure 3a and b. analysis of g-banded chromosomes showed an interstitial deletion of p13 to p21 in the proximal short arm of chromosome 3, figures 4 and 5. patient died at the age of 5 and 1/2 months due to escherichia coli sepsis. facial features of the patient with 3p deletionsupine position features of the patientlateral view molar tooth sign in magnetic resonance imaging, absence of vermis with partial agenesis of corpus collosum magnetic resonance imaging showing the molar tooth sign an interstitial deletion of p13 to p21 in the proximal short arm of chromosome 3 to our knowledge, total of 11 cases of the proximal interstitial deletion of the short arm of chromosome 3 have been published, deletion of the short arm of chromosome 3 in association with js was not reported. js is an autosomal-recessive disorder, characterized by hypotonia, ataxia, global developmental delay and molar tooth sign on magnetic resonance imaging. a variety of other abnormalities described in children with js, including abnormal breathing, abnormal eye movements, a characteristic facial appearance, delayed language, hypersensitivity to noise, autism, ocular and oculomotor abnormalities, meningoencephaloceles, microcephaly, low-set ears, polydactyly, retinal dysplasia, kidney abnormalities (renal cysts), soft-tissue tumor of the tongue, liver disease and duodenal atresia. even within siblings the phenotype may vary, making it difficult to establish clinical diagnostic boundaries of js. dagmar wieczorek et al. reported a case of interstitial deletion of the short arm of chromosome 3 and charge like phenotype association. our patient has overlapping chromosomal breaking points and strikingly similar facial appearance; in addition, our patient had bilateral hydronephrosis, but no coloboma of iris. neri et al. reported a proximal 3p deletion phenotype including four major manifestations; the first being a characteristic facial phenotype was characterized by a low fore head, epicanthic folds hypertelorism, broad nasal bridge, short stubby nose with anteverted nares, short philtrum, small mouth, micrognathia, low set and dysplastic ears. thus, in comparison with proximal 3p deletion, our patient has recognizable facial phenotype. in 3 of the 5 reports with the proximal break points in 3p 13, the patient of kogame and kudo and wyandt et al. illustrated, but show a different facial phenotype, neri et al. reported 3 major manifestations of the proximal 3p deletion phenotype are limitations of joint movements, deformities, including ulnar deviation of the hands, comptodactyly and calcaneous feet and delayed psychomotor development. other reported anomalies such as heart defects and intestinal malformations including agenesis of gall bladder, posteriorly placed anus and meckel's diverticulum seems to be non-specific. being also present in other chromosomal rearrangements, they do not defining the proximal interstitial 3p phenotype. in addition to the clinical manifestations mentioned above, which fit into the spectrum of an interstitial deletion of 3p, our patient had bilateral choanal atresia, congenital heart disease (patent ductus arteriosus, atreal and ventricular septal defects). she had also growth retardation, bilateral hydronephrosis, low set, dysplastic protruding ears, absence of cerebellar vermis and partial agenesis of corpus collosum, which is consistent with js. lin et al. reported a case of direct interstitial duplication of chromosome 4 from 4q28.1 to 4q35 associated with bilateral choanal atresia. the child also had dysmorphic features including a broad nasal bridge, telecanthus, downward slanting palpebral fissures, prominent ears, and mild bilateral clinodactyly of the fifth fingers and bilateral hypoplasia of the second to fifth toenails. there was also a slightly dilated renal collecting system. at the age of 2.5 years, he had moderate global developmental delay, short, wide, tapering fingers and short toes with hypo plastic toenails. reported a case of a 22-month-old boy with developmental and psychomotor retardation as well as craniofacial dysmorphism, including a cleft lip. analysis of g-banded chromosomes of the propositus showed a de novo interstitial deletion of the short arm of chromosome 3, del (3) (p13p11). hertz reported de novo interstitial deletion of the short arm of chromosome 3 prenatally diagnosed in a male fetus, karyotype 46, xy, del (3) (pter p14.2::p11qter). the fetus had craniofacial dysmorphisms, a single transverse palmar crease, ulnar deviation in the wrists, cardiovascular anomalies, a slight ureteric dilatation and a mobile caecum. short et al. reported deletions of 3p usually involve the terminal portion (3p25). an interstitial deletion of a proximal 3p segment (3p14) was detected at amniocentesis. the clinical and cytogenetic characteristics of this case and of three previously published cases are reviewed. reported a girl with delayed growth in body height and weight, retarded psychomotor development, facial dysmorphism, high-arched palate, extension defects of elbows, and a probable hearing impairment is presented. a chromosome investigation by both conventional and high-resolution banding techniques revealed an apparently pure interstitial deletion of the proximal segment of the short arm of chromosome 3 (46, xx, del (3) (p11 p14.2) de novo). the paternal karyotype is 47, xyy. the clinical features of the patient are compared with those of two previously reported cases in the literature with an interstitial 3p deletion. he was found to have an interstitial deletion of band p14 from the proximal short arm of chromosome 3. examination of the father's chromosomes indicates an inserted para centric inversion in chromosome 3 as the probable origin of the deletion in the child. from the above discussion we believe that our patient with the typical phenotype features of 3p (p13-p21) has a unique findings in association the js, which is characterized by the dysgenesis of the cerebellar vermis with the brain stem malformation comprising the molar tooth sign in magnetic resonance imaging.

we report a case of 4 weeks old girl with a de novo interstitial deletion of the short arm of chromosome 3 (p13-p21) and clinical findings typical of proximal 3p deletion together with heart defects, choanal atresia, ear anomalies, central nervous system anomalies, renal anomalies and associated joubert's syndrome (js). family history is unremarkable and parenteral chromosomes were normal. the clinical manifestations of the patient are compared with those of 11 patients previously described with a proximal 3p deletion. the additional js features associated with this syndrome were described. this is the first case report in english literature describing 3p deletion associated with additional js features.

PMC3761952

pubmed-1292

allergic disease is an increasingly prevalent problem affecting up to one-third of the general population in industrialized countries. immunotherapy is a treatment modality that can modify the immunological response of the allergy sufferer so that the affected individual will stop reacting to involved allergens. immunotherapy is indicated for the treatment of allergic rhinitis (ar) and asthma, and it may prevent development of asthma in patients with ar [1, 2]. immunotherapy can be administered by different routes amongst which we find injectable and oral vaccines. injectable vaccines refers to the classical subcutaneous injection immunotherapy (scit) usually known as allergy shots. oral vaccines refer to sublingual immunotherapy (slit) where the allergens are administered as drops to the sublingual area even though the term oral vaccines may also include allergy tablets. the purpose of this study is to compare the efficacy of treatment results in patients with nasal allergies, with or without asthma, that were treated with either one or the other of these two treatment modalities: scit or slit. there is a voluminous body of scientific evidence that proves that these two treatment modalities are efficacious for the management of allergic conditions but the issue of these two modalities having similar efficacy has not yet been fully addressed. a review of the literature reveals only a few articles that directly address this issue [410]. in five of these reports [59] scit and slit is found to have better results, and one report finds both equally effective for ar patients but scit more effective for asthmatic patients. in our own experience, slit and scit appear to be of similar efficacy in this report the efficacy of one will be compared against the other. scit is a well-established treatment modality that has been successfully used for many decades and is relatively well tolerated. slit is also a very old treatment modality (earliest description is from 1900) and yet, while commonly used in europe, it is still not well established in the usa. over the last 20 years the european medical community produced a large amount of high-quality evidence suggesting that slit is safer than scit [14, 15]. while no single case of mortality has ever been reported with slit [12, 16] this is not the case with scit [17, 18]. this study constitutes a retrospective, consecutive chart review of allergy patients treated by the author at his private office. inclusion criteria were as follows: a patient of any age with nasal allergies with or without asthma that was treated with immunotherapy for at least for 6 months and had at least 2 complete evaluations. a complete evaluation implies symptom scoring, evaluation of medication use, and determination of the peak flow meter (pfm) value. these evaluations are done every 36 months as treatment progresses. because evaluations depend on patient's cooperation not all the patients had the same number of evaluations, but any patient that was considered a candidate had to have 2 evaluations as a minimum. we compared the first evaluation (pretreatment) and the last evaluation the patient had just at the time of inclusion for the study. the symptoms in the pretreatment evaluation and the amount of medications the patient was taking at that time reflect how the patient was doing without immunotherapy treatment. ethical considerations. subjects ' privacy was respected by collecting and recording data in such a way that the subjects could not be identified, directly or indirectly, through identifiers linked to the subject. in other words, a patient's confidentiality would be protected by entering data in a simple spreadsheet with nonspecific identifiers as patient no. 1, patient no. 2, and so forth with subsequent refiling of the patient's chart, according to usual procedure. the content of the spread sheet became anonymous and ready for statistical analysis. after discussing with patient about their allergies and advising about environmental modification maneuvers a discussion about treatment options including immunotherapy follows. in our office scit or slit is used to treat patients with inhalant allergies with or without bronchial involvement. the decision to use one or the other economical considerations, living far from the office, busy schedule, or needle phobia, are examples of when a patient may chose slit. having severe asthma, being a very young patient or having medical problems that may render administration of scit risky are examples of why the treating physician will advise slit. all patients were tested using a fivefold intradermal dilution skin test (idt) as taught by the aaoa [20, 21]. the test includes several panels: dust, dander, epidermals, molds, and pollens for our geographic area (table 1). standardized antigens were used for testing and treatment whenever these were available; otherwise weight/volume antigen extracts were used. after identifying the minimally reactive antigen concentration (meaning first reactive wheal) for each of the patient's reactive allergens, scit vials or slit bottles were formulated including all of the positive results (reactive allergens in the intradermal test) in the treatment mixture. patients on slit were treated according to a previously published protocol where the dose is slowly advanced from 1 drop per day to 5 drops per day until attaining the most concentrated mixture in the slit bottle. the formulation was the same for both injectable and oral vaccines. while the concentration of antigens is exactly the same for both scit and slit but slit is administered daily, patients on slit will receive a larger amount of antigen each week than those treated with scit. the slit bottles are mixed with 7.5 ml. if we consider a single allergen, for example, dermatophagoides pteronyssinus (dp), standardized dust mite dp has a concentration of 10,000 au/ml containing 68 mcg/ml of der p 1 and 71 mcg/ml of der p 2 antigens. if the minimally reactive antigen concentration occurred at dilution no. 3 and dose was advanced until mixing a vial from manufacturer's concentrate, the cumulative dose this patient would receive weekly by scit would be 200 au per week, while a patient treated by slit would receive 464 au per week. as stated before, the initial allergen concentration in both scit and slit is the same: 80 au/ml as in both circumstances the extract (with 10.000 au/ml) will be diluted 125 times. after one year of treatment the patient on scit would receive 9680 au and the patient treated by slit would receive 21149 au or 2.18 times more allergen. a chi-square test was applied for the following allergens: dust mite, cat, roach, mold, tree-pollens, grass-pollens, and weed-pollens for both groups, scit and slit. asthma diagnosis was based on the presence of recurrent cough, chest tightness, sob, or wheezing, having a spirometry consistent with airflow obstruction or having the symptoms respond to the administration of a short-acting broncho-agonist (saba). recorded symptoms included runny nose, sneezing, nasal obstruction, itchy eyes, itchy ears, cough, shortness, and wheezing. these were scored according to fell's method with a numerical analog from 0 through 3 as follows: 0=symptom not present, 2=symptom is moderate, 3=symptom is severe. medication use was also evaluated on a similar numerical scale as follows: 0=medication is not being used, 1=medication is being used once a week or less, 2=medication is being used 23 times per week, 3=medication is being used 4 or more times per week. medications were generically grouped as allergy pills, intranasal steroids (inss), and short-acting broncho-agonists (sabas) in the case of asthmatic patients. the value of the pfm determination was used as the parameter to be recorded at each patient's encounter. ninety-three charts met the inclusion criteria, 50 on scit and 43 on slit. among the 50 patient's on scit, 20 (40%) were male, 30 (60%) female ranging in age from 2.33 to 75 years (mean 45 17.8 sd). this compared to 43 patients on slit of whom 21 (49%) were male, 22 (51%) female ranging in age from 1.66 to 75 years (mean 35 20.8 sd). analysis of covariance for the dependent variables for which a significant pre/posttreatment by treatment modality interaction effects was obtained did not reveal gender or age to account for significant dependent variable variance; in other words the results were not affected by age or gender so both groups can be considered homogeneous. a chi-square test was applied for the following allergens: dust mite, cat, roach, mold, tree-pollens, grass-pollens, and weed-pollens. results indicate that there are no statistical differences between both groups (at the p<0.05 level); therefore in their reactivity to allergens both groups can also be considered homogeneous. there were 3 children<12 years on scit (mean 7.8 years) versus 11 on slit (mean 6.9 years). ten (20%) scit patients had asthma versus 12 (28%) on slit. thus a greater percentage of asthmatics (12/22 or 55%) and more children under 12 years of age (11/14 or 79%) were on slit. length of treatment for the scit group was 12 to 86 (mean 31 18.7 sd) months and for the slit group was 10 to 32 (mean 19 6.3 sd) months. for all patients the pre- and posttreatment averages for each symptom, medication use, and pf value were statistically compared through the use of repeated measure analysis of variance (anova). the results for the two treatment modalities (scit versus slit) were also compared using the between-subjects factor of the anova (table 2). the same analyses were completed for medication use (table 3). for the pf evaluation the pre- and post-treatment values were compared (table 4). in table 2 the mean value for each symptom score before treatment and at the time of data collection the result of the test of significance is shown for each symptom within each treatment modality (paired t-test). lastly, the result of the statistical analysis comparing symptom improvement with one or the other treatment modality is shown. shortness of breath and wheezing had significant improvements at p<0.05 for both treatment modalities. the remaining symptoms had a significant improvement at p<0.001 for both treatment modalities. wheezing and coughing were the only symptom scores which seemed to respond better to either scit (coughing slightly better, p=0.037) or slit (wheezing slightly better, p=0.024), though both symptoms significantly improved regardless of treatment modality. both scit and slit provided equally significant reduction in use of medication (p<0.001) including allergy pills, ins, and, to a slightly lesser but still significant degree, saba (table 3) but without no significant difference between both treatment modalities. pf value before treatment and at the time of the last patient evaluation is shown in table 4. both treatment modalities were equally effective in achieving a significant increase in pf values (p<0.001) but there was no significant difference between both treatment modalities. this paper is a retrospective chart review and as such lacks the rigor of a prospective randomized study with a placebo control group which is very difficult to do in a private office setting. while an analysis of covariance is useful, it is not a perfect solution. a future, larger-scale study should be planned to include the above design characteristics. we observed that patients usually come to the office already using one or more allergy medications. this study, like others, demonstrates that immunotherapy, whether scit or slit, will lead to the reduction of medication use for ar and/or asthma. it was not the purpose of this paper to evaluate the effect of medications on allergy symptoms but rather to compare the effects of scit versus slit on medication use. both treatment modalities resulted in the reduction of antihistamines, inhaled nasal steroids, and sabas. the slight imbalances in demographic characteristics between the groups on scit versus slit were not statistically significant and did not affect the statistical results. the reason why there are more young patients and more asthmatic patients in the slit group can be explained by the fact that slit is safer and easier to administer therefore it is suggested more frequently for these difficult-to-manage patients. indeed we would have expected a much more pronounced difference; yet fewer than expected chose slit because it is not covered by insurance. patients on scit have been treated for a longer period of time because slit was added to our practice later than scit. the improvement of the asthmatic symptoms wheezing and sob and the decrease in saba use were significant at p<0.05 yet because of sample size this is not as strong as the improvement in other symptoms or medications that had an improvement at the level of p<0.001. the advantage for scit in treating coughing is real, but the effect size (eta-squared) is only 0.025, meaning that it only accounts for 2.5% of the variance in pre- versus posttreatment differences, which is not much. the advantage of slit in treating wheezing may have been influenced by our own bias of suggesting slit use to asthmatic patients as a safer treating modality. it is therefore more likely that patients with higher symptom scores were present in the slit group. our findings demonstrate that slit is not only effective in controlling symptoms in nasal allergy patients with or without asthma, in decreasing medication use in such patients, and in improving parameters of pulmonary function, but it also appears that slit is as effective as scit these findings are in agreement with those published in the european literature [26, 27] but certainly this presentation lacks the scientific validity of other reports that present a prospective, randomized, controlled study; therefore this presentation we hope will serve as a stimulus for centers with the capability to undertake such a study to continue with this line of research. this would help the fda to finally recognize slit as an effective and safe treatment modality. if slit became an fda-approved treatment modality (and hopefully) reimbursed by insurance companies many more patients might be receptive to immunotherapy which is a treatment capable of altering the immunological mechanisms responsible for the development of allergic conditions. pf values for asthma control should be taken as a guideline only because the predicted lung function has a high degree of variability with significant differences in pf values according to presence or not of lung disease, smoking, age, sex, and even patient's social environment [2931]. having the advantage of providing results quickly, and requiring little training (from the patient as well as from the technical staff), the pfm device is useful to monitor progress during immunotherapy. it is most useful when the changes in pf values are compared to the initial value of each patient, recorded at the time of treatment initiation. for the purpose of this study individual improvement with therapy is not reported, but rather an overall trend, thus the use of pfm provides a gross indicator of change. some of our patients were children, and it is expected they grow during treatment. certainly using a pfm as a tool to determine improvement in pulmonary function adds uncertainty as to whether the improvement in pf value is related to clinical improvement or to the growth of the patient during treatment. in this we have demonstrated that the pf value in patients treated by immunotherapy increases regardless of age or asthmatic condition. in our experience, the use of slit with multiple antigens has enabled us to treat patients that otherwise would have not received immunotherapy, or would have not continued to receive immunotherapy, like asthmatic patients with poorly controlled asthma, patients that had severe arm reactions, very young patients to whom it is difficult to administer shots or patients whose schedules prevent them from being compliant. these results suggest that scit and slit exhibit similar efficacy. slit objectively improves symptom scores for asthma and ar while decreasing medication usage of allergy medications and sabas. given the increased risk and difficulty in treating asthmatic and young patients, these results would suggest that slit should be considered as the main treatment modality for these patients, considering scit only for treatment failures. the results of this study are in agreement with the european literature and therefore would support the inclusion of slit in the routine management of the allergic disease.

while it is generally accepted that subcutaneous injection immunotherapy (scit) and sublingual immunotherapy (slit) are both efficacious, there is not yet a significant amount of information regarding their comparative efficacy. in this paper, we performed a retrospective chart review and compared treatment results in two groups of patients (both with nasal allergies with or without asthma) that were treated either with scit or slit. both treatment modalities were found to be of similar efficacy.

