Susceptibility to diabetes is widely distributed in normal class IIu haplotype rats

Abstract Aims/hypothesis. We did experiments to explore the pathways putatively leading to Type I (insulin-dependent) diabetes mellitus, and their association with the MHC locus, the major genetic determinant of disease susceptibility.?Methods. Normal MHC congenic rat strains that do not spontaneously develop diabetes or any other autoimmune syndrome were injected with the interferon-alpha inducer polyinosinic-polycytidylic acid (Poly IC).?Results. Insulitis and diabetes developed only in strains expressing Class II^u genes and was independent of the Class I haplotype. Poly IC induced islet cell Class I hyperexpression, up regulation of pancreatic endothelial intercellular adhesion molecule-1 and vascular adhesion molecule-1 and a T-cell and macrophage infiltration of the pancreatic interstitium in all rat strains studied, including diabetes-resistant strains. Poly IC also induced the generation of diabetes-transferring spleen cells in most Class II^u haplotype rats, including the diabetes-resistant WF rat.?Conclusion/Interpretation. The minimum requirements for autoimmune diabetes development in the rat include: RT1 Class II^u genes, a T-cell repertoire containing beta-cell autoreactive T cells and a triggering event which breaks tolerance by the local up regulation of pancreatic endothelial adhesion receptors. Even when all of the minimum requirements have, however, been met, most Class II^u rats do not develop diabetes in response to autoimmune stimuli. It is clear, nonetheless, that susceptibility to diabetes is widely distributed in the RT1 ^u rat. [Diabetologia (2000) 43: 890–898] Received: 8 February 2000 and in revised form: 27 March 2000     

The role of viruses in Type I diabetes: two distinct cellular and molecular pathogenic mechanisms of virus-induced diabetes in animals

Type I (insulin-dependent) diabetes mellitus results from the progressive loss of pancreatic beta cells. Environmental factors are believed to play an important part in the development of Type I diabetes by influencing the penetrance of diabetes susceptibility genes. As one environmental factor, the virus has long been considered to play a part in this disease. To date 13 different viruses have been reported to be associated with the development of Type I diabetes in humans and in various animal models. The most clear and unequivocal evidence that a virus induces diabetes in animals comes from studies on the d variant of the encephalomyocarditis (EMC-D) virus in mice and the Kilham rat virus (KRV) in rats. The infection of genetically susceptible strains of mice with a high titre of EMC-D virus results in the development of diabetes within 3 days. This is largely due to the rapid destruction of beta cells by the replication of the virus within the beta cells. In contrast, the infection of mice with a low titre of EMC-D virus results in a limited replication of the virus before the induction of neutralizing anti-virus antibody and the subsequent recruitment of activated macrophages. The Src kinases, particularly hck, play an important part in the activation of macrophages and the subsequent production of tumour necrosis factor (TNF)-α, interleukin (IL)-1β and nitric oxide (NO), leading to the destruction of beta cells which results in the development of diabetes. The Kilham rat virus causes autoimmune diabetes in diabetes resistant (DR)-BB rats without infection of beta cells. The infection of DR-BB rats with KRV results in the disruption of the finely tuned immune balance of Th1-like CD45RC⁺CD4⁺ and Th2-like CD45RC^-CD4⁺ T cells, leading to the selective activation of beta-cell-cytotoxic effector T cells. [Diabetologia (2001) 44: 271–285]     

Complete mutation scan of the human Fas ligand gene: Linkage studies in Type I diabetes mellitus families

Aims/hypothesis: Type I (insulin-dependent) diabetes mellitus is the result of a T-cell regulated selective destruction of pancreatic beta cells. There is evidence that the apoptosis inducing T-cell effector, Fas ligand (FasL) could be involved in the pathogenesis of Type I diabetes, probably because FasL-mediated apoptosis is important in maintaining peripheral self-tolerance and in down-regulating an immune response. We therefore evaluated the human FasL gene FASL on chromosome 1q23 as a candidate susceptibility gene for Type I diabetes. Methods: The entire FASL (promoter, exons 1–4 and 3'UTR) was scanned for polymorphisms using single strand conformational polymorphism-heteroduplex analysis and direct sequencing. Results: We identified two novel polymorphisms, a g-C843T and a g-A475T, in a negative regulatory region of the promoter. A Danish Type I diabetes family collection of 1143 subjects comprising 257 families (420 affected and 252 unaffected offspring) was typed for the g-C843T polymorphism and for a FASL microsatellite. Haplotypes were established and data were analysed using the extended transmission disequilibrium test. Conclusion/interpretation: We found no overall evidence for linkage in the presence of association of the FASL polymorphism to Type I diabetes and conclude that FASL does not contribute to the genetic susceptibility to Type I diabetes. [Diabetologia (2002) 45: 134–139] Received: 4 July 2001 and in revised form: 12 September 2001     

