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Hepatitis C virus (HCV), which affects between 2 and 4 million individuals in the United States and over 120 million worldwide, is a leading cause of cirrhosis and hepatocellular carcinoma and claims the lives of over 350,000 people each year. At present, no vaccine exists for HCV, and antiviral therapies are only effective in a fraction of cases. Hepatitis C infection occurs exclusively in humans and chimpanzees; rodent cells are completely protected against infection. This lack of a small-animal model that faithfully recapitulates the life cycle of hepatitis C is a major barrier to the development of improved treatments or even a vaccine to HCV. Now, Dorner et al. have identified a cocktail of four critical human molecules needed to establish HCV infection in the liver of mice. In a recent Nature article, they use recombinant adenoviral technology to deliver these four factors—CD81, occludin (OCLN), scavenger receptor type B Class I (SCARB1), and claudin (CLDN1)—to establish a humanized mouse model for hepatitis C viral infection. Infecting mice with HCV is hard and tedious work, and requires some clever tools to monitor infection. To this end, Dorner et al. first created a bicistronic HCV genome that expressed the genetic material of hepatitis C as well as CRE recombinase. Genetically altered but immunocompetent mice that synthesize nuclear green fluorescent protein in the presence of CRE recombinase could thus be used to monitor infection. After systemic administration of adenoviral vectors encoding the four human proteins described above, HCV-infected cells would fluoresce green. Using this approach, one in five mouse hepatocytes expressing CD81, OCLN, SCARB1, and CLDN1 were successfully infected with HCV. The beauty of this system lies in its potential ability to inform scientists about the biology of hepatitis C. More importantly, this small-animal model provides a platform to test vaccination and treatment strategies. As proof of principle, the group showed that passive immunization with antibodies to CD81 was able to decrease infection by 70%, whereas active immunization with recombinant vaccinia virus protected 20% of HCV-challenged animals. Hepatitis C has been described as both a silent and smoldering public health epidemic. It is silent no longer; Dorner and colleagues are using the first humanized mouse model of HCV infection to shine (fluorescent green) light on this important problem. M. Dorner et al ., A genetically humanized mouse model for hepatitis C virus infection. Nature 474 , 208–211 (2011). [[Abstract]][1] [1]: http://www.nature.com/nature/journal/v474/n7350/full/nature10168.html

Although obesity contributes to the development of type 2 diabetes, a key side effect of one class of antidiabetic drugs—the thiazolidinediones (TZDs)—is weight gain. TZDs are agonists of the nuclear receptor peroxisome proliferator–activated receptor–γ (PPAR-γ); they improve peripheral

Spermatogenesis in mammals is a lengthy and complicated process involving meiotic divisions of spermatogonial stem cells to form first spermatids and then mature sperm. Given this, it is not surprising that it has been very difficult to achieve successful spermatogenesis in a culture dish. Sato et

The previous discovery of circulating free fetal DNA in maternal blood during pregnancy has opened a door for the development of noninvasive prenatal genetic screening techniques. However, the amount of free fetal DNA that can be identified in a peripheral blood sample is miniscule (3 to 6% of circulating free DNA), and finding it among this excess of maternal DNA is akin to finding a needle in the proverbial haystack. A major limitation has been the ability to both enrich a peripheral blood sample for fetal DNA and accurately discriminate between maternal and fetal cells. Interestingly, fetal cells appear to have a unique epigenetic footprint—meaning, the DNA has specific methylation (–CH3) patterns—which could make discrimination easier between mother and fetus. Now, Papageorgiou and colleagues have used these methylation differences to reliably diagnose trisomy 21, the genetic basis of Down’s syndrome. The authors first identified a specific pattern of differentially methylated regions on the fetal chromosome 21 that predicted either a normal or trisomy 21 pregnancy. Using a technique termed methylated DNA precipitation (MeDiP), they next added an antibody to maternal blood samples that specifically recognizes methylated regions on a gene promoter and analyzed these differences using quantitative polymerase chain reaction (PCR). This approach provided the necessary one-two punch of discrimination and enrichment, leading to the correct identification of 14 Down’s syndrome cases and 26 normal cases in a blind sampling of 40 pregnant women. Noninvasive prenatal diagnostic techniques such as this might soon obviate the need for more invasive procedures, including amniocentesis (extraction of amniotic fluid) and sampling of chorionic villi. For many women, these traditional methods harbor an unacceptably high risk of miscarriage. Continued refinement is needed for large-scale application, however, and the method applied here might not be suitable to screen for every genetic disorder. For instance, in order for MeDiP to work, the fetal gene of interest must be hypermethylated in comparison to the maternal gene. Still, Papageorgiou et al. are moving forward to develop similar methods that can be used to detect aneuploidies of chromosomes 13, 18, X, and Y, thereby expanding prenatal diagnostic options for expectant moms. E. A. Papageorgiou et al ., Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21. Nat. Med . 6 March 2011 (10.1038/nm.2312). [[Abstract]][1] [1]: http://www.nature.com/nm/journal/vaop/ncurrent/abs/nm.2312.html

