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Over the last two decades, increased availability of human pancreatic tissues has allowed for major expansions in our understanding of islet biology in health and disease. Indeed, studies of fixed and frozen pancreatic tissues, as well as efforts using viable isolated islets obtained from organ donors, have provided significant insights toward our understanding of diabetes. However, the procedures associated with islet isolation result in distressed cells that have been removed from any surrounding influence. The pancreas tissue slice technology was developed as an in situ approach to overcome certain limitations associated with studies on isolated islets or fixed tissue. In this Perspective, we discuss the value of this novel platform and review how pancreas tissue slices, within a short time, have been integrated in numerous studies of rodent and human islet research. We show that pancreas tissue slices allow for investigations in a less perturbed organ tissue environment, ranging from cellular processes, over peri-islet modulations, to tissue interactions. Finally, we discuss the considerations and limitations of this technology in its future applications. We believe the pancreas tissue slices will help bridge the gap between studies on isolated islets and cells to the systemic conditions by providing new insight into physiological and pathophysiological processes at the organ level. Article Highlights: Human pancreas tissue slices represent a novel platform to study human islet biology in close to physiological conditions. Complementary to established technologies, such as isolated islets, single cells, and histological sections, pancreas tissue slices help bridge our understanding of islet physiology and pathophysiology from single cell to intact organ. Diverse sources of viable human pancreas tissue, each with distinct characteristics to be considered, are available to use in tissue slices for the study of diabetes pathogenesis. [ABSTRACT FROM AUTHOR]

Proinsulin is an abundant protein that is selectively expressed by pancreatic beta cells and has been a focus for development of antigen-specific immunotherapies for type 1 diabetes (T1D). In this study, we sought to comprehensively evaluate reactivity to preproinsulin by CD4 T cells originally isolated from pancreatic islets of organ donors having T1D. We analyzed 187 T cell receptor (TCR) clonotypes expressed by CD4 T cells obtained from six T1D donors and determined their response to 99 truncated preproinsulin peptide pools, in the presence of autologous B cells. We identified 14 TCR clonotypes from four out of the six donors that responded to preproinsulin peptides. Epitopes were found across all of proinsulin (insulin B-chain, C-peptide, and A-chain) including four hot spot regions containing peptides commonly targeted by TCR clonotypes derived from multiple T1D donors. Of importance, these hot spots overlap with peptide regions to which CD4 T cell responses have previously been detected in the peripheral blood of T1D patients. The 14 TCR clonotypes recognized proinsulin peptides presented by various HLA class II molecules, but there was a trend for dominant restriction with HLA-DQ, especially T1D risk alleles DQ8, DQ2, and DQ8-trans. The characteristics of the tri-molecular complex including proinsulin peptide, HLA-DQ molecule, and TCR derived from CD4 T cells in islets, provides an essential basis for developing antigen-specific biomarkers as well as immunotherapies. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is characterized by insulin deficiency resulting from the selective destruction of pancreatic β-cells by self-reactive T cells. Recent evidence demonstrates that innate immune responses substantially contribute to the pathogenesis of T1D, as they represent a first line of response to danger/damage signals. Here we discuss evidence on how, in a relapsing-remitting pattern, pancreas remodeling, diet, microbiota, gut permeability, and viral/bacterial infections induce the accumulation of leukocytes of the innate arm of the immune system throughout the pancreas. The subsequent acquisition and presentation of endocrine and exocrine antigens to the adaptive arm of the immune system results in a chronic progression of pancreatic damage. This process provides for the generation of self-reactive T-cell responses; however, the relative weight that genetic and environmental factors have on the etiopathogenesis of T1D is endotype imprinted and patient specific. With this Perspectives in Diabetes, our goal is to encourage the scientific community to rethink mechanisms underlying T1D pathogenesis and to consider therapeutic approaches that focus on these processes in intervention trials within new-onset disease as well as in efforts seeking the disorder's prevention in individuals at high risk. [ABSTRACT FROM AUTHOR]

Type 1 diabetes risk can reliably be predicted by markers of autoimmunity, but approaches to prevent or modify the underlying disease process are needed. We posit this void fundamentally results from a limited understanding of immune-islet cell interactions within the pancreas and relevant immune organs, contributions of β-cells to their own demise, and epigenetic predispositions affecting both immune and islet cells. Because biopsy of the human pancreas and pancreatic lymph nodes carries risk and the pancreas begins to autodigest soon after death, detailed cellular and molecular phenotyping of the human type 1 diabetes pancreas is lacking, limiting our understanding of the mechanisms of β-cell loss. To address these challenges, the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases established the Human Pancreas Analysis Program (HPAP) to procure human type 1 diabetes pancreata for an extensive array of tissue-based, cellular, and epigenetic assays aimed at critical knowledge gaps in our understanding of the local immune attack and loss of β-cells. In this Methodology Review, we describe how HPAP is performing detailed islet and immune cell phenotyping and creating publicly available data sets with the goals of an improved understanding of type 1 diabetes and the development of more effective treatments to prevent or reverse the disease. [ABSTRACT FROM AUTHOR]

