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Background: Accelerators in medical gloves are a common cause of allergic contact dermatitis among healthcare workers., Objective: A systematic review of medical and nursing literature, patch testing reports, and chemical analyses of gloves was conducted to assess accelerator contents reported in the literature and to identify accelerator-free gloves., Methods: A systematic literature search was performed in OVID Medline and OVID EMBASE. Hand-searching of reference lists of articles in the field and author input generated the remainder of articles assessed., Results: We present an inventory of accelerator contents of gloves and accelerator-free glove options as reported in the literature as a clinical reference tool to assist allergen-free glove selection for individuals suffering from allergic contact dermatitis due to rubber accelerators., Limitations: Pertinent limitations of our review include lack of predefined study exclusion criteria and screening of the studies identified in the search by 1 review author only., Conclusion: The glove inventory we provide summarizes the available literature regarding medical and surgical glove accelerator content, describing gloves both by brand and manufacturer as well as by accelerators.

This submission highlights the importance of careful history taking along with a strong index of suspicion for an exogenous cause of dermatitis. In particular, it highlights the importance of being aware of the latest trends and exposure risks for contact dermatitis.

Grenz rays, or minimally penetrating X-rays, are known to be an effective treatment of certain recalcitrant immune-mediated skin diseases, but their use in modulating allograft rejection has not been tested. We examined the capacity of grenz ray treatment to minimize islet immunogenicity and extend allograft survival in a mouse model. In a preliminary experiment, 1 of 3 immunologically intact animals demonstrated long-term acceptance of their grenz ray treated islet allograft. Further experiments revealed that 28.6% (2 of 7) grenz ray treated islet allografts survived >60 d. A low dose of 20Gy, was important; a 4-fold increase in radiation resulted in rapid graft failure, and transplanting a higher islet mass did not alter this outcome. To determine whether increased islet allograft survival after grenz treatment would be masked by immunosuppression, we treated the recipients with CTLA-4 Ig, and found an additive effect, whereby 17.5% more animals accepted the graft long-term versus those with CTLA-4 Ig alone. Cell viability assays verified that islet integrity was maintained after treatment with 20Gy. As well, through splenocyte infiltration analysis, donor CD4+ T cell populations 24-hours after transplant were decreased by more than16-fold in recipients receiving irradiated islets compared with control. Donor CD8+ T cell populations, although less prevalent, decreased in all treatment groups compared with control. Our results suggest that brief treatment of isolated islets with low energy grenz rays before allotransplantation can significantly reduce passenger leukocytes and promote graft survival, possibly by inducing donor dendritic cells to differentiate toward a tolerogenic phenotype.

Background: Neomycin contact sensitization rates in North America range from 7% to 13%, whereas in Europe they average approximately 1.9%., Objectives: Given that topical neomycin products are no longer readily available in Canada, the aim of this study was to examine what influence this may have had on neomycin sensitization rates in the 3 western provinces., Methods: On the basis of an observation originally communicated by L. M. Parsons and C. Zhang of the University of Calgary, which suggested significantly reduced rates of neomycin sensitization in Calgary, Alberta, Canada, a multicenter study of patch test results from 5690 patient charts was undertaken. Data from 3 other western Canadian Universities (the University of Saskatchewan, the University of Alberta, and the University of British Colombia) were analyzed. Data were available from 2001 to 2013 for the University of Saskatchewan (except 2006), whereas the University of Alberta and the University of British Columbia had data from 2009 to 2013. Descriptive statistics, trend analysis, and risk estimates were determined using SPSS version 20., Results: Sensitization rates for neomycin have decreased in western Canada and are now similar to those of Europe., Conclusions: This trend is likely influenced by the reduced availability of over-the-counter and prescription neomycin products in Canada., (© The Author(s) 2015.)

The American Contact Dermatitis Society recognizes the interest in the evaluation and management of metal hypersensitivity reactions. Given the paucity of robust evidence with which to guide our practices, we provide reasonable evidence and expert opinion-based guidelines for clinicians with regard to metal hypersensitivity reaction testing and patient management. Routine preoperative evaluation in individuals with no history of adverse cutaneous reactions to metals or history of previous implant-related adverse events is not necessary. Patients with a clear self-reported history of metal reactions should be evaluated by patch testing before device implant. Patch testing is only 1 element in the assessment of causation in those with postimplantation morbidity. Metal exposure from the implanted device can cause sensitization, but a positive metal test does not prove symptom causality. The decision to replace an implanted device must include an assessment of all clinical factors and a thorough risk-benefit analysis by the treating physician(s) and patient.