PMC3317104

pubmed-1293

neisseria meningitidis is one of the most frequent causes of meningitis and septicemia worldwide [1, 2], being not only responsible for 1020% of specific meningococcal-related mortality but also the cause of (particularly pediatric) long-term morbidity as it leads to permanent neurological sequelae and disabilities in an additional 20% [35]. furthermore, the pediatric mortality rate among children with sepsis is over 20% [6, 7]. the incidence of meningococcal meningitis is greatest amongst children, adolescents, and adults aged up to 29 years, but young children are the most susceptible. the risk of n. meningitidis infection is particularly high in some regions of the world but, despite the introduction of innovations in health care, morbidity and mortality rates are high in both developed and undeveloped countries, and prevention is therefore a priority. the bacteria colonise the nasopharyngeal tract of human hosts and are spread from subject to subject via air droplets. transmission rates vary and are also related to individual risk factors such as age and/or underlying medical and social conditions (e.g., primary or secondary immunodeficiencies, a history of travel, and overcrowded living condition). twelve different serogroups are known, but most invasive meningococcal diseases are caused by one of the six capsular groups a, b, c, w, x, and y. a number of excellent conjugate vaccines against serogroups a, c, w, and y have been licensed, and the introduction of conjugate meningococcal c vaccine (menc) has led to a rapid and sustained reduction in the incidence of invasive menc disease across all age groups in italy [8, 9]. however, a vaccine against capsular group b (menb), which has now become responsible for most cases in italy and the rest of the world [7, 9], has long eluded vaccinologists, particularly because of the problems associated with the b polysaccharide [1012]. unlike the highly immunogenic polysaccharides of serogroups a, c, w, and y, the serogroup b polysaccharidic capsule contains a polysialic acid whose antigenic structure resembles the cell surface glycoproteins of human neurological tissue, and this has proved to be a formidable challenge. the new protein-based vaccine against menb (4cmenb; bexsero, novartis vaccines and diagnostics, siena, italy) has now overcome this barrier by using a cocktail of four main immunogenic components: two recombinant fusion proteins (neisseria heparin-binding antigen [nhba-gna1030] and factor h binding protein [fhbp-gna2091]), recombinant neisseria adhesion a (nada), and detergent-treated outer membrane vesicles (omvs) derived from the nz98/254 new zealand meningococcal outbreak strain in which porin a (pora 1.4) is the major immunodominant antigen. these components were identified using reverse vaccinology, a technique that analyses the whole bacterial genome in order to predict meningococcal antigens (exposed on the pathogen's surface or secreted) that can act as vaccine targets. nhba is a surface b-barrel lipoprotein that binds to the anticoagulant heparin and induces protective immunity in human hosts [1517], and fhbp is a surface-exposed protein that allows binding exclusively to human fh, mediates host serum resistance, and induces bactericidal antibodies upon host detection [15, 19]. nada is a surface adhesin and invasin whose interactions with abundantly expressed human heat shock protein 90 (hsp90) also induce bactericidal antibodies [21, 22]. finally, pora (one of the two b-barrel porin proteins produced by n. meningitides) assists in opsonophagocytic activity and is also involved in host actin reorganisation during infection, which depends on its ability to nucleate actin filaments. this four-component meningococcal serogroup b vaccine (4cmenb), the first successful vaccine against the endemic form of this cause of serious bacterial meningitis and septicemia, has been in development for almost 20 years and has recently been approved for the active immunisation of subjects aged 2 months [9, 10] by licensing authorities in europe, canada, and australia. a bivalent fhbp recombinant vaccine (also known as lp2086; trumenba, pfizer inc., philadelphia, pa, usa) has been developed since 2006 and has now been approved by the us food and drug administration for use in 10-to-25-year olds. this vaccine appeared safe in a phase 3 study in approximately 5,600 healthy individuals 10 to 25 years of age and immunogenic and safe when coadministered with routine meningococcal a, c, y, and w and tetanus, diphtheria, and pertussis (tdap) vaccines in a phase 2 study in more than 2,600 healthy individuals 10 to 12 years of age. studies on this vaccine are ongoing in europe and approval from european medicines agency is expected in 2017. the aim of this review is to discuss the immunogenicity, safety, and tolerability of 4cmenb vaccine in infants and toddlers, and the efficacy of different vaccination strategies. an important challenge for the licensing of 4cmenb vaccine was the difficulty in showing its activity against epidemiologically relevant strains of n. meningitidis [27, 28]. after its development and phases i and ii trials, some large-scale randomised phase iii studies were planned in order to assess its efficacy and describe adverse reactions, but due to the rarity of the diseases caused by n. meningitides serotype b (which have annual rates of 0.55 per 100,000 people) laboratory-based methods were developed with the aim of predicting the vaccine's effectiveness and coverage. in europe, the vaccine was licensed on the basis of a correlate of protection calculated using a titre of human serum bactericidal activity (hsba) that is present in convalescent patients which was shown to be protective in us army recruits. hsba assay is a recognised in vitro surrogate for evaluating protective immunity against n. meningitidis, and an adequate response is a crucial criterion for licensing vaccines against serogroup b meningococci. in order to justify the inclusion of each antigen in the formulation, it is necessary to run four hsba assays on each serum sample, each using a n. meningitidis strain expressing the target antigen independently from the others in order to evaluate immunogenicity of each component of the vaccine, for example, a meningococcal strain that uniquely expresses nada but not factor-h-binding protein or neisseria heparin-binding antigen. subsequent phase iii studies of 4cmenb in children used a more conservative titre of 5, which ensures that the level is>4 with 95% confidence taking into account within-assay variability. the safety and immunogenicity of 4cmenb vaccine has been studied when administered at the same time as other routine infant vaccines (diphtheria, tetanus, acellular pertussis, inactivated poliovirus, hepatitis b, haemophilus influenzae type b [dtap-ipv-hepb/hib], and 7-valent pneumococcal conjugate vaccine [pcv7]), and it has been found that the antibody responses to the routine vaccines are equivalent to those observed when the routine vaccines are given alone in the case of all of the antigens except for the pertactin component of acellular pertussis and pneumococcal serotype 6b. however, this laboratory observation seems to be of no clinical significance, and published data also suggest that the incidence of pneumococcal disease due to serotype 6b is low in the countries in which pcv7 vaccination is used. other studies have investigated the persistence of bactericidal antibodies in young children after primary immunisation and the level of immunogenicity after a preschool booster [29, 30]. the levels of bactericidal antibodies after primary 4cmenb vaccination at the ages of two, four, six, and 12 months had waned when measured at 4044 months, but an anamnestic response was observed following a booster dose given at the age of 4044 months. similarly, bactericidal antibody levels in infants who originally received 4cmenb during late infancy (6, 8, and 12 months) had also waned when measured at the age of 40 months but, once again, there was an anamnestic response to a booster dose given at 40 months. the participating infants were observed for 30 minutes after each vaccine administration, and their parents were given a diary card on which to record the occurrence and severity of solicited local (i.e., injection site tenderness, erythema, induration, and swelling) and systemic reactions (i.e., changes in eating habits, sleepiness, vomiting, diarrhea, irritability, unusual crying, rash, and increased/decreased body temperature) and any other adverse events, during the following seven days. the rates of local and systemic reactions were similar to those seen following other routine infant and early childhood vaccinations, but injection site pain was consistently reported more frequently, especially by older children. fever was more frequent in the children who received 4cmenb together with other routine infant vaccines. it mainly occurred during the first 24 hours after administration but, as in case of other vaccines, it has been found that the prophylactic administration of paracetamol before and 46 hours after vaccination significantly reduces postvaccination fever without affecting immunological responses [31, 32]. the pivotal and phase iib studies found that the most frequently reported local reaction of tenderness affected 87% of the 4cmenb injection sites, 80% of the dtap- ipv-hepb/hib sites, and 79% of the pcv7 sites when all three vaccines were administered together. the frequency of reported tenderness after dtap-ipv-hepb/hib and pcv7 injections when they were administered without 4cmenb was respectively 59% and 53%, whereas when dtap-ipv-hepb/hib and pcv7 injections were administered with 4cmenb it was respectively 68% and 62%. the reported rates of local reactions to 4cmenb were slightly higher than those related to routine vaccines, but the majority were transient, most intense on the day after vaccination, and resolved within a week. although the systemic reactions that occurred when 4cmenb was administered concomitantly with routine vaccines can not be specifically attributed to one or other of the vaccines, it is possible to assess the overall profile. the occurrence of 4cmenb-related seizures is rare: the combined data of infant studies including>20,000 vaccinations in the primary 4cmenb study arm indicate an overall rate 0.1 febrile seizures/1000 vaccinations on the day of vaccination or the day after and no events in the control arm. they are similarly rare in toddlers: 0.4 events/1000 vaccinations (95% confidence interval (ci): 0.051.46) after a total of 11,000 4cmenb vaccinations administered with or without routine vaccines, as against 0.3 event/1000 visits (95% ci: 0.041.05) in the case of those receiving routine vaccines alone [10, 33]. six suspected cases of kawasaki disease reported during the course of two infant studies (four in the pivotal trial and two in the phase iib study) were evaluated by an independent external expert panel in order to assess whether they were true kawasaki cases and whether they were vaccine related. analysis of the kawasaki cases indicates an annual incidence of 72/100,000 person-years (95% ci: 23169) after 4cmenb vaccinations, as against 56/100,000 person-years (95% ci: 1311) after routine vaccinations alone. the overall data from different studies indicate that the frequency of febrile seizures, the incidence of kawasaki disease, and the proportion of infants using antipyretics are similar to those observed during clinical licensure programmes. however, as the number of exposed infants is still too small to exclude any relationship with rare adverse events, further postmarketing surveillance is necessary. the new 4cmenb vaccine may not protect against all invasive meningococcal b strains because the antigens included in the vaccine are expressed by only some of the strains in circulation. however, it is not known what protection 4cmenb vaccine provides against invasive meningococcal disease (imd) because it depends on the vaccine antigens expressed by the meningococcal strains in any given geographical area and their cross-reactivity with the antigens included in the vaccine. epidemiological and microbiological data regarding the circulating meningococcal strains are important in order to predict the theoretical coverage provided by 4cmenb vaccine and assess its impact on disease burden. further postmarketing surveillance will allow a more precise estimate of the effectiveness of 4cmenb. however, although an hsba can be used to demonstrate whether the vaccine induces antibodies capable of killing meningococcal strains, the presence of four antigens means that it is more complex than in the case of other meningococcal vaccines (i.e., menc). moreover, the genetic diversity of serogroup b strains means that not all of them have the genes coding for each of the antigens, and their expression may vary over time or from place to place. for these reasons, the meningococcal antigen typing system (mats) is used to measure bacterial antigen expression in order to predict whether bactericidal serum is capable of killing particular strains [3436]. this method is characterised by both phenotypic and genotypic analyses: the expression of the individual antigens that cross-react with the corresponding vaccine antigen is quantified using polyclonal antibodies against nhba, nada, and fhbp in an enzyme-linked immunosorbent assay (elisa), and dna sequence homology to the variable region sequence of the vaccine strain pora gene is assessed, in order to estimate coverage in a specific region [32, 33]. it has not yet been proved that there is a correlation between the mats results and real vaccination coverage, but the predicted protection based on the expression of at least one matched antigen ranges from 73% to 87%. the mats has been applied to isolates of 1,052 menb strains causing imd in europe submitted to reference laboratories in france, germany, italy, norway, and the uk between 2007 and 2008, and the analysis demonstrated estimated efficacy values ranging from 73% in the uk to 87% in italy. furthermore, a study conducted in canada between 2006 and 2009 analysed 157 menb isolates collected from children and adults with imd and found that the potential coverage of 4cmenb vaccine was 7590%. on the basis of the mats elisa findings, the authors predicted that 66% of the circulating strains were covered by at least one vaccine antigen although none were covered by all four. a new meningococcal serotype x has recently been isolated in africa, against which no vaccine is currently available. however, some authors have recently used the mats and bactericidal assays of 11 serogroup x isolates taken from nine african and two french patients and found that 4cmenb vaccine could have a good coverage against the strains from africa but not those from france. in regions where meningococcal strains are appropriately monitored, the mats can evaluate the real effectiveness of 4cmenb vaccine, and the development of changes in the menb serogroup over time. it is also a very useful means of monitoring the emergence of new menb mutants due to selective vaccination pressure. although the development of a meningococcal b serogroup vaccine was slow and difficult, the new bexsero 4cmenb vaccine has recently been licensed in the eu, australia, and canada. the introduction of any new vaccine is never easy, but it is especially complex in this case. the success of a vaccination programme is based on both cost-effectiveness and public acceptance and although meningococcal b infection is a cause for concern in the general population, the acceptability of the vaccine by parents is influenced by worries concerning its potential side effects, its real effectiveness, and the consequences of its coadministration with other routine vaccines in terms of the number of injections and possible immunological interference. for example, its acceptance may be reduced by the fact that it has been associated with increased rates of fever when coadministered with the routine vaccinations provided during infancy on the basis of the national immunisation schedules. one recent study of parental attitudes to 4cmenb showed that 82.5% of the interviewees wanted their children vaccinated. the most frequent concerns were side effects including fever (41.3%) and adequate vaccine testing (11.7%), but 26% of the parents said that they had no concerns. moreover, as in the case of other vaccinations (e.g., hpv), the authors found that most parents (81.7%) were more likely to accept the vaccination if their immunisation providers recommended it. the vaccination schedule should take into account the age of the subjects most frequently affected by meningococcal disease and the epidemiology of meningococcal infections. one recent study aimed at defining the optimal age for administering 4cmenb to children has shown that the incidence is highest in those aged<5 years (particularly those in their in the first year of life, when deaths are more frequent) and, on the basis of this finding, the authors suggested that vaccination should be started in the first year of life, with a catch-up dose being given at the age of five years. other recent studies have evaluated the cost-effectiveness of a potential menb vaccination programme [43, 44]. one study carried out in the netherlands estimated that an infant menb vaccination programme would prevent 14% of cases over the lifetime of a birth cohort and concluded that this was not cost-effective and although another study carried out in the uk estimated that routine vaccination would prevent 2756% of cases over the lifetime of a birth cohort, the authors also considered it not cost-effective. finally, a recent study of the economic impact of menb vaccination in canada found that the menb vaccination programme exceeds the generally cost-effectiveness thresholds and therefore should not be considered economically advantageous. nevertheless, 4cmenb is now being used in canada and, upon parents ' request, also in all of the countries and it has been licensed and its use in at-risk populations has been implemented. it has also been announced that it may be introduced into the uk's routine infant immunisation schedule using a 2+1 regimen although only a 3+1 regimen with the concomitant use of paracetamol is currently licensed, and some other countries are reconsidering their cost-effectiveness calculations by also considering its possible impact on carrier status. with the availability of the bivalent rlp2086 vaccine for the adolescent age, it will be important to compare vaccination strategies that cover different age groups as well as to understand the impact of the two vaccines against imd overall, meningococcal disease due to serogroups different from menb and meningococcal carriage in the nasopharynx. n. meningitidis is still one of the major causes of sepsis and meningitis among children worldwide and is associated with a high mortality rate. considerable efforts have therefore been made to prevent meningococcal disease by means of vaccination, and two effective conjugate vaccines have recently been licensed and led to good results (men c and menacyw). however, it proved to be very difficult to develop a vaccine against serogroup b because of the poor immunogenicity of its capsular polysaccharide, even when conjugated with a carrier that could also induce an autoimmune response, until experts used reverse vaccinology to identify appropriate antigenic recombinant proteins. the overall findings of various studies have shown that the administration of three doses of 4cmenb to young children (alone or with routine vaccines) does not interfere with immune responses, and most have found that its safety and tolerability are acceptable. however, its coadministration with other vaccines does lead to increased reactogenicity (particularly fever) and so such coadministration should be combined with paracetamol given both before and after vaccination. the new 4cmenb vaccine represents an important opportunity to fight pediatric imds, but its introduction should take into account the need to maintain the appropriate use of meningococcal conjugate vaccines that cover serogroups other than b, community opinion, and cost-effectiveness data. moreover, it will be important to compare 4cmenb potential efficacy with that of bivalent rlp2086 vaccine. finally, it is very important to continue surveillance in order to monitor the emergence of new meningococcal b strains in order to identify any that are not susceptible to 4cmenb.

neisseria meningitidis is a gram-negative pathogen that actively invades its human host and leads to the development of life-threatening pathologies. one of the leading causes of death in the world, n. meningitidis can be responsible for nearly 1,000 new infections per 100,000 subjects during an epidemic period. the bacterial species are classified into 12 serogroups, five of which (a, b, c, w, and y) cause the majority of meningitides. the three purified protein conjugate vaccines currently available target serogroups a, c, w, and y. serogroup b has long been a challenge but the discovery of the complete genome sequence of an menb strain has allowed the development of a specific four-component vaccine (4cmenb). this review describes the pathogenetic role of n. meningitidis and the recent literature concerning the new meningococcal vaccine.