Objective: We tested the ability of a type 1 diabetes (T1D) genetic risk score (GRS) to predict progression of islet autoimmunity and T1D in at-risk individuals.Research Design and Methods: We studied the 1,244 TrialNet Pathway to Prevention study participants (T1D patients' relatives without diabetes and with one or more positive autoantibodies) who were genotyped with Illumina ImmunoChip (median [range] age at initial autoantibody determination 11.1 years [1.2-51.8], 48% male, 80.5% non-Hispanic white, median follow-up 5.4 years). Of 291 participants with a single positive autoantibody at screening, 157 converted to multiple autoantibody positivity and 55 developed diabetes. Of 953 participants with multiple positive autoantibodies at screening, 419 developed diabetes. We calculated the T1D GRS from 30 T1D-associated single nucleotide polymorphisms. We used multivariable Cox regression models, time-dependent receiver operating characteristic curves, and area under the curve (AUC) measures to evaluate prognostic utility of T1D GRS, age, sex, Diabetes Prevention Trial-Type 1 (DPT-1) Risk Score, positive autoantibody number or type, HLA DR3/DR4-DQ8 status, and race/ethnicity. We used recursive partitioning analyses to identify cut points in continuous variables.Results: Higher T1D GRS significantly increased the rate of progression to T1D adjusting for DPT-1 Risk Score, age, number of positive autoantibodies, sex, and ethnicity (hazard ratio [HR] 1.29 for a 0.05 increase, 95% CI 1.06-1.6; P = 0.011). Progression to T1D was best predicted by a combined model with GRS, number of positive autoantibodies, DPT-1 Risk Score, and age (7-year time-integrated AUC = 0.79, 5-year AUC = 0.73). Higher GRS was significantly associated with increased progression rate from single to multiple positive autoantibodies after adjusting for age, autoantibody type, ethnicity, and sex (HR 2.27 for GRS >0.295, 95% CI 1.47-3.51; P = 0.0002).Conclusions: The T1D GRS independently predicts progression to T1D and improves prediction along T1D stages in autoantibody-positive relatives. [ABSTRACT FROM AUTHOR]

Although B cells reactive with islet autoantigens are silenced by tolerance mechanisms in healthy individuals, they can become activated and contribute to the development of type 1 diabetes. We previously demonstrated that high-affinity insulin-binding B cells (IBCs) occur exclusively in the anergic (BND) compartment in peripheral blood of healthy subjects. Consistent with their activation early in disease development, high-affinity IBCs are absent from the BND compartment of some first-degree relatives (FDRs) as well as all patients with autoantibody-positive prediabetes and new-onset type 1 diabetes, a time when they are found in pancreatic islets. Loss of BND IBCs is associated with a loss of the entire BND B-cell compartment consistent with provocation by an environmental trigger or predisposing genetic factors. To investigate potential mechanisms operative in subversion of B-cell tolerance, we explored associations between HLA and non-HLA type 1 diabetes-associated risk allele genotypes and loss of BNDs in FDRs. We found that high-risk HLA alleles and a subset of non-HLA risk alleles (i.e., PTPN2 [rs1893217], INS [rs689], and IKZF3 [rs2872507]), relevant to B- and T-cell development and function are associated with loss of anergy. Hence, the results suggest a role for risk-conferring alleles in perturbation of B-cell anergy during development of type 1 diabetes. [ABSTRACT FROM AUTHOR]

Despite decades of research in humans and mouse models of disease, substantial gaps remain in our understanding of pathogenic mechanisms underlying the development of type 1 diabetes. Furthermore, translation of therapies from preclinical efforts capable of delaying or halting β-cell destruction has been limited. Hence, a pressing need exists to identify alternative animal models that reflect human disease. Canine insulin deficiency diabetes is, in some cases, considered to follow autoimmune pathogenesis, similar to NOD mice and humans, characterized by hyperglycemia requiring lifelong exogenous insulin therapy. Also similar to human type 1 diabetes, the canonical canine disorder appears to be increasing in prevalence. Whereas islet architecture in rodents is distinctly different from humans, canine pancreatic endocrine cell distribution is more similar. Differences in breed susceptibility alongside associations with MHC and other canine immune response genes parallel that of different ethnic groups within the human population, a potential benefit over NOD mice. The impact of environment on disease development also favors canine over rodent models. Herein, we consider the potential for canine diabetes to provide valuable insights for human type 1 diabetes in terms of pancreatic histopathology, impairment of β-cell function and mass, islet inflammation (i.e., insulitis), and autoantibodies specific for β-cell antigens. [ABSTRACT FROM AUTHOR]