We investigated whether a prevalent epitope of the β-cell-specific autoantigen islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP206-214) reaches regional Ag-presentation pathways via unprocessed polypeptide chains, as free IGRP206-214 peptide or via preformed IGRP206-214/K(d) complexes. This was accomplished by expressing bacterial artificial chromosome transgenes encoding wild-type (stable) or ubiquitinated (unstable) forms of IGRP in IGRP-deficient NOD mice carrying MHC class I-deficient β-cells, dendritic cells, or B cells. We investigated the ability of the pancreatic lymph nodes of these mice to prime naive IGRP206-214-reactive CD8(+) T cells in vivo, either in response to spontaneous Ag shedding, or to synchronized forms of β-cell necrosis or apoptosis. When IGRP was made unstable by targeting it for proteasomal degradation within β-cells, the cross-priming, autoimmune-initiating potential of this autoantigen (designated autoantigenicity) was impaired. Yet at the same time, the direct presentation, CTL-targeting potential of IGRP (designated pathogenicity) was enhanced. The appearance of IGRP206-214 in regional Ag-presentation pathways was dissociated from transfer of IGRP206-214 or IGRP206-214/K(d) from β cells to dendritic cells. These results indicate that autoantigenicity and pathogenicity are separable and inversely related properties and suggest that pathogenic autoantigens, capable of efficiently priming CTLs while marking target cells for CTL-induced killing, may have a critical balance of these two properties., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Polymorphisms in MHC class II molecules, in particular around β-chain position-57 (β57), afford susceptibility/resistance to multiple autoimmune diseases, including type 1 diabetes, through obscure mechanisms. Here, we show that the antidiabetogenic MHC class II molecule I-A(b) affords diabetes resistance by promoting the differentiation of MHC-promiscuous autoreactive CD4(+) T cells into disease-suppressing natural regulatory T cells, in a β56-67-regulated manner. We compared the tolerogenic and antidiabetogenic properties of CD11c promoter-driven transgenes encoding I-A(b) or a form of I-A(b) carrying residues 56-67 of I-Aβ(g7) (I-A(b-g7)) in wild-type nonobese diabetic (NOD) mice, as well as NOD mice coexpressing a diabetogenic and I-A(g7)-restricted, but MHC-promiscuous T-cell receptor (4.1). Both I-A transgenes protected NOD and 4.1-NOD mice from diabetes. However, whereas I-A(b) induced 4.1-CD4(+) thymocyte deletion and 4.1-CD4(+)Foxp3(+) regulatory T-cell development, I-A(b-g7) promoted 4.1-CD4(+)Foxp3(+) Treg development without inducing clonal deletion. Furthermore, non-T-cell receptor transgenic NOD.CD11cP-I-A(b) and NOD.CD11cP-IA(b-g7) mice both exported regulatory T cells with superior antidiabetogenic properties than wild-type NOD mice. We propose that I-A(b), and possibly other protective MHC class II molecules, afford disease resistance by engaging a naturally occurring constellation of MHC-promiscuous autoreactive T-cell clonotypes, promoting their deviation into autoregulatory T cells.

Juvenile or type 1 diabetes (T1D) involves autoimmune-mediated destruction of insulin-producing β cells in the islets of Langerhans in the pancreas. Lack of insulin prevents the absorption and metabolism of glucose throughout the body by interfering with cell signaling. Cytokines have been shown to play a key role in β cell destruction and regulation of autoimmunity in T1D. The multiple roles of cytokines in T1D pathogenesis, regulation, and regeneration of β cells presents both promise and challenge for their use in immunotherapy. We found that mycobacterial adjuvants induce various regulatory T cells in the non-obese diabetic (NOD) mouse model of T1D. Cytokines produced by these cells not only regulate innate and adaptive immunity but also prevent the development of diabetes and partially restored normoglycemia in diabetic NOD mice. We discovered that adjuvant immunotherapy upregulated Regenerating (Reg) genes in the islets and induced interleukin 22 (IL-22)-producing Th17 cells. IL-22 is known to upregulate Reg gene expression in islets and could potentially induce regeneration of β cells and prevent their apoptosis. Therefore, cytokines both induce and regulate T1D and have the potential to regenerate and preserve insulin-producing β cells in the islets.

Objective: The T helper 17 (Th17) population, a subset of CD4-positive T-cells that secrete interleukin (IL)-17, has been implicated in autoimmune diseases, including multiple sclerosis and lupus. Therapeutic agents that target the Th17 effector molecule IL-17 or directly inhibit the Th17 population (IL-25) have shown promise in animal models of autoimmunity. The role of Th17 cells in type 1 diabetes has been less clear. The effect of neutralizing anti-IL-17 and recombinant IL-25 on the development of diabetes in NOD mice, a model of spontaneous autoimmune diabetes, was investigated in this study., Research Design and Methods and Results: Although treatment with either anti-IL-17 or IL-25 had no effect on diabetes development in young (<5 weeks) NOD mice, either intervention prevented diabetes when treatment was started at 10 weeks of age (P < 0.001). Insulitis scoring and immunofluorescence staining revealed that both anti-IL-17 and IL-25 significantly reduced peri-islet T-cell infiltrates. Both treatments also decreased GAD65 autoantibody levels. Analysis of pancreatic lymph nodes revealed that both treatments increased the frequency of regulatory T-cells. Further investigation demonstrated that IL-25 therapy was superior to anti-IL-17 during mature diabetes because it promoted a period of remission from new-onset diabetes in 90% of treated animals. Similarly, IL-25 delayed recurrent autoimmunity after syngeneic islet transplantation, whereas anti-IL-17 was of no benefit. GAD65-specific ELISpot and CD4-positive adoptive transfer studies showed that IL-25 treatment resulted in a T-cell-mediated dominant protective effect against autoimmunity., Conclusions: These studies suggest that Th17 cells are involved in the pathogenesis of autoimmune diabetes. Further development of Th17-targeted therapeutic agents may be of benefit in this disease.

Insulin-specific immune responses appear early in preclinical type 1 diabetes (T1D), and bovine insulin in cow's milk-based infant formulas has been suggested to be of importance in induction of the primary response to insulin in humans. To characterize insulin-specific T-cell reactivity we studied T-cell responses to 10 insulin peptides derived from bovine (BI) and human insulin (HI) in 42 children with recently diagnosed T1D, 47 children with multiple autoantibodies and 111 autoantibody-negative control children with risk-associated HLA alleles. Proliferation responses detected in antigen-stimulated peripheral blood mononuclear cells did not differ between the three groups when the comparison was performed without considering HLA genotypes. However, significant differences were observed, when children with the high-risk genotype HLA (DRB1*03)-DQA1*05-DQB1*02/DRB1*0401-DQA1*03-DQB1*0302 were analyzed separately. The responses to the peptides including amino acids A1-12 derived from B1 and H1 were significantly higher in children with T1D (P=0.008, P=0.004, for B1 and H1, respectively) and in children with diabetes-associated autoantibodies (P=0.002 and P=0.001, respectively) than in control children. Positive responses (stimulation indices SI> or =3) were seen more frequently in T1D children than in controls (4/7 vs 2/19; P=0.03 and 4/7 vs 1/19; P=0.01 for B1 and H1, respectively). T-cell response to the insulin peptide A1-12 is enhanced in clinical and preclinical T1D associated with the high-risk HLA-genotype emphasizing the importance of this epitope.