PMC4553319

pubmed-1294

hepatitis c virus (hcv) infection is a leading cause of end-stage liver disease and hepatocellular carcinoma [1, 2]. hcv-related liver disease is the most common indication for orthotopic liver transplantation in the usa and northern europe and for living donor liver transplantation (ldlt) in japan [3, 4, 5]. unfortunately, liver transplantation is not a cure for hcv infection, and the occurrence of graft reinfection with hcv is universal, leading to progression of liver fibrosis and occasionally to graft loss at rates higher than in transplant patients not infected with hcv [5, 6]. in japan, because of long-standing legal difficulties associated with cadaveric donation, ldlt is the main type of liver transplantation for end-stage liver disease. the sustained virologic response (svr) rate was 40% in donors of ldlt treated with pegylated interferon (peginterferon) plus ribavirin, although adverse events were commonly observed even in nontransplanted patients receiving this treatment. reported that the dose reduction rate and the discontinuation rate of peginterferon plus ribavirin treatment was 40 and 42%, respectively. recent progress in the development of direct-acting antivirals (daas) against hcv has made it possible to eradicate hcv effectively and to shorten the treatment duration compared to the previous standard of care, i.e. peginterferon plus ribavirin. treatment with the hcv ns5b polymerase inhibitor sofosbuvir plus ribavirin for 12 weeks was shown to lead to high rates of svr (97%) in japanese patients infected with chronic hcv genotype 2. asian-pacific countries, including japan, account for 50% of all chronic hepatitis b virus (hbv) infection globally. given the prevalence of hbv, ldlt from hepatitis b core antibody-positive donors to recipients was occasionally performed with passive immunization with hyperimmune hepatitis b immunoglobulin (hbig) plus nucleos(t)ide analogs. here, we report on an ldlt recipient who was reinfected with hcv genotype 2a after receiving a graft from a hepatitis b core antibody-positive donor. hcv genotype 2a was eradicated by a 12-week treatment with sofosbuvir plus ribavirin with hbig plus entecavir, one of the nucleos(t)ide analogs, for the successful prevention of hbv reappearance. a 66-year-old japanese woman developed liver failure due to cirrhosis and hcv genotype 2a infection. she was a treatment-nave patient, but her il28b rs8099917 was a favorable genotype (tt). two years prior to ldlt, she has been diagnosed with liver cirrhosis due to hcv infection without liver biopsy. she had been infected with hcv after having received a blood transfusion during childbirth at the age of 30 years. at ldlt, she had peripheral edema with a meld (model for end-stage liver disease) score of 12. ldlt with a right liver graft from a hepatitis b core antibody-positive donor was performed in february 2015. five months after ldlt, the hcv rna level was 5.8 log iu/ml, and she was diagnosed with graft reinfection with hcv genotype 2a. combination treatment with 400 mg daily of sofosbuvir and 400 mg daily of ribavirin was commenced. her height, body weight, and body mass index were 1.56 m, 50 kg, and 20.5, respectively. her laboratory data before treatment are shown in table 1. the hcv rna level before treatment was 5.8 log iu/ml. after ldlt, she received triple immunosuppressive therapy consisting of tacrolimus (3 mg daily), mycophenolate mofetil, and basiliximab with passive immunization with hbig and 0.5 mg daily of entecavir. four weeks after initiating the combination treatment with sofosbuvir and ribavirin, hcv rna levels were undetectable. she completed this treatment for 12 weeks and achieved svr at 24 weeks following the termination of this treatment (svr24) (fig. 1). there was no evidence of hbv reactivation. because her hemoglobin level was 12.2 g/dl before the commencement of this treatment, an oral iron preparation was also started. after 2 months of treatment, her hemoglobin level fell to 10.8 mg/dl. then, the dose of ribavirin was decreased to 200 mg daily, and her hemoglobin level improved to 12.8 g/dl (fig., she did not develop evidence of bone marrow suppression, such as observed in peginterferon-plus-ribavirin treatment. no serious adverse events were observed. during treatment, the trough level of tacrolimus remained stable. we presented a female patient with living donor-related graft reinfection with hcv genotype 2a who was treated with a combination of sofosbuvir and ribavirin for 12 weeks. although several daas have drug-drug interactions, no changes in this patient's drug protocol, which included immunosuppressants and combination treatment of sofosbuvir plus ribavirin, were necessary in this case. peginterferon plus ribavirin with or without daas may be attempted, but the use of peginterferon is restricted by severe side effects and inadequate efficacy. in addition, interferon has an immune-mediated cytotoxicity, occasionally causing allograft dysfunction. interferon-free therapy is a viable treatment option and improves treatment efficacy [15, 16, 17, 18, 19, 20]. this case suggests that daas are well tolerated and effective for the eradication of hcv from post-liver transplantation patients. in the present case of ldlt, graft reinfection with hcv genotype 2 the patient achieved svr24, although she had anemia as an adverse event, and anti-hcv treatment was continued without blood transfusion. curry et al. reported that common adverse events of combination treatment with sofosbuvir and ribavirin were fatigue (in 38% of patients), headache (23%), and anemia (21%) after liver transplantation. charlton et al. reported that the most common adverse events were fatigue (30%), diarrhea (28%), headache (25%), and anemia (20%) during a 24-week combination treatment course of sofosbuvir plus ribavirin after liver transplantation. in the present study, the patient received a liver graft from a hepatitis b core antibody-positive donor, and, in general, hbv from these donors is transmitted to recipients at a high rate. therefore, passive immunization with hbig plus treatment with entecavir were provided to prevent hbv reactivation. curing hcv infection with daas in hbv/hcv coinfection and monitoring for hbv reactivation should be performed, because daas against hcv do not have any effect on hbv replication, unlike interferon. in the present case, the patient was treated with hbig and entecavir, and there was no evidence of hbv reactivation. recently, more effective regimens with daas against hcv have been reported [22, 23]. these regimens may make it possible to shorten the duration of treatment and to make it easier to achieve svr. the use of interferon-free regimens is possible for the eradication of hcv in post-ldlt patients with grafts from hepatitis b core antibody-positive donors, as was demonstrated by our patient, who was successfully treated with hbig and entecavir to prevent hbv reactivation. in conclusion, 12-week treatment with sofosbuvir plus ribavirin is relatively safe and highly effective for the eradication of hcv genotype 2 in ldlt patients. the other authors declare that there is no conflict of interest regarding the publication of this manuscript.

direct-acting antivirals (daas) are relatively safe and highly effective for the eradication of hepatitis c virus (hcv) in liver transplant recipients. in this case study, we present a female with a graft reinfected with hcv genotype 2 who was treated with a combination of sofosbuvir and ribavirin after living donor liver transplantation (ldlt). because the graft was from a hepatitis b core antibody-positive donor, passive immunization with hyperimmune hepatitis b immunoglobulin (hbig) and entecavir were also provided to prevent hepatitis b virus (hbv) reactivation. it became clear that the combination of sofosbuvir and ribavirin promptly led to a sustained virologic response and that this combination was safe to treat graft reinfection with hcv genotype 2 after ldlt. adverse events caused by daas were not observed, except for slight anemia. hbig and entecavir were useful in the prevention of hbv reactivation. in conclusion, the present case indicated that daa treatment for graft reinfection with hcv is safe and effective in ldlt from hepatitis b core antibody-positive donors.