Fas and Fas ligand expression in inflamed islets in pancreas sections of patients with recent-onset Type I diabetes mellitus

Abstract Aims/hypothesis. Type I (insulin-dependent) diabetes results mainly from T-cell-mediated autoimmune destruction of pancreatic beta cells. Cytotoxic T lymphocytes destroy target cells via a perforin-based or Fas-based mechanism. Our previous study indicated that the Fas-Fas ligand (FasL) pathway is required for the development of autoimmune diabetes in the NOD mouse. We now investigated whether or not the Fas-FasL system is involved in the beta-cell destruction in human Type I diabetes. Methods. We immunohistochemically analysed pancreas biopsy specimens of 13 recent-onset patients. Results. Pancreatic islets were identified but showed various degrees of reduction in beta-cell volume in all patients. Out of 13 patients 6 had insulitis. In these 6 patients Fas was expressed in both the islets and infiltrating cells but not in either cell type in the 7 other patients without insulitis. Double immunostaining showed that Fas was positive in 92.2 to 97.7 % of beta cells but only in 17.6 to 46.7 % of alpha cells in Fas-positive, insulin-remaining islets. We found FasL was expressed exclusively in islet-infiltrating cells in patients with insulitis. Double immunostaining revealed that the most prevalent phenotype of FasL-positive cells was CD8, which was followed by macrophages and CD4. Conclusion/interpretation. The interaction between Fas on beta cells and FasL on infiltrating cells might trigger selective apoptotic beta-cell death in inflamed islets, leading to immune-mediated Type I diabetes. [Diabetologia (1999) 42: 1332–1340] Received: 3 March 1999 and in final revised form: 9 July 1999     

Nicotinamide protects human beta cells against chemically-induced necrosis, but not against cytokine-induced apoptosis

Summary Nicotinamide intervention trials are presently undertaken to prevent Type I (insulin-dependent) diabetes in high risk subjects. They are based on studies in rodents reporting nicotinamide protection against beta-cell injury in vitro and in vivo. This study examines whether nicotinamide can protect human beta cells in vitro. At concentrations (2 and 5 mmol/l) to protect rat beta cells against necrosis by streptozotocin or hydrogen peroxide, nicotinamide prevents hydrogen peroxide-induced necrosis of human beta cells. As with rat beta cells, nicotinamide fails to protect human beta cells against apoptosis induced by a combination of the cytokines interleukin-1β , interferon-γ and tumour necrosis factor-α. In rat beta cells, nicotinamide (2 to 20 mmol/l) was also found to induce apoptosis, in particular during the days following its protection against necrosis; this cytotoxic effect was not observed with human beta cells. These data demonstrate that nicotinamide can protect human beta cells against radical-induced necrosis, but not against cytokine-induced apoptosis. This effect is not associated with a delayed apoptosis as in rat beta cells. [Diabetologia (1999) 42: 55–59] Received: 7 July 1998 and in revised form: 10 September 1998     