The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine.

NOD mice with knockout of both native insulin genes and a mutated proinsulin transgene, alanine at position B16 in preproinsulin (B16:A-dKO mice), do not develop diabetes. Transplantation of NOD islets, but not bone marrow, expressing native insulin sequences (tyrosine at position B16) into B16:A-dKO mice rapidly restored development of insulin autoantibodies (IAAs) and insulitis, despite the recipients' pancreatic islets lacking native insulin sequences. Splenocytes from B16:A-dKO mice that received native insulin-positive islets induced diabetes when transferred into wild-type NOD/SCID or B16:A-dKO NOD/SCID mice. Splenocytes from mice immunized with native insulin B chain amino acids 9-23 (insulin B:9-23) peptide in CFA induced rapid diabetes upon transfer only in recipients expressing the native insulin B:9-23 sequence in their pancreata. Additionally, CD4(+) T cells from B16:A-dKO mice immunized with native insulin B:9-23 peptide promoted IAAs in NOD/SCID mice. These results indicate that the provision of native insulin B:9-23 sequences is sufficient to prime anti-insulin autoimmunity and that subsequent transfer of diabetes following peptide immunization requires native insulin B:9-23 expression in islets. Our findings demonstrate dependence on B16 alanine versus tyrosine of insulin B:9-23 for both the initial priming and the effector phase of NOD anti-islet autoimmunity.

Pancreas transplantation in type 1 diabetes patients could result in (re)activation of allo- and autoreactive T lymphocytes. Anti-thymocyte globulin (ATG) induction treatment is a successful, but broadly reactive anti-lymphocyte therapy used in pancreas and islet transplantation. A more selective alternative is daclizumab, a monoclonal antibody directed against the interleukin-2 receptor (CD25) on activated lymphocytes. We tested the hypothesis that daclizumab is more selective and has less immunological side effects than ATG. Thirty-nine simultaneous pancreas-kidney transplantation patients with type 1 diabetes were randomized for induction therapy with ATG or daclizumab. Auto- and recall immunity was measured cross-sectionally by lymphocyte stimulation tests with a series of auto- and recall antigens in 35 successfully transplanted patients. T cell autoimmunity to islets was low in both groups, except for a marginal but significantly higher reactivity against glutamic acid decarboxylase (GAD)65 in daclizumab-treated patients. The memory responses to recall antigens were significantly higher in the daclizumab-treated group compared to ATG-treated patients, specifically against purified protein derivative (PPD) (anti-bacterial immunity), Haemophilus influenzae virus matrix protein-1 (anti-viral immunity) and p53 [anti-tumour (auto)immunity]. These data imply that daclizumab is more specifically affecting diabetes-related immune responses than ATG. The autoimmunity is affected effectively after daclizumab induction, while memory responses towards bacterial, viral and tumour antigens are preserved.

Autoimmune (Hashimoto's) thyroiditis is a chronic inflammatory disease which affects >3% of the population and shows an increasing prevalence with increasing age. Anti-thyroid autoantibodies, particularly against thyroperoxidase (also known as thyroid peroxidase or TPO), occur commonly in humans with autoimmune thyroid disease, and assays for anti-TPO autoantibodies are used in clinical diagnosis. In contrast anti-TPO autoantibodies have not been observed in classical mouse models of autoimmune thyroiditis, except in cases where mice were deliberately immunized with TPO. In the past, detection of anti-TPO autoantibodies in mice has relied on an indirect immunofluorescence assay (iIFA) which screens for thyroid follicle membrane staining in frozen sections of mouse thyroid glands. Since recent transgenic mouse models of autoimmune thyroiditis spontaneously develop anti-TPO autoantibodies, an assay other than serial dilution and iIFA would be useful to detect and quantify these autoantibodies. In this paper we describe such an assay, based on the capacity of autoimmune mouse sera to bind to the extracellular domain of mouse TPO which was produced in a radioactively labeled form using a coupled in vitro transcription/translation system. The same approach, using human TPO, could provide a highly sensitive method to detect anti-TPO autoantibodies in humans.

Type 1 diabetes is characterized by T cell-mediated autoimmune destruction of pancreatic beta cells. Several studies have suggested an association between Coxsackie enterovirus seroconversion and onset of disease. However, a direct link between beta cell viral infection and islet inflammation has not been established. We analyzed pancreatic tissue from six type 1 diabetic and 26 control organ donors. Immunohistochemical, electron microscopy, whole-genome ex vivo nucleotide sequencing, cell culture, and immunological studies demonstrated Coxsackie B4 enterovirus in specimens from three of the six diabetic patients. Infection was specific of beta cells, which showed nondestructive islet inflammation mediated mainly by natural killer cells. Islets from enterovirus-positive samples displayed reduced insulin secretion in response to glucose and other secretagogues. In addition, virus extracted from positive islets was able to infect beta cells from human islets of nondiabetic donors, causing viral inclusions and signs of pyknosis. None of the control organ donors showed signs of viral infection. These studies provide direct evidence that enterovirus can infect beta cells in patients with type 1 diabetes and that infection is associated with inflammation and functional impairment.