Type 1 diabetes results from chronic autoimmune destruction of insulin-producing β-cells within pancreatic islets. Although insulin is a critical self-antigen in animal models of autoimmune diabetes, due to extremely limited access to pancreas samples, little is known about human antigenic targets for islet-infiltrating T cells. Here we show that proinsulin peptides are targeted by islet-infiltrating T cells from patients with type 1 diabetes. We identified hundreds of T cells from inflamed pancreatic islets of three young organ donors with type 1 diabetes with a short disease duration with high-risk HLA genes using a direct T-cell receptor (TCR) sequencing approach without long-term cell culture. Among 85 selected CD4 TCRs tested for reactivity to preproinsulin peptides presented by diabetes-susceptible HLA-DQ and HLA-DR molecules, one T cell recognized C-peptide amino acids 19-35, and two clones from separate donors responded to insulin B-chain amino acids 9-23 (B:9-23), which are known to be a critical self-antigen-driving disease progress in animal models of autoimmune diabetes. These B:9-23-specific T cells from islets responded to whole proinsulin and islets, whereas previously identified B:9-23 responsive clones from peripheral blood did not, highlighting the importance of proinsulin-specific T cells in the islet microenvironment. [ABSTRACT FROM AUTHOR]

There is an ongoing need to develop strategic combinations of therapeutic agents to prevent type 1 diabetes (T1D) or to preserve islet β-cell mass in new-onset disease. Although clinical trials using candidate therapeutics are commonly based on preclinical studies, concern is growing regarding the reproducibility as well as the potential clinical translation of reported results using animal models of human disorders. In response, the National Institutes of Health Immune Tolerance Network and JDRF established a multicenter consortium of academic institutions designed to assess the efficacy and intergroup reproducibility of clinically applicable immunotherapies for reversing new-onset disease in the NOD mouse model of T1D. Predicated on prior studies, this consortium conducted coordinated, prospective studies, using joint standard operating procedures, fixed criteria for study entry, and common reagents, to optimize combined anti-CD3 treatment plus interleukin-1 (IL-1) blockade to reverse new-onset disease in NOD mice. We did not find that IL-1 blockade with anti-IL-1β monoclonal antibody or IL-1trap provided additional benefit for reversing new-onset disease compared with anti-CD3 treatment alone. These results demonstrate the value of larger, multicenter preclinical studies for vetting and prioritizing therapeutics for future clinical use. [ABSTRACT FROM AUTHOR]

Descriptions of insulitis in human islets throughout the natural history of type 1 diabetes are limited. We determined insulitis frequency (the percent of islets displaying insulitis to total islets), infiltrating leukocyte subtypes, and β-cell and α-cell mass in pancreata recovered from organ donors with type 1 diabetes (n = 80), as well as from donors without diabetes, both with islet autoantibodies (AAb(+), n = 18) and without islet autoantibodies (AAb(-), n = 61). Insulitis was observed in four of four donors (100%) with type 1 diabetes duration of ≤1 year and two AAb(+) donors (2 of 18 donors, 11%). Insulitis frequency showed a significant but limited inverse correlation with diabetes duration (r = -0.58, P = 0.01) but not with age at disease onset. Residual β-cells were observed in all type 1 diabetes donors with insulitis, while β-cell area and mass were significantly higher in type 1 diabetes donors with insulitis compared with those without insulitis. Insulitis affected 33% of insulin(+) islets compared with 2% of insulin(-) islets in donors with type 1 diabetes. A significant correlation was observed between insulitis frequency and CD45(+), CD3(+), CD4(+), CD8(+), and CD20(+) cell numbers within the insulitis (r = 0.53-0.73, P = 0.004-0.04), but not CD68(+) or CD11c(+) cells. The presence of β-cells as well as insulitis several years after diagnosis in children and young adults suggests that the chronicity of islet autoimmunity extends well into the postdiagnosis period. This information should aid considerations of therapeutic strategies seeking type 1 diabetes prevention and reversal. [ABSTRACT FROM AUTHOR]

The destruction of beta cells in type 1 diabetes (T1D) results in loss of insulin production and glucose homeostasis. Treatment of non-obese diabetic (NOD) mice with immune-depleting/modulating agents (e.g., anti-CD3, murine anti-thymocyte-globulin (mATG)) can lead to diabetes reversal. However, for preclinical studies with these and other agents seeking to reverse disease at onset, the necessity for exogenous insulin administration is debated. Spontaneously diabetic NOD mice were treated with a short-course of mATG and insulin provided as drug therapy or by way of allogeneic islet implants. Herein we demonstrate that exogenous insulin administration is required to achieve disease reversal with mATG in NOD mice. Unexpectedly, we also observed that provision of insulin by way of allogeneic islet implantation in combination with mATG leads to a pronounced reversal of diabetes as well as restoration of tolerance to self-islets. Expansion/induction of regulatory cells was observed in NOD mice stably cured with mATG and allogeneic islets. These data suggest that transient provision of allogeneic insulin-producing islets might provide a temporary window for immune depletion to be more effective and instilling stable tolerance to endogenous beta cells. These findings support the use of a never before explored approach for preserving beta cell function in patients with recent onset T1D. [ABSTRACT FROM AUTHOR]

