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Extracellular factors that regulate the growth and differentiation of cell lineages in the pancreatic primordia are poorly understood. Identification of these factors for pancreatic islet Β-cells could open new avenues [...]

A dendritic cell (DC) imbalance with a marked deficiency in CD4−8+ DC occurs in non-obese diabetic (NOD) mice, a model of human autoimmune diabetes mellitus. Using a NOD congenic mouse strain, we find that this CD4−8+ DC deficiency is associated with a gene segment on chromosome 4, which also encompasses non-MHC diabetes susceptibility loci. Treatment of NOD mice with fms-like tyrosine kinase 3 ligand (FL) enhances the level of CD4−8+ DC, temporarily reversing the DC subtype imbalance. At the same time, fms-like tryosine kinase 3 ligand treatment blocks early stages of the diabetogenic process and with appropriately timed administration can completely prevent diabetes development. This points to a possible clinical use of FL to prevent autoimmune disease.

The Melbourne Prediabetes Family Study, a prospective study of first-degree relatives of people with type 1 diabetes (T1D), provided an opportunity to examine late-onset autoimmune diabetes within the context of a family history of T1D. We compared genetic, immunologic, and clinical features in relatives of people with T1D, who developed early- versus late-onset diabetes.

Cell lineage development is a finely tuned process of proliferation and differentiation, survival and apoptosis, that is regulated by numerous extracellular signals. Here we review some of the extracellular signals—including insoluble cell-cell and extracellular matrix-cell interactions, as well as soluble factors—that appear critical for pancreatic β-cell development. Knowledge of how these signals control the development of pancreatic endocrine stem/precursor cells into fully functional insulin-secreting βcells is a platform for the restoration of β-cell function and the cure therapy of type 1 diabetes.

ABSTRACTRotavirus infection in children at risk of developing type 1 diabetes has been temporally associated with development of pancreatic islet autoantibodies. In this study, nonobese diabetic mice were shown to be susceptible to rhesus rotavirus infection and pancreatic islets from nonobese diabetic mice, nonobese diabetes-resistant mice, fetal pigs, and macaque monkeys supported various degrees of rotavirus growth. Human rotaviruses replicated in monkey islets only. This islet susceptibility shows that rotavirus infection of the pancreas in vivo might be possible.

Pancreatic islet xenotransplantation has been advocated as a way of overcoming the shortage of human donor tissue for the treatment of type 1 diabetes. However, the potent immune response against xenografts is a major barrier to their use. We show that a short course of the anti‐CD45RB antibody, MB23G2, prolongs survival of fetal pig pancreas grafts in mice. To investigate this effect further we used an i.p. xenograft model in which both donor pig cells and host inflammatory cells can be expediently recovered and analyzed. Graft prolongation was associated with reduced T cell and macrophage infiltration, and reduced production of both Th1 and Th2 cytokines at the graft site. Graft survival was further increased and T cell infiltration further reduced by combining anti‐CD45RB antibody with co‐stimulation blockade. The primary effect of anti‐CD45RB antibody may be on CD4 T cells, in keeping with the marked reduction in T cell cytokine production in both spleen and graft sites. This concurs with previous studies in allogeneic models that indicate that this antibody perturbs T cell responses by modifying signaling via the TCR. In addition, anti‐CD45RB treatment led to reduced expression of LFA‐1 and CD62 ligand (CD62L) on CD4 T cells, independent of antigenic challenge. LFA‐1 may enhance co‐stimulation, and both LFA‐1 and CD62L are involved in T cell trafficking. Their reduced expression provides an explanation why the T cell pool is reduced in lymph nodes. We conclude that modulation of inflammation against xenografts by anti‐CD45RB antibody is due to effects on both T cell priming and trafficking.

The innate immune system existed prior to the emergence of adaptive immunity in sharks and higher vertebrates. Homologues of many mammalian innate immune‐system elements such as the toll‐like receptors exist in species as distant as Drosophila. Selective pressure has led to the development of highly conserved, soluble, and cell‐surface receptors that recognize functionally essential molecules shared by microbial pathogens. It is thought that molecular patterns that exquisitely distinguish pathogenic cells from mammalian cells are recognized. Therefore, it would seem unlikely that innate immune‐system elements should recognize mammalian tissues. However, there is increasing evidence to suggest that this is the case and that innate immunity promotes rejection of transplanted mammalian tissues, particularly those from other species (xenografts). Evidence for innate recognition of mammalian grafts, the nature of this recognition, and the bi‐directional interactions between innate and adaptive immunity that contribute to graft rejection are discussed in this review, with the emphasis on nonvascular xenografts.

