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Background: The incidence of type 1 diabetes has increased worldwide, particularly in younger children and those with lower genetic susceptibility. These observations suggest factors in the modern environment promote pancreatic islet autoimmunity and destruction of insulin-producing beta cells. The Environmental Determinants of Islet Autoimmunity (ENDIA) Study is investigating candidate environmental exposures and gene-environment interactions that may contribute to the development of islet autoimmunity and type 1 diabetes. Methods/design: ENDIA is the only prospective pregnancy/birth cohort study in the Southern Hemisphere investigating the determinants of type 1 diabetes in at-risk children. The study will recruit 1,400 unborn infants or infants less than six months of age with a first-degree relative (i.e. mother, father or sibling) with type 1 diabetes, across five Australian states. Pregnant mothers/infants will be followed prospectively from early pregnancy through childhood to investigate relationships between genotype, the development of islet autoimmunity (and subsequently type 1 diabetes), and prenatal and postnatal environmental factors. ENDIA will evaluate the microbiome, nutrition, bodyweight/composition, metabolome-lipidome, insulin resistance, innate and adaptive immune function and viral infections. A systems biology approach will be used to integrate these data. Investigation will be by 3-monthly assessments of the mother during pregnancy, then 3-monthly assessments of the child until 24 months of age and 6-monthly thereafter. The primary outcome measure is persistent islet autoimmunity, defined as the presence of autoantibodies to one or more islet autoantigens on consecutive tests. Discussion: Defining gene-environment interactions that initiate and/or promote destruction of the insulin-producing beta cells in early life will inform approaches to primary prevention of type 1 diabetes. The strength of ENDIA is the prospective, comprehensive and frequent systems-wide profiling from early pregnancy through to early childhood, to capture dynamic environmental exposures that may shape the development of islet autoimmunity. [ABSTRACT FROM AUTHOR] more...

Evaluating risk of developing type 1 diabetes (T1D) depends on determining an individual's HLA type, especially of the HLA DRB1 and DQB1 alleles. Individuals positive for HLA-DRB1*03 (DR3) or HLA-DRB1*04 (DR4) with DQB1*03:02 (DQ8) have the highest risk of developing T1D. Currently, HLA typing methods are relatively expensive and time consuming. We sought to determine the minimum number of single nucleotide polymorphisms (SNPs) that could rapidly define the HLA-DR types relevant to T1D, namely, DR3/4, DR3/3, DR4/4, DR3/X, DR4/X, and DRX/X (where X is neither DR3 nor DR4), and could distinguish the highest-risk DR4 type (DR4-DQ8) as well as the nonT1D--associated DR4-DQB1*03:01 type. We analyzed 19,035 SNPs of 10,579 subjects (7,405 from a discovery set and 3,174 from a validation set) from the Type 1 Diabetes Genetics Consortium and developed a novel machine learning method to select as few as three SNPs that could define the HLA-DR and HLA-DQ types accurately. The overall accuracy was 99.3%, area under curve was 0.997, true-positive rates were .0.99, and false-positive rates were,0.001. We confirmed the reliability of these SNPs by 10fold cross-validation. Our approach predicts HLA-DR/DQ types relevant to T1D more accurately than existing methods and is rapid and cost-effective. [ABSTRACT FROM AUTHOR] more...

Background We investigated whether graft produced anti-human CD2, mediated by adenovirus (Adv) transduction of pig neonatal islet cell clusters ( pNICC), would protect xenografts in a humanized mouse model from immune attack and whether such immunosuppression would remain local. Methods A mouse anti-human CD2 Ab (CD2hb11) previously generated by us was genetically engineered to produce chimeric and humanized versions. The three forms of CD2hb11 were named dilimomab (mouse), diliximab (chimeric) and dilizumab (humanized). All 3 forms of CD2hb11 Ab were tested for their ability to bind CD3+ human T cells and to inhibit a human anti-pig xenogeneic mixed lymphocyte reaction (MLR). They were administered systemically in a humanized mouse model in order to test their ability to deplete human CD3+ T cells and whether they induced a cytokine storm. An adenoviral vector expressing diliximab was generated for transduction of p NICC. Humanized mice were transplanted with either control-transduced p NICC or diliximab-transduced p NICC and human T cells within grafts and spleens were enumerated by flow cytometry. Results Dilimomab and diliximab inhibited a human anti-pig xenogeneic response but dilizumab did not. All 3 forms of CD2hb11 Ab bound human T cells in vitro though dilimomab and diliximab exhibited 300-fold higher avidity than dilizumab. All 3 anti-CD2 Abs could deplete human CD3+ T cells in vivo in a humanized mouse model without inducing upregulation of activation markers or significant release of cytokines. Humanized mice transplanted with diliximab-transduced p NICC afforded depletion of CD3+ T cells at the graft site leaving the peripheral immune system intact. Conclusions Local production of a single Ab against T cells can reduce graft infiltration at the xenograft site and may reduce the need for conventional, systemic immunosuppression. [ABSTRACT FROM AUTHOR] more...