Background: Despite the success of the Edmonton protocol for human islet transplantation, an alternate source of islet tissue must be developed if beta-cell replacement therapy is to see widespread application. Neonatal porcine islets (NPI) represent one potential source of tissue. When human or rodent islets are transplanted, the majority of cells undergo hypoxia-induce apoptosis soon after the grafts are placed in the recipient. In the present study, we investigated whether NPI were similarly sensitive to hypoxia., Methods: NPI were exposed to hypoxia and hypoxia/reoxygenation using an in vitro hypoxic chamber. Afterwards, viability, frequency of apoptosis, and beta-cell function were evaluated. NPI and adult porcine islets were transplanted into chemically diabetic, immunodeficient mice and graft apoptosis was assessed 24 hours and seven days posttransplant., Results: NPI demonstrated a remarkable capacity to resist apoptosis and maintain insulin secretion despite severe stresses such as hypoxia/reoxygenation. One day after transplantation, NPI grafts showed limited apoptosis, confined to rare strongly insulin positive cells. In contrast, adult porcine islet grafts underwent widespread apoptosis. Western blotting revealed that NPI express high levels of at least one potent endogenous antiapoptotic protein (XIAP)., Conclusions: The majority of cells within transplanted human islets undergo apoptosis soon after portal infusion. In contrast, NPI have the capacity to resist this early posttransplant apoptosis, with likely reduced antigen release and diminished immune stimulation. NPI appear to contain a population of insulin-low to insulin-negative pre-beta-cells, which are resistant to hypoxia-induced apoptosis and still capable of differentiating into mature beta-cells.

We analyzed double native insulin gene knockout NOD mice with a mutated (B16:alanine) proinsulin transgene at multiple ages for the development of insulin autoantibodies, insulitis, and diabetes. In contrast to mice with at least one copy of a native insulin gene that expressed insulin antibodies, only 2 out of 21 (10%) double native insulin gene knockout mice with a mutated insulin transgene developed insulin autoantibodies. Of 21 double insulin knockout mice sacrificed between 10 to 48 weeks of age, only 5 showed minimal insulitis versus 100% of wild-type NOD and more than 90% of insulin 1 knockout mice. Consistent with robust suppression of insulin autoantibodies and insulitis, no double insulin knockout mice developed diabetes. In that the B9-23 peptide with B16A is an altered peptide ligand inducing Th2 responses, we analyzed transfer of splenocytes into NOD.SCID mice. There was no evidence for regulatory T cells able to inhibit transfer of diabetes by diabetogenic NOD splenocytes. Insulin peptide B9-23 is likely a crucial target for initiation of islet autoimmunity and further mutation of the sequence will be tested to attempt to eliminate all anti-islet autoimmunity.

Proinsulin is a key Ag in type 1 diabetes, but the mechanisms regulating proinsulin immune tolerance are unknown. We have shown that preproinsulin-2 gene-deficient mice (proins-2(-/-)) are intolerant to proinsulin-2. In this study, we analyzed the mechanisms underlying T cell-mediated tolerance to proinsulin-2 in 129/Sv nonautoimmune mice. The expression of one proinsulin-2 allele, whatever its parental origin, was sufficient to maintain tolerance. The site of proinsulin-2 expression relevant to tolerance was evaluated in thymus and bone marrow chimeras. CD4+ T cell reactivity to proinsulin-2 was independent of proinsulin-2 expression in radiation-sensitive bone marrow-derived cells. A wt thymus restored tolerance in proins-2(-/-) mice. Conversely, the absence of the preproinsulin-2 gene in radioresistant thymic cells was sufficient to break tolerance. Although chimeric animals had proinsulin-2-reactive CD4+ T cells in their peripheral repertoire, they displayed no insulitis or insulin Abs, suggesting additional protective mechanisms. In a model involving transfer to immunodeficient (CD3epsilon(-/-)) mice, naive and proinsulin-2-primed CD4+ T cells were not activated, but could be activated by immunization regardless of whether the recipient mice expressed proinsulin-2. Furthermore, we could not identify a role for putative specific T cells regulating proinsulin-2-reactive CD4+ T in transfer experiments. Thus, proinsulin-2 gene expression by radioresistant thymic epithelial cells is involved in the induction of self-tolerance, and additional factors are required to induce islet abnormalities.

In humans, spontaneous autoimmune attack against cardiomyocytes often leads to idiopathic dilated cardiomyopathy (IDCM) and life-threatening heart failure. HLA-DQ8 transgenic IAb knockout NOD mice (NOD.DQ8/Ab(0); DQA1*0301, DQB1*0302) develop spontaneous anticardiomyocyte autoimmunity with pathology very similar to human IDCM, but why the heart is targeted is unknown. In the present study, we first investigated whether NOD/Ab(0) mice transgenic for a different DQ allele, DQ6, (DQA1*0102, DQB1*0602) would also develop myocarditis. NOD.DQ6/Ab(0) animals showed no cardiac pathology, implying that DQ8 is specifically required for the myocarditis phenotype. To further characterize the cellular immune mechanisms, we established crosses of our NOD.DQ8/Ab(0) animals with Rag1 knockout (Rag1(0)), Ig H chain knockout (IgH(0)), and beta(2)-microglobulin knockout (beta(2)m(0)) lines. Adoptive transfer of purified CD4 T cells from NOD.DQ8/Ab(0) mice with complete heart block (an indication of advanced myocarditis) into younger NOD.DQ8/Ab(0) Rag1(0) animals induced cardiac pathology in all recipients, whereas adoptive transfer of purified CD8 T cells or B lymphocytes had no effect. Despite the absence of B lymphocytes, NOD.DQ8/Ab(0)IgH(0) animals still developed complete heart block, whereas NOD.DQ8/Ab(0)beta(2)m(0) mice (which lack CD8 T cells) failed to develop any cardiac pathology. CD8 T cells (and possibly NK cells) seem to be necessary to initiate disease, whereas once initiated, CD4 T cells alone can orchestrate the cardiac pathology, likely through their capacity to recruit and activate macrophages. Understanding the cellular immune mechanisms causing spontaneous myocarditis/IDCM in this relevant animal model will facilitate the development and testing of new therapies for this devastating disease.