The odds of surviving a bleeding stroke [intracerebral hemorrhage (ICH)] are poor; the mortality rate approaches 60% one year after the event. With little in the way of effective therapy, those that do survive are often left neurologically impaired. Diabetes and hyperglycemia are independent risk

To compare prescription drug cost savings under the most commonly selected Medicare Part D prescription plan in 2006 with savings under the Medicare standard benefit and with drug costs assuming no coverage in an elderly cohort of patients.Inception cohort study.An academic medical center.Four hundred seventy-two patients aged 65 and older who were followed as part of a larger study assessing stroke prevention in patients with atrial fibrillation.Prescription drug expenditures were calculated for each patient in the cohort under three conditions: the 2006 AARP-endorsed prescription drug plan, the Medicare standard benefit, and no prescription drug coverage.Total prescriptions drug costs were lower under the AARP plan, yet patients paid a similar percentage of total costs under the AARP plan and the Medicare standard benefit. Using different cost assessments, 27% to 46% of patients entered the "doughnut hole" in the AARP plan, and 3% to 11% emerged to receive catastrophic coverage.Both the AARP-sponsored and standard Medicare Part D prescription drug benefit programs offer significant savings to enrollees. A greater savings is achieved under the private AARP drug insurance plan, largely due to greater discounts reflected in the negotiated drug prices. A substantial portion of enrollees enter but do not emerge from the coverage gap.

Background— Warfarin is effective in the prevention of stroke in atrial fibrillation but is under used in clinical care. Concerns exist that published rates of hemorrhage may not reflect real-world practice. Few patients ≥80 years of age were enrolled in trials, and studies of prevalent use largely reflect a warfarin-tolerant subset. We sought to define the tolerability of warfarin among an elderly inception cohort with atrial fibrillation. Methods and Results— Consecutive patients who started warfarin were identified from January 2001 to June 2003 and followed for 1 year. Patients had to be ≥65 years of age, have established care at the study institution, and have their warfarin managed on-site. Outcomes included major hemorrhage, time to termination of warfarin, and reason for discontinuation. Of 472 patients, 32% were ≥80 years of age, and 91% had ≥1 stroke risk factor. The cumulative incidence of major hemorrhage for patients ≥80 years of age was 13.1 per 100 person-years and 4.7 for those P =0.009). The first 90 days of warfarin, age ≥80 years, and international normalized ratio (INR) ≥4.0 were associated with increased risk despite trial-level anticoagulation control. Within the first year, 26% of patients ≥80 years of age stopped taking warfarin. Perceived safety issues accounted for 81% of them. Rates of major hemorrhage and warfarin termination were highest among patients with CHADS 2 scores (an acronym for congestive heart failure, hypertension, age ≥75, diabetes mellitus, and prior stroke or transient ischemic attack) of ≥3. Conclusions— Rates of hemorrhage derived from younger noninception cohorts underestimate the bleeding that occurs in practice. This finding coupled with the short-term tolerability of warfarin likely contributes to its underutilization. Stroke prevention among elderly patients with atrial fibrillation remains a challenging and pressing health concern.

The sarcoendoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl l-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-d,l-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca2+ imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca2+ and decreased ER Ca2+ levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b protein stability is decreased under inflammatory conditions through NO- and AMPK-dependent pathways and provide novel insight into pathways leading to altered β-cell calcium homeostasis and reduced β-cell survival in diabetes.

Heat shock protein 90 (HSP90) is a molecular chaperone that regulates diverse cellular processes by facilitating the activities of a wide variety of protein clients. Recent collaborative studies from our lab have shown that serum HSP90 levels are elevated in newly diagnosed type I diabetic patients, thus distinguishing HSP90 as a potential biomarker for this autoimmune disease. The clinical onset of type 1 diabetes (T1D) is known to be associated with insulitis, the infiltration of inflammatory cells into the pancreatic islets. Therefore, to test the hypothesis that inflammatory stress results in HSP90 release from pancreatic islets, we treated primary human cadaveric islets from non-diabetic donors with a cocktail of inflammatory cytokines IFN-γ, TNF-α, and IL-1β and found that increased levels of HSP90 are secreted by cytokine-treated islets. Flow cytometry of dispersed pancreatic islets suggests beta cells may be responsible for the release of this chaperone in response to cytokine treatment. Further studies with cytokine-treated human beta cell lines βLOX5 and 1.1B4 support this finding. In contrast, beta cell lines do not secrete HSP90 upon treatment with thapsigargin, suggesting HSP90 is not released in response to beta cell endoplasmic reticulum stress, which is also known to precede the onset on T1D. Studies are underway to determine the exact mechanism by which HSP90 is released in response to cytokine stress as well as to elucidate the role of HSP90 in T1D etiology.

Background and Purpose— Numerous studies have documented under use of warfarin particularly among elderly patients. A better understanding of the discrepancy between trials and clinical practice will help inform stroke prevention strategies in this vulnerable age group. The study objective was to prospectively assess the use of antithrombotic therapy among a contemporary cohort of patients with atrial fibrillation at the time of hospital discharge. In addition to baseline characteristics, we sought to define the physician-cited reason for not prescribing warfarin for each patient. Methods— Patients with atrial fibrillation were prospectively identified and followed to hospital discharge. Enrolled patients were ≥65 years of age, not taking warfarin on admission, and had their longitudinal care provided at our institution. Predictors of warfarin use were determined and physician-cited contraindications were compared across age groups. Results— Fifty-one percent (n=206) of patients were discharged on warfarin: 75% of those 65 to 69 years of age, 59% 70 to 79, 45% 80 to 89, and 24% age ≥90 years. Of the remaining 199 patients, 83% had ≥2 major risk factors for stroke, and 98% were felt to have contraindications including nearly 25% who were unable to tolerate warfarin in the past. Among patients age ≥80, falling was the most often physician-cited reason for not prescribing warfarin (41%) followed by hemorrhage (28%). Conclusion— Our findings suggest that many elderly patients at high risk for stroke may not be optimal candidates for anticoagulant therapy. There is a pressing need for alternative stroke prevention strategies for this expanding patient population.

Glucose homeostasis reflects the integrated efficiencies of the islet β-cell to secrete insulin and of metabolically active tissues to transduce insulin signals. Conceptually, defects in either insulin secretion or insulin signaling can lead to glucose intolerance or frank diabetes. In the case of type 2 diabetes (T2D), the primary etiology of the disease is not only of academic significance but has important implications for the design of therapeutics. In recent years, there has been increasing evidence from rodent and human studies pointing to a primary defect in β-cell function in T2D. The results of genome-wide association studies provide genetic credence to the notion that defects in insulin secretion may be primarily causative of disease (1). Insulin secretion by the β-cell occurs in two distinct phases after an intravenous glucose load, with an acute phase occurring within minutes and a chronic phase persisting through the end of the glucose challenge. Among the earliest defects observed in humans and mice with T2D or at risk for T2D is the predominant loss of the acute phase of insulin secretion (2,3), and restoration of the acute phase can improve glycemic control in T2D subjects (4). At present, only two major classes of drugs used clinically in T2D are capable of enhancing insulin release: sulfonylureas and incretins. Sulfonylureas cause closure of ATP-sensitive K+ channels, resulting in β-cell depolarization and calcium influx from voltage-dependent calcium channels, and leads to insulin granule exocytosis. The obvious advantage of sulfonylureas in T2D is enhanced insulin secretion, but a prominent drawback is that secretion occurs …