Insights from prospective, longitudinal studies of individuals at risk for developing type 1 diabetes have demonstrated that the disease is a continuum that progresses sequentially at variable but predictable rates through distinct identifiable stages prior to the onset of symptoms. Stage 1 is defined as the presence of β-cell autoimmunity as evidenced by the presence of two or more islet autoantibodies with normoglycemia and is presymptomatic, stage 2 as the presence of β-cell autoimmunity with dysglycemia and is presymptomatic, and stage 3 as onset of symptomatic disease. Adoption of this staging classification provides a standardized taxonomy for type 1 diabetes and will aid the development of therapies and the design of clinical trials to prevent symptomatic disease, promote precision medicine, and provide a framework for an optimized benefit/risk ratio that will impact regulatory approval, reimbursement, and adoption of interventions in the early stages of type 1 diabetes to prevent symptomatic disease. [ABSTRACT FROM AUTHOR]

The article explores evolving concepts underlying the pathogenesis of type 1 diabetes. Particular focus is given to autoimmunity as the formative cause of diabetes during the 1980s, as well as efforts to preserve C-peptide production in clinical trials. Topics discussed include human immune response, disease subtypes, and inflammatory process in the pancreas. The role of coxsackievirus in the pathogenesis of type 1 diabetes and human pancreatic islets are also mentioned.

Impaired regulatory B cell (Breg) responses are associated with several autoimmune diseases in humans; however, the role of Bregs in type 1 diabetes (T1D) remains unclear. We hypothesized that naturally occurring, interleukin-10 (IL-10)-producing Bregs maintain tolerance to islet autoantigens, and that hyperglycemic nonobese diabetic (NOD) mice and T1D patients lack these potent negative regulators. IgVH transcriptome analysis revealed that islet-infiltrating B cells in long-term normoglycemic (Lnglc) NOD, which are naturally protected from diabetes, are more antigen-experienced and possess more diverse B-cell receptor repertoires compared to those of hyperglycemic (Hglc) mice. Importantly, increased levels of Breg-promoting CD40(+) B cells and IL-10-producing B cells were found within islets of Lnglc compared to Hglc NOD. Likewise, healthy individuals showed increased frequencies of both CD40(+) and IL-10(+) B cells compared to T1D patients. Rituximab-mediated B-cell depletion followed by adoptive transfer of B cells from Hglc mice induced hyperglycemia in Lnglc human CD20 transgenic NOD mouse models. Importantly, both murine and human IL-10(+) B cells significantly abrogated T-cell-mediated responses to self- or islet-specific peptides ex vivo. Together, our data suggest that antigen-matured Bregs may maintain tolerance to islet autoantigens by selectively suppressing autoreactive T-cell responses, and that Hglc mice and individuals with T1D lack this population of Bregs. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Consistent with studies in NOD mice, early clinical trials addressing whether depletion of B cells by the Rituximab CD20-specific antibody provides an effective means for type 1 diabetes reversal have produced promising results. However, to improve therapeutic efficacy, additional B-cell--depleting agents, as well as attempts seeking diabetes prevention, are being considered. RESEARCH DESIGN AND METHODS--Autoantibodies, including those against insulin (IAAs), are used to identify at-risk subjects for inclusion in diabetes prevention trials. Therefore, we tested the ability of anti-CD20 to prevent diabetes in NOD mice when administered either before or after IAA onset. RESULTS--The murine CD20-specific 18B12 antibody that like Rituximab, depletes the follicular (FO) but not marginal zone subset of B cells, efficiently inhibited diabetes development in NOD mice in a likely regulatory T-cell--dependent manner only when treatment was initiated before IAA detection. One implication of these results is that the FO subset of B cells preferentially contributes to early diabetes initiation events. However, most important, the inefficient ability of anti-CD20 treatment to exert late-stage diabetes prevention was found to be attributable to downregulation of CD20 expression upon B cell entry into pancreatic islets. CONCLUSIONS--These findings provide important guidance for designing strategies targeting B cells as a potential means of diabetes intervention. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is considered a disorder whose pathogenesis is autoimmune in origin, a notion drawn in large part from studies of human pancreata performed as far back as the 1960s. While studies of the genetics, epidemiology, and peripheral immunity in T1D have been subject to widespread analysis over the ensuing decades, efforts to understand the disorder through analysis of human pancreata have been far more limited. We have reviewed the published literature pertaining to the pathology of the human pancreas throughout all stages in the natural history of T1D. This effort uncovered a series of findings that challenge many dogmas ascribed to T1D and revealed data suggesting the marked heterogeneity in terms of its pathology. An improved understanding and appreciation for pancreatic pathology in T1D could lead to improved disease classification, an understanding of why the disorder occurs, and better therapies for disease prevention and management. [ABSTRACT FROM AUTHOR]