The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II-related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (<3,400 generations). These results indicate that the differential disease associations of these two DR3 haplotypes are due to sequence variation outside this central 158-kb segment, and that shuffling of ancestral blocks via recombination is a potential mechanism whereby certain DR-DQ allelic combinations, which presumably have favoured immunological functions, can spread across haplotypes and populations., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

We detected insulin2 mRNA but not insulin1 in thymus using real-time PCR analysis. Transgenic expression of a mutated insulin message (alanine rather than tyrosine at insulin B chain amino acid 16) was variably induced in thymus of four transgenic founder strains. The transgenic message levels were as high or higher than native insulin2 message. Lack of the insulin2 gene resulted in the enhancement of anti-insulin autoantibodies (regular NOD vs insulin2-knockout NOD, P<0.001) and in the presence of the B16:A insulin transgenes, levels of insulin autoantibodies remained elevated (regular NOD vs insulin2-knockout NOD with B16:A insulin, P<0.01). Diabetes acceleration by the knockout of the insulin2 gene was not influenced by the presence of the B16:A insulin transgenes. These data suggest that the B16:A insulin does not compensate for lack of native insulin expression in thymus. If lack of thymic insulin message of the insulin2 knockout is the cause of diabetes acceleration, this suggests that native insulin B:9-23 sequences may be crucial in thymus for insulin mediated immunomodulation. Further experiments varying native insulin message expression in thymus is necessary for direct comparison, but the current study provides additional evidence of the potential important role of a specific insulin B chain epitope.

The Edmonton Protocol for treatment of type 1 diabetes requires islets from two or more donors to achieve euglycemia in a single recipient, primarily because soon after portal infusion, the majority of the transplanted cells undergo apoptosis due to hypoxia and hypoxia reperfusion injury. X-linked inhibitor of apoptosis protein (XIAP) is a potent endogenous inhibitor of apoptosis that is capable of blocking the activation of multiple downstream caspases, and XIAP overexpression has previously been shown to enhance engraftment of a murine beta-cell line. In this study, human islets transduced with a XIAP-expressing recombinant adenovirus were resistant to apoptosis and functionally recovered following in vitro stresses of hypoxia and hypoxia with reoxygenation (models reperfusion injury). Furthermore Ad-XIAP transduction dramatically reduced the number of human islets required to reverse hyperglycemia in chemically diabetic immunodeficient mice. These results suggest that by transiently overexpressing XIAP in the immediate posttransplant period, human islets from a single donor might be used to effectively treat two diabetic recipients.

The progression of immune responses is generally associated with an increase in the overall avidity of antigen-specific T cell populations for peptide-MHC. This is thought to result from preferential expansion of high-avidity clonotypes at the expense of their low-avidity counterparts. Since T cell antigen-receptor genes do not mutate, it is puzzling that high-avidity clonotypes do not predominate from the outset. Here we provide a developmental basis for this phenomenon in the context of autoimmunity. We have carried out comprehensive studies of the diabetogenic CD8 T cell population that targets residues 206-214 of the beta cell antigen islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP(206-214)) and undergoes avidity maturation as disease progresses. We find that the succession of IGRP(206-214)-specific clonotypes with increasing avidities during the progression of islet inflammation to overt diabetes in nonobese diabetic mice is fueled by autoimmune inflammation but opposed by systemic tolerance. As expected, naive high-avidity IGRP(206-214)-specific T cells respond more efficiently to antigen and are significantly more diabetogenic than their intermediate- or low-avidity counterparts. However, central and peripheral tolerance selectively limit the contribution of these high-avidity T cells to the earliest stages of disease without abrogating their ability to progressively accumulate in inflamed islets and kill beta cells. These results illustrate the way in which incomplete deletion of autoreactive T cell populations of relatively high avidity can contribute to the development of pathogenic autoimmunity in the periphery.

Recent advances in clinical islet transplantation have allowed patients with type 1 diabetes to become insulin independent, but the procedure is limited since islets from two donors per recipient are typically required. This limitation arises because within a few days of the islets being embolized into the portal circulation, at least half of the transplanted beta-cells have undergone apoptotic cell death triggered by hypoxic and chemokine/cytokine-mediated stress. We hypothesized that the survival of beta-cells in the early post-transplant period would be enhanced if naturally occurring inhibitor of apoptosis proteins (IAPs) were transiently overexpressed in the grafts. In the present study, we used a growth-regulatable beta-cell line (betaTC-Tet) as a model for beta-cells within islets, and examined whether adenovirally delivered XIAP (X-linked IAP-a highly potent IAP) could enhance beta-cell survival. In vitro, XIAP-expressing betaTC-Tet cells were markedly resistant to apoptosis in an ischemia-reperfusion injury model system and following exposure to cytokines. When Ad-XIAP transduced betaTC-Tet cells were transplanted subcutaneously into immunodeficient mice, the grafts were able to reverse diabetes in 3 days, vs. 21 days for Ad-betaGal transduced cells. This approach may allow more efficient use of the limited existing supply of human islets.