Objective: The effectiveness of tolerizing immunotherapeutic strategies, such as anti-CD40L or dendritic cells (DCs), is greater when administered to young nonobese diabetic (NOD) mice than at peak insulitis. RelB(lo) DCs, generated in the presence of an nuclear factor-κB inhibitor, induce T-regulatory (Treg) cells and suppress inflammation in a model of rheumatoid arthritis. Interleukin (IL)-1β is overexpressed in humans and mice at risk of type 1 diabetes, dysregulates Treg cells, and accelerates diabetes in NOD mice. We investigated the relationship between IL-1β production and the response to RelB(lo) DCs in the prediabetic period.Research Design and Methods: We injected RelB(lo) DCs subcutaneously into 4- or 14-week-old NOD mice and tracked the incidence of diabetes and effect on Treg cell function. We measured the expression of proinflammatory cytokines by stimulated splenocytes and unstimulated islets from mice of different ages and strains and proliferative and cytokine responses of T effectors to Treg in vitro.Results: Tolerizing RelB(lo) DCs significantly inhibited diabetes progression when administered to 4-week-old but not 14-week-old mice. IL-1β production by NOD splenocytes and mRNA expression by islets increased from 6 to 16 weeks of age when major histocompatibility complex (MHC)-restricted islet antigen presentation to autoreactive T-cells occurred. IL-1 reduced the capacity of Treg cells to suppress effector cells and promoted their conversion to Th17 cells. RelB(lo) DCs exacerbated the IL-1-dependent decline in Treg function and promoted Th17 conversion.Conclusions: IL-1β, generated by islet-autoreactive cells in MHC-susceptible mice, accelerates diabetes by differentiating Th17 at the expense of Treg. Tolerizing DC therapies can regulate islet autoantigen priming and prevent diabetes, but progression past the IL-1β/IL-17 checkpoint signals the need for other strategies. [ABSTRACT FROM AUTHOR] more...

OBJECTIVE--Insulin resistance and other features of the metabolic syndrome have been causally linked to adipose tissue macrophages (ATMs) in mice with diet-induced obesity. We aimed to characterize macrophage phenotype and function in human subcutaneous and omental adipose tissue in relation to insulin resistance in obesity. RESEARCH DESIGN AND METHODS--Adipose tissue was obtained from lean and obese women undergoing bariatric surgery. Metabolic markers were measured in fasting serum and ATMs characterized by immunohistology, flow cytometry, and tissue culture studies. RESULTS--ATMs comprised CD11c[sup +]CD206[sup +] cells in "crown" aggregates and solitary CD11c[sup -]CD206[sup +] cells at adipocyte junctions. In obese women, CD11c[sup +] ATM density was greater in subcutaneous than omental adipose tissue and correlated with markers of insulin resistance. CD11c[sup +] ATMs were distinguished by high expression of integrins and antigen presentation molecules; interleukin (IL)-1β, -6, -8, and -10; tumor necrosis factor-α and CC chemokine ligand-3, indicative of an activated, proinflammatory state. In addition, CD11c[sup +] ATMs were enriched for mitochondria and for RNA transcripts encoding mitochondrial, proteasomal, and lysosomal proteins, fatty acid metabolism enzymes, and T-cell chemoattractants, whereas CD11c[sup -] ATMs were enriched for transcripts involved in tissue maintenance and repair. Tissue culture medium conditioned by CD11c[sup +] ATMs, but not CD11c[sup -] ATMs or other stromovascular cells, impaired insulin-stimulated glucose uptake by human adipocytes. CONCLUSIONS--These findings identify proinflammatory CD11c[sup +] ATMs as markers of insulin resistance in human obesity. In addition, the machinery of CD11c[sup +] ATMs indicates they metabolize lipid and may initiate adaptive immune responses. [ABSTRACT FROM AUTHOR] more...

The homeodomain transcription factor Pdx1 is essential for pancreas development. To investigate the role of Pdx1 in the adult pancreas, we employed a mouse model in which transcription of Pdx1 could be reversibly repressed by administration of doxycycline. Repression of Pdx1 in adult mice impaired expression of insulin and glucagon, leading to diabetes within 14 days. Pdx1 repression was associated with increased cell proliferation predominantly in the exocrine pancreas and upregulation of genes implicated in pancreas regeneration. Following withdrawal of doxycycline and derepression of Pdx1, normoglycemia was restored within 28 days; during this period, Pdx1(+)/Ins(+) and Pdx(+)/Ins(-) cells were observed in association with the duct epithelia. These findings confirm that Pdx1 is required for beta-cell function in the adult pancreas and indicate that in the absence of Pdx1 expression, a regenerative program is initiated with the potential for Pdx1-dependent beta-cell neogenesis. [ABSTRACT FROM AUTHOR] more...