Apoptosis and disease progression in the spontaneously diabetic BB/S rat

Aims/hypothesis. Type I (insulin-dependent) diabetes mellitus is an autoimmune disease culminating in pancreatic beta-cell destruction. A role for apoptosis in this destruction has been suggested, although controversy exists over the identity of the apoptotic cells and the time of onset of apoptosis. This study investigates the extent and timing of islet cell apoptosis in vivo in the spontaneously diabetic BB/S rat. Methods. Pancreatic biopsies were taken from 30 diabetes-prone and 6 diabetes-resistant BB/S rats matched for age. Animals were serially biopsied before, during and after development of diabetes and apoptotic cells analysed in serial sections. The diabetes-prone group included animals (n = 6) that had insulitis but did not develop diabetes. Results. Apoptosis was not detected in any pancreatic sections from diabetes resistant animals at any age investigated or from any animal before 50 days of age. By 68 days, apoptosis was, however, detectable in both the diabetes-prone group and in the group that had insulitus but did not develop diabetes and this correlated with a decrease in pancreatic insulin staining and a development of insulitis. There was a further increase in apoptosis in the diabetes-prone group at 85 days, which coincided with the time of onset of diabetes (84 days). In addition, there was a sixfold increase in intra-islet apoptosis between 68 and 85 days in the diabetes-prone group and at 85 days intra-islet apoptosis was threefold higher in the diabetes-prone group than in the group that had insulitus but did not develop diabetes. At 107 days, apoptosis (total and intra-islet) was higher in the group that had insulitus but did not develop diabetes (OND-DP) than in either the diabetes resistant (DR) or diabetes-prone (DP) groups. Conclusion/interpretation. We have shown significant islet cell apoptosis in the pancreas of diabetes-prone BB/S rats, which coincides with the appearance of insulitis and the onset of diabetes. We have also detected differences in the levels of apoptosis between diabetic and non-diabetic animals and suggest that such differences could be an important determinant of disease progression in this animal model of Type I diabetes. [Diabetologia (2001) 44: 320–324] Received: 15 May 2000 and in revised form: 31 October 2000     

Prevalence of coeliac disease in siblings of patients with Type I diabetes is related to the prevalence of DQB1*02 allele

Aims/hypothesis: Coeliac disease is more prevalent among patients with Type I (insulin-dependent) diabetes mellitus and coeliac disease-related antibodies have been reported to increase in frequency in their first-degree relatives. Our aim was to find out if coeliac disease is more common among siblings of children with Type I diabetes than in the normal population. Methods: IgA endomysium antibodies were measured by indirect immunofluorescence in 550 subjects (mean age 11.8 years, range 3.1–26.9 years) with a sibling with Type I diabetes. We performed jejunal biopsy on as many subjects with positive antibodies as agreed. HLA-DQB1 genotyping was done in 427 subjects. Results: Endomysium antibodies were positive in nine subjects (1.6 %). Jejunal biopsy was diagnostic for coeliac disease in five out of seven patients. An additional patient with coeliac disease, one already on a gluten-free diet, was identified by questionnaire. The prevalence of coeliac disease was 1.1 %. Five of six patients with coeliac disease had HLA-DQB1^*02 allele, compared with 118 of 421 of those without coeliac disease (p = 0.009). The sixth patient was positive for HLA-DQB1^*0302 allele, which was also found in 241 of 421 of those without coeliac disease (p = 0.4). Conclusion/interpretation: We found the prevalence of coeliac disease among siblings of children with Type I diabetes to be similar to figures reported from recent population-based studies and to be correlated with the prevalence of coeliac disease associated HLA-DQB1 alleles. We propose that routine screening for coeliac disease among all first-degree relatives of patients with Type I diabetes is not warranted. [Diabetologia (2001) 44: 1051–1053] Received: 11 January 2001 and in revised form: 27 April 2001     

Apoptosis and beta-cell destruction in pancreatic islets of NOD mice with spontaneous and cyclophosphamide-accelerated diabetes

Summary Autoimmune-mediated destruction of pancreatic islet beta cells leads to insulin-dependent diabetes in non-obese diabetic (NOD) mice. Although both direct cytotoxic T cell- and indirect cytokine-, nitric oxide- or free radical-mediated mechanisms induce beta-cell apoptosis in vitro, beta-cell death in vivo in spontaneous autoimmune diabetes is not well-characterized. Furthermore, whether beta cells die gradually, or rapidly in the late pre-clinical stage, is a question of current interest. To investigate beta-cell death in vivo, we measured the frequency and intra-islet localisation of apoptosis, defined as DNA strand breaks by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) technique, during spontaneous and cyclophosphamide-accelerated diabetes in NOD mice. In spontaneous diabetes, the frequency of apoptosis in islets correlated with the progression of beta-cell destruction with age. Although apoptosis was detected at low frequency within the reduced insulin-positive islet area of pre-diabetic mice at 90 days of age, it was rarely co-localised to beta cells. After acceleration of beta-cell destruction with cyclophosphamide, the frequency of apoptosis reached maximum at 12 days, at which time 3.2 % of apoptotic cells were beta cells. Apoptosis was most frequent in the insulin-negative islet area comprised of mononuclear cell infiltrate and was localized to CD8⁺ T cells. The rarity of detectable apoptotic beta cells in spontaneous pre-diabetic mice with pronounced insulitis and reduced insulin-positive islet areas most likely reflects the rapid clearance of apoptotic beta cells. Our findings are more consistent with gradual destruction of non-renewable beta-cells in spontaneous diabetes, than with their rapid, accelerated destruction (as after cyclophosphamide) in the late pre-clinical stage. [Diabetologia (1998) 41: 1381–1388] Received: 6 May 1998 and in revised form: 14 July 1998     

Sustained beta cell apoptosis in patients with long-standing type 1 diabetes: indirect evidence for islet regeneration?