PMC5043295

pubmed-1295

lithium ion batteries are the power source of choice for most mobile electronic devices. these systems generally work using the insertion and removal of lithium ions into host materials, resulting in redox and structural changes during the electrochemical cycling. lifepo4 adopts the olivine structure type (( mg, fe)2sio4, orthorhombic),with feo6 corner-linked octahedra in the bc plane and lio6 octahedra forming edge-sharing chains on the b axis. hence the li ions reside within 1d channels, allowing their extraction and insertion during charge and discharge via the reaction shown in figure 1. equation for charge and discharge of lifepo4 and structural diagrams of the lifepo4 and fepo4 active materials with iron atoms shown in orange, small gray phosphorus atoms, large blue lithium atoms, and red oxygen atoms. both structures adopt space group pnma, with lattice parameters of a=10.3290(3), b=6.0065(2), and c=4.6908(2) for lifepo4 and a=9.8142(2), b=5.7893(2), and c=4.7820(2) for fepo4. the discharge profile is characterized by a flat plateau at 3.45 v vs li. this flat potential discharge reaction is characteristic of the coexistence of two phases, lifepo4 and fepo4, each having a narrow compositional stability range in contrast with nonstoichiometric electrode materials such as lixcoo2 that generally show sloping profiles. the first in situ x-ray diffraction (xrd) study of lifepo4 was presented by andersson et al. using a coffee bag this cell can be placed directly in the x-ray beam and diffraction is observed in transmission mode through the coffee bag. the study clearly showed the phase change reaction and monitored the growth of the heterosite (fepo4) phase as the triphylite (lifepo4) phase diminishes during charge, with the reverse occurring on discharge. the intensities of the peaks were found to be in good agreement with those anticipated from the charge passed during cycling. several other designs for in situ diffraction studies have since been reported including a recent study that characterized a meta stable new phase formation at high rates in large particle size lifepo4. since some of the first commercial uses of lifepo4 have emerged for high power applications such as power tools, many different preparations of lifepo4 have been explored to improve the material s performance in order to allow for practical use at high rates. these have focused on control of its particle size, doping on the li and fe site, and various coating methodologies. these strategies have been largely successful on the particle and agglomerate levels, reducing solid state diffusion times, phase boundary strain, and electronic resistance. given the above successes in improving the discharge of single particles and agglomerates such that intraparticle equilibration processes are not rate limiting, attention has recently focused on the effects of ion transport restrictions in composite electrodes containing dispersed active material, an electron conducting additive, and electrolyte. in the case where electronic conductivity is not rate limiting, discharge should begin at the electrode/separator interface where ion transport restrictions are at a minimum. with nonstoichiometric electrode materials, where the equilibrium potential decreases continuously with discharge, the progression of discharge from the front to the back of the electrode can be described by ambipolar diffusion of lithium ions and electrons according to the delevie description of a porous electrode. here the active material is represented by a series of capacitances distributed along the electrode thickness. these are connected to the bulk electrolyte through the infused electrolyte within the pores, and to the current collector via the electron conducting additive, so that parts of an electrode that are at different states of charge are continuously equilibrated during discharge. the interface between charged and discharged material is diffuse, and further diffusion occurs after the current has been switched off. importantly, the driving force for ambipolar diffusion is the increase in the potential with the state of charge. this is notably absent in the case of stoichiometric materials such as fepo4/lifepo4 where the potential is constant for most of the composition range, as shown by a long plateau in the discharge curve. ambipolar (ion-electron) diffusion should not occur in these materials; instead, the interface between charged and discharged parts of the electrode should be linked directly to the passage of charge and should stop moving once the current stops despite the sharp change in the average concentration of lithium. we recently described this phenomenon as the sharp discharge front (sdf) effect, and supported our hypothesis with electrochemical discharge data that responded to changes of electrolyte conductivity and salt diffusion as predicted, but previously had no direct evidence for the distribution of discharged material within the electrode. the above example cites just one case among many where a direct observation of the profile of the extent of discharge with depth into the electrode thickness could provide valuable insight into the discharge process and verification of simulated discharge phase profiles. in situ neutron depth profiling can resolve variations in lithium concentration through the thickness of an electrode via the energy profile of particles formed as a result of neutron capture by li. cross-sectional imaging by neutron absorption or tem can also provide valuable information on variations through the electrode. the use of x-ray diffraction allows direct observation of the phase distribution of the active materials during charge and discharge. this information is different from the lithium distribution, which would include any lithium in the electrolyte, and can be collected rapidly in situ providing the possibility of time resolution. variation of the incidence angle provides a depth sensitivity as absorption of x-ray photons results in a limited path length so with low incident angles the diffraction signal comes largely from close to the surface. the lifepo4/fepo4 system provides a model composite electrode, which should provide a sharp and controllable discharge front. positive electrodes for in situ cycling were formed by mixing appropriate amounts of carbon-coated lifepo4 (hydro-quebec) and acetylene black conducting additive (shawinigan black), then mixing in a polytetrafluoroethylene (ptfe) binder (6c n, dupont). the resulting mixture was calendared to a controlled film thickness of 100 m and punched to produce circular electrodes with a diameter of 8 mm. the electrodes were dried overnight at 120 c under vacuum, before being transferred to an argon-filled glovebox. two compositions of lifepo4 electrodes were used for the testing: (a) 50% lifepo4, 40% acetylene black, and 10% ptfe by mass and (b) 25% lifepo4, 60% acetylene black, and 15% ptfe by mass. currents were calculated to achieve complete charge or discharge in a fixed time period based on theoretical capacity, e.g. c/2=complete charge or discharge in 2 h and 2c=0.5 h. scanning electron microscopy (sem) used a jeol jsm-6500 fegsem with 15 kv accelerating voltage and secondary electron imaging powders or whole electrodes were mounted on conducting carbon tape and imaged without any further coating. initial xrd patterns were collected with a bruker d2 phaser (cu k x-rays) and rietveld refinement of the data used the gsas package. the electrochemical cell used for the in situ work is based on the commonly used swagelok cell design and therefore consists largely of cheap, commercially available parts. this cell can be constructed readily in any laboratory and does not require the use of specialized equipment or the use of toxic beryllium metal windows. the few bespoke parts used in its assembly are easily fabricated with use of basic tools. furthermore, it is simple to assemble and clean after use, and the positive current collector, which doubles as the x-ray window, is disposable and easily replaced. the cell consists of three main sections as shown schematically in figure 2a and as a photograph in figure 2b: (1) a 12 m thick aluminum foil acts as the positive current collector. this was attached to a stainless steel washer with black silicone rubber adhesive (loctite type 5910this was found to be inert in the environment of an operational cell). the washer was similarly attached to the swagelok nut used to seal the cell. (2) a 12.7 mm diameter stainless steel piston was used as the negative current collector. this was placed under tension with a steel spring held in place with a 12.7 mm diameter stainless steel rod, the bottom of which was machined to fit into a standard goniometer head for mounting onto the beamline. (3) ptfe sheaths, ferrules, and a nut sealing the bottom of the cell were used to avoid short-circuits, including during handling of the cell. (a) schematic and (b) image of the electrochemical cell used in the in situ xrd studies. the layered battery assemblies consisted of a composite positive electrode, two electrolyte-soaked 12.7 mm diameter separators (whatman gf/f grade glass fiber with eight drops of 1.0 mol dm lipf6 in 1:1 ethylene carbonate/dimethyl carbonate (novolyte technologies)) and a lithium metal negative electrode formed by compressing lithium (99.9%, aldrich) into a hemispherical mold and punching to a diameter of 11 mm. the shaped lithium negative electrode allowed an even pressure to be applied across the diameter of the stack, countering the effect of deformation of the aluminum window under pressure and allowing a similar electrochemical performance to be achieved to that observed with the same electrode material in a standard swagelok cell with flat electrodes. to study the electrode composition as a function of depth and state of charge the diffraction geometry shown schematically in figure 3 was used. the sample was mounted on the hexapod stage at beamline i07 of the diamond light source with use of an insulating mount and visually centered in the xy plane such that the highest point on the curved surface of the window was aligned with the center of the diffractometer cirles. the x-ray beam height was approximately 80 m and a scan in the vertical direction monitoring the direct beam intensity was used to position the sample such that the beam center was level with the top of the 100 m thick positive electrode. hence 40 m of the beam passed through the back of the sample with 0 incidence angle and 60% of the electrode (the part facing the separator and negative electrode) was not contributing to the observed xrd pattern. the sample was then tilted to increase the incidence angle and the penetration depth into the electrode. additional information about sample mounting on the beamline is included in the supporting information. schematic showing the effect of changing the angle of incidence on the penetration of the 80 m high x-ray beam into the positive electrode at 0 (left) and 4 (right) angle of incidence. in situ xrd patterns were collected with 20 kev beam energy (= 0.620) and an exposure time of 1 s using a pilatus 100k area detector at a camera length of 497 mm such that a 7 range could be collected in a single frame with high resolution. this restricted the accessible 2 range but facilitated fast acquisition times so that the phase change reaction could be observed at high rates. data were continuously collected with a series of different incidence angles between 0 and 6, although ultimately the analysis focused on data collected at 0 and 4. collecting 1 s patterns at 0, 0.5, 1, 2, 3, 4, and 6 incidence angles resulted in a 20 s cycle of measurements (patterns could be recorded with acceptable quality in 0.1 s but the sample position adjustment to effect the different angles of incidence was the rate limiting step). the peak width was between 0.05 and 0.06 at both incidence angles hence the variation in peak width with incident angles was not found to be significant. with a battery performing to theoretical capacity at our fastest rate of 20c under these conditions 9 patterns could be collected at each angle during the 3 min discharge. the peak heights of the most intense bragg reflections for lifepo4 at 15.7 and fepo4 at 16.3 were then used to provide a measure of the electrode phase composition at each angle of incidence. this structural change was correlated to the state of charge by using time stamps in both the electrochemical and xrd data files. the peak heights were extracted by using a matlab macro with a baseline correction. strictly peak area is proportional to the phase fraction, but since peak widths of the lifepo4 and fepo4 phases were similar in any given pattern the intensity was taken as a good indication of the relative phase contents. commercially sourced lifepo4 was used in this work to take advantage of its optimized performance, achieving a capacity of approximately 150 ma h g with a good cycle life and rate capability. powder xrd studies showed it to contain single phase lifepo4 (supporting information, figure s1). rietveld refinement by using the standard triphylite model in space group pnma resulted in a good fit with a=10.32227(12), b=6.00341(6), and c=4.69092(7), similar values to those described in the literature. to obtain a good fit it was necessary to employ a preferred orientation parameter allowing for a small increase in intensity along 010 (march dollase preferential orientation ratio of 0.9105(12) along 010). the sem (figure s1, supporting information) showed well-formed crystallites that are slightly elongated along one axis and with an approximate size of 200 nm. since preferred orientation is only being observed along one axis it is likely that the large flat face observable on some crystallites is becoming aligned with the xrd sample surface during sample preparation and that this face is the {010} plane of the crystallites. figure 4 shows xrd patterns obtained during the slow (c/2) charge and discharge of a lifepo4 half cell. the electrochemical response is characteristic of the coexistence of two phases with a flat plateau observed in the voltage capacity profile during charge and discharge. in the fully discharged (or as-constructed) state the characteristic triphylite lifepo4 phase can be clearly identified and as expected after charging (due to the near theoretical capacity extraction) a complete conversion to the heterosite fepo4 phase was observed. in the partially charged or discharged condition we can clearly observe a mixture of these two phases in the diffraction data with the phase contributions to the pattern corresponding to the state of charge (specifically peaks at 16.2 and 13.7 corresponding to lifepo4 and those at 15.7 and 13.6 pertaining to fepo4). it can be observed that peak overlap is quite limited due to the narrow xrd reflections and the significant differences in lattice parameters between the lifepo4 and fepo4 phases. charge and discharge curve for lifepo4 at a rate of c/2 with stacked diffraction patterns (4 incident angle) showing the linked structural changes between the lifepo4 and fepo4 phases. a significant enhancement in the 020 reflection relative to the expected intensity distribution based on the literature powder patterns is observed in both lifepo4 and fepo4, a larger elongation of the 020 reflections than observed in the powder pattern of the starting material (figure s1, supporting information). here the calendaring process used to make the electrodes is likely to have induced this orientational effect. importantly the degree of 020 preferred orientation was observed to be very similar in lifepo4 and fepo4 due to the topotactic transformation between them. the discharge performance of the batteries constructed for in situ testing using 25% and 50% active material with rates between 2 and 20 c is summarized in figure 5b, d (a constant charging rate of 2c was used for all experiments irrespective of the discharge rate to ensure the condition of the electrode at the start of discharge was as similar as possible). at relatively slow rates of discharge a characteristic flat discharge plateau was observed around 3.45 v vs li in both cases. as the rate was increased the discharge changes to a negative gradient linear profile as noted in our previous publication and explained by an ohmic potential drop in the electrolyte within the composite electrode to the nearest delithiated particle, increasing with the distance from the separator to the discharge front. discharges at rates greater than 5 c showed sharp end points at capacities well short of those obtained at low rates, similar to our previous work where an explanation was given in terms of severe lithium salt polarization at high rates due to a low lithium ion transference number. for electrodes containing only 25% and 50% lifepo4 (used in this work to ensure that x-rays could pass from the front to the back of the electrode without total absorption during the penetration path length) the capacity was well maintained up until rates of around 10c (figure 5a, c), with a slight reduction in capacity easily explained by premature termination of the discharge resulting from an arbitrary choice of potential limit that did not account for the discussed increase in ir drop. at rates greater than 10c we observed that sharp end points are reached which are premature of that anticipated entirely from ir drop and were hence consistent with the electrolyte limitation discussed earlier. the good retention of capacity at high rates observed in these electrodes results from a reduced amount of lifepo4; we chose relatively dilute amounts of active material to ensure that x-rays could pass from the front to the back of the electrode without total absorption during the penetration path length. if we assume that at the highest rates the electrodes are discharging under the sdf model discussed in our previous work then we can calculate effective diffusion coefficients of lithium ions in both electrodes using eq 1.1where dod is the degree of discharge, f is c rate, [lix]0 is the concentration of salt ions in the electrode, t is the transport number (assumed to be 0.3 in this case), [li] is the concentration of li ions stored in the active material within the electrode, d is the diffusion coefficient, and l is the thickness of the electrode. the effective diffusion coefficients for the salt in these structures were found to be 1.7 10 and 2.2 10 m s for the 25% and 50% electrodes, respectively. these values are significantly larger than we reported for an electrode with 75% active material in our previous work (10) and can be explained by increased porosity and a lower tortuosity of the diffusion paths. electrochemical performance of 25% (a, b) and 50% lifepo4 (c, d) electrodes shown as electrode capacity retention as a function of rate (left) and discharge capacity vs potential (right). slow charging rates and constant voltage top up periods were used to ensure full charging of the battery. based on the electrochemical performance an almost complete discharge of the cell at all rates is likely for the electrode containing 25% active material and therefore relatively small concentration gradients should be observed. for the 50% active material electrode much larger gradients are likely to be observed, especially at high rates. during the electrochemical measurements presented in figure 5, xrd patterns were collected at a number of incidence angles to probe the structural changes as a function of state of discharge rate. our analysis focuses on data collected at 0 and 4. grazing incidence xrd is widely used to increase the effective sample thickness in the study of thin films. applying this technique to battery electrodes can provide an effective method to profile any differences in phase behavior as a function of depth in the electrode by varying the proportion of the signal that is scattered from the side on which the beam impinges. the absorption of x-ray photons is a significant consideration in this geometry as with a 4 incidence angle the path length through the electrode will be increased from the 100 m electrode thickness to 1400 m. based on calculated x-ray absorption characteristics of the electrode components (supporting information, table s1) only about 6% of photons are expected to reach the front face of the electrode so the observed phase concentrations will be significantly biased toward contributions from material close to the current collector. the battery could be assembled the other way up to reverse this bias, but that geometry would also contain a compromise in that the beam would have to pass through the negative electrode and the separator, which would increase the contribution to the diffraction patterns from these components. importantly the absorption profiles of the two electrode compositions discussed herein are similar (table s1, supporting information) and so direct comparisons between their behavior can be made, while keeping in mind the bias in the data toward the back of the electrodes. the intensity of the 020 reflection of fepo4 is approximately equal to that of the 020+211 reflections of lifepo4 in an equimolar mixture of these phases, hence the intensities of these reflections were used as a semiquantitative measure of the content of the relevant phase. figure 6 shows the variation in the phase fraction of fepo4 based on the intensity of the 020 reflection during discharge at various rates. as expected the intensity changes occur over shorter time periods as the discharge rate is increased. with 25% active material in the electrode the intensity profile at all rates is similar using a 0 or 4 incidence angle. it is striking, however, that these profiles diverge with 50% active material and that the observed intensity of the fepo4 020 reflection is stronger with 4 incidence angle than it is with 0 incidence angle at all rates above 4c. this divergence shows that the back of the electrode is undergoing less discharge at these rates than the region being sampled closer to the front of the electrode. the divergence is largest at 8c and 10c, and is observed to decrease again at 20c. this indicates that the region of the electrode with the largest variation in composition is moving further from the side of the electrode from which the x-ray is impinging and hence closer to the electrolyte-soaked separator. the variation in fepo4 phase fraction expressed as the fepo4 020 peak height (relative to the combined heights of the fepo4 020 and the lifepo4 020+211) with time during cell discharge for electrodes prepared with 50% and 25% lifepo4 and linear fits to the data. figure 7a, b shows the gradients of the linear fits to the data in figure 6 plotted versus current density and c rate for both the 25% and 50% electrodes, respectively. this plot emphasizes the divergence in the observed intensity of the fepo4 020 reflection of the 50% active material electrode during fast discharge. a particular strength of our approach of rapidly collecting a series of incidence angles is that these data were collected on a single electrode so are directly comparable. the rate of change of the intensity of the fepo4 020 reflection during discharge as a function of current density and c rate for (a) 25% and (b) 50% lifepo4-containing electrodes. points circled in red highlight those which indicate significant concentration gradients within the electrode. the variations in phase behavior that we have observed during fast discharge are consistent with the effects described earlier based on observations from the electrochemical performance. in the electrode containing 25% active material the electrochemical data show only a slight increase in the negative gradient of the discharge plateau indicating that some small ionic diffusion gradients may exist in the electrode resulting in some preferential discharge of material near the bulk electrolyte as observed in figure 7a (a notable deviation from this trend is seen at 20c which may be due to a low number of data points increasing the bias from experimental scatter). in the 50% electrode, little or no variation was seen in the gradients at the front and back of the electrode with rates of 4c or slower. however, at elevated rates there is a sharp deviation in the rate of change between the front and back of the electrode suggesting that a significantly different limitation is controlling the electrode composition. this effect is consistent with the salt concentration polarization aspect of our previously reported sdf model, and originates from the insufficient concentration of lithium ions in the electrolyte stored within the electrode (see table 1). during the discharge of the battery lithium ions must be transported from the bulk electrolyte to the active material by migration and diffusion. at slow rates these mass transfer processes are sufficient to allow complete discharge of the battery, but at higher rates they are severely limiting. calculated assuming that the solid material is approximately 80% of the volume and that the free space (filled with electrolyte) is 20% (approximated from ref (48)). densities were taken as 3.6, 2, and 2.2 g cm for lifepo4, carbon black, and ptfe, respectively. it was also assumed that changes in composition of the active material did not change the packing of the solids and therefore the occupied volume. the concentration of the lipf6 in the electrolyte solution used was 1 m. in the flat discharge plateau region of lifepo4 the discharge reaction can be written as: the fepo4 and lifepo4 phases coexist within the electrode structure, so if one region of the electrode has an insufficient supply of lithium ions then a slower discharge will occur in one region and a faster rate of discharge in another. at high rates of discharge lithium ions this is followed by a mass transport process driven by diffusion from the bulk electrolyte. the lithium ions thus transported will react with the first particles of electrode material encountered which will be in the region of the electrode facing the separator. hence this region will fully discharge and the electrode region near the current collector will be undercharged (as observed at high rates in the 50% active material electrode). this is shown schematically in figure 8; when there is no limitation on the electrolyte during discharge, an even concentration in the electrode can be seen and when an electrolyte limitation is in effect, a preferential discharge occurs at the electrode close to the bulk electrolyte and results in an incomplete discharge. schematic showing how the discharge proceeds in an electrode where there are no limitations on the discharge from the electrolyte and also where there is a severe limitation as a result of insufficient transport of li ions (usually resulting from a high ratio of lithium ion vacancies to lithium ions in solution and when the electrode is discharged at high rates). this schematic negates the inclusion of any conductive additive or binder and assumes no electronic limitations. during the in situ cycling experiments described above, in which the electrodes were cycled sequentially at rates of 2, 4, 6, 8, 10, and 20 c, a reduction in the crystallinity of the active electrode was observed. note that the peak widths of lifepo4 and fepo4 were observed to be similar in any given pattern throughout the study so this broadening does not affect the phase faction calculations presented above. however, xrd patterns recorded at the start of discharge at each rate for the 25% electrode do show a clear increase in peak width (figure. 9a). to check whether this breakdown could be caused by x-ray beam damage we recorded patterns at the end of a 2c discharge while the battery was under open circuit conditions for 40 min. these results showed no discernible degradation of the active material as a function of time exposed to the beam, and since none of our measurements exceeded 5 h total collection time it seems unlikely that beam damage is a significant factor. we also investigated the behavior using ex situ measurements of cells cycled with the same regime used during this in situ test, and two cells cycled at 10c and 2c for the same number of total cycles. a significant broadening of the fepo4 reflections was observed in the materials which were cycled at a range of different rates (figure 9b) but very little extra broadening was observed with either of the fixed cycling rates. xrd patterns at the start of charge during a sequence of cycles at different rates (2c 20c), showing the increased peak width (a) and the full width half-maximum value of the fepo4 020 reflection in patterns recorded ex situ with a fresh electrode, an electrode cycled at a number of different rates (as per the left-hand image), an electrode cycled 10 times at 10c, and an electrode cycled 10 times at 2c (b). we also examined sem images of the electrodes before (figure 10a) and after (figure 10b) cycling using the multi-rate regime employed for the in situ measurements described above. no obvious breakdown in particle size can be seen in these images and we therefore suggest that the line broadening is a result of increasing disorder within the crystallites. this disordering seems to be limited to cases where multiple cycling rates have been applied. sem images of electrodes containing 25% lifepo4 before (a) and after (b) cycling under the test regime employed in diamond. the results presented above have tracked the formation of concentration gradients within electrodes under high rates of discharge. the limitations seen are consistent with effects corresponding to an insufficient transport of li ions from the bulk electrolyte through the electrode structure as predicted by sdf theory. this confirms that the rate performance of many modern materials used in battery technologies is not a result of the intrinsic properties of the material itself but rather the matrix in which it is stored. in this study, this effect is observed in electrodes with relatively dilute amounts of active material where the x-ray absorption is not obscuring measurement. however, these gradients should be much more pronounced in electrodes with higher concentrations of active material in the electrode (such as those conventionally used in research laboratories with 75% active material) and thus the electrochemical rate performance of these systems will be significantly hindered. the effect should also be far more pronounced in electrode materials which have a much higher volumetric capacity, where the requirement of lithium ions by the material stored in the electrode during discharge will be much larger. this means that to realize the full rate potential of battery materials the following strategies should be considered: (1) dilution of the active material (which reduces the stored energy density); (2) reducing the electrode thickness; (3) increasing the concentration of lithium ions in the electrolyte: the electrolyte concentrations used herein are typical for conventional liquid electrolytes but there are recent reports of more concentrated electrolytes which retain high diffusion rates, these would allow an increase in the amount of lithium initially in the pores c.f. values given in table 1; and (4) increasing the diffusion coefficient of lithium in the electrolyte. a new method for the in situ study of battery materials that allows for the visualization of concentration gradients formed in electrodes during discharge is introduced. a significant difference in the performance of the material dependent on whether it is near the bulk electrolyte or current collector is observed. at higher rates of discharge (> 10 c) the electrode material near the current collector changes at a much slower rate compared with the material close to the bulk electrolyte in cells containing a high concentration of lithium ion vacancies. it is believed that this effect is the major limitation in the rate performance of electrodes in conventionally prepared batteries. in parallel we observed a significant breakdown in crystallinity of the lifepo4 during the electrochemical measurements. it was shown that the breakdown is far more significant when the battery is cycled at a range of different rates rather than for the same number of cycles at either a high or low rate. ideally, to obtain the maximum rate out of a battery material of a given particle size and ionic and electronic conductivity we need an electrolyte that can supply the ions at high rates. in some cases the particles of the electrode material may be significantly large or the ionic or electronic conductivities sufficiently small such that electrolyte is not limiting the discharge rate. nevertheless, modern synthesis techniques such as sol gel and hydrothermal routes mean that synthesis of materials on the nanometre scale is routinely achieved which means that commonly the rate limiting step in the discharge of the battery material even with intrinsically poor electronic and ionic conductivities is the li ion transport through the electrolyte.

the phase changes that occur during discharge of an electrode comprised of lifepo4, carbon, and ptfe binder have been studied in lithium half cells by using x-ray diffraction measurements in reflection geometry. differences in the state of charge between the front and the back of lifepo4 electrodes have been visualized. by modifying the x-ray incident angle the depth of penetration of the x-ray beam into the electrode was altered, allowing for the examination of any concentration gradients that were present within the electrode. at high rates of discharge the electrode side facing the current collector underwent limited lithium insertion while the electrode as a whole underwent greater than 50% of discharge. this behavior is consistent with depletion at high rate of the lithium content of the electrolyte contained in the electrode pores. increases in the diffraction peak widths indicated a breakdown of crystallinity within the active material during cycling even during the relatively short duration of these experiments, which can also be linked to cycling at high rate.