Cytotoxic CD8 T lymphocytes (CTL) are effectors of pancreatic islet β-cell destruction in type 1 diabetes but, with the exception of a single report, CTL to islet antigen peptides have not been identified. We used autologous blood monocyte-derived dendritic cells to elicit HLA-A2-restricted CTL to a peptide, MVWESGCTV (aa 797–805), that is contiguous with a dominant CD4 T-cell epitope in the islet antigen tyrosine phosphatase IA-2. IA-2 peptide-specific CTL activity measured as 51Cr release from autologous lymphoblasts was detected in 2/6 islet antibody-positive relatives at high risk for type 1 diabetes but also in 2/6 closely HLA-matched controls. All subjects had CTL activity to an HLA-A2-restricted Epstein–Barr virus peptide. CTL to the IA-2 self-peptide were therefore not disease-specific, consistent with other evidence that autoreactive T cells are present in healthy individuals.

Background: The ability to manipulate the development of pancreatic insulin-producing βcells has implications for the treatment of type 1 diabetes. Previously, we found that laminin-1, a basement membrane trimeric glycoprotein, promotes β-cell differentiation. We have investigated the mechanism of this effect, using agents that block the receptors for laminin-1, α6integrin, and α-dystroglycan (α-DG). Materials and Methods: Dissociated cells from 13.5-day postcoitum (dpc) fetal mouse pancreas were cultured for 4 days with laminin-1, with and without monoclonal antibodies and other agents known to block integrins or α-DG. Fetuses fixed in Bouin’s solution or fetal pancreas cells fixed in 4% paraformaldehyde were processed for routine histology and for immunohistology to detect hormone expression and bromodeoxyuridine (BrdU) uptake. Results: Blocking the binding of laminin-1 to α6integrin with a monoclonal antibody, GoH3, abolished cell proliferation (BrdU uptake) and doubled the number of βcells. Inhibition of molecules involved in α6integrin signaling (phosphotidylinositol 3-kinase, F-actin, or mitogen-activated protein kinase) had a similar effect. Nevertheless, βcells appeared to develop normally in α6integrin-deficient fetuses. Blocking the binding of laminin-1 to α-DG with a monoclonal antibody, IIH6, dramatically decreased the number of βcells. Heparin, also known to inhibit laminin-1 binding to α-DG, had a similar effect. In the presence of heparin, the increase in βcells in response to blocking α6integrin with GoH3 was abolished. Conclusions: These findings reveal an interplay between α6integrin and α-DG to regulate laminin-1-induced β-cell development. Laminin-1 had a dominant effect via α-DG to promote cell survival and β-cell differentiation, which was modestly inhibited by α6signaling.

Objectives: To determine the utility of various autoantibodies in predicting progression to clinical diabetes in first‐degree relatives of patients with type 1 diabetes mellitus. Participants: 3315 first‐degree relatives of patients with type 1 diabetes (1161 parents, 1206 siblings and 948 offspring) recruited through diabetes clinics, private endocrinologists, Diabetes Australia and the Juvenile Diabetes Foundation. Main outcome measures: Prevalence of islet cell antibodies (ICA) levels ≥20 JDFu, insulin autoantibodies (IAA) levels > 100nU/ml, and antibodies to glutamic acid decarboxylase (GADAb) and tyrosine phosphatase IA2 (IA2Ab); change in cell function over time; and development of clinical diabetes. Results: 2.6% of relatives had elevated ICA levels, 1.3% had elevated IAA levels and 0.3% had both. High ICA levels were significantly more frequent in siblings than in offspring or parents, and were more frequent in relatives younger than 20 years. GADAb were detected in 68% and IA2Ab in 57% of relatives with elevated ICA and/or IAA levels. Diabetes developed in 33 relatives (25 siblings, 2 offspring and 6 parents). Before diagnosis of clinical diabetes, high ICA levels were detected in 18 (58%), high IAA levels in 7 (23%), both in 5 (15%), and either in 19 (61%); GADAb were detected in 26 (84%), IA2Ab in 13 (42%), both in 11 (35%), and either in 28 (90%). First phase insulin release (FPIR) less than 50mU/L was very strongly associated with progression to diabetes. In relatives with FPIR initially greater than 50mU/L who eventually developed diabetes, there was a gradual and continuous reduction in FPIR over time before diagnosis. Conclusions: Type 1 diabetes can be diagnosed in the preclinical stage. The recently described antibodies to glutamic acid decarboxylase and tyrosine phosphatase IA2 appear superior to ICA as screening tools for the preclinical diagnosis of type 1 diabetes.