Aims/hypothesis Type 1 diabetes is widely held to result from an irreversible loss of insulin-secreting beta cells. However, insulin secretion is detectable in some people with long-standing type 1 diabetes, indicating either a small population of surviving beta cells or continued renewal of beta cells subject to ongoing autoimmune destruction. The aim of the present study was to evaluate these possibilities.Materials and methods Pancreatic sections from 42 individuals with type 1 diabetes and 14 non-diabetic individuals were evaluated for the presence of beta cells, beta cell apoptosis and replication, T lymphocytes and macrophages. The presence and extent of periductal fibrosis was also quantified.Results Beta cells were identified in 88% of individuals with type 1 diabetes. The number of beta cells was unrelated to duration of disease (range 4–67 years) or age at death (range 14–77 years), but was higher (p<0.05) in individuals with lower mean blood glucose. Beta cell apoptosis was twice as frequent in type 1 diabetes as in control subjects (p<0.001), but beta cell replication was rare in both groups. The increased beta cell apoptosis in type 1 diabetes was accompanied by both increased macrophages and T lymphocytes and a marked increase in periductal fibrosis (p<0.001), implying chronic inflammation over many years, consistent with an ongoing supply of beta cells.Conclusions/interpretation Most people with long-standing type 1 diabetes have beta cells that continue to be destroyed. The mechanisms underlying increased beta cell death may involve both ongoing autoimmunity and glucose toxicity. The presence of beta cells despite ongoing apoptosis implies, by definition, that concomitant new beta cell formation must be occurring, even after long-standing type 1 diabetes. We conclude that type 1 diabetes may be reversed by targeted inhibition of beta cell destruction.     

Progression to insulin-requiring diabetes in seronegative prediabetic subjects: the role of two HLA-DQ high-risk haplotypes

Aims/hypothesis: Most Caucasians with Type I (insulin-dependent) diabetes mellitus develop an autoimmune form of diabetes known as Type IA diabetes, based on the presence of humoral responses to islet autoantigens. Alleles at the HLA locus account for the strongest susceptibility to this form of diabetes, which requires insulin therapy. Because a number of patients who develop insulin-requiring diabetes are islet autoantibody negative, the HLA class II haplotypes, DQA1 ^* 0501-DQB1*0201 and DQA1 ^* 0301-DQB1*0302, were evaluated to assess whether they are an independent risk factor for progression to insulin requirement in first-degree relatives of Type I diabetic patients. Methods: Both HLA-DQ genotyping and islet cell autoantibody assessment (insulin, GAD65, IA-2 autoantibodies and cytoplasmic islet cell antibodies) were evaluated prospectively in 74 relatives of Type I diabetic patients who developed diabetes treated with insulin (prediabetics) and in 426 control subjects who did not develop insulin-requiring diabetes. Based on the presence of DQA1 ^* 0501-DQB1*0201 and/or DQA1 ^* 0301-DQB1*0302, the number of HLA-DQ high-risk haplotypes was assigned as 0, 1 or 2. Results: A higher prevalence of 2 HLA-DQ high-risk haplotypes was present in seronegative prediabetic subjects as compared to non-diabetic autoantibody negative first-degree relatives (33.3 % vs 10.1 % respectively; p < 0.05). Moreover, in seronegative relatives who developed insulin-requiring diabetes, the presence of 2 HLA-DQ high-risk haplotypes conferred an increased cumulative risk of developing insulin requirement of 27 % at 12.5 years of follow-up, compared to a risk of 6 % for non-diabetic relatives who were antibody-negative and had 0 or 1 HLA-DQ high-risk haplotypes (Log rank p = 0.01). Conclusion/interpretation: These data provide evidence for a phenotype, which is associated with the absence of conventional islet autoantibodies at initial screening, while usually remaining seronegative, and the presence of 2 HLA-DQ high-risk haplotypes with progression to clinical Type I diabetes after a prolonged follow-up. Given the fact that in humans the highest risk-conferring locus associated and linked to the disease is the HLA cluster, and that HLA-DQ molecules play a key role in the development of autoimmune diabetes, our observations imply that as yet unidentified immunologic abnormalities could well exist in seronegative relatives at risk of developing clinical diabetes and carrying 2 HLA-DQ high-risk haplotypes. [Diabetologia (2002) 45: 66–76] Received: 29 May 2001 and in revised form: 28 September 2001     