Several studies have shown that gut bacteria have a role in diabetes in murine models. Specific bacteria have been correlated with the onset of diabetes in a rat model. However, it is unknown whether human intestinal microbes have a role in the development of autoimmunity that often leads to type 1 diabetes (T1D), an autoimmune disorder in which insulin-secreting pancreatic islet cells are destroyed. High-throughput, culture-independent approaches identified bacteria that correlate with the development of T1D-associated autoimmunity in young children who are at high genetic risk for this disorder. The level of bacterial diversity diminishes overtime in these autoimmune subjects relative to that of age-matched, genotype-matched, nonautoimmune individuals. A single species, Bacteroides ovatus, comprised nearly 24% of the total increase in the phylum Bacteroidetes in cases compared with controls. Conversely, another species in controls, represented by the human firmicute strain CO19, represented nearly 20% of the increase in Firmicutes compared with cases overtime. Three lines of evidence are presented that support the notion that, as healthy infants approach the toddler stage, their microbiomes become healthier and more stable, whereas, children who are destined for autoimmunity develop a microbiome that is less diverse and stable. Hence, the autoimmune microbiome for T1D may be distinctly different from that found in healthy children. These data also suggest bacterial markers for the early diagnosis of T1D. In addition, bacteria that negatively correlated with the autoimmune state may prove to be useful in the prevention of autoimmunity development in high-risk children. [ABSTRACT FROM AUTHOR]

OBJECTIVE--In type 1 diabetes, allogeneic pancreatic islet transplant restores insulin production, but life-threatening immunosuppression is required to avoid graft rejection. Induction of antigen (Ag)-specific tolerance by cell therapy with regulatory T-cells (Tregs) represents an attractive alternative approach but its therapeutic efficacy in islet transplant remains to be determined. Among the different subsets of CD4+ Tregs, the T inducible regulatory type 1 (Tr1) cells can be generated from naive T-cells in the presence of interleukin-10 (IL-10) and represent one promising therapeutic choice. This study was designed to define the efficacy of Tr1-cell therapy in preclinical models of islet transplant. RESEARCH DESIGN AND METHODS--Non-Ag-specific polyclonal Tr1 cells and donor Ag-specific Tr1 cells were transferred, in the absence of any pharmacological treatment, in two distinct mouse models of islet transplant. The two models differed in their therapeutic stringency, based on the mean rejection time of untreated mice that underwent a transplant. RESULTS--Transfer of polyclonal Tr1 cells engendered graft tolerance only in the nonstringent mouse model. Conversely, cell therapy with Ag-specific Tr1 cells induced an IL-10-dependent tolerance in the stringent mouse model of islet transplant. The therapeutic advantage of Ag-specific Tr1 cells over polyclonal Tr1 cells was due to their donor Ag specificity. CONCLUSIONS--These results demonstrate that Tr1-cell therapy leads to tolerance in settings of islet transplant and that its therapeutic efficacy is highly dependent on the antigen specificity of these cells. Diabetes 59:433-439, 2010 [ABSTRACT FROM AUTHOR]

Although alloxan-resistant (ALR) mice share 70% of the NOD genome, ALR islets are remarkably resistant to oxidative stress and autoimmunity. Using 2D PAGE comparative analysis, we have characterized 55 proteins that are differentially expressed between the NOD and ALR islet proteome. Ten proteins were found to be highly expressed in the NOD islets. Typically, proteins associated with pancreatic inflammation and autoimmunity, such as amylase and carboxypeptidase, are highly expressed in the NOD islets. Forty-five proteins showed significantly higher expression in the ALR islets. Among these, 30 are proteins implicated in the regulation of intracellular stress including heat-shock proteins, disulfide isomerase–associated proteins, ROS detoxification enzymes, and apoptotic regulators. Our results clearly demonstrate that the 30% unique ALR genome encodes protective determinants expressed at islet levels, which render the islets of this strain of mice resistant to oxidative stress and autoimmunity. [ABSTRACT FROM AUTHOR]

The cause of cystic fibrosis-related diabetes (CFRD) remains unknown, but cystic fibrosis transmembrane conductance regulator (CFTR) mutations contribute directly to multiple aspects of the cystic fibrosis phenotype. We hypothesized that susceptibility to islet dysfunction in cystic fibrosis is determined by the lack of functional CFTR. To address this, glycemia was assessed in CFTR null (CFTR-/-), C57BL/6J, and FVB/NJ mice after streptozotocin (STZ)-induced β-cell injury. Fasting blood glucose levels were similar among age-matched non-STZ-administered animals, but they were significantly higher in CFTR-/- mice 4 weeks after STZ administration (288.4 ± 97.4, 168.4 ± 35.9, and 188.0 ± 42.3 mg/dl for CFTR-/-, C57BL/6J, and FVB/NJ, respectively; P < 0.05). After intraperitoneal glucose administration, elevated blood glucose levels were also observed in STZ-administered CFTR-/- mice. STZ reduced islets among all strains; however, only CFTR-/- mice demonstrated a negative correlation between islet number and fasting blood glucose (P = 0.02). To determine whether a second alteration associated with cystic fibrosis (i.e., airway inflammation) could impact glucose control, animals were challenged with Aspergillus fumigatus. The A. fumigatus-sensitized CFTR-/- mice demonstrated similar fasting and stimulated glucose responses in comparison to nonsensitized animals. These studies suggest metabolic derangements in CFRD originate from an islet dysfunction inherent to the CFTR-/- state. Diabetes 55:1939-1945, 2006 [ABSTRACT FROM AUTHOR]