PMC3998516

pubmed-1296

in september 2007, the american college of obstetricians and gynecologists (acog) in its committee's opinion recommended that labium minus operations can be performed to alter the size or shape (labioreduction) for the following medical indications: labial hypertrophy or asymmetrical labial growth secondary to congenital conditions, chronic irritation, or excessive androgenic hormones. additionally, the acog committee opinion suggested that clinicians who receive request from patients for such procedures should discuss with the patient the reason for her request and perform an evaluation for any physical signs or symptoms that may indicate the need for surgical intervention. also, acog viewed that women should be discouraged from cosmetic gynecologic surgery based upon variation of the anatomical appearance of female external genitalia. there are several surgical techniques that have been applied for labia minora labioplasty such as straightforward or partial amputation, central v-plasty (the wedge resection) and its modification, deepithelialized labioreduction, central wedge nymphectomy with a 90-degree z-plasty, inferior wedge resection and superior pedicle flap reconstruction, and laser labioplasty [28]. existing surgical techniques of clinical applications for labioreduction of the labia minora had been detailed by ostrzenski elsewhere [9, 10]. reviewing labium minus labioreduction techniques and performing some of those techniques, ostrzenski was guided to establish hypothesis that a surgical intervention for labium minus labioreduction should offer reduction of the height and length, should establish symmetry, should preserve natural color and contour of the labium minus, and should restore or create natural appearance of the labium minus frenulum (posterior edge of the fossa navicularis). in the observational prospective, multiple time case series clinical study, this hypothesis was tested, with study's objectives, to develop and to present a newly developed surgical intervention of labium minus labioreduction to determine applicability of this procedure, to evaluate aesthetic surgical outcomes, and to assess potential complications of this procedure. today, woman's demands for labia minora labioplasty either for aesthetic motives or medical indication(s) are growing; therefore, such a new surgical intervention is very important not only for gynecologist but also for cosmetic-plastic surgeons, urologists, and general surgeons who perform labium minus labioreduction. two out of three subjects presented with physical symptoms associated with the labium minus enlargements and one subject presented with dissatisfying appearance of her labia minora. the first subject was a 22-year-old caucasian woman, g0p0, unmarried, and a college student, who has been sexually active. the disproportionately protuberant labia minora were responsible for her symptoms such as persistent irritation leading to discomfort during physical activities and following voiding and defecation. blood flow during menses significantly increased irritations and discomfort due to difficulties in maintaining personal hygiene related to enlargement of the labia minora. also, she reported superficial dyspareunia, which was caused by twitching and inadvertently pulling the enlarged labia minora into the vaginal pool. although she learned how to separate her labia minora enough to introduce a penis into her vagina to minimize superficial dyspareunia on insertion, during the act she could not control her discomfort caused by labia minora being brought into the vaginal introitus and the distal vagina. the second subject was a 27-year-old caucasian woman, g3 p3003, married, high school teacher, and sexually active. the symptoms included discomfort associated with rubbing while walking or wearing close-fitting underwear and superficial dyspareunia, which precludes her from reaching an orgasm during sexual intercourse. the third subject was a 22-year-old caucasian woman, g0p0, single, and a professional ballet dancer. this abnormality forced her to use specially designed compression underwear during her practices and performances. she does not report any physical discomfort when she does not wear compressive underwear on the vulvar area. however, when she wears it, she has significant discomfort, particularly, during her professional dancing. this condition caused a negative body image perception, which led to decreased self-confidence and a social phobia and anxiety. the subject requested to reduce the volume of both labia minora, which she has considered to be responsible for her deteriorated professional, social, and emotional well-beings. aesthetic dissatisfaction with the subject's external genitalia led to social embracement and emotional disturbances. she felt extreme embarrassment not only during her professional dancing but also during her intimate life due to significant and disproportional overgrown of the labia minora. the subject requested to reduce the length and height and to create symmetrical and uniform appearance of the labia minora. all three women were subjected to newly developed fenestration labioplasty with inferior flap transposition under local infiltration. a search for the existing literatures was carried out from 1900 to may 2010, using medical subject headings (mesh) and keywords of fenestration labioplasty, fenestration labioreduction, cosmetic gynecology, labial reduction, labioreduction, labia minora labioplasty, labioplasty, labia minora procedures, female genitalia, labial hypertrophy, vaginal rejuvenation, vaginoplasty, designer vaginoplasty, designer vagina, and labium minus which were selected and used in a search on isi web of science (including conferences proceedings; 1950 pubmed), acognet, proquest, ovid, cochrane collection, the lancet on line collection, mdconsultant, new england journal of medicine, american college of physician on line resources, highwire journal, and citation index reference, and a manual search was utilized. an informed consent was structured in accordance with the existing recommendation of the american college of obstetricians and gynecologists. additionally, all women authorized ostrzenski to use their clinical data and digital photo images of their genital organs for publishing in medical peer-reviewed journals. the procedure was executed under local anesthesia without conscious sedation. a thick layer of lidocaine-prilocaine (2.5%/2.5%) cream was applied to the labia minora and immediately adjacent areas bilaterally; the region was covered with sterile gauze for 1 hour, the last 30 minutes before procedures an ice pack was added to this area. upon removing the ice pack, the remaining anesthetic cream was wiped off and the operative field was prepped with betadine solution. half way between the posterior commissure and the upper part of the anus in the middle and the ischopubic ramus, just under the superficial transverse perineal muscle, 510 ml of plain 1% lidocaine was injected with the 27 g 1/2 inch needle and 10 cc syringes (terumo, elkton, md, usa) in one side for local anesthesia. the superficial part of the deep branch of the perineal nerve and the posterior labial nerves were infiltrated with one injection and provided with adequate local anesthesia for this procedures. neither conscious sedation nor pudendal block was used. upon determining the size of labia minora volume being reduced, the base of the lower margin of incision was determined and outlining of the amount of the tissues being removed was marked in the shape of a bicycle helmet within the anterior labial surface; see figures 1, 5, and 6. also, in this process the arch of the new labium was determined; see figure 1. shape was accomplished and excised, see figures 2, 5(b), and 6(a). by doing so, the labium was divided into two fragments: the superior strip was partially detached from the rest of the labium and the inferior part at the base of the labium minus. immediately, the superior strip of the labium was sutured to the lower base edge of the labium minus. the anterior labial lamina and posterior labial lamina were sutured on both sites separately without suturing the erectile tissues between the labial laminae. below the arch, the labium inferior flap is gradually reduced in the wedge shape (the proximal part of the flap being bigger and then the distal segment) gradually getting smaller and thinner to create natural look of the labia. the distal labium part of the inferior flap is modeled in the arch shape to meet in the midline with the opposite distal labium just above the posterior commissure. such a tissue transposition creates the labium minus frenulum (posterior border of the fossa navicularis). the length of the inferior flap of the labium is trimmed and sutured in the same manner as presented above. the procedure was executed bilaterally in all the subjects; see figures 1, 2, 3, and 4. postoperatively, discomfort was controlled with external application of dermoplast, an antiseptic and pain relieving spray (medtech, jackson, wy, usa). the electronic and manual searches failed to identify fenestration labioreduction with inferior flap transposition or similar surgical intervention. therefore, this presentation is the first description in the scientific-clinical literature of a fenestration labioreduction with inferior flap transposition technique. the disproportionately protuberant, enlarged, and asymmetrical labia minora were confirmed in each subject. all subjects reported feelings of decreased body image perception, being sexually inadequate and undesirable, and decreased self-image and confidence. two women reported symptoms of persistent irritation leading to discomfort during physical activities, following voiding, defecation, and getting worse during menses as a result of difficulties in maintaining personal hygiene and reported superficial dyspareunia during sexual intercourse. the third subject presented with aesthetic dissatisfaction from her appearance of enlarged, asymmetrical labia minora. the newly developed operation of fenestration labioreduction with inferior flap transposition was applied without intraoperative, short- and long-term complications. in all subjects, the fenestration labioreduction with inferior flap transposition operation reduced the height and length, established symmetry, preserved natural color and contour of the labium minus, and restored or created natural appearance of the labium frenulum (posterior edge of the fossa navicularis). postoperatively, medical and emotional symptoms and signs resolved; pleasing surgical outcomes exceeded subjects ' aesthetic expectations. additionally, body self-image and confidence improved meaningfully in all subjects. none of the subjects verbally reported feeling of regret and described reduction of the emotional tension, which was generated by conflict of being different and dilemma of feeling of being helpless. social openness improved and intimate interaction increased, and their body image perception improved following the operation. there were no intraoperative, short-, or long-term complications recorded in all three subjects. the average time of surgery measured from the initial incision to completion of fenestration with inferior flap transposition was 36 minutes. all subjects engaged in vaginal sexual intercourse with their respective male partners 6 weeks following the surgery. this clinical study's results indicated that practitioners should look at the enlarged labia minora not only for physical symptoms (irritation, difficulties in maintaining personal hygiene, discomfort during physical activities, and discomfort during voiding or defecation) but also from the prospective of sexual dysfunction (pain during vaginal sexual intercourse) or emotional disturbances related to this condition (feeling inadequate, decreased feeling of body image perception, and being embarrassed socially). therefore, not only clinical symptoms but also an aesthetic aspect plays significant roles in the labium minus enlargement. implementation of fenestration labium minus labioreduction with inferior flap transposition demonstrates the use of clinical settings and eliminates potential for denuding the posterior vaginal introitus. analyzing existing surgical techniques for labioreduction such as central v-plasty, central v-plasty, and central wedge nymphectomy with 90 z-plasty and w-plasty one can draw a conclusion that these surgical techniques will leave transverse single or multiple scars on longitudinal organ such as the labium minus [2, 4, 7]. an inferior wedge resection technique leaves completely denuded areas around posterior and lateral vaginal introitus, which lead to high superficial dyspareunia and unaccepted high rates of wound separations [5, 8]. consequently, this technique will not only compromise aesthetic outcomes but also can be responsible for sexual dysfunction (superficial dyspareunia). other labioreduction surgical interventions such as labial partial amputation, deepithelialized reduction labioplasty, inferior wedge resection and superior pedicle flap reconstruction will be appropriate for clinical implementations [2, 3, 6, 8]. liao et al. and ostrzenski presented detailed evaluation of each surgical technique relating to labioreduction of the labia minora [9, 10, 13]. differences between existing surgical procedures and the fenestration labioreduction with inferior flap transposition (flft) technique are significant. none of existing surgical interventions will encompass reduction of the height and length, established symmetry, preserved natural color and contour of the labia minora, and restored or created natural appearance of the labium frenulum (posterior edge of the fossa navicularis) in one procedure and only the fenestration labioreduction with inferior flap transposition technique incorporates all of them. when compared to partial labial amputation, flft preserved natural color and contour of the labium minus and restored or created natural appearance of the posterior edge of the fossa navicularis and partial amputation does not [2, 3]. deepithelialized reduction labioplasty can only be offered for very thin and elongated labia minora, since it makes the labia minora much thicker or bulky at the base and flft will not do it. deepithelialized reduction labioplasty can only reduce the height of the labia minora; flft will reduce both the height and the length of the labia minora. deepithelialized reduction labioplasty will not restore or create the posterior boarder of the fossa navicularis and flft will. the inferior wedge resection and superior pedicle flap reconstruction will not restore or create the posterior boarder of the fossa navicularis and flft will. the inferior wedge resection and superior pedicle flap reconstruction will create unnatural appearance of the proximal labia connection and flft will not. the inferior wedge resection and superior pedicle flap reconstruction has tendency to stretch the superior pedicle flap and flft has not. the inferior wedge resection and superior pedicle flap reconstruction often creates undesirable permanent wrinkling and irregularity at the proximal approximation of the incision and the flft procedures are free of it. by all means, ostrzenski does not make any suggestion that flft is the only procedure that should be used in all cases. therefore, the clinical judgment should be exercise, and all four relevant procedures (labial partial amputation, deepithelialized reduction labioplasty, and inferior wedge resection and superior pedicle flap reconstruction, and fenestration labioreduction with inferior flap transposition) should be taken into account and the surgical intervention which suits patient's needs should be selected. small power of the study can be considered as a weakness; however, to test the established hypothesis the numbers of cases were sufficient to determine surgical applicability of flft. the importance of this study's results strongly suggest that not only aesthetic pleasing results can be accomplished in well-selected women by applying this surgical intervention but also the results imply that the clinical symptoms as well as emotional disturbances related to the enlarged labium minus can be eradicated. in this study group, fenestration labioreduction with inferior flap transposition surgical intervention can be executed effortlessly without complications and the method can be reproduced; aesthetically, the new operation achieves very pleasing results and the procedure improves physical-, emotional, and social well being.

objectives. to test applicability of the new surgical concept for labioreduction of the labia minora. study design. the observational, prospective, case series study was designed. subjects. three consecutive subjects were included. methods. the application of new surgical intervention was tested. main outcome measures. a primary outcome measured applicability of the fenestration labioplasty and secondary measures was used to evaluate surgical resolution of medical, emotional, and social symptoms; aesthetic outcomes; and potential complications. results. symptomatic, asymmetrical, and enlarged labia minora were associated with aesthetic dissatisfaction from deformations of the labia minora. the new operation was applied easily and without complications. the procedure reduced height and length, accomplished symmetries, preserved natural color and contour, and accomplished the labium minus expected appearance. postoperatively, physical, emotional, social symptoms and signs resolved, pleasing surgical outcomes exceeded subjects ' aesthetic expectations. meaningfully, self-image and self-confidence improved in all subjects. no feelings of regrets were reported. emotional tensions were reduced, social openness improved, intimate interaction increased, and their body image perception improved following the operation. conclusion. in this study group, fenestration labioreduction with inferior flap transposition was easy to execute without complications and the method was reproducible; the new operation achieves pleasing aesthetic results and the procedure improves physical, emotional, and social wellbeing.