Immunology in diabetes: an update

Type 1 (insulin-dependent) diabetes mellitus is strongly associated with autoimmune phenomena connected to the loss of β-cells in the pancreatic islets. Despite considerable progress in our understanding of genetic susceptibility factors and islet autoimmunity preceding the clinical onset of Type 1 diabetes there are considerable gaps in our knowledge. First, the etiology is unclear. It is speculated that multiple etiological factors may initiate a common pathogenic pathway which results in immune-mediated β-cell destruction. In 1998 we will need to learn more about the possible importance of gestational infections, as well as isolation of viral DNA or RNA from the blood of new-onset patients or marker-positive individuals. The scan of the whole genome has provided a smorgasbord of genetic regions which confer diabetes risk either alone or in combination. HLA remains the major genetic risk factor, and while HLA peptide binding information is considerable, we understand less of autoantigen processing and presentation. Cloned autoantigens and their use in standardized autoantibody assays have improved our ability to identify individuals at risk for diabetes. The diagnostic sensitivity and specificity of autoantibody markers for Type 1 diabetes are high as are their predictive values. We need methods to combine autoantibodies with genetic risk factors. The identification of individuals in different stages of their pathogenesis, including patients with so-called slowly progressive Type 1 diabetes (SPIDDM, LADA etc.), allow approaches to novel therapeutic interventions. Insulin is currently the therapeutic agent of choice and although spontaneous insulin-dependent diabetes in the NOD mouse and the BB rat can be prevented by immune suppression or modulation, this has not yet been possible in humans. The 1998 research on the interaction between environmental factors and susceptibility genes to initiate β-cell specific autoreactivity should allow the development of therapies for prevention, and perhaps a cure, of insulin-dependent (Type 1) diabetes. © 1998 John Wiley & Sons, Ltd.     

IFNγ/TNFα synergism in MHC class II induction: effect of nicotinamide on MHC class II expression but not on islet-cell apoptosis

Aims/hypothesis: Interferon-γ (IFNγ) and TNFα synergistically induce pancreatic beta-cell apoptosis. Apart from their direct effect, we studied the possible indirect immunological role of IFNγ/TNFα synergism on pancreatic beta-cell death by investigating MHC class II induction by cytokines. The effect of nicotinamide on the cytokine-induced MHC class II expression and pancreatic beta-cell death was also studied. Methods: Immunocytochemistry, flow cytometry and RNase protection assay were used to study MHC class II expression. Immunoblotting was done to study downstream signals of IFNγ. The effects of nicotinamide on islet-cell apoptosis and diabetes mellitus were examined using MTT assay and adoptive transfer model. Results: IFNγ alone induced MHC class II expression on a small number of insulinoma cells. TNFα alone did not induce MHC class II expression, but enhanced IFNγ-induced MHC class II expression. MHC class II expression by cytokine(s) was due to the induction of class II transactivator (CIITA). Nicotinamide reduced MHC class II expression by cytokine(s) but did not protect insulinoma-cell apoptosis by IFNγ and TNFα in combination or protect against the development of diabetes mellitus after adoptive transfer of diabetogenic lymphocytes. Conclusion/interpretation: IFNγ and TNFα synergistically induced MHC class II expression on insulinoma cells through the induction of CIITA; nicotinamide reduced the expression of cytokine-induced MHC class II expression on insulinoma cells through its effect on CIITA expression; and the preventive effect of nicotimamide on Type I (insulin-dependent) diabetes mellitus is probably due to its effect of MHC class II expression rather than that on islet cell apoptosis. [Diabetologia (2002) 45: 385–393] Received: 13 August 2001 and in revised form: 22 October 2001     

MFP14, a multifunctional emerging protein with immunomodulatory properties, prevents spontaneous and recurrent autoimmune diabetes in NOD mice