The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islet cells may allow enhanced posttransplantation survival of islet allografts and inhibition of recurrent autoimmune destruction of these cells in type 1 diabetes. However, transient transgene expression and the tendency to induce host inflammatory responses have limited previous gene delivery studies using viral transfer vectors. We demonstrate here that recombinant adeno-associated virus (rAAV) serotype 2, a vector that can overcome these limitations, effectively transduces both human and murine pancreatic islet cells with reporter genes as well as potentially important immunoregulatory cytokine genes (interleukin-4, interleukin-10), although a very high multiplicity of infection (10,000 infectious units/islet equivalent) was required. This requirement was alleviated by switching to rAAV serotype 5, which efficiently transduced islets at a multiplicity of infection of 100. Although adenovirus (Ad) coinfection was required for efficient ex vivo expression at early time points, islets transduced without Ad expressed efficiently when they were transplanted under the renal capsule and allowed to survive in vivo. The rAAV-delivered transgenes did not interfere with islet cell insulin production and were expressed in both beta- and non-beta-cells. We believe rAAV will provide a useful tool to deliver therapeutic genes for modulating immune responses against islet cells and markedly enhance longterm graft survival. [ABSTRACT FROM AUTHOR]

Coxsackievirus infections have been proposed as an environmental trigger for the development of T-cell-mediated autoimmune (type 1) diabetes by either providing a molecular mimic of the candidate pancreatic beta-cell autoantigen GAD or inducing bystander inflammation in the pancreas. In this study in the NOD mouse model, we found that infection with a pancreatrophic coxsackievirus isolate can accelerate type 1 diabetes development through the induction of a bystander activation effect, but only after a critical threshold level of insulitic beta-cell-autoreactive T-cells has accumulated. Thus, coxsackievirus infections do not appear to initiate beta-cell autoreactive immunity but can accelerate the process once it is underway. These findings indicate that the timing of a coxsackievirus infection, rather than its simple presence or absence, may have important etiological implications for the development of T-cell-mediated autoimmune type 1 diabetes in humans. [ABSTRACT FROM AUTHOR]

OBJECTIVE-Evidence supporting an association between complement (C) and type 1 diabetes (T1D) includes the identification of C-fixing islet cell autoantibodies in T1D sera and genetic associations with the major histocompatibility complex III C4 region on chromosome 6. Therefore, we investigated whether C activation was present in pancreata from those with or at increased risk (positive for T1D associated autoantibodies) for T1D. RESEARCH DESIGN AND METHODS-Immunohistochemical techniques were used to measure the C degradation product C4d in organ donor pancreata frompatients with T1D and type 2 diabetes and autoantibody-positive and autoantibody-negative subjects. RESULTS-Median C4d antigen density differed across the groups (P, 0.0001) and was highest in patients with T1D. C4d immunostaining localized to the blood vessel endothelium and extracellular matrix surrounding blood vessels and exocrine ducts. Receiver operating characteristic analysis resulted in 81.8% sensitivity and 94.4% specificity for C4d staining. CONCLUSIONS-These data suggest that C activation is occurring within pancreata from patients with T1D and C4d may be a biomarker for T1D. [ABSTRACT FROM AUTHOR]

The article focuses on the islet cell cytoplasmic autoantibody (ICA). ICA's have widely been accepted as the gold standard for confirming the diagnosis of type 1 diabetes since their initial description in the early 1970s. The identification of these autoantibodies in serum samples are made by indirect immunofluorescence testing using blood group O human pancreas as a substrate. Many research has been made utilizing biochemical autoantibodies, which indicates that the presence of biochemical autoantibodies, especially when multiple in number within an individual, confers increased risk for development of type 1 diabetes.

Several immunotherapies have demonstrated endogenous insulin preservation in recent-onset type 1 diabetes (T1D). We considered the primary results of rituximab, abatacept, teplizumab, alefacept, high-dose antithymocyte globulin (ATG), low-dose ATG, and low-dose ATG ± granulocyte-colony-stimulating factor trials in an attempt to rank the effectiveness of the agents studied. C-peptide 2-h area under the curve means were modeled using analysis of covariance. The experimental treatment group effect for each study, compared with its internal control, was estimated after adjusting for baseline C-peptide and age. Percentage increase in C-peptide over placebo and the absolute difference within study were calculated to compare and contrast effect size among interventions. Low-dose ATG (55% and 103%) and teplizumab (48% and 63%) ranked highest in C-peptide preservation at 1 and 2 years, respectively. Low-dose ATG and teplizumab show the greatest impact on C-peptide preservation among recent new-onset T1D studies; these should be further explored as core immunotherapies in the T1D prevention setting. [ABSTRACT FROM AUTHOR]