PMC3945180

pubmed-1297

the sporting career of an athlete depends not only on how soon he can return back to his sporting activity but also on the level of return to sports, with the least long-term complications. rupture of anterior cruciate ligament (acl) results in a mechanically unstable joint, resulting in difficulty in athletic performance, increased risk of subsequent meniscal injury, and increased risk of early degenerative joint disease. acl reconstruction is recommended in athletes to help restore knee stability for return to pivoting sports. many different techniques using a variety of grafts with varying fixation techniques have continued to evolve to restore the stability to an acl-deficient knee. numerous papers and meta-analyses have shown similar results by different graft materials using multiple graft fixation techniques [49]. however, the results on return to sports after acl reconstruction have varied [1012]. although short-term evaluation is critical for assessment with regard to return to sports, an assessment 5 to 6 years after surgery is essential to determine the medium to long-term effect of surgery on maintaining knee joint stability, range of motion, restoring patient satisfaction while on field, returning to stressful pivoting sports, and development of complications if any. the ability to return to sports after acl reconstruction is governed not only by postoperative knee function but also by various other factors like social reasons, psychological impediments like fear of reinjury, and even monetary factors especially in sports persons of developing countries. there is a dearth of western literature regarding return to sports after surgery that is relevant to indian context making it difficult to counsel our patients regarding their eventual return to sports. to the best of our knowledge there is no study that evaluates the mid- to long-term results regarding return to sports after acl reconstruction in indian sports persons. the purpose of this study was to analyze the functional outcome in competitive level sports persons at 5 years after acl reconstruction. our hypothesis was that reliable and sustainable results could be achieved over time using the arthroscopic technique of acl reconstruction. additional goals were to assess function in the acl reconstructed knee, return to sports and level of sporting activity, patient satisfaction, identification of complications if any, and the factors or reasons in those who either stopped sports or showed a fall in their sporting levels. between 2002 and 2005, records of 96 patients who underwent arthroscopic acl reconstruction by the single surgeon (first author) were procured. patients with concomitant meniscal and chondral lesions were included whilst excluding those with multiligament injuries. 62 persons could be contacted out of whom 48 persons agreed to come for followup examination and interview. the mean age of our patients was 23.6 years (range 20.4 to 28.7 years). all were involved in competitive level sports at district and state level including 6 who were national level athletes. the sports played were wrestling (32 patients), kabaddi (8), athletics (6), and cricket (2). all had symptomatic and repeated episodes of instability despite conservative treatment and had wished to return to competitive sports that involved pivoting, cutting, and side stepping actions before proceeding to surgical procedure. all patients of our cohort underwent arthroscopic acl reconstruction using single incision transtibial technique by a single surgeon. standard titanium interference screws were employed for fixation of patellar tendon graft with additional cortical screw post on the tibial side. for the hamstring grafts endobutton (smith&nephew, mass, usa) was used for fixation on the femoral side whilst using a biodegradable screw with tendon staple on the tibial side. 20 patients who had meniscal or chondral lesions or both were subgrouped and were compared with those patients who only underwent arthroscopic acl reconstruction the post operative program was standardized in all cases that involved quadriceps and hamstring isometric setting exercises, progressing to closed chain exercises and range of motion physiotherapy with the aim of regaining full range of motion by 6 weeks. partial weight bearing was allowed at 3-4 weeks and light running on even ground, cycling, semi squats, and step exercises after 6 weeks. at 16 weeks, in addition to the strengthening exercises, sports-specific physiotherapy was instituted. return to sports involving pivoting, cutting, or side stepping was permitted at 6 months after surgery if the patient had close to full range of motion and muscle strength. the patients were clinically examined and completed the subjective international knee documentation committee (ikdc) questionnaires, the lysholm knee form, and the tegner activity scale (tas). the ikdc subjective score is a questionnaire with different subjective factors such as symptoms, sports activities, and ability to function. the objective ikdc grading has 7 parameters related to the knee, reflecting both impairments and disability. the worst grading for the first three key parameters, that is, presence of effusion, knee range of motion, and ligament stability, determines the final ikdc grade. there are 4 grades a, b, c, and d implying, respectively, normal, nearly normal, abnormal, and severely abnormal. the lysholm knee score quantitates knee function, symptoms, and disability in a scale of 1 to 100 points, with 100 implying the best results and 1 the worst results. the tegner activity scale depicts the level of sporting activity and allows us to compare and document the preinjury activity level with the present activity level. all the patients in our cohort had preinjury tas level of 7 or more, which indicates that they were involved in competitive sports. at the time of review, they were asked whether they were still playing sports and whether they had returned to their preinjury levels of sporting activity. return to sports was defined as returning to the same preinjury type and level of sports. those patients who either stopped sports or showed a decrease in level of participation were asked to tell the reasons for the same. the group of patients who returned to the same level of sports was compared with the group of patients who either stopped sports completely or decreased their level of sporting activity. statistical analyses using chi-square with yates ' correction and one way analysis of variance (anova) test for independent samples were performed to compare results in patient groups to determine if the reasons for not returning to sports had any significant correlation to the documented objective and subjective scales. at 5-year followup, the mean lysholm score was 86.4 (sd=8.8). 84.6% of patients had normal or nearly normal objective ikdc grade (a or b), while the remaining 15.4% had ikdc grade c (abnormal). the median preinjury tegner scale was 8 (sd=1.1), and the median 5 years after acl reconstruction tegner scale was 7 (sd=1.8). 8 patients out of the 48 that were reviewed at 5 years had left sports completely due to reasons other than sports. these included social reasons like marriage, getting into police and military services, and monetary reasons. out of the remaining 40, 22 patients had returned to the preinjury levels of sports and 18 showed a decrease in their sporting levels. of the 18 patients when asked for reasons for fall in sporting levels, 12 refered to fear of reinjuring the same or contra-lateral knee as the prime reason for the same. 6 patients refered to persisting knee pain, instability, annoying clicks, and numbness around the joint as reasons for a fall in their sporting abilities. table 2 shows the results of various scores in the patient subgroups according to table 1. we found that at 5-year followup, the subgroup of patients that had returned to preinjury level of sporting activity (45.8%) had the best scores this was in contrast to patients who showed a fall in their sporting levels because of painful and unstable knee (12.5%). lysholm 74.3, subjective ikdc 64.6, and objective ikdc grade a and b 33.3%. those patients who decreased their sporting levels due to fear of re-injuring their knee (same or contra-lateral) (25%) showed that they had intermediate scores lysholm 82.3, subjective ikdc 76.7, and ikdc grade a and b 75%. by statistical analyses, the difference in the outcome scores in the aforesaid three categories of patients was found to be statistically significant objective ikdc, subjective ikdc, and lysholm scale (p< 0.05) (table 2). when we see their scores we find that lysholm was 88.9, subjective ikdc 86.4, and ikdc a and b 100%. they cited social reasons like marriage, monetary factors, and getting into police and military services as the main reasons for not continuing with sports despite having scores that were comparable with patients who returned to their preinjury levels of sporting levels (table 2). by this study we have reviewed the functional results at 5 years after arthroscopic acl reconstruction in a cohort of competitive level sports persons. the results that were quantitated by lysholm, subjective and objective ikdc, and tegner activity scale were comparable to those in previously published studies [15, 16]. in our study, subjective assessment with particular attention to return to sports and at what level was given more attention than objective findings, type of graft used, fixation method used, instrumented testing of joint stability, and investigations like roentogram. noyes et al. proposed the rule of thirds for chronic acl injury managed conservatively with rehabilitation and physiotherapy. they stated that one third of their patients resumed their previous recreational activities without reconstruction, one third managed by modifying their activity level and one third required reconstruction because of recurrent giving-way episodes even in day-to-day activities.. showed in their followup of competitive hand ball players that 91% of players treated without reconstruction could return to their preinjury activity level compared to 58% in the reconstructed group., however, found that at 6 years after acl injury, only 46% of their patients treated without reconstruction could return to preinjury sports. kostogiannis et al. indicated that many in their cohort who returned to sports at the same tegner level without reconstruction avoided contact sports as advised by the rehabilitation team. these kind of conflicting results in the literature create confusion in the mind of the attending surgeon who is counseling the injured sports person for acl reconstruction. however, the consensus rests on the suggestion that an athlete who wishes to return to his preinjury level should undergo reconstruction, especially competitive athletes or individuals engaging in pivoting sports [11, 12]. the literature is also full of a variety of grafts and fixation devices that are employed for arthroscopic acl reconstruction [49]. however most of them show similar results regarding stability, patient function, and final outcome. the median tas before injury in our patients was 8 and at five-year review it was 7. this is comparable to that of 84.3 of matsumoto et al. and charlton et al. 84.6% of patients had normal or nearly normal objective ikdc grade (a or b). return to sports is one of the most important outcome measures of a successful acl reconstructive procedure. in our study, 22 (45.8%) of the patients who underwent subjective and objective analyses at 5 years after their acl reconstruction had returned to their preinjury levels of sporting activities. in the literature the data for return to sports shows a wide variation51% (maletis et al.), 53% (kvist et al.), 65% (gobbi and francisco), 71.4% (smith et al.), 92% (nakayama et al.), and 100% (fabbriciani et al.). the literature also shows that competitive level athletes are more likely to return to the same level of sports after acl reconstruction as compared to recreational level athletes. this may be one of the factors that account for a wide variation of percentage of return to sports as depicted in the literature. report a 100% return of their cohort of 18 competitive level rugby players to the same level of sports after acl reconstruction at 6-month and at 2-year followup. the motivation to return to sports is very high especially in competitive sports persons after surgery. reported that 81% of their patients who were competitive athletes returned to sports within 1 year of surgery. however, at mean followup at 43 months after surgery, this dropped to 71% of their initial cohort. another interesting point was that 21.8% were still in sports despite major functional impairment in the operated knee. this study highlights the fact that a very high motivational factor may be the reason for a high return ration in competitive athletes. also there is a significant fall in percentage when reviewed at 1 and at approximately 3 years after surgery. thus assessment regarding return to sports should not only be a shortterm one but should also look at mid- to long-term results vis-visa return to sports. however, in our study of competitive athletes the return to sports was only 45.8%, due to reasons other than sports that include social reasons, monetary reasons, and fear of reinjury to the same or contra-lateral knee. in our study, if we exclude the 8 (16.6%) cases who had a stable symptom-free knee but had left sports due to social and other reasons, our results show that 55% of cases returned to preinjury levels of sports. but all these are western literature, where there are a dedicated team of sports-specific physiotherapists, a sports-specific psychologist to counsel the patients, and ample funding from government, and private sources to support the surgical costs, physiotherapy and rehabilitation of the injured sports person. fear of reinjuring their knees and going through the surgery again, a prolonged period of physiotherapy, and remaining off the competitive field of sports proves to be a detrimental factor in the minds of our patients. this factor was found to be a major factor that led to fall in sporting levels in 12 of the 18 patients who showed a fall in their sporting levels at 5-year followup after arthroscopic acl reconstruction. the same has been observed by kvist et al. and lee et al.. reported that 66.1% of their patients experienced fear of re-injury at 9.3 months. reported that 72% of their patients who did not return to their preinjury levels of sporting activities feared instability. a striking point was that the majority (70%) of them had no objective knee instability. in our study, fear of re-injuring the same or contralateral knee was a major factor in 12 of the 18 cases who showed a fall in sporting activities at 5-year review. it has been observed also in our study where the results of various scores in the sub group of patients who had fear of re-injury were not poor but were intermediate (table 2) that is they were better than those who had a painful and unstable knee but worse than those who had returned to their preinjury sporting levels. our study highlights an area that is often forgotten in the rehabilitation and evaluation after acl injury or reconstruction. no attempts are made to find the reasons for fear of disability to return to sports. plausible factors that have not been evaluated are, for example, impaired knee proprioception and neuromuscular control possibly resulting in both decreased performance and increased fear of re-injury. the number of injured knee structures, objective knee stability, time between injury and acl reconstruction, and follow-up time are important factors that may influence performance. the long rehabilitation time and difficulties to regain a position in the sports team may affect motivation and cease the athlete's competitive career in favour of social and family life. further prospective research combining assessments of psychological variables and functional tests is warranted in order to fully elucidate why patients return or not to their preinjury level and to fully establish the reasons. the tampa scale of kinesiophobia (tsk) has been used by kvist et al. to quantify the fear of re-injury due to physical activity. their study reports a 53% return to preinjury level of sports after 3 to 4 years of acl reconstruction. a high score on tsk scale implying a greater fear of re-injury and pain correlated with patients who did not return to preinjury level of sports. the other group of 6 out of 18 patients who showed a fall in sporting levels pointed a painful and unstable knee as reasons for the same. this has been reflected very well in their outcome scores (table 2) which show poor subjective and objective scores. when we look at the three groups those who returned to preinjury level, those who showed a fall due to a painful, unstable knee, and those who showed a fall in levels due to fear of re-injury we find that the difference in the scores of the three groups was significant statistically. possible factors that have been suggested for this are impaired knee proprioception and neuromuscular control leading to decreased performance and increased fear of re-injury. our study highlights that the psychosocial issues that are relevant to the social milieu of the athlete are very important and affect the overall results of the surgery with respect to return to sports. moreover, we found that there was a psychological fear in the mind of the athlete that his knee is weak and he can reinjure it more easily than the normal knee. the whole thing makes him afraid of rerupturing the graft as well as injuring the ligament in the contralateral knee as well. 8 of our patients left sports completely although on assessment their knees had good outcome scores (table 2). a thorough and in-depth counseling by the surgeon at the time of index surgery besides social and family support mechanisms including regular sport-specific physiotherapy and psycho therapy session prove to be of great help in this regard. our study has limitations in the form of a short sample size and a high drop-out rate of followup at 5 years. a young, active population that undergoes this surgery has high relocation rates due to study and employment reasons. long-term studies related to orthopedic sports medicine well document this problem of loss to follow-up. a national or regional level acl registry to follow up cases after surgery is called for in the current scenario. despite the limitations, our study should prove useful to orthopedicians who operate and treat sports persons as they counsel them for surgery regarding the likelihood of eventual return to sports.

introduction. the purpose of this study was to analyze the functional outcome in competitive level athletes at 5 years after acl reconstruction with regard to return to sports and the factors or reasons in those who either stopped sports or showed a fall in their sporting levels. methods. 48 competitive athletes who had undergone arthroscopic assisted acl reconstruction with a minimum follow up of at least 5 years were successfully recalled and were analyzed. results. 22 patients had returned to the preinjury levels of sports and 18 showed a decrease in their sporting levels. of the 18 patients, 12 referred to fear of reinjuring the same or contra-lateral knee as the prime reason for the same while 6 patients reported persisting knee pain and instability as reasons for a fall in their sporting abilities. the difference in the scores of these groups was statistically significant. 8 patients out of the 48 had left sports completely due to reasons other than sports, even though they had good knee outcome scores. conclusion. fear of reinjury and psychosocial issues that are relevant to the social milieu of the athlete are very important and affect the overall results of the surgery with respect to return to sports.