Type 1 diabetes (T1D) is characterized by chronic systemic inflammation that could be involved in the enhanced susceptibility to sepsis. The IL-1b/IL-1bR axis drives inflammation in a MyD88-dependent manner, but the molecular mechanisms involved in IL-1b actions in macrophages during T1D are unknown. We hypothesized that leukotriene (LT) B4 promotes IL-1b responsiveness in T1D by enhancing MyD88 expression. Peritoneal macrophages (PMs) from T1D mice expressed higher levels of MyD88 and its transcription factor STAT-1 than PMs from nondiabetic mice. 5-LO expression, LTB4 synthesis and IL-1b levels were also increased in both PM and serum from T1D mice. In vitro and in vivo insulin treatment restored LTB4 and STAT-1/MyD88 levels. Genetic and pharmacologic inhibition of the LTB4/BLT1 axis abolished MyD88 and STAT-1 induction in diabetic mice. ChIP assay showed that AP-1/cJun drove basal STAT-1 levels in an LTB4-dependent manner in T1D. We speculated that high LTB4 levels contribute to enhanced susceptibility to sepsis in T1D. Compared to WT or untreated T1D mice, genetic and pharmacologic inhibition of 5-LO prolonged T1D mice survival after polymicrobial sepsis; associated with decreased blood bacteria and reduced production of proinflammatory cytokines. We conclude that LTB4, by increasing MyD88 and IL-1b levels in a c-Jun/STAT-1 dependent manner, is the driving force involved in inflammation associated with T1D that contributes to high mortality associated to sepsis T1D.

Perturbations in endoplasmic reticulum (ER) homeostasis can evoke stress responses leading to aberrant glucose and lipid metabolism. ER dysfunction is linked to inflammatory disorders, but its role in the pathogenesis of autoimmune type 1 diabetes (T1D) remains unknown. We identified defects in the expression of unfolded protein response (UPR) mediators ATF6 (activating transcription factor 6) and XBP1 (X-box binding protein 1) in β cells from two different T1D mouse models and then demonstrated similar defects in pancreatic β cells from T1D patients. Administration of a chemical ER stress mitigator, tauroursodeoxycholic acid (TUDCA), at the prediabetic stage resulted in a marked reduction of diabetes incidence in the T1D mouse models. This reduction was accompanied by (i) a significant decrease in aggressive lymphocytic infiltration in the pancreas, (ii) improved survival and morphology of β cells, (iii) reduced β cell apoptosis, (iv) preserved insulin secretion, and (v) restored expression of UPR mediators. TUDCA′s actions were dependent on ATF6 and were lost in mice with β cell-specific deletion of ATF6. These data indicate that proper maintenance of the UPR is essential for the preservation of β cells and that defects in this process can be chemically restored for preventive or therapeutic interventions in T1D.

Response: We appreciate the thoughtful comments of Testa et al, particularly regarding the pivotal issue of stroke prevention among elderly individuals with atrial fibrillation. In our study, patients with planned cardioversion were treated with warfarin consistent with current guidelines.1 Antiplatelet agents would not be used in this setting. Based on risk factors, the patients in our study were predominantly classified as high risk for stroke. Aspirin in this group would not be considered a surrogate for warfarin. For each patient who was not discharged on warfarin, we ascertained the reason directly either from the discharge record or the treating physician. Among patients considered to have …