Alpha-1 Antitrypsin (AAT) is a serine protease inhibitor having immunomodulatory properties that can prevent diabetes onset in NOD mice as well as prolong islet allograft survival in a non-autoimmune murine model. In this study, we explored the effect of chronic administration of two different clinical formulations of AAT (Prolastin and Aralast) on allogeneic (C57BL/6) islet graft survival in spontaneously diabetic (nonfasting glycemia >350 mg/dl) female NOD mice. Islets were implanted under the kidney capsule. Treatments consisted of intraperitoneal saline (control), Prolasfin® (either 1 or 2 mg/mouse) or Aralast® (2mg/mouse) given on d -1,0,3 and every 3 d thereafter. Control mice rejected islet allografts 10.0±2.5 d (mean±SD) after transplantation (n=9). Graft survival was 8.7±1.5 d in the group receiving Prolastin 1mg (n=9) and 14±5.6 d in the group receiving 2mg (n=2). The group treated with Aralast 2mg also showed prolongation of graft survival (13±5.8 d; p=0.03). Notably, we observed that repeated administrations of either AAT formulation at the indicated doses led to sudden death of a proportion of treated animals. This phenomenon occurred invariably following the 5th AAT injection (3/8 mice in the Prolastin 1 mg, 1/3 in Prolastin 2mg, and 2/5 in the Aralast 2mg group) and treatment discontinuation in the remaining animals prevented additional deaths. Collectively, our preliminary data indicates that some prolongation of islet allograft survival in the stringent NOD mouse model can be achieved with AAT monotherapy. The unexpected death of a proportion of treated animals may be consequent to immunization to human AAT and lethal hypersensitivity. Interestingly, this phenomenon was not observed in a non-antoimmune mouse swain (C57BL/6) despite extended (>100 d) AAT treatment. Studies aiming at further understanding the mechanisms underlying this observation are underway. [ABSTRACT FROM AUTHOR]

In type 1 diabetes patients, islet-infiltrating cytotoxic T lymphocytes (CTL) and natural killer (NK) cells release cytotoxic granule contents (perforin and granzymes) into the inter- or intra-cellular space and induce islet cell apoptosis. Granzyme B, the major killing enzyme, is a serine proteinase. Alpha 1 antitrypsin (AAT) is a well-known serine proteinase inhibitor and plays important roles in innate immunity and protection against tissue damage. We have recently shown that AAT protects against cytokine- or streptozotocin-induced islet cell death and prevents type 1 diabetes in NOD mice. However, the inhibitory effects of AAT on granzyme B activity and CTL/NK killing remains elusive. In this study, we tested the direct effect of AAT on granzyme B activity using a cell-free assay system. Results from these experiments indicated that AAT inhibited granzyme B activity significantly and in a dose dependent manner (P<0.5 and <0.01 at 0.5 and 5 mg/ml, respectively). In order to determine the effect of AAT on NK cell killing, we have performed an in vitro cell killing assay using NK cells (NK92-ci) as effector cells and fluorescently labeled K562 cells as targets at various effector to target ratios (2.5:1,5:1 or 10:1). These experiments showed that in the presence of AAT (0.5 and 1 mg/ml), NK cell killing activity were decreased significantly (30 to 60%, P<0.5). The inhibitory effect was evident at all ratios and in AAT-dose dependent manner. Collectively, these results imply a novel protective mechanism of AAT. This mechanism may be operative in protecting against CTL or NK cell mediated islet destruction since AAT gene is expressed in islet cells. Future studies will further elucidate this mechanism. [ABSTRACT FROM AUTHOR]

Presents the recommendations of the Immunology of Diabetes Society on the assessment of risk of type 1 diabetes in unaffected relatives of patients. Basis of the assessment of risk of type 1 diabetes in relatives; Determination of insulin autoantibodies; Application of genetic testing; Use of metabolic testing.

Background: Previous efforts to preserve β cell function in individuals with type 1 diabetes (T1D) have focused largely on the use of single immunomodulatory agents administered within 100 days of diagnosis. Based on human and preclinical studies, we hypothesized that a combination of low-dose anti-thymocyte globulin (ATG) and pegylated granulocyte CSF (G-CSF) would preserve β cell function in patients with established T1D (duration of T1D >4 months and <2 years). Methods: A randomized, single-blinded, placebo-controlled trial was performed on 25 subjects: 17 subjects received ATG (2.5 mg/kg intravenously) followed by pegylated G-CSF (6 mg subcutaneously every 2 weeks for 6 doses) and 8 subjects received placebo. The primary outcome was the 1-year change in AUC C-peptide following a 2-hour mixed-meal tolerance test (MMTT). At baseline, the age (mean ± SD) was 24.6 ± 10 years; mean BMI was 25.4 ± 5.2 kg/m²; mean A1c was 6.5% ± 1.1%; insulin use was 0.31 ± 0.22 units/kg/d; and length of diagnosis was 1 ± 0.5 years. Results: Combination ATG/G-CSF treatment tended to preserve β cell function in patients with established T1D. The mean difference in MMTT-stimulated AUC C-peptide between treated and placebo subjects was 0.28 nmol/l/min (95% CI 0.001-0.552, P = 0.050). A1c was lower in ATG/G-CSF-treated subjects at the 6-month study visit. ATG/G-CSF therapy was associated with relative preservation of Tregs. Conclusions: Patients with established T1D may benefit from combination immunotherapy approaches to preserve β cell function. Further studies are needed to determine whether such approaches may prevent or delay the onset of the disease. Trial Registration: Clinicaltrials.gov NCT01106157. Funding: The Leona M. and Harry B. Helmsley Charitable Trust and Sanofi. [ABSTRACT FROM AUTHOR]