Abstract Aims/hypothesis. To test the effects of multifunctional protein 14 (MFP14), which shares structural homology with heat shock proteins (HSPs), on the development of Type I (insulin-dependent) diabetes mellitus in NOD mice. Methods. MFP14 was given to euglycaemic female NOD mice from either the 4^th to the 25^th or from the 12^th until the 35^th week, or commencing one day before islet transplantation and until the reappearance of hyperglycaemia. Pancreata from NOD mice treated with multifunctional protein 14 for 14 consecutive weeks until 18 weeks of age were examined histologically for insulitis. Anti-CD3 and/or lipopolysaccharide (LPS)-induced blood levels of interferon (IFN)-γ, interleukin (IL)-4, IL-10, IL-12 and tumour necrosis factor (TNF)-α were measured by ELISA in 10 week-old female NOD mice treated for 6 consecutive weeks with either MFP14 or PBS. Unless otherwise stated, multifunctional protein 14 was administered daily 5 times a week at a dose of 25 μg. Control mice received PBS or, in selected experiments, heat-inactivated MFP14. Results. MFP 14 treated mice had a significantly lower incidence of spontaneous diabetes compared to control mice. The MFP14 was equally effective both upon early and late prophylaxis and the protection persisted until week 50 in mice treated from weeks 4 to 25. Insulitis was significantly reduced by the MFP14. The MFP14 also delayed recurrence of hyperglycaemia in syngeneic islet-transplanted NOD mice. Although MFP14 reduced anti-CD3 and/or LPS-induced blood levels of IFN-γ, TNF-α and IL-12 it increased IL-4 and IL-10. Conclusion/interpretation. The MFP14 could be a possible candidate for the prevention or early treatment of human Type I (insulin-dependent) diabetes mellitus. [Diabetologia (2001) 44: 839–847] Received: 2 November 2000 and in revised form: 14 February 2001     

Genetics and pathogenesis of type 1 diabetes: prospects for prevention and intervention

Type 1 diabetes is etiologically a multifactorial disease caused by a complex interaction of genetic and environmental factors, with the former consisting of multiple susceptibility genes. Identification of genes conferring susceptibility to type 1 diabetes would clarify etiological pathways in the development and progression of type 1 diabetes, leading to the establishment of effective methods for prevention and intervention of the disease. Among multiple susceptibility genes, HLA and INS are particularly important because of their contribution to tissue specificity in the autoimmune process. DRB1*04:05‐DQB1*04:01 is associated with autoimmune type 1 diabetes, idiopathic fulminant type 1 diabetes and anti‐islet autoimmunity in autoimmune thyroid diseases, suggesting that this haplotype is associated with beta‐cell specificity in autoimmune diseases. Genes involved in the expression of insulin in the thymus contribute to beta‐cell‐specific autoimmune mechanisms in type 1 diabetes. These genes and pathways are important targets for tissue‐specific prevention and intervention of type 1 diabetes. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2011.00176.x, 2011)     

Autoantibodies against a novel 51 kDa islet antigen and glutamate decarboxylase isoforms in autoimmune polyendocrine syndrome type I

Summary Beta-cell function and islet cell antibodies were studied in six patients with autoimmune polyendocrine syndrome type I. All suffered from mucocutaneous candidiasis, five had adrenocortical insufficiency and three hypoparathyroidism. All sera contained high titres of antibodies staining islets of Langerhans. Reactivity against glutamate decarboxylase, predominantly the 65 kDa isoform, was detected by immunoprecipitations and Western blots in five of the six sera, and all six sera immunoprecipitated a 51 kDa antigen from [³⁵S]methionine labelled rat islet cell lysates. No reactivity against this latter antigen was found in sera of patients with Type 1 (insulin-dependent) diabetes mellitus (n=9), Graves' disease (n=5), autoimmune gastritis (n=4), idiopathic Addison's disease (n=7), or stiffman syndrome (n=2). The 51 kDa antigen was also detected by Western blots using homogenates of rat islets and autoimmune polyendocrine syndrome type I patient sera, whereas no such reactivity was found with homogenates of testes, adrenals, small intestine, spleen, exocrine pancreas or brain. Moreover, the 51 kDa antigen was present in the rat insulinoma cell line RINm 5F but not in the SV-40 transformed, monkey kidney cell line COS, when examined by immunoprecipitations of [³⁵S]-methionine labelled cell lysates and by Western blots. None of the patients with autoimmune polyendocrine syndrome type I had symptoms of diabetes and their insulin responses to glucose challenge were normal. The data illustrate that patients with autoimmune polyendocrine syndrome type I present an autoimmune response against islets of Langerhans, which is apparently different from that associated with classic Type 1 diabetes. As most of the autoantigens in many autoimmune diseases are enzymes involved in important functions in the affected organs, it is possible that the anti-51 kDa antibodies are directed against a protein with important functional activity in the islet.     