A fundamental question about the pathogenesis of spontaneous autoimmune diabetes is whether there are primary autoantigens. For type 1 diabetes it is clear that multiple islet molecules are the target of autoimmunity in man and animal models. It is not clear whether any of the target molecules are essential for the destruction of islet beta cells. Here we show that the proinsulin/insulin molecules have a sequence that is a primary target of the autoimmunity that causes diabetes of the non-obese diabetic (NOD) mouse. We created insulin 1 and insulin 2 gene knockouts combined with a mutated proinsulin transgene (in which residue 16 on the B chain was changed to alanine) in NOD mice. This mutation abrogated the T-cell stimulation of a series of the major insulin autoreactive NOD T-cell clones. Female mice with only the altered insulin did not develop insulin autoantibodies, insulitis or autoimmune diabetes, in contrast with mice containing at least one copy of the native insulin gene. We suggest that proinsulin is a primary autoantigen of the NOD mouse, and speculate that organ-restricted autoimmune disorders with marked major histocompatibility complex (MHC) restriction of disease are likely to have specific primary autoantigens.

We hypothesize that NOD mice without native insulin, but with an altered insulin B:9-23 sequence, will be completely protected from diabetes/insulitis if insulin B:9-23 is an essential T cell epitope. To investigate this hypothesis, we have established initial insulin 1- and 2-negative NOD mice with a transgene directing production of preproinsulin with alanine at position B:16 rather than the native tyrosine of both insulin 1 and insulin 2. Sets of primers for PCR-based assays have been created and validated. They are able to distinguish the presence or absence of the insulin gene knockouts and of both native insulin 1 and insulin 2 (and thus distinguish heterozygous versus homozygous knockouts), as well as the presence of the altered insulin transgene, B:16 alanine preproinsulin. Four B:16 alanine transgenic founders were produced directly in NOD mice and, by intercrossing, initial live native insulin-negative B:16 alanine transgenic mice have been generated.

The future systematic mapping of variants that confer susceptibility to common diseases requires the construction of a fully informative polymorphism map. Ideally, every base pair of the genome would be sequenced in many individuals. Here, we report 4.75 Mb of contiguous sequence for each of two common haplotypes of the major histocompatibility complex (MHC), to which susceptibility to >100 diseases has been mapped. The autoimmune disease-associated-haplotypes HLA-A3-B7-Cw7-DR15 and HLA-A1-B8-Cw7-DR3 were sequenced in their entirety through a bacterial artificial chromosome (BAC) cloning strategy using the consanguineous cell lines PGF and COX, respectively. The two sequences were annotated to encompass all described splice variants of expressed genes. We defined the complete variation content of the two haplotypes, revealing >18,000 variations between them. Average SNP densities ranged from less than one SNP per kilobase to >60. Acquisition of complete and accurate sequence data over polymorphic regions such as the MHC from large-insert cloned DNA provides a definitive resource for the construction of informative genetic maps, and avoids the limitation of chromosome regions that are refractory to PCR amplification., (Copyright 2004 Cold Spring Harbor Laboratory Press)

Deciphering mechanisms involved in failure of self tolerance to preproinsulin-2 is a key issue in type 1 diabetes. We used nonautoimmune 129SV/Pas mice lacking preproinsulin-2 to study the immune response to preproinsulin-2. In these mice, a T cell response was detected after immunization with several preproinsulin-2 peptides and confirmed by generating hybridomas. Activation of some of these hybridomas by wild-type (wt) islet cells or recombinant murine proinsulin-2 demonstrated that two epitopes can be generated from the naturally expressed protein. Although T cells from wt mice responded to preproinsulin-2 peptides, we could not detect a response to the naturally processed epitopes in these mice. Moreover, after immunization with recombinant whole proinsulin-2, a T cell response was detected in preproinsulin-2-deficient but not in wt mice. This suggests that islet preproinsulin-2-autoreactive T cells are functionally eliminated in wt mice. We used a transplantation model to evaluate the relevance of reactivity to preproinsulin-2 in vivo. Wild-type preproinsulin-2-expressing islets transplanted in preproinsulin-2-deficient mice elicited a mononuclear cell infiltration and insulin Abs. Graft infiltration was further increased by immunization with preproinsulin-2 peptides. Preproinsulin-2 expression thus shapes the immune response and prevents self reactivity to the islet. Moreover, islet preproinsulin-2 primes an immune response to preproinsulin-2 in deficient mice.

A line of nonobese diabetic (NOD) mice expressing the human diabetes-associated HLA-DQ8 transgene in the absence of mouse IAbeta failed to show spontaneous insulitis or diabetes, but rather developed dilated cardiomyopathy, leading to early death from heart failure. Pathology in these animals results from an organ- and cell-specific autoimmune response against normal cardiomyoctes in the atrial and ventricular walls, as well as against very similar myocytes present in the outermost muscle layer surrounding the pulmonary veins. Progression of the autoimmune process could be followed by serial ECG measurements; irradiation of young animals significantly delayed disease progression, and this effect could be reversed by adoptive transfer of splenocytes taken from older animals with complete heart block. Disease progression could also be blocked by cyclosporin A treatment, but was accelerated by injection of complete Fruend's adjuvant. The constellation of findings of spontaneously arising destructive focal lymphocytic infiltrates within the myocardium, rising titers of circulating anticardiac autoantibodies, dilation of the cardiac chambers, and gradual progression to end-stage heart failure bears a striking resemblance to what is seen in humans with idiopathic dilated cardiomyopathy, a serious and often life-threatening medical condition. This transgenic strain provides a highly relevant animal model for human autoimmune myocarditis and postinflammatory dilated cardiomyopathy.

