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Type 1 diabetes is a cell-mediated autoimmune disease characterized by autoantibody and peripheral blood mononuclear cell (PBMC) reactivity to islet cell proteins. Type 2 diabetes is not an autoimmune disease but rather results from both insulin resistance and a nonautoimmune insulin secretory defect. There is, however, a group of phenotypic type 2 diabetic patients who have islet autoantibodies that are similar to those of type 1 diabetic patients. In this study, we investigated, using cellular immunoblotting, whether type 2 diabetic patients positive for islet autoantibodies have PBMC responses to islet proteins. We observed that autoantibody negative (Ab-) type 2 diabetic patients (n = 9) and normal control subjects (n = 12) demonstrated PBMCs responsive to 0-3 molecular weight regions. In contrast, autoantibody positive (Ab+) type 2 diabetic patients (n = 11) demonstrated PBMC responses to 3-18 molecular weight regions, similar to that of type 1 diabetic patients (responsive to 4-18 molecular weight regions). PBMCs from over 90% of the Ab+ type 2 and type 1 diabetic patients were observed to proliferate to islet proteins in the vicinity of 97 kDa. In contrast, 65-90% of type 1 diabetic patients had responsive PBMCs for islet proteins in most of the molecular weight regions, whereas <60% of the Ab+ type 2 diabetic patients had PBMCs responsive to the same molecular weight proteins. Ab+ type 2 diabetic patients appear to be heterogeneous with respect to cellular reactivity to islet proteins. Some subjects demonstrate PBMC responses similar to those of "classic" type 1 diabetic patients, whereas others have PBMC responses potentially distinct from type 1 diabetic patients. [ABSTRACT FROM AUTHOR]

In human islet transplantation, insulin independence decreases over time. We previously showed that amyloid deposition following transplantation of islets from human islet amyloid polypeptide (hIAPP) transgenic mice resulted in ß-cell loss and that rosiglitazone treatment decreased islet amyloid deposition and preserved ß-cell area in the endogenous pancreas of hIAPP transgenic mice. Thus, we sought to determine if rosiglitazone treatment decreases islet amyloid deposition and the associated ß-cell loss after islet transplantation. Streptozocin-diabetic mice were transplanted with 100 islets from hIAPP transgenic (T) mice or nontransgenic (NT) littermates under the kidney capsule and received either rosiglitazone (R) in drinking water or plain drinking water (C). The resultant groups (NTC [n = 11], NTR [n = 9], TC [n = 14], and TR [n = 10]) were followed for 12 weeks after which the graft was removed and processed for histology. Amyloid was detected in nearly all T islet grafts (TC = 13/14, TR = 10/10) but not in NT grafts. Rosiglitazone did not alter amyloid deposition (% graft area occupied by amyloid; TC: 2.15 ± 0.7, TR: 1.72 ± 0.66; P = .86). % ß-cell/graft area was decreased in the TC grafts compared to NTC (56.2 ± 3.1 vs 73.8 ± 1.4; P < .0001) but was not different between TC and TR groups (56.2 ± 3.1 vs 61.0 ± 2.9; P = .34). Plasma glucose levels before and after transplantation did not differ between NTC and TC groups and rosiglitazone did not affect plasma glucose levels post-islet transplantation. Rosiglitazone did not decrease amyloid deposition in hIAPP transgenic islet grafts. Therefore, rosiglitazone treatment of recipients of amyloid forming islets may not improve transplantation outcomes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This study was undertaken to determine which type 1 diabetes-associated autoantibodies and what clinical characteristics are most useful to identify patients with type 1(1/2) diabetes. We studied 125 patients, recently diagnosed clinically with type 2 diabetes for the presence of islet cell antibodies (ICA), insulin autoantibodies (IAA), antibodies to glutamic acid decarboxylase(GADAb), and IA-2a (IA-2Ab). Patients with a diagnosis of type 2 diabetes who met all of the following criteria at diagnosis were studied: age > or = 30 years, no history of ketonuria or ketoacidosis, and not requiring insulin treatment. Thirty-six patients (29%) were positive for at least 1 antibody. Thirty-two (26%) were ICA positive and 20 (16%) GADAb positive. Insulin autoantibodies and IA-2Ab occurred less frequently in 2 (1.6%) and 8 (6.4%) patients, respectively. There was no significant difference in the ages at diagnosis between the Ab(+) and Ab(-) patients, age in years (range) 47.2 (32 to 64) versus 51.2 (31 to 77), respectively, P =.06. Body mass index (BMI) was different in the 2 groups, with Ab(+) patients being less obese, BMI (range) 28.3 kg/m(2) (17.6 to 54.9) versus 32.0 kg/m(2) (19.2 to 68.8), respectively, P =.01. Clinical presentation of diabetes was more commonly symptomatic with polyuria and polydipsia in Ab(+) patients, while in Ab(-) patients, diagnosis was more often incidental, P =.002. However, more than 95% of patients overlapped in both age and BMI irrespective of antibody status. Similarly, 42% of Ab(+) patients had their diabetes diagnosed incidentally, while 29% of Ab(-) patients presented with polyuria and polydipsia. We therefore conclude that screening with antibodies, mainly ICA and GAD, but not age, BMI, or clinical presentation should be used to identify type 1(1/2) diabetes., (Copyright 2001 by W.B. Saunders Company)