Recent work on the pathogenesis of type 1 diabetes has led to an evolving recognition of the heterogeneity of this disease, both with regards to clinical phenotype and responses to therapies to prevent or revert diabetes. This heterogeneity not only limits efforts to accurately predict clinical disease but also is reflected in differing responses to immunomodulatory therapeutics. Thus, there is a need for robust biomarkers of beta cell health, which could provide insight into pathophysiological differences in disease course, improve disease prediction, increase the understanding of therapeutic responses to immunomodulatory interventions and identify individuals most likely to benefit from these therapies. In this review, we outline current literature, limitations and future directions for promising circulating markers of beta cell stress and death in type 1 diabetes, including markers indicating abnormal prohormone processing, circulating RNAs and circulating DNAs.

Context Clinical onset of type 1 diabetes (Stage 3 T1D) is preceded by a presymptomatic phase characterized by multiple islet autoantibodies with normal glucose tolerance (Stage 1 T1D). Objective The aim was to explore the metabolic phenotypes of β-cell function and insulin sensitivity and clearance in normoglycemic youth with Stage 1 T1D and compare them with healthy nonrelated peers during a 3-hour oral glucose tolerance test (OGTT). Methods Twenty-eight lean youth, 14 with ≥2 islet autoantibodies (cases) and 14 healthy controls underwent a 3-hour 9-point OGTT with measurement of glucose, C-peptide, and insulin. The oral minimal model was used to quantitate β-cell responsiveness (φtotal) and insulin sensitivity (SI), allowing assessment of β-cell function by the disposition index (DI=φtotal×SI). Fasting insulin clearance (CL0) was calculated as the ratio between the fasting insulin secretion rate (ISR) and plasma insulin levels (ISR0/I0), while postload clearance (CL180) was estimated by the ratio of AUC of ISR over the plasma insulin AUC for the 3-hour OGTT (ISRAUC/IAUC). Participants with impaired fasting glucose, impaired glucose tolerance, or any OGTT glucose concentration ≥200 mg/dL were excluded. Results Cases (10.5 years [8, 15]) exhibited reduced DI (P < .001) due to a simultaneous reduction in both φtotal (P < 0.001) and SI (P = .008) compared with controls (11.5 years [10.4, 14.9]). CL0 and CL180 were lower in cases than in controls (P = .005 and P = .019). Conclusion Presymptomatic Stage 1 T1D in youth is associated with reduced insulin sensitivity and lower β-cell responsiveness, and the presence of blunted insulin clearance.

Aim Type 1 diabetes (T1D) is a chronic autoimmune disease leading to progressive loss of pancreatic beta cells. Interferon (IFN)-α plays a critical role in the crosstalk between pancreatic beta cells and the immune system in early insulitis. In human beta cells IFNα signals through JAK1 and TYK2, leading to endoplasmic reticulum stress, inflammation and HLA class I overexpression. IFNα, acting synergistically with IL-1β, induces apoptosis. Polymorphisms in TYK2 that decrease its activity are associated with protection against T1D, and we hypothesized that pharmacological inhibitors that specifically target TYK2 could protect human beta cells against the deleterious effects of IFNα. Materials and methods Two TYK2 inhibitors provided by Nimbus Lakshmi were tested in human insulin-producing EndoC-βH1 cells and human islets to evaluate their effect on IFNα signalling, beta-cell function and susceptibility to viral infection using RT-qPCR, western blot, immunofluorescence, ELISA and nuclear dyes. Results The two TYK2 inhibitors tested prevented IFNα-induced human beta-cell gene expression in a dose-dependent manner. They also protected human islets against IFNα + IL-1β-induced apoptosis. Importantly, these inhibitors did not modify beta-cell function or their survival following infection with the potential diabetogenic coxsackieviruses CVB1 and CVB5. Conclusions The two TYK2 inhibitors tested inhibit the IFNα signalling pathway in human beta cells, decreasing its pro-inflammatory and pro-apoptotic effects without sensitizing the cells to viral infection. The preclinical findings could pave the way for future clinical trials with TYK2 inhibitors for the prevention and treatment of type 1 diabetes.