HLA-DM stabilizes peptide-receptive class II alphabeta dimers and facilitates the capture of high affinity peptides, thus influencing the peptide repertoire presented by class II molecules. Variations in DM levels may therefore have a profound effect on the antigenic focus of T cell-mediated immune responses. Specifically, DM expression may influence susceptibility and resistance to autoimmune diseases. In this study the role of DM in HLA-DR4-restricted presentation of an insulin-dependent diabetes mellitus autoantigen, glutamate decarboxylase (GAD), was tested. Presentation of immunodominant GAD epitope 273-285 was regulated by endogenous DM levels in human B lymphoblasts. T cell responses to exogenous GAD as well as an endogenous cytoplasmic form of this Ag were significantly diminished with increasing cellular expression of DM. Epitope editing by DM was observed only using Ag and not small synthetic peptides, suggesting that this process occurred within endosomes. Results with cytoplasmic GAD also indicated that peptides from this compartment intersect class II proteins in endocytic vesicles where DM editing was facilitated. Changes in DM levels within APC may therefore influence the presentation of autoantigens and the development of autoimmune disorders such as type I diabetes.

To better understand loss of self-tolerance in diabetes-prone NOD mice, we are generating ICA69 transgenes under control of the tetracycline-regulated tet07 minimal promoter. In vitro pilot studies showed leaky transgene expression, but addition of beta-globin genomic insulator flanks prevented leakage and dramatically enhanced transgene expression even in transient transfection, with excellent suppression by Doxycycline. In vivo, the accidental loss of insulator flanks during transgene insertion in one transgenic NOD founder, tet1, re-established leakiness with high level, exclusive ICA69-transgene expression in stromal elements of thymus and spleen. This led to persistent deletion of T cells targeting the immunodominant ICA69 epitope, Tep69, but emergence of T cell pools targeting cryptic ICA69 epitopes not normally generated in sufficient density to select and maintain ICA69-autoreactive T cells. This subtle modification of T cell repertoires reduced insulitis, and protected from diabetes in transgenics and in wild-type mice carrying irradiated tet1 thymus grafts. The low pathogenicity of T cells targeting cryptic epitopes likely reflects the fact that the major ICA69 determinant presented in the islet milieu remains Tep69, while cryptic epitopes are under-represented. Deviation of T cell autoreactivity from major to cryptic target epitopes in tet1 mice provides a fortuitous model to explain previously observed diabetes protection by immunotherapy or autoantigen transgenes despite apparent failure to achieve tolerance to the full length islet antigens.

Autoimmune (type 1) diabetes mellitus results from the destruction of insulin-producing pancreatic beta cells by T lymphocytes. Prediction of cell-mediated autoimmune diseases by direct detection of autoreactive T cells in peripheral blood has proved elusive, in part because of their low frequency and reduced avidity for peptide MHC ligands. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

The expression of pro(insulin) in the thymus may lead to the negative selection of pro(insulin) autoreactive T cells and peripheral tolerance to this autoantigen in type 1 diabetes (T1D). We investigated whether proinsulin is expressed in the thymus of young nonobese diabetic (NOD) mice, whether T cells from naive NOD female mice at weaning are reactive to mouse proinsulin, and the role of proinsulin as a pathogenic autoantigen in T1D. Proinsulin II mRNA transcripts were detected in the thymus of 2-wk-old NOD mice at similar levels to other control strains. Despite this expression, proinsulin autoreactive T cells were detected in the periphery of 2- to 3-wk-old naive NOD mice. Peripheral T cells reactive to the insulin, glutamic acid decarboxylase 65 (GAD65), GAD67, and islet cell Ag p69 autoantigens were also detected in these mice, indicating that NOD mice are not tolerant to any of these islet autoantigens at this young age. T cell reactivities to proinsulin and islet cell Ag p69 exceeded those to GAD67, and T cell reactivity to proinsulin in the spleen and pancreatic lymph nodes was directed mainly against a p24-33 epitope that spans the B chain/C peptide junction. Intraperitoneal immunization with proinsulin perinatally beginning at 18 days of age delayed the onset and reduced the incidence of T1D. However, s.c. immunization with proinsulin initiated at 5 wk of age accelerated diabetes in female NOD mice. Our findings support the notion that proinsulin p24-33 may be a primary autoantigen epitope in the pathogenesis of T1D in NOD mice.

Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

Activated T cells and their naive precursors display different functional avidities for peptide/MHC, but are thought to have identical antigenic repertoires. We show that, following activation with a cognate mimotope (NRP), diabetogenic CD8(+) T cells expressing a single TCR (8.3) respond vigorously to numerous peptide analogs of NRP that were unable to elicit any responses from naive 8.3-CD8(+) T cells, even at high concentrations. The NRP-reactive, in vivo activated CD8(+) cells arising in pancreatic islets of nonobese diabetic mice are similarly promiscuous for peptide/MHC, and paradoxically this promiscuity expands as the aviditiy of the T cell population for NRP/MHC increases with age. Thus, activation and avidity maturation of T lymphocyte populations can lead to dramatic expansions in the range of peptides that elicit functional T cell responses.