Background: Fetal pancreatic tissue has been suggested to be less immunogenic than adult islets. Thus, transplantation of human fetal pancreatic tissue as treatment for type 1 diabetes has been gaining interest. To investigate this question, we tested the peripheral blood mononuclear cell (PBMC) responses from different subject populations to human adult islet proteins (AIP) versus human fetal pancreatic proteins (FPP)., Methods: PBMC responses to FPP and AIP from normal controls (n=14), newly diagnosed type 1 diabetes patients (n=5), long-term type 1 diabetes patients (n=9), and subjects at-risk for development of type 1 diabetes (n=3) were studied., Results: We observed that normal controls demonstrated PBMC reactivity to 0-3 molecular weight regions (mwr) for both the AIP (mean+/-SD, 0.8+/-1.1) and the FPP (0.6+/-0.7). In contrast, newly diagnosed type 1 diabetic patients (<1 year) demonstrated PBMC responses to 9-16 mwr for the AIP (12.8+/-2.5) and 0-14 mwr for the FPP (6.8+/-5.0). The PBMCs from long-term type 1 diabetes patients (> 3 years) were responsive to 2-11 mwr for AIP (6.0+/-2.8) and 0-11 mwr for FPP (4.9+/-4.0). Three nondiabetic ICA positive subjects at-risk for development of type 1 diabetes demonstrated positive PBMC reactivity to 9-18 mwr for the AIP (12.7+/-3.9) and 4-18 mwr for the FPP (10.0+/-5.9)., Conclusions: We conclude that human fetal pancreatic proteins are not significantly less stimulatory than human adult islet proteins to PBMCs of subjects with or at risk for type 1 diabetes.

Background: Type 1 diabetes is a cell-mediated autoimmune disease. Successful transplantation of human fetal pancreatic tissue into type 1 diabetic patients must address both autoimmunity and allograft rejection. We investigated whether humoral and cellular responses to islet antigens could be demonstrated in the peripheral blood of type 1 diabetic subjects receiving human fetal pancreatic tissue transplants., Methods: We investigated peripheral blood mononuclear cell (PBMC) responses, using cellular immunoblotting, and autoantibody responses to islet proteins, before transplant and at 3-month intervals after transplant, of nine long-term type 1 diabetes patients (mean disease duration of 21 years) receiving human fetal pancreatic tissue subcutaneously into the abdominal wall without immunosuppression., Results: Before transplant, all nine subjects were islet cell autoantibody (ICA)-negative and seven out of nine subjects were glutamic acid decarboxylase autoantibody (GADAb)-positive. After transplant, all subjects became ICA(+) and the two patients who were GADAb(-) before transplant became GADAb(+) after transplant. Maximum PBMC reactivity to separated human fetal pancreatic proteins was observed in four patients 3 months after transplant, in one patient at 6 months, in two patients at 9 months, and in one patient at 12 months after transplant. One subject, who had PBMC reactivity to multiple islet proteins before transplant, continued to respond to multiple islet proteins throughout the study., Conclusions: We conclude that the development in the peripheral blood of ICA, GADAb, and PBMC reactivity to human fetal pancreatic proteins in the trans plant recipients is most consistent with reactivation of the type 1 diabetes disease process.

Type 1 diabetes is an autoimmune disease characterized by immune-mediated destruction of the pancreatic beta cells. Insulin autoantibodies (IAAs) develop in many subjects at high risk for developing type 1 diabetes prior to onset of clinical disease and exposure to exogenous insulin, whereas insulin antibodies (IAs) commonly develop in patients treated with exogenous insulin. To investigate whether the binding characteristics of IAA and IA are similar, we measured eight different binding characteristics of IAAs from 19 insulin-naive first-degree relatives of type 1 diabetes patients and compared these to the binding characteristics of IAs from 19 type 1 diabetes patients treated with exogenous insulin. IAA and IA samples were matched for percentage insulin binding. Scatchard analysis revealed that IAAs have a two-slope representation similar to IAs-that is, two populations of antibodies, a high-affinity low-capacity population and a low-affinity high-capacity population. Binding properties of the two respective populations were found to be similar for IAAs and IAs. Sipps' equation was used to generate a Hill plot and produce an index of heterogeneity, which showed further similarity between IAAs and IAs. These results suggest that the IAAs found in subjects at increased risk for type 1 diabetes, like IAs, are likely to be polyclonal in nature. The similarities between IAAs and IAs support the hypothesis that both are induced by insulin presentation to the immune system.