Background: Insulin (1) and glutamic acid decarboxylase (GAD) (2) are both autoantigens in insulin-dependent diabetes mellitus (IDDM), but no molecular mechanism has been proposed for their association. We have identified a 13 amino acid peptide of proinsulin (amino acids 24–36) that bears marked similarity to a peptide of GAD65 (amino acids 506–518) (G. Rudy, unpublished). In order to test the hypothesis that this region of similarity is implicated in the pathogenesis of IDDM, we assayed T cell reactivity to these two peptides in subjects at risk for IDDM. Materials and Methods: Subjects at risk for IDDM were islet cell antibody (ICA)-positive, first degree relatives of people with insulin-dependent diabetes. Peripheral blood mononuclear cells from 10 pairs of at-risk and HLA-DR matched control subjects were tested in an in vitro proliferation assay. Results: Reactivity to both proinsulin and GAD peptides was significantly greater among at-risk subjects than controls (proinsulin; p< 0.008; GAD: p< 0.018). In contrast to reactivity to the GAD peptide, reactivity to the proinsulin peptide was almost entirely confined to the at-risk subjects. Conclusions: This is the first demonstration of T cell reactivity to a proinsulin-specific peptide. In addition, it is the first example of reactivity to a minimal peptide region shared between two human autoimmune disease-associated self antigens. Mimicry between these similar peptides may provide a molecular basis for the conjoint autoantigenicity of proinsulin and GAD in IDDM.

Autoantibodies to glutamic acid decarboxylase (GAD) are present in humans before and after the onset of clinical insulin-dependent diabetes (IDD). The non-obese diabetic (NOD) mouse, a model of human IDD, develops mononuclear cell infiltration of the pancreatic islets ('insulitis') associated particularly in females with T cell-mediated destruction of the islet β cells. In NOD mice of both sexes we detected serum antibodies to GAD (GAD Ab) that precipitate mouse brain GAD enzymatic activity. Antibodies in NOD sera also precipitate a M_r 65,000 protein from Triton X-100 extracts of ³⁵S-methionine-labelled NOD islets, identical in size to that precipitated by a monoclonal antibody to GAD. GAD Ab were not detected in other mouse strains. There were significant differences in the frequency, level and age at initial detection of GAD Ab between females of the NOD/Lt and NOD/WEHI lines, previously shown to have a higher and lower incidence of diabetes, respectively. Comparing NOD/Lt (n=26) and NOD/WEHI (n=20) females, in which diabetes occurred in 38% and 20% by 150 days, the frequency of elevated GAD Ab was 50 vs. 80%, the mean maximum GAD Ab levels 21.1 vs. 30.6% and the mean age at which GAD Ab were first detected 94 vs. days. No significant differences in these parameters were observed between male mice of either line. There was a significant negative correlation between the level of GAD Ab and the degree of insulitis in female mice from both lines. GAD Ab were not a prerequisite for the development of diabetes. In 7 of 10 female mice the onset of diabetes was preceded by a decrease of GAD Ab levels into the normal range. These findings indicate that, while GAD is a target of autoimmunity in the NOD mouse, GAD Ab do not necessarily correlate with the development of diabetes. Indeed, the difference between the two NOD lines and the inverse relationship with insulitis suggests that a strong humoral response to GAD may be associated with a less destructive pathology, as proposed in humans 'at-risk' for IDD. Copyright 1994, 1999 Academic Press