Blood glucose normalization upon transplantation of human embryonic pancreas into beta-cell-deficient SCID mice

Aims/hypothesis: Transplanting human pancreatic islet beta cells could represent a radical new treatment of Type I (insulin-dependent) diabetes mellitus. However, beta cells available for grafting are scarce and finding new sources of such cells would be crucial for any cell therapy for diabetes. Undifferentiated precursor cells present in the human embryonic pancreas could represent such a source. Methods: We grafted human embryonic pancreases (6–9 weeks of development) that contain very few beta cells onto NOD/scid mice. Results: The human pancreatic tissue grew, increasing in weight 200 times within six months and endocrine cells differentiated, the number of human beta cells being increased by a factor 5000. Finally, the developed human endocrine tissue was mature enough to control the glycaemia of mice deficient in endogenous beta cells. Conclusion/interpretation: Human embryonic pancreas represent a source of immature cells that can proliferate and differentiate into mass beta cells after transplantation. Transplantation of human embryonic pancreas into NOD/scid mice is a useful model for understanding the development of the human pancreas during prenatal life. [Diabetologia (2001) 44: 2066–2076] Received: 12 June and in revised form: 17 July 2001     

Antibodies to tissue transglutaminase C in Type I diabetes

Abstract Aims/hypothesis. Silent coeliac disease is a gluten driven autoimmune disease which is relatively frequent in patients with Type I (insulin-dependent) diabetes mellitus. To determine the extent of gluten associated autoimmunity in Type I diabetes, autoantibodies to tissue transglutaminase C, a major autoantigen in coeliac disease, were measured in patients with new-onset Type I diabetes. Methods. We measured IgG and IgA tissue transglutaminase C autoantibodies using human recombinant antigen and radio-binding assays in a cohort of 287 patients with new-onset Type I diabetes, 119 with Type II (non-insulin-dependent) diabetes mellitus and in 213 control subjects. Results. We found IgA and IgG tissue transglutaminase C antibodies in 24 (8 %) patients with Type I diabetes; 97 (33 %) patients had IgG antibodies only and 1 IgA antibodies only. Antibody concentrations were highest in those with both IgA and IgG antibodies. Only 2 (2 %) patients with Type II diabetes and 2 (1 %) control subjects had either IgG or IgA tissue transglutaminase C antibodies. Patients with HLA DRB1 ^* 04 alleles had the highest prevalence of IgG tissue transglutaminase C antibodies. Conclusion/Interpretation. These data show that almost 10 % of patients have autoimmunity typical of coeliac disease and that another 30 % have low level tissue transglutaminase C antibody binding. This high prevalence suggests either involvement of the gut in the pathogenesis of Type I diabetes or that transglutaminase is a secondary autoantigen resulting from beta-cell destruction. [Diabetologia (1999) 42: 1195–1198] Received: 15 April 1999 and in revised form: 8 June 1999     

Induction and prevention of type 1 diabetes mellitus by viruses.

Genetic factors and environmental factors are thought to be involved in the pathogenesis of insulin-dependent diabetes mellitus Type 1. Viruses, as one environmental factor, may act as primary injurious agents to beta cells or as triggering agents for autoimmunity. Some viruses such as EMC-D and Coxsackie B4 can induce Type 1 diabetes by infecting and destroying beta cells in genetically susceptible mice. In addition, certain species of monkey, such as Patas, show elevated blood glucose levels and depressed insulin secretion after infection with Coxsackie B4 virus. An occasional case of Type 1 diabetes mellitus appears to be associated with the infection of beta cells with Coxsackie B viruses. In addition, Coxsackie B4 virus may also generate viral antigen-specific cytotoxic T cells which may cross-react with a beta cell-specific autoantigen leading to autoimmune Type 1 diabetes. In the case of viral triggering of autoimmune Type 1 diabetes, certain viruses (eg, retrovirus in NOD mice and rubella virus in hamsters and humans) may alter a normally existing beta cell antigen into an immunogenic form or might induce a new antigen, leading to beta cell-specific autoimmune insulin dependent diabetes mellitus. In addition, other viruses (eg, Kilham's rat virus in DR-BB rats) could generate antigen-specific T effector cells which may cross-react with a beta cell-specific autoantigen. In contrast to the induction of diabetes, viruses can prevent the development of diabetes. Inoculation of DP-BB or NOD mice with lymphocytic choriomeningitis virus reduced the incidence of diabetes or prevented the disease by disordering particular lymphocyte subsets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low bcl-2 expression and increased spontaneous apoptosis in T-lymphocytes from newly-diagnosed IDDM patients