Islet transplantation can restore glucose homeostasis in those with type 1 diabetes; however, most recipients eventually lose graft function. A noninvasive method to monitor islets following transplantation would enable assessment of their survival and aid the development of therapeutics to prolong graft survival. Here, we show that recombinant lentivirus can be used to engineer human islets to express a positron emission tomography (PET) reporter gene. Following transplantation into mice, transduced islets could be imaged in vivo using microPET and a radiolabeled probe approved by the FDA for clinical use in humans. The magnitude of signal from engineered islets implanted into the axillary cavity reflected the implanted islet mass. Signals from implanted islets decreased by approximately one-half during the first few weeks following transplantation, which may reflect islet cell death shortly after transplantation. Thereafter, the magnitude of signals from the implanted islets remained fairly constant when the recipients were repetitively reimaged over 90 days. Histological analysis of the implants showed healthy islets with PET reporter-expressing cells distributed throughout the islet architecture. These studies suggest that PET imaging of lentivirus-transduced islets could provide a safe and feasible method for long-term monitoring of islet graft survival.Molecular Therapy (2006) 14, 851–856; doi: 10.1016/j.ymthe.2006.08.007 [ABSTRACT FROM AUTHOR]

Type 1 diabetes acceleration in nonobese diabetic (NOD) mice through coxsackievirus B4 (CVB4) infection requires a preexisting critical mass of autoreactive T cells in pancreatic islets, and in the absence of this insulitic threshold, CVB4 infection leads to long-term disease protection. To understand this acceleration and protection process, we challenged 8- and 12-week-old NOD mice containing a disruption in interleukin-4 (IL-4) or gamma interferon (IFN-γ) genes (NOD IL-4-/- and NOD IFN-γ-/-, respectively) with a diabetogenic, pancreatropic Edwards strain of CVB4. The elimination of IL-4 did not alter the rate of insulitis or diabetes development in NOD mice, while the elimination of IFN-γ delayed these events several weeks. CVB4 infection in 8-week-old mice only significantly accelerated the onset of diabetes in a subset of standard, but not IL-4- or IFN-γ,-deficient, NOD mice. Long-term diabetes protection was established in standard NOD mice as well as in the NOD IFN-γ-/-mice that did not rapidly develop disease following CVB4 infection at 8 weeks of age. When mice were infected at 12 weeks of age, the onset of diabetes was accelerated in NOD IL-4-/- mice, while neither acceleration nor long-term protection was elicited in NOD IFN-γ-/- mice. No differences were observed in the kinetics of CVB4 clearance in pancreases from NOD, NOD IL-4-/-, and NOD IFN-γ-/- mice. Collectively, these results suggest that at the insulitis threshold at which CVB4 infection can first accelerate the onset of diabetes in NOD mice, IL-4 as well as IFN-γ contributes to this pathogenic process. The protective mechanism against diabetes elicited in NOD mice infected with CVB4 prior to the development of a critical threshold level of insulitis requires neither IL-4 nor IFN-γ. [ABSTRACT FROM AUTHOR]

Recent reports indicate successful transduction of pancreatic islets using recombinant adeno-associated viral (rAAV) vectors. This advance offers new possibilities in rendering islets resistant to rejection and recurrence of autoimmune destruction in the setting of islet transplantation as treatment of type 1 diabetes. Most gene delivery approaches using islets have thus far involved transduction with a single gene. However, the concomitant delivery of more than one gene encoding cytoprotective and/or immunoregulatory molecules may offer superior clinical utility. Here, we have generated a bicistronic rAAV (serotype 2) vector incorporating a viral internal ribosome entry site (IRES), derived from polio virus type 1, to allow for translation of two coupled cDNAs from a single mRNA transcript. Our study demonstrates the ability of this vector to produce significant expression of two reporter proteins in human and mouse islets in vitro. This expression did not interfere with β-cell function. Transduction was maintained in vivo following transplantation of mouse islets. These data are the first report of efficient islet cell transduction with two genes using a single bicistronic rAAV vector and have direct implications for strategies aimed at enhancing islet transplant survival. [Copyright &y& Elsevier]