Background: Individuals at risk for insulin-dependent diabetes mellitus (IDDM), with an affected first-degree relative, can be identified by the presence of islet cell antibodies (ICA). ICA-positive relatives progress at variable rates to IDDM and identification of those at highest risk is a prerequisite for possible preventative treatment. Those who develop IDDM may exhibit less genetic heterogeneity than their ICA-positive or ICA-negative relatives. Specific human leucocyte antigen (HLA) genes predispose to IDDM but could also influence the rate of progression of preclinical disease. Therefore, by comparing HLA antigen frequencies between first-degree relatives, we sought to identify HLA genes that influence progression to IDDM. Materials and Methods: HLA antigen frequencies were compared in 68 IDDM, 53 ICA-positive, and 96 ICA-negative first-degree relatives from 40 Caucasoid families. Predictions were tested in a panel of 270 unrelated IDDM subjects. HLA typing was performed serologically (HLA class I and II) and by sequence-specific oligotyping (11th International Histocompatibility Workshop protocol) (HLA class II). ICA tests were measured by an internationally standardized indirect immunofluorescence assay. Results: In general, known susceptibility class II HLA antigens increased in frequency and known protective class II HLA antigens decreased in frequency, from ICA-negative to ICA-positive to IDDM relatives. Thus, DR4 and DQ8 increased whereas DR2 and DQ6 decreased; DR3 and DQ2 increased from ICA-negative to ICA-positive relatives, but not further in IDDM relatives. The high-risk DR3, 4 phenotype increased across the three groups; DR4,X was unchanged, and DR3,X and DRX,X both decreased. The HLA class I antigen, A24, occurred more frequently in ICA-positive relatives who developed IDDM and, in 270 unrelated IDDM subjects, was associated with an earlier age at diagnosis of IDDM in those with the lower risk class II phenotypes DR4,4 and DR3,X. Conclusions: HLA-DR3 and DQ2 predispose to islet autoimmunity, but not development of clinical IDDM in the absence and DR4 and DQ8. DR4 and DQ8 predispose to the development of clinical IDDM in ICA-positive relatives, in combination with DR3-DQ2 and other haplotypes but not when homozygous. HLA-A24 is significantly associated with rapid progression to IDDM in ICA-positive relatives and with an earlier age at clinical diagnosis. Analysis of IDDM families reveals that HLA genes not only predispose to islet autoimmunity but influence progression to clinical disease. The findings have implications for identifying high-risk relatives as candidates for possible preventative therapy.

The class II major histocompatibility complex molecule I-Ag7 is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its β chain. To identify the requirements for peptide binding to I-Ag7 and thereby potentially pathogenic T cell epitopes, we analyzed a known I-Ag7-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9–27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-Ag7 M12-Y20/ K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-Ag7 deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-Ag7–restricted T cell epitopes or eluted from I-Ag7. Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

Cellular immune hyporesponsiveness can be induced by the presentation of soluble protein antigens to mucosal surfaces. Most studies of mucosa-mediated tolerance have used the oral route of antigen delivery and few have examined autoantigens in natural models of autoimmune disease. Insulin is an autoantigen in humans and nonobese diabetic (NOD) mice with insulindependent diabetes mellitus (IDDM). When we administered insulin aerosol to NOD mice after the onset of subclinical disease, pancreatic islet pathology and diabetes incidence were both significantly reduced. Insulin-treated mice had increased circulating antibodies to insulin, absent splenocyte proliferation to the major epitope, insulin B chain amino acids 9–23, which was associated with increased IL-4 and particularly IL-10 secretion, and reduced proliferation to glutamic acid decarboxylase, another islet autoantigen. The ability of splenocytes from insulin-treated mice to suppress the adoptive transfer of diabetes to nondiabetic mice by T cells of diabetic mice was shown to be caused by small numbers of CD8 γδ T cells. These findings reveal a novel mechanism for suppressing cell-mediated autoimmune disease. Induction of regulatory CD8 γδ T cells by aerosol insulin is a therapeutic strategy with implications for the prevention of human IDDM.

Two examples of fatal poisoning by the weedkiller paraquat are presented. The first patient deliberately took several hundred millilitres of the solution, and died 36 hours later from intractable peripheral circulatory failure. The second patient accidentally swallowed about 30 ml of the solution, and died 23 days later from a progressive proliferative reaction in the lungs, with hyaline membrane formation. There was indirect evidence of disseminated intravascular coagulation, but this condition was relieved with heparin therapy, and cyclophosphamide treatment may have helped the patient to survive longer than any other patient with this condition. The histological appearances of the lung resembled those of oxygen poisoning, and support the theory that oxygen is a factor in the toxic reaction to paraquat.