Abstract: We previously described differences in the 50% protective dose and isotype-specific antibody secreting cell (ASC) responses to US and Russian influenza A cold-adapted (ca) donor strains in the lungs of BALB/c mice [Wareing MD, Watson JM, Brooks MJ, Tannock GA. Immunogenic and isotype-specific responses to Russian and US cold-adapted influenza A vaccine donor strains A/Leningrad/134/17/57, A/Leningrad/134/47/57, and A/Ann Arbor/6/60 (H2N2) in mice. J Med Virol 2001;65(1):171–7]. A/Leningrad/134/17/57(Len/17-ca) was shown to be a superior immunogen to A/Leningrad/134/47/57-ca (Len/47-ca), which, in turn, was superior to A/Ann Arbor/6/60-ca (AA-ca) but no other comparative data exist. In order to extend our findings and determine a means for selecting the most immunogenic ca influenza A vaccine, the intracellular cytokine responses by CD4+ and CD8+ T cells to AA-ca, Len/47-ca and Len/17-ca and their respective wild-type parental viruses were compared in mice. Day 5 after infection with Len/17-ca, when levels of IL-2, -4 and -10 were highest in the mediastinal lymph nodes (MLN) and lungs, was chosen as the optimum time to harvest lymphocytes and 72h was determined to be the optimum re-stimulation period for lymphocytes by APCs. Under these conditions, the frequency of CD4+ and CD8+ cells expressing cytokines was highest in the lungs compared with the MLN. A dominant IL-6 response was induced, although all virus strains induced a Th1/Th2 cytokine profile. While the CD8+ cytokine response appeared non-specific, the cytokine response elicited in the lungs by CD4+ cells to Len/17-ca-inoculation was greater than that induced by Len/47-ca, or AA/ca. The CD4+ cytokine response in the lungs may be a useful measure of immunogenicity to determine the most effective influenza reassortant for inclusion in vaccines. [Copyright &y& Elsevier] more...

The epidermal growth factor (EGF) family is implicated in the development and function of multiple cells and organs, including the pancreas. We used a serum-free, low-cell density culture system to investigate the effect of EGFs on fetal pancreas cells. By RT-PCR, the EGF receptors ErbB 1–3 were detected in the developing mouse pancreas between embryonic day (E) 13.5 and E17.5, whereas ErbB4 was not detected until E17.5. The presence but not absence of the basement membrane glycoprotein laminin-1, betacellulin, and to a lesser extent EGF, transforming growth factorα, heparin binding EGF, and epiregulin induced E15.5 pancreatic cells to proliferate and form cystoid and solid colonies. These results demonstrate that laminin-1 and EGF signaling pathways interact to promote pancreas development. [ABSTRACT FROM AUTHOR] more...

The ability to measure proliferation of autoantigen-specific T cells is critical for the evaluation of cellular immune function. Using a novel, sensitive, CFSE-based assay, we were able to directly quantitate autoantigenspecific CD4+ T cell proliferation. However, peripheral blood cells from healthy, pre-diabetic and diabetic donors exhibited overlap in responses to glutamic acid decarboxylase (GAD65) and proinsulin (PI). This indicates that autoantigen-induced CD4+ T cell proliferation in a functionally complex cell population may not discriminate disease in the general population. Clear discrimination was found between diabetic and healthy sibs, suggesting the need to standardize the genetic and environmental background. In addition, the ability of the CFSE assay to allow analysis of the phenotype and function of autoantigen-responsive T cells may improve discrimination. [ABSTRACT FROM AUTHOR] more...

OBJECTIVE -- Mucosal administration of insulin retards development of autoimmune diabetes in the nonobese diabetic mouse model. We conducted a double-blind crossover study in humans at risk for type 1 diabetes to determine if intranasal insulin was safe, in particular did not accelerate β-cell destruction, and could induce immune effects consistent with mucosal tolerance. RESEARCH DESIGN AND METHODS -- A total of 38 individuals, median age 10.8 years, with antibodies to one Or more pancreatic islet antigens (insulin, GAD65, or tyrosine phosphatase-like insulinoma antigen 2) were randomized to treatment with intranasal insulin (1.6 mg) or a carrier solution, daily for 10 days and then 2 days a week for 6 months, before crossover. The primary outcome was β-cell function measured as first-phase insulin response (FPIR) to intravenous glucose at 0, 6, and 12 months and then yearly; the secondary outcome was immunity to islet antigens, measured monthly for 12 months. RESULTS -- No local or systemic adverse effects were observed. Diabetes developed in 12 participants with negligible β-cell function at entry after a median of 1.1 year. Of the remaining 26, the majority had antibodies to two or three islet antigens and FPIR greater than the first percentile at entry, as well as β-cell function that generally remained stable over a median follow-up of 3.0 years. Intranasal insulin was associated with an increase in antibody and a decrease in T-cell responses to insulin. CONCLUSIONS -- Results from this pilot study suggest that intranasal insulin does not accelerate loss of β-cell function in individuals at risk for type 1 diabetes and induces immune changes consistent with mucosal tolerance to insulin. These findings justify a formal trial to determine if intranasal insulin is immunotherapeutic and retards progression to clinical diabetes. [ABSTRACT FROM AUTHOR] more...