PMC4063161

pubmed-1298

most inhibitors occur at an early age and usually within the first 50 exposure days. as such however, it should be acknowledged that there are no clear data that demonstrate an increased risk of inhibitor when switching fviii concentrate in patients prior to 50 exposure days. certain clinical scenarios have been associated with an increased risk of inhibitor development, and for some patients with haemophilia a, switching products requires careful consideration. haemophilia patients with a history of inhibitors, including those in whom the inhibitor has been eradicated with immune tolerance induction (iti), may relapse and constitute a group of individuals at higher risk of inhibitor development. there may be similar concerns for haemophilia patients with a family history of inhibitor and/or a higher risk mutation in the f8 gene. as such, there may be reluctance on the part of the physician and the patient to consider switching products when they have been shown to be tolerant of their current therapeutic product. if such individuals are to have the opportunity to benefit from advances in therapy such as those with increased safety profiles or extended duration of action, they would need to consider switching products. in this situation, it should be noted that there is no evidence of increased risk of inhibitor development 6. lastly, intensive treatment (including surgery) is reported to be associated with an increased risk of inhibitor development 7. as such, patients scheduled to have elective orthopaedic surgery should remain on their current product and switching in the intraoperative or early postoperative period should be avoided. however, for all patients, following discussions with patients or their caregivers, a product switch may be undertaken if there is a clinical need; there are no absolute contraindications for switching. for patients for whom product switching may be appropriate, a reluctance to switch products may be associated with concerns regarding the potential negative outcomes of such a switch. in addition, some patients with haemophilia often develop a strong psychological link with their current product 2. to investigate patient concerns regarding switching, a semi-structured, non-random, brief, online survey was conducted using the web research platform surveymonkey. participants from seven national haemophilia organisations (argentina, brazil, chile, santo domingo, mexico, nicaragua and spain) were informally invited (by e.r.) through social media during 15 days in april 2013. survey participation was voluntary and a total of 46 participants (of whom 27.5% were parents of a child with haemophilia) anonymously completed the online survey (response rate 85%). ethical standards for online behavioural research were strictly followed and all participants gave their electronic consent before taking the survey. data were provided regarding haemophilia a (n=37) and b (n=9), of which the majority of patients had severe haemophilia (n=27), and some patients had been diagnosed with an inhibitor (n=9). of note, 57% of the respondents believed that the probability of inhibitor development was high or very high when switching product. moreover, when asked to list up to five concerns related to switching product, inhibitor development appeared first in the list (25.3% of respondents), followed by potential product side effects (21.8%), product effectiveness (17.2%), safety/purity (17.2%), and finally, product quality and longevity (4.6%). the original survey and complete report in spanish are available by email request to eduardo.remor@uam.es. to explore concerns regarding product switching by healthcare professionals, a recently conducted delphi consensus exercise was undertaken to canvass expert opinion on the topic 6. briefly, the delphi process is a structured group communication in which a complex problem is considered by a group of experts. the procedure usually begins with a face-to-face meeting to set the context of the communication, after which experts input their thoughts/opinions through several rounds of questions and answers. the delphi panel noted that currently available studies are often retrospective, characterised by a mixture of methodological approaches, and frequently lack appropriate control groups. given this background, and the modest amount of data available on product switching, the delphi process provided an alternative approach to addressing the complex problem of assessing the risk of immunogenicity associated with product switching. the group addressed 14 separate items relating to the issue of product switching and the risk of inhibitor development and reached a high level of consensus on most items. they, too, concluded that much of current clinical practice regarding treatment switching in haemophilia was not based on evidence, but on the fear of developing an inhibitor 6. treatment-related factors, including treatment intensity as briefly mentioned above, therapeutic regimen (i.e. prophylaxis vs. on-demand treatment) and product type have been proposed as possible influences on inhibitor development 712. for example, a systematic literature review concluded that inhibitor incidence was lower in patients treated with one pd-fviii vs. those who had used multiple pd-fviii concentrates or a single rfviii product 12. although a more recent systematic review using multi-way analysis of variance concluded that source of concentrate did not significantly influence inhibitor development 11, suggestions of an increased incidence of inhibitor development and treatment with rfviii products, and also those with a b-domain deletion/modification, may continue to contribute to patient and physician reluctance to switch to new rfviii products. three early studies in previously untreated patients (pups) suggested that the incidence of inhibitor development was less for those treated with pd-fviii than in patients treated with rfviii 1315. however, significant differences in inhibitor development were only observed in two of these three studies 13,14. in the uk study, inhibitors developed more frequently in patients initially treated with rfviii when compared with pd-fviii (p=0.006) 13. in a french cohort study, the risk of inhibitor development was reported to be higher in patients treated with rfviii than those treated with pd-fviii, regardless of other risk factors (e.g. f8 genotype, history of inhibitors in patients with a family history of haemophilia, age at first fviii infusion) 14. however, in sweden, no significant increase in the incidence of inhibitors was reported for haemophilia a patients in the 1990s who were mainly treated with recombinant products (n=10/48, total incidence 21%), as compared with the 1980s (n=9/52, 17%), when patients received intermediate/high-purity plasma-derived concentrates 15. the concerted action on neutralising antibodies in severe haemophilia a (canal) study was a retrospective, multi-centre cohort study designed to further describe the relationship between treatment and inhibitor development in 366 pups with severe haemophilia (residual fviii activity<2%) born between 1990 and 2000 9,16. a total of 82 patients (26%) developed clinically relevant inhibitors; of 181 patients first treated with rfviii product, 53 (29%) developed inhibitors, while inhibitors were reported in 29 of the 135 (21%) patients treated with pd-fviii, and the relative risk (rr) of inhibitors in pd-fviii vs. rfviii products was 0.8 (95% confidence interval [ci], 0.51.3) 16. in addition, switching between fviii products did not appear to increase the risk of inhibitor development (rr, 1.1; ci, 0.61.6). hence, the canal study results do not support previous findings suggesting an increased risk of inhibitor development with rfviii products, nor that switching products may influence inhibitor development 16. more recently, the potential influence of rfviii vs. pd-fviii product type on inhibitor development was also explored in the rodin (research of determinants of inhibitor development) study, which used data from the pednet registry that comprised 29 centres in europe, canada and israel. overall, there was no difference in inhibitor risk between pd-fviii and rfviii products (adjusted hazard ratio 0.96; 95% ci, 0.621.49), and switching between different fviii products was not associated with an increased risk of inhibitor development (adjusted hazard ratio 0.99; 95% ci, 0.631.56). however, a significantly increased risk of inhibitor development was found to be associated with second-generation (produced in baby hamster kidney [bhk] cells) vs. third-generation full-length rfviii products (adjusted hazard ratio 1.60; 95% ci, 1.082.37) 7. although this latter finding is intriguing, there is no clear biological explanation for the difference in inhibitor development between second- vs. third-generation full-length rfviii. concerns regarding a potential increase in immunogenicity associated with b-domain deleted rfviii were raised by an early italian study of previously treated patients (ptps) 17. of 25 low-risk ptps, one patient developed an inhibitor after switching from pd-fviii to b-domain-deleted rfviii 17. results from a more recent meta-analysis by aledort and colleagues of prospective clinical studies on product switching appeared to demonstrate an increased risk of inhibitor development with b-domain-deleted rfviii in ptps 8. however, good results for meta-analyses come from inclusion of good data 18, and in this respect, of the two studies that contributed the most to the final odds ratio for the aledort meta-analysis, one consisted only of case reports 19 and the other contained only the prospective arm from the italian study 17. the ongoing european haemophilia safety surveillance (euhass), a prospective adverse event reporting system, is exploring the incidence of inhibitors in pups and ptps and the potential factors that may be contributing to inhibitor development. data reported from the first 2 years of the study, provided by 64 haemophilia centres from 27 european countries (caring for 22 242 patients), showed that the inhibitor rate in pups with severe haemophilia a was 25% overall, with a similar incidence of inhibitors in patients treated with rfviii (25%) as compared with those treated with pd-fviii (27%) 20. for ptps, no significant difference was observed in inhibitor incidence between different rfviii products (including full-length and b-domain-deleted products) 20. a more recent evaluation of data now available from the first 3 years of euhass has confirmed that in pups, there are no significant differences in inhibitor development between pd-fviii and rfviii products, or between different rfviii products 21. of note, the second-generation full-length rfviii product associated with increased inhibitor incidence in the rodin study was also one produced in bhk cells; together, these findings may lead to speculations that products may be associated with inhibitor development in pups. however, such speculation should be made with caution as many other variables may contribute to immunogenicity. although early studies suggested a potential for increased inhibitor incidence in patients treated with rfviii and b-domain deleted product, the findings summarised above from canal, rodin and euhass do not provide support for these earlier suppositions. to date, studies on three national product switches (ireland, canada and the uk) have been published (table1) 2224. while all three studies examined product switching, the product switches were different, and only the uk study investigated inhibitor incidence in both switchers and non-switchers. summary of data from the three national product switches one patient had previously documented inhibitors, and one child who had been on prophylaxis with kogenate developed an inhibitor during intense therapy for treatment of an acute bleed. patients remaining on kogenate. the difference in inhibitor incidence rates between switchers and non-switchers was not significant (p=0.12). the national irish product switch resulted from a national tender process in 2006 in which all patients with haemophilia a changed their fviii treatment product en masse to a plasma and albumin-free recombinant full-length fviii product (advate) 22. in this study, case records of irish ptps were retrospectively reviewed to evaluate the risk of inhibitor formation following this treatment switch. only one of the 96 patients without a previous history of inhibitors developed an inhibitor following the switch. however, as this patient had only received three exposure days prior to the switch 22, the inhibitor might also have developed if the patient had remained on his previous treatment. in addition, there were no cases of recurrent inhibitor formation in any of 16 patients with previously documented inhibitors. the canadian national product switch surveillance study comprised 460 haemophilia a paediatric and adult patients from 17 canadian comprehensive haemophilia care centres, of whom 274 had evaluable data 24. this study was conducted by the inhibitor subcommittee of the association of hemophilia clinic directors of canada to evaluate inhibitor development in patients with haemophilia a following the switch to a second-generation rfviii product (table1). an inhibitor was detected in four of the 274 (1.5%) evaluable patients at the time of the switch, but no additional patients with inhibitors were reported afterwards 24. this finding highlights the importance of studying patients prospectively and testing for inhibitors before and after switching. a national tendering exercise conducted in the uk in 20092010 required half of patients receiving rfviii to change rfviii brands 23. based on the contractual requirements of the exercise, patients were randomly selected for switching in each local treatment centre. centres were requested to test all patients for inhibitors prior to the switching date and 6-monthly thereafter. a total of 1217 patients with severe haemophilia a lacking an inhibitor history were analysed; 535 patients switched rfviii product and 682 patients did not. four patients who switched, two from kogenate and two from advate, developed inhibitors; in two cases, the inhibitors were transient, while for the other two cases, patients were rapidly tolerised. the incidence of inhibitors reported (7.5/1000 treatment per years) did not significantly differ from the 5.31/1000 treatment year incidence observed during the 20-year period preceding the study (p=0.24), although the study was underpowered. of note, among the 682 non-switchers, one patient developed inhibitors. as these three studies employed different methodologies and studied heterogeneous patient populations, the findings of each can not be directly compared, nor can the data be pooled for a combined analysis. however, all three studies suggest that switching is not associated with an increased risk of inhibitor formation relative to the very low background frequency of immunogenicity of these products, although the results are not conclusive and should be interpreted with caution. the implementation of prospective, controlled surveillance programmes on switching and not switching is imperative, as there is insufficient evidence currently available to support the development of clear best practice in addition, such surveillance programmes will provide researchers with the data needed to address the many unanswered questions regarding the patient-related and treatment-related factors that contribute to the risk of inhibitor development. most importantly, for the full potential of surveillance programmes to be realised, all data on inhibitor development and products received should be submitted to a centralised, unbiased database to establish a baseline on the current inhibitor risk and include all new patients, regardless of which product they receive. this is the only way to ensure that the field is able to utilise all of the data in the service of our patients. given the limitations of the existing evidence base, we can make a number of recommendations for the conduct of future studies on product switching. first, inhibitor testing should be performed before and after the switch to determine if any new inhibitors detected may be in association with switching to the new treatment. similarly, inhibitor testing should also be performed before and after intensive treatment/surgery. concerning routine patient care, educational materials addressing patients concerns about switching are needed because some of patients concerns are not supported by the actual evidence about the consequences of switching products. more importantly, physicians are encouraged to discuss directly with patients and parents their therapeutic approach and the other treatment options that are available before a more urgent need arises to consider switching. doing so may increase patient satisfaction with treatment and foster more informed and positive attitudes when and if the need arises to address switching to a new product. in the future, it may become feasible in routine practice to calculate an inhibitor risk score and identify patients at high risk, thus aiding the evaluation of which patients to consider for switching treatments. among patients with haemophilia (and their physicians), there is often a reluctance to switch factor concentrates because of concerns about increasing the risk of inhibitors. however, current evidence does not suggest that switching products significantly influences inhibitor development. with the forthcoming arrival of new haemophilia treatments, elena santagostino has received speaker fees for meetings organised by bayer, baxter, pfizer, csl behring, novo nordisk, biotest, kedrion, octapharma and grifols, acted as paid consultant for bayer, pfizer, csl behring, novo nordisk and grifols and has received unrestricted research grants from novo nordisk and pfizer. victor jimnez-yuste has received reimbursement for attending symposia/congresses and/or honoraria for speaking and/or honoraria for consulting and/or funds for research from baxter, bayer, csl behring, grifols, novo nordisk, octapharma and pfizer. thierry lambert has acted as a board member for baxter, bayer, csl behring, novo nordisk and pfizer. rolf ljung has during the last five years received consultancy/speaker fees from novo nordisk, bayer, baxter and octapharma. massimo morfini has served as a consultant and invited speaker for novo nordisk a/s, csl behring, wyeth/pfizer, baxter and bayer. gnter auerswald, gary benson, gerry dolan and silva zupani alek have no conflict of interests to declare.

patients with haemophilia a (and their physicians) may be reluctant to switch factor viii (fviii) concentrates, often due to concerns about increasing the risk of inhibitors; this reluctance to switch may contribute to patients missing the clinical benefits provided by the arrival of new factor viii products. this topic was explored at the eleventh zrich haemophilia forum. clinical scenarios for which product switching may be cause for concern were discussed; when there is a clinical need, there are no absolute contraindications to switching, but some patients (e.g. previously untreated patients and those undergoing elective surgery) may require more careful consideration. both patient and physician surveys indicate that the reluctance to switch, and the fear of inhibitor development, does not appear to be evidence based. the evaluation of more recent data did not support previous studies suggesting that particular products (e.g. recombinant vs. plasma-derived and full length vs. b-domain modified) may be associated with increased risk. in addition, data from three national product switches showed that switching was not associated with increased inhibitor risk, but highlighted the need for regular inhibitor testing and for a centralised, unbiased database of inhibitor incidence. to conclude, current evidence does not suggest that switching products significantly influences inhibitor development.

PMC4407931

pubmed-1299

in view of growing awareness of the side-effects of some orthodox medicines, people are turning more towards alternative medicines with fewer and less-toxic side-effects, and homeopathy has become a major complementary and alternative medicine (cam) in many countries today. in homeopathy, microdoses of very high dilutions (potentized) of natural substances are generally preferred over mother tinctures (crude extracts) [2-4] for stronger and longer-effects. the initial drug substance is generally dissolved in an aqueous solution of ethanol (mostly 70%) and is potentized in gradual steps of dilution with agitation or succussion. on a centesimal scale, when 1 ml of mother tincture is diluted with 99 ml of an aqueous solution of ethanol (vehicle of drug) and given 10 mechanical jerks, potency 1c is produced. when 1 ml of 1c is again diluted with 99 ml of an aqueous solution of ethanol and given 10 jerks, the potency 2c is produced, and so on. therefore, when the drug has attained potency 12c, it has been diluted to 10 (beyond avogadro s limit), and the existence of even a single molecule of the original drug substance becomes highly improbable. although some researchers have demonstrated the existence of nanoparticles of the original drug in such ultra-highly diluted homeopathic drugs [5, 6], the efficacy is often questioned by rationalists, as the precise mechanism of drug action has still not been firmly established. therefore, we became interested in the study of any perceivable differences in the actions between dilutions below avogadro s limit (6c, diluted 10 times) and above avogadro s limit (30c, diluted 10 times) in living cells, in vitro, and we attempted to understand the possible signalling pathway of their action. non-small cell lung cancer (nsclc) is the most prevalent accounting for-80% of all lung cancer cases. in addition, benzo[a]pyrene is one of the major polycyclic aromatic hydrocarbons found in cigarette smoke and is responsible for inducing lung tumours in smokers. patients with lung cancer are commonly treated with conventional modalities including chemotherapy, radiation therapy, etc. which also affect normal cells., are now gaining importance in the cure/amelioration of many difficult- to-cure diseases including cancer. first, do the two potentized homeopathic drugs, condurango 6c and 30c show any ability to induce apoptosis in nsclc, and do they act via reactive oxygen species (ros) generation and mitochondrial membrane potential (mmp)-depolarization. second, dose if condurango 30c have more apoptosis-inducing ability than condurango 6c. nci-h460 (h460) human nsclc cells were procured from national centre for cell science (nccs), pune, india and cultured in rpmi-1640 media, supplemented with 10%-fetal bovine serum (fbs) and 1%-antibiotic-antimicotic solution. h460 cells (110 /well) were treated with different concentrations of condurango 6c and 30c (0.5l/100l media- 5l/100l media) and with placebos (successed 70% alcohol-vehicle for the drugs) as a control for 24 hours and 48 hours. the concentrations at which both the drugs showed nearly 50% cell death were determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assays. h460 cells were also treated with half maximal inhibitory concentration (ic50) doses of condurango 6c and 30c and were compared against placebo-treated and untreated cells. after 24 hours and 48 hours of treatment, the cells were observed and photographed under an inverted phase-contrast microscope (axiscope plus 2, zeiss, germany). for further confirmation of morphological changes, if any, a scanning electron microscopy (sem) study was done using an s530-hitachi scanning electron microscope. reactive oxygen species (ros)-accumulation is generally known to occur at early hours of apoptosis. cells were treated with condurango 6c and 30c (ic50 doses) and with their respective placebos for 2 hours, 6 hours, 12 hours, 18 hours, and 24 hours, to estimate the specific time-point at which maximum ros accumulated. after treatment, cells were incubated with 2,7-dichlorodihydrofluorescein diacetate (h2 dcfda) (5 mm) and ros was estimated by using fluorimetry (perkinelmer, usa). fluorescence microscopy was done at the specific time-point(s) when ros-generation was maximum. the changes in mmp were recorded at 2 hours, 6 hours, 12 hours, 18 hours, and 24 hours of treatment with ic50 doses of both drugs were compared against drug-untreated cells by using rhodamine-123 and the cells were analyzed by using fluorescence microscope (leica, dmls). changes in mmp were also measured using flowcytometry (facs, aria iii, bd bioscience) at the specific time-point(s) at which the mmp showed the maximum decrease. cells were stained separately with 4,6-diamidino-2-phenylindole (dapi) (10gml) and acridine orange/ethidium bromide (ao/eb) (1mg/ml) to visualize changes in nuclear morphology. after 48 hours of treatment, stained cells were observed and photographed under a fluorescence microscope (leica, dmls). dna-fragmentation was assayed using the conventional phenol/chloroform method and was visualized under a uv-transilluminator (ultracam digital imaging, genei, india). dna strand breakage was analysed flowcytometrically by labeling the treated and the untreated cells with 5-bromo- 2-deoxyuridine 5-triphosphate (br-dutp) by using bd facs verse. the percent of cells in each phase (subg0/g1, g0/g1, s, and g2/m) was quantified flowcytometrically using bd facs verse. rt-pcr analysis was done using primers of bax, bcl2, cytochrome-c, caspase-3 and gapdh. the cells were treated with ic50 dose of both drugs for 18 hours in the case of bax-bcl2, 24 hours for cytochrome-c, and 48 hours for caspase-3 and poly (adp-ribose) polymerase (parp). the protein activities were measured by using indirect- enzyme linked immunosorbent assay (elisa) and western blot analyses. quantifications of developed proteins after the western blot analyses were done densitometrically by using image j software. localization of caspase-3 was done by using an immunofluorescence study and was photographed under fluorescence microscope. the observers were blinded during observation as to whether they were observing the control and/or drug-treated materials. data were analyzed, and the signicance of differences between the mean values was determined by using a one-way analysis of variance (anova) with fisher s least significant difference (lsd) post hoc tests by using spss 14-software (spss inc, chicago, il, usa). ic50 values for 48 hours treatment with condurango 6c (3.57l/100l) and condurango 30c (2.43l/100l) against h460 were selected for the entire study (fig 1) the cell viability of both placebo-treated cells at maximum dose (5l/100l) was found to be very close to that of the untreated (control) ones. cells were exposed to condurango 6c and 30c for 24 hours and 48 hours (0.5l/100l-5l/100l dose) with respective placebos (5l/100l). results are expressed as mean percent of cell viability standard deviation (sd, n=6). signicance levels are presented as,*p<0.05 vs. untreated cells andp<0.05 vs. both placebo-treated cells. the light microscopy (fig. 3) studies revealed that cell morphologies remained unaltered with intact cell membrane and cellular extensions in both untreated and placebo-treated cells. however, condurango 6c-treated cells showed gradual deformation with cellular shrinkage; condurango 30c-treated cells became gradually smaller and rounded with distorted nuclei and damaged cellular extensions, depicting the great apoptosis-inducing potential of condurango 30c. the fluorescence intensity in optical density (od) was too low in untreated and placebo-treated cells at different hour intervals. a significant increase in the fluorescence intensity was observed at 18 hours in both condurango 6c and 30c-treated cells (fig. fluorescence microscopy showed a greater h2 dcfda-intensity in drug-treated cells at 18 h (fig. signicance levels are presented as*p<0.05 and***p<0.001 between untreated and condurango 6c and 30c-treated cells and between untreated and placebo-treated cells. results showed up-regulation and downregulation of bax and bcl2, respectively, at 18 hours in response to both drug treatments as compared to drug-untreated cells (fig. condurango 30c-treated sample showed significantly increased bax and decreased bcl2-expressions than condurango 6c and placebo-treated samples. signicance*p<0.05 untreated (ut) vs condurango 6c and 30c, p<0.05 placebo (6c) vs condurango 6c andp<0.05 placebo (30c) vs condurango 30c. figure 6a shows bright green fluorescence in drug-un-treated and cells at different time-points while condurango 6c and 30c-treated cells showed a gradual decrease in the fluorescence intensity with increasing time, but a marked decrease occurred at 24 hours, suggesting that mmp depolarizes the maximum at 24h that trigger cytochrome- c release from mitochondria. an increased expression of cytochrome-c was found in response to drugs at 24 hour of treatment (fig. condurango 30c-treated cells showed more significant upregulation of cytochrome-c than condurango 6c-treated ones. signicance levels are presented as*p<0.05 untreated (ut) vs condurango 6c and 30c, p<0.05 placebo (6c) vs condurango 6c andp<0.05 placebo (30c) vs condurango 30c. a gradual increase in the dapi-fluorescence intensity was observed in condurango 6c and 30c-treated cells at 48 hours of treatment (fig. further, ao/eb- staining showed both a change in the fluorescence pattern from green (normal-cellular dna) to orange (nicked-cellular dna) and an increase in the fluorescence of eb in drug-treated cells (fig. figure8c shows the formation of dna-laddering in both drug-treated samples, especially in condurango 30c-treated samples, compared to the dna in the drug-untreated groups. a gradual increase in the number of dutp-nicks was noted in drug-treated cells compared to drug-untreated cells (fig. this strongly suggests the abilities of both drugs to generate dna-nicks that induce apoptosis. quantitative evaluation revealed the percent of tunel-positive nuclei was greater in condurango 30c-treated cells. the sub-diploid cell population at subg0/g1 was more highly increased in the condurango 6c and 30c-treated cells than in the placebo-treated and untreated cells (fig. rt-pcr and elisa data showed upregulation of caspase-3 after condurango 6c and 30c treatment as compared to untreated samples (fig. the western blot analysis revealed that condurango 30c had more capacity to increase caspase-3 expression significantly by forming cleaved fragments (20kda and 12kda). p<0.05 untreated (ut) vs condurango 6c and 30c, p<0.05 placebo (6c) vs condurango 6c andp<0.05 placebo (30c) vs condurango 30c. the immunofluorescence study demonstrated a gradual increase in the localization of caspase-3 within cell-cytosol in drug-treated cells, especially in condurango 30c-treated cells (fig. parp activation, the end process of the caspase-3-mediated pathway is designated by the formation of two cleaved fragments of 116kda (inactive) and 89kda (active). the western blot analysis showed the formation of two fragments in condurango 6c and 30c-treated cells at 48 h of treatment, which was nearly absent in drug-untreated samples (fig. band-intensities confirmed gradual down-regulation of the 116kda fragment and significant up-regulation of the 89kda fragment, especially in the condurango 30c-treated cells. present findings would demonstrate that exposure of nsclc- h460 cells to ic50 doses of both condurango 6c and 30c for 48 hours resulted in apoptotic cell-death. further, the effects of condurango 30c, which actually was highly- diluted, caused relatively more palpable alterations in all parameters of this study than did condurango 6c. this phenomenon did not apparently follow the general pharmacological rule that the effect of a drug increases linearly with its concentration, but was in line with the claim of the higher the dilution, the stronger the effect as per homeopathic doctrine. incidentally, apparent evidence for discernible effects produced by two dilutions, one below and one above avogadro s limit an enigma to many rationalists who believe in the accepted laws of physical sciences, as well as pharmacological sciences. however, claims are accumulating that homeopathic potencies beyond avogadro s limit show demonstrable beneficial/ curative effects against different diseases including cancer [20-22]. in fact, our earlier in vivo findings on benzo[a] pyrene-induced lung cancer in rats also convincingly demonstrated the ameliorative effect of condurango 30c, for which we tried to verify that the potentized remedy showed anti-cancer potential in nsclc cells. apoptosis induction in cancerous cells is often targeted as one of the key events of cancer chemotherapy. thus, one approach was to look at the generation of ros and to ascertain if it had a specific role in the induction of apoptosis. the time-course studies showed elevation of ros, mmp-depolarization and cytochrome-c release at 18 hours through 24 hours of treatment, indicating that these events were earlier than apoptotic execution at 48 hours. our result for cytochrome-c release at 24 hours of treatment further suggested that these events occurred due to an alteration in the mitochondrial structure that could trigger apoptosis. along with mitochondrial dysfunctions, ros generation may be initiated at early hours of drug exposure via bax- bcl2 modulation. in this study, we found increased and decreased expression of bax and bcl2, respectively, at 18 hours in the drug-treated series. however, surprisingly bax-bcl2 expressions were more significant in the condurango 30c-treated samples, which is a good indication of the higher efficacy of a more highly diluted homeopathic drug. another indication of apoptosis is internucleosomal dna breakdown. results of dapi and ao/eb-staining would suggest the formation of dna nicks, as further confirmed by dna-laddering in drug-treated cells. tunel-positivity in both drug-treated cells as compared to placebo- treated cells would further strengthen ros-dependent dna-breakage-mediated apoptotic events. interestingly quantitatively more dna damage was found in condurango 30c-treated cells, than was found in condurango 6c. increased ratio of bax-bcl2 is well known to stimulate the release of cytochrome-c from mitochondria to promote activation of caspase-9 that binds to apaf-1 to lead to caspase-3 and parp-activation. results provide clear indication of caspase-3 activation in drug-treated cells. increased expression and formation of cleavage of caspase-3 and parp after drug exposure confirmed apoptosis induction via caspase-3-mediated- intrinsic pathway. however, noticeably, condurango 30c-treated cell showed more significant modulation of caspase-3 and parp cleavage than did condurango 6c-treated cells. these strongly support again the idea that more potentized condurango 30c had greater ability to induce apoptosis in nsclc-h460. if the similar results are taken into consideration, one possible conclusion is that homeopathic drugs may act by interacting with certain high affinity receptors that regulate expressions of specific genes, but more work is necessary to identify some missing links. the present study has gain significance because it attempts to determine if potentized forms of condurango, below (6c) and above (30c) avogadro s limit, which are occasionally used to treat digestive problems and stomach cancer [22, 24], had abilities to inhibit lung cancer progression, in vitro. the various changes noted in this study can only be brought about by the activities of certain genes and by epigenetic modifications, which would support the hypothesis [3, 23] that ultra-highly-diluted drugs might somehow manage to correct expressions of relevant genes, the regulation of which had failed in cancer cells. interestingly, many factors, including the presence of nanoparticles [5, 25], interaction between containers (e.g., silica from glass or a polymer) and drug molecules, etc., are now under scrutiny and are implicated in modifications of the structural orientation, the size, and the physico-chemical properties of resultant homeopathic drugs even in absence of any original molecule [6, 26]. the overall results of this study suggest that condurango 30c has more apoptosis-inducing ability than condurango 6c, which is consistent with the claim made in the homeopathic doctrine.