Summary The bcl-2 gene product has been shown to regulate apoptotic cell death, and its dysregulation has been shown to induce several abnormalities in the immune system. No data exist regarding bcl-2 expression in autoimmune diseases, such as human insulin-dependent diabetes mellitus (IDDM). We investigated bcl-2 protein expression by testing T lymphocytes from 15 newly-diagnosed (<3 weeks) IDDM patients in comparison to 10 age-matched control subjects. The expression of bcl-2 on CD3⁺ lymphocyte subsets was investigated after membrane permeabilization by two- or three-colour immunofluorescence. When the percentage and mean fluorescence intensity (MFI) of bcl-2⁺/CD3⁺ cells from normal individuals and patients were compared, we found that bcl-2 expression within the CD3⁺ and CD4⁺ CD45R0⁺ T-cell populations was reduced significantly in IDDM patients (46.8±15.4 vs 79.6±11.7; 25.7±3.8 vs 47.15±5.7, respectively; p<0.001). To establish whether low bcl-2 expression in T cells from newly-diagnosed patients reflects their susceptibility to death by an apoptotic process, we also evaluated DNA staining with propidium iodide in CD3⁺ lymphocyte suspension after a (24–72 h) culture period (spontaneous apoptosis). We found that IDDM patients have higher levels of spontaneous apoptosis (mean±SEM: 24 h=4.6±0.8; 48 h=9.9±1; 72 h=12.8±1.1) than control subjects (24 h=1.8±0.4; 48 h=4.6±0.4; 72 h=5.7±0.3; p<0.02–0.001). Our study suggests that recent onset IDDM is characterised by reduced bcl-2 expression, which in turn may be associated with the increased spontaneous apoptosis we observed.Abbreviations MFI Mean fluorescence intensity - mAb monoclonal antibody - PMA phorbol myristate - PHA phytoemagglutinin - Con A concanavalin-A - PKC protein kinase C - ICA islet cell autoantibody - IAA insulin autoantibody - PE phycoerythrin - PI propidium iodide - FSC forward scatter - SSC side scatter - PBMC peripheral blood mononuclear cells     

Diabetes in the parents of children with Type I diabetes

Aims/hypothesis. Previous studies have reported an excess of Type II (non-insulin-dependent) diabetes mellitus in parents of children with Type I (insulin-dependent) diabetes mellitus. We set out to characterise the clinical and immunogenetic features of diabetes in parents of affected children, and to test the hypothesis that there is no excess of Type II diabetes within this population. Methods. Clinical details were collected from 3164 parents of 1641 children with Type I diabetes participating in the Bart's-Oxford study of childhood diabetes. Islet cell antibodies, antibodies to GAD and IA-2, and HLA class II genotype were determined in a subset of this group. Individuals were assigned a classification of Type I diabetes on the basis of clinical features and measurement of islet autoantibodies. Results. Of 184 parents with diabetes, 138 (75 %) were on insulin. At least one islet autoantibody was detected in 90 (59 %) of 152 parents tested, and of 116 who were HLA-typed, 23 (20 %) had the highest risk genotype HLA-DRB1^*03-DQA1*0501-DQB1^*0201 / DRB1*04-DQA1^*0301-DQB1*0302. Of 46 non-insulin-treated parents, 12 had islet autoantibodies. Of all parents, 141 (4.5 %) were therefore classified as having Type I diabetes, and 31 (0.98 %) as Type II diabetes; 12 could not be classified because of missing data or samples. Conclusion/interpretation. Autoimmune diabetes can present late and without immediate need for insulin treatment in parents of children with the disease. Previous studies have categorised this as Type II diabetes. Our study suggests that there is no excess of non-autoimmune diabetes in the families of children with Type I diabetes. [Diabetologia (2002) 45: ▪–▪] Received: 7 November 2001 and in revised form: 3 January 2002