Background: The inability to diagnose early rejection of an islet allograft has previously proved to be a major impediment to progress in clinical islet transplantation. The need to detect early rejection will become even more relevant as new tolerance-inducing protocols are evaluated in the clinic. We explored three novel approaches toward development of early diagnostic markers of islet rejection after islet allotransplantation., Methods: (a) Canine islet allograft transplant recipients were immunosuppressed for 1 month, then therapy was withdrawn. Serum glutamic acid decarboxylase antigen (GAD65), an endogenous islet protein, was monitored daily with a CO2 release assay. (b) Rodent islets were genetically engineered to express a unique foreign protein (beta-galactosidase) by using adenoviral vectors, and after allograft transplantation, the viral-specific protein was measured in serum using optical luminescence. (c) Rodents receiving islet allografts were immunosuppressed temporarily, and daily glucose tolerance tests were followed until graft failure occurred., Results: (a) Although serum monitoring of GAD65 antigen demonstrated elevated levels preceding loss of graft function in preliminary studies, the effect was not reproducible in all animals. (b) Genetically engineered rodent islets demonstrated normal insulin kinetics in vitro (insulin stimulation index 2.57+/-0.2 vs. 2.95+/-0.3 for control islets, P=ns), and purified viral protein products had a stable half-life of 8 hr in vivo. After islet allotransplantation, there were two peak elevations in serum viral proteins, confirming that an intra-islet "sentinel signal" could be detected serologically during acute rejection. There was no lead-time ahead of hyperglycemia, however. (c) Daily sequential intravenous glucose tolerance (IVGT) tests demonstrated evidence of allograft dysfunction (decline in KG) with a 2-day lead time to hyperglycemia (2.58+/-0.3 vs. 1.63+/-0.2%/min, respectively, P<0.001), with an accuracy of 89%, sensitivity of 78%, and specificity of 95%., Conclusions: Of the three diagnostic tests, metabolic assessment with an abbreviated IVGT was the most effective method of demonstrating early islet dysfunction due to rejection.

Islet transplantation offers the prospect of good glycemic control without major surgical risks. After our initial report of successful islet transplantation, we now provide further data on 12 type 1 diabetic patients with brittle diabetes or problems with hypoglycemia previous to 1 November 2000. Details of metabolic control, acute complications associated with islet transplantation, and long-term complications related to immunosuppression therapy and diabetes were noted. Insulin secretion, both acute and over 30 min, was determined after intravenous glucose tolerance tests (IVGTTs). The median follow-up was 10.2 months (CI 6.5-17.4), and the longest was 20 months. Glucose control was stable, with pretransplant fasting and meal tolerance-stimulated glucose levels of 12.5+/-1.9 and 20.0+/-2.7 mmol/l, respectively, but decreased significantly, with posttransplant levels of 6.3+/-0.3 and 7.5+/-0.6 mmol/l, respectively (P < 0.006). All patients have sustained insulin production, as evidenced by the most current baseline C-peptide levels 0.66+/-0.06 nmol/l, increasing to 1.29+/-0.25 nmol/l 90 min after the meal-tolerance test. The mean HbA1c level decreased from 8.3+/-0.5% to the current level of 5.8+/-0.1% (P < 0.001). Presently, four patients have normal glucose tolerance, five have impaired glucose tolerance, and three have post-islet transplant diabetes (two of whom need oral hypoglycemic agents and low-dose insulin (<10 U/day). Three patients had a temporary increase in their liver-function tests. One patient had a thrombosis of a peripheral branch of the right portal vein, and two of the early patients had bleeding from the hepatic needle puncture site; but these technical problems were resolved. Two patients had transient vitreous hemorrhages. The two patients with elevated creatinine levels pretransplant had a significant increase in serum creatinine in the long term, although the mean serum creatinine of the group was unchanged. The cholesterol increased in five patients, and lipid-lowering therapy was required for three patients. No patient has developed cytomegalovirus infection or disease, posttransplant lymphoproliferative disorder, malignancies, or serious infection to date. None of the patients have been sensitized to donor antigen. In 11 of the 12 patients, insulin independence was achieved after 9,000 islet equivalents (IEs) per kilogram were transplanted. The acute insulin response and the insulin area under the curve (AUC) after IVGTT were consistently maintained over time. The insulin AUC from the IVGTT correlated to the number of islets transplanted, but more closely correlated when the cold ischemia time was taken into consideration (r = 0.83, P < 0.001). Islet transplantation has successfully corrected labile type 1 diabetes and problems with hypoglycemia, and our results show persistent insulin secretion. After a minimum of 9,000 IEs per kilogram are provided, insulin independence is usually attained. An elevation of creatinine appears to be a contraindication to this immunosuppressive regimen. For the subjects who had labile type 1 diabetes that was difficult to control, the risk-to-benefit ratio is in favor of islet transplantation.

Biochemical and functional studies have demonstrated major histocompatibility complex (MHC) class II-restricted presentation of select epitopes derived from cytoplasmic antigens, with few insights into the processing reactions necessary for this alternate pathway. Efficient presentation of an immunodominant epitope derived from glutamate decarboxylase (GAD) was observed regardless of whether this antigen was delivered exogenously or via a cytoplasmic route into human histocompatibility leukocyte antigen class II-DR4(+) antigen-presenting cells. Presentation of exogenous as well as cytoplasmic GAD required the intersection of GAD peptides and newly synthesized class II proteins. By contrast, proteolytic processing of this antigen was highly dependent upon the route of antigen delivery. Exogenous GAD followed the classical pathway for antigen processing, with an absolute requirement for endosomal/lysosomal acidification as well as cysteine and aspartyl proteases resident within these organelles. Presentation of endogenous GAD was dependent upon the action of cytoplasmic proteases, including the proteasome and calpain. Thus, translocation of processed antigen from the cytoplasm into membrane organelles is necessary for class II-restricted presentation via this alternate pathway. Further trimming of these peptides after translocation was mediated by acidic proteases within endosomes/lysosomes, possibly after or before class II antigen binding. These studies suggest that processing of exogenous and cytoplasmic proteins occurs through divergent but overlapping pathways. Furthermore, two cytoplasmic proteases, the proteasome and calpain, appear to play important roles in MHC class II-restricted antigen presentation.