objectives: in homeopathy, it is claimed that more homeopathically-diluted potencies render more protective/curative effects against any disease condition. potentized forms of condurango are used successfully to treat digestive problems, as well as esophageal and stomach cancers. however, the comparative efficacies of condurango 6c and 30c, one diluted below and one above avogadro s limit (lacking original drug molecule), respectively, have not been critically analyzed for their cell-killing (apoptosis) efficacy against lung cancer cells in vitro, and signalling cascades have not been studied. hence, the present study was undertaken. methods:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (mtt) assays were conducted on h460-non-small-cell lung cancer (nsclc) cells by using a succussed ethyl alcohol vehicle (placebo) as a control. studies on cellular morphology, cell cycle regulation, generation of reactive oxygen species (ros), changes in mitochondrial membrane potential (mmp), and dna-damage were made, and expressions of related signaling markers were studied. the observations were done in a blinded manner. results:both condurango 6c and 30c induced apoptosis via cell cycle arrest at subg0/g1 and altered expressions of certain apoptotic markers significantly in h460 cells. the drugs induced oxidative stress through ros elevation and mmp depolarization at 18-24 hours. these events presumably activated a caspase-3-mediated signalling cascade, as evidenced by reverse transcriptase- polymerase chain reaction (rt-pcr), western blot and immunofluorescence studies at a late phase (48 hours) in which cells were pushed towards apoptosis. conclusion:condurango 30c had greater apoptotic effect than condurango 6c as claimed in the homeopathic doctrine.

PMC4331988

pubmed-1300

gestational diabetes mellitus (gdm) is defined as abnormal glucose tolerance that is first identified or diagnosed during pregnancy. it is estimated that approximately 2% to 5% of all pregnancies in korean women are complicated by gdm. the clinical significance of gdm is that it increases the risk of adverse pregnancy outcomes. in a recent large-scale multinational prospective study, increased maternal glucose concentration during pregnancy was significantly associated with increased neonatal birth weight, primary cesarean delivery, neonatal hypoglycemia, and increased placental c-peptide levels. after parturition, about 15% of gdm women had persistent diabetes at an early postpartum period of 2 months. women with a previous history of gdm are at increased risk of future development of type 2 diabetes mellitus (t2 dm). the risk of t2 dm is 3.5 times greater in women with a history of gdm compared to the general population in koreans. in addition, offspring of gdm women are also at risk of developing obesity and t2 dm. the incidence of gdm is expected to rise, as it will parallel the increasing rate of obesity and t2 dm. furthermore, the recent recommendation from the international association of diabetes and pregnancy study group has lowered the diagnostic threshold of gdm and is expected to increase the incidence of gdm. gdm women have an increased positive family history of t2 dm. compared to pregnant women with normal glucose tolerance, gdm women have a significantly greater parental history of t2 dm (13.2% vs. 30.1%, p<0.001). in addition, both gdm women and their offspring are at increased risk of future development of t2 dm. the heritability estimate of t2 dm was reported to be quite high (h=0.69) in a recent study performed in europeans. these findings are indirect evidence that gdm has familial tendency. however, there is no study that has specifically evaluated the heritability of gdm using familial clustering or twins. it would be important to estimate the heritability of gdm and compare it with that of t2 dm in women. during normal pregnancy, women experience increased adiposity and weight gain, which begin near mid-pregnancy and progress throughout the third trimester. in this period, insulin resistance ensues as a consequence of multiple factors, including increased production of placental growth hormone, estrogen, and tumor necrosis factor [14, 15]. pregnant women with normal glucose tolerance can increase their -cell insulin secretion in response to this increased insulin resistance during pregnancy. although the mechanism of increased -cell insulin secretion during pregnancy is not fully understood, it is reported that prolactin, which increases during pregnancy, can repress islet menin levels and stimulate -cell proliferation in mice. in addition, recent reports suggest that -cell serotonin signaling is also a major determinant of -cell mass during pregnancy. it has been suggested from several clinical studies that gdm women have limited insulin secretion capacity that can not compensate for the increased insulin resistance. similar to gdm, t2 dm is also characterized by relative deficiency in insulin secretion in the face of increased insulin resistance. various factors, including increased age, obesity, high-fat diet, and sedentary lifestyle, can induce insulin resistance, and those who do not have sufficient -cell insulin secretory capacity are more likely to develop t2 dm. therefore, it is said that a large proportion of gdm women are experiencing future t2 dm in advance. based on the findings that gdm women are at high risk of t2 dm and both gdm and t2 dm share similar pathophysiologies, it is reasonable to assume that they might also share similar genetic risk factors. soon after the initial reports of t2 dm genome-wide association (gwa) studies [18-21], several studies investigated whether genetic variants that were identified through gwa studies of t2 dm were also associated with the risk of gdm [22, 23]. genotyped variants in or near cdkal1, cdkn2a/2b, fto, hhex, igf2bp2, slc30a8, tcf7l2, kcnj11, and pparg in 869 gdm women and 632 carefully selected nondiabetic control subjects. they found that genetic variants in cdkal1 and cdkn2a/2b were highly associated with the risk of gdm (p<1 10). in addition, variants in hhex, igf2bp2, slc30a8, and tcf7l2 were all nominally associated with gdm (p<0.05). among a total of 18 genetic variants studied, 9 reached a nominal significance level (p<0.05). in fig. 1, the risk allele frequency as well as the odds ratios of known t2 dm variants are compared among a control group (n=632, men: women=287: 345), t2 dm group (n=761, men: women=354: 407), and gdm group (n=869) in koreans [22, 24]. for most of the variants, there was an increasing trend of risk allele frequencies from control to t2 dm and from t2 dm to gdm.. also found that variants in tcf7l2, cdkal1, and tcf2 were significantly associated with the risk of gdm in europeans, consisting of 283 gdm women and 2,446 glucose-tolerant control women. variants in kcnq1, which were first identified in an east asian t2 dm gwa study, were also significantly associated with the risk of gdm in koreans (p<0.05). recently, it was reported that tph1 and htr2b play a crucial role in regulating pancreatic -cell mass during pregnancy in a mouse model and that their genetic alterations could result in gdm. a total 6 genetic variants in htr2b and 11 variants in tph1 were identified and genotyped in korean gdm women and control subjects. although there were no significant associations of these variants with the risk of gdm, they were associated with measures of obesity and weight gain during pregnancy. following these candidate approach studies was a two-staged gwa study that was performed in korean gdm women. a total of 468 gdm women and 1,242 nondiabetic control women were genotyped using affymetrix genome-wide human snp array 5.0. variants that passed the prespecified p-value threshold in the stage 1 genome scan were further genotyped in 931 gdm women and 783 nondiabetic control women. two variants, one located in an intron of cdkal1 (rs7754840) and one near mtnr1b (rs10830962), were associated with a risk of gdm at a genome-wide significance level (p=6.65 10 and p=2.49 10, respectively). although variants in mtnr1b have been previously reported to be associated with increased fasting glucose levels, the study was the first to report that mtnr1b variants are associated with gdm at a genome-wide significance level. one of the limitations was that it was not sufficiently powered to find truly novel genetic variants that were only specific in gdm. it should be noted that current gwa and gwa meta-analysis performed in t2 dm recruits more than 100,000 cases and controls. one of the interesting findings was that genetic variants of t2 dm were enriched in gdm subjects. among the 34 confirmed t2 dm genetic variants, 8 were associated with a risk of gdm. in addition, when the -coefficients (or odds ratio) of the variants derived from the logistic regression were compared between gdm and t2 dm, there was a significant positive correlation of -coefficients between the two. these findings suggest that gdm and t2 dm might share similar genetic backgrounds, at least in part. gwa studies have opened a new era in diabetes research. our knowledge on the genetic predisposition of gdm as well as t2 dm is expected to increase even faster as next-generation sequencing technology is applied to this field. there should be even larger gwa studies on gdm, and gwa meta-analyses should be available. in this way, we could find variants that have smaller effect sizes but are more specific to gdm than t2 dm. in order to understand the genetic determinants of glucose and insulin concentration during pregnancy, a fast way is to see whether genetic variants that are known to affect glucose or insulin concentration in the normal population also affect glucose or insulin concentration during pregnancy. in addition, a gwa study on glucose or insulin concentration during pregnancy should also be helpful. in this way, we would be able to better understand the pathophysiology of gdm. by definition, gdm encompasses women with pre-exiting t2 dm, maturity onset diabetes of the young (mody), or even type 1 diabetes patients that were not diagnosed previously. in particular, about 15% of gdm patients remain diabetic at early postpartum periods, and a significant portion of these subjects might fall into the category of mody. the contribution of genetic variants known to cause mody, such as gck and hnf1a, in gdm has been reviewed in recent literature. however, whole-exome sequencing will provide us with better bird's eye view on the contribution of mody genes in gdm. in addition, it might be able to find novel mody genes in those who have persistent diabetes after gdm pregnancy and also have a strong family history of diabetes. one of the first steps in translating the genetic information into clinical practice would be to predict the future development of t2 dm in gdm women. gdm women are at particularly high risk of developing t2 dm and require preventive measures and early screening of t2 dm. genetic information might improve our prediction of t2 dm in women with a history of gdm. this is an area of active research [31, 32], and we are looking forward to studies that use genotype risk scores in predicting t2 dm in gdm women. a similar approach could also be applied in predicting gdm, as more genetic variants associated with gdm are expected to be revealed. the functional consequences of the current common genetic variants identified through gwa studies of gdm are not well understood yet. it is not known whether they are markers in linkage disequilibrium with nearby causal variants or whether they have unknown but relevant functional roles. next-generation sequencing might give answers to these questions, but a huge number of samples and much effort will be required. gdm women are at increased risk of developing t2 dm and have familial clustering of t2 dm. a common pathophysiology that is shared by gdm and t2 dm is impaired compensatory increase in insulin secretion to overcome the increased insulin resistance. the gwa study of gdm enabled us to investigate the common genetic risk factors of gdm, and it revealed that gdm and t2 dm share similar genetic backgrounds, at least in part (fig. although this information has significantly improved our insight in the pathogenesis of gdm, there are more unanswered questions remaining that should be explicitly expressed. using the technology of next-generation sequencing in addition, we hope that personalized genomic medicine could be available using the advances in the genetics of gdm and t2 dm.

gestational diabetes mellitus (gdm) is a complex metabolic disorder of pregnancy that is suspected to have a strong genetic predisposition. it is associated with poor perinatal outcome, and both gdm women and their offspring are at increased risk of future development of type 2 diabetes mellitus (t2 dm). during the past several years, there has been progress in finding the genetic risk factors of gdm in relation to t2 dm. some of the genetic variants that were proven to be significantly associated with t2 dm are also genetic risk factors of gdm. recently, a genome-wide association study of gdm was performed and reported that genetic variants in cdkal1 and mtnr1b were associated with gdm at a genome-wide significance level. current investigations using next-generation sequencing will improve our insight into the pathophysiology of gdm. it would be important to know whether genetic information revealed from these studies could improve our prediction of gdm and the future development of t2 dm. we hope further research on the genetics of gdm would ultimately lead us to personalized genomic medicine and improved patient care.

PMC3543924