Insulin-dependent diabetes (IDDM) results from autoimmune destruction of pancreatic beta cells mediated predominantly by cellular effector mechanisms. To date, investigators have studied a limited number of islet cell proteins stimulatory to T cells. However, before development of clinical IDDM, the majority of the beta cells are impaired or destroyed. Thus, numerous proteins from lysed beta cells would be accessible to the immune system of the patient. Our goal was to investigate the PBMC reactivity of IDDM patients to the full spectrum of fractionated human pancreatic islet cell proteins to determine whether numerous islet cell proteins or a select few would be recognized. We observed that PBMCs from IDDM patients responded reproducibly (mean stimulation index, >2.0) to the proteins in all m.w. regions, whereas the mean stimulation index for controls from all m.w. regions was <2.0. Using three different islet protein preparations, PBMC responses of IDDM patients (n = 30) and controls (n = 39) to the islet cell proteins were significantly different. Dose responses were also demonstrated for the lymphocyte reactivity of the IDDM patients (n = 29) vs controls (n = 56) to the islet cell preparations. Proteins, presumably irrelevant to the IDDM disease process, from a human osteosarcoma cell line and normal human spleen cells did not stimulate PBMCs from IDDM patients or controls. Moreover, IDDM patients and controls responded similarly to mitogens and tetanus toxoid. These studies show that at the time of diagnosis of IDDM, PBMCs from IDDM patients are stimulated by a wide array of islet cell proteins.

The macrophage-derived cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) have direct effects on pancreatic beta cells and have been hypothesized to play important roles in the autoimmune beta cell lesion of type I diabetes because of two major effects on beta cells: altered insulin secretion and beta cell cytotoxicity. High doses of IL-1 are cytotoxic to beta cells and strongly inhibit insulin release; high-dose IL-1 plus TNF acts synergically to suppress further the insulin release. In contrast, we observed that the predominant effect of low-dose IL-1 and TNF when administered separately was the stimulation of insulin release. We therefore asked whether the combination of low-dose IL-1 plus TNF would act synergistically to stimulate or suppress insulin release. Studies were performed on cultured rat islets and both insulin release and cytotoxicity (51Cr release) were measured. After 2 days of culture, increasing doses of IL-1-25, 50, 75 and 100 ng/l--caused progressively increased cytotoxicity and impaired insulin release. In contrast, the lowest dose of IL-1 tested, 10 ng/l, increased insulin release but was still slightly cytotoxic. Tumor necrosis factor at doses of 10, 25, 62.5, 75 and 100 micrograms/l also was slightly cytotoxic but increased insulin release. The augmented insulin release declined progressively with increasing TNF dose. However the combination of insulin stimulatory doses of IL-1 (10 ng/l) and TNF (62.5 micrograms/l) suppressed insulin release. The effects of these two cytokines on insulin release demonstrated a similar pattern after 4 and 6 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-dependent diabetes is an autoimmune disease specifically targeting the pancreatic beta cells and several observations, both experimental and clinical, suggest that the interaction of the immune system with the beta cells is in part determined by the functional state of the target cells, increased beta cell activity resulting in augmented immunologic mechanisms and vice versa for suppressed beta cell activity and decreased immune attack. In this study we investigated whether cytokine induced islet cell cytotoxicity in vitro was in part dependent on the functional state of the beta cells. Cytotoxicity of cultured rat islets was induced by IL-1 (100 pg/ml) and TNF (62.5 ng/ml) individually and in combination and beta cell activity was modulated by culturing the islets in media containing 3.3, 5.5, 11, and 20 mmol/liter glucose. Both IL-1 and TNF were cytotoxic when administered individually and the combination of IL-1 and TNF was more cytotoxic than either cytokine alone. Maximum cytotoxicity was observed at 11 mmol/liter glucose with cytotoxicity being reduced at 5.5 mmol/liter glucose and further reduced at 3.3 mmol/liter glucose. Interestingly, the degree of cytotoxicity was lower in 20 mmol/liter glucose compared to 11 mmol/liter. These results firmly establish that islet cytotoxicity of IL-1 and TNF is highly dependent on the functional state of the beta cells. This suggests that during the IDDM disease process as some beta cells are destroyed, the compensatory increased activity of the remaining beta cells may increase their susceptibility to cytokine attack. Furthermore, our observations provide rational support for the use of beta cell rest as intervention therapy for IDDM.

Cellular immune responses are influential for protection against intracellular bacteria such as brucellae. Therefore, identification of Brucella abortus antigens that activate primed bovine lymphocytes is fundamental for discerning the breadth of cellular response in bovine brucellosis. Potentially antigenic components of B. abortus S19 were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by nitrocellulose blotting. Specific one-dimensional blot segments induced proliferation of peripheral blood lymphocytes from all 25 of the vaccinated cattle tested and were defined as immunodominant. Individual proteins that stimulated lymphocyte proliferation were further characterized by two-dimensional cellular immunoblotting by two different approaches. Individual one-dimensional stimulatory blot segments were eluted, concentrated, and then subjected to two-dimensional cellular immunoblotting. Alternatively, entire two-dimensional gels containing all of the B. abortus components were blotted and nitrocellulose sections containing individual proteins were assayed for lymphocyte activation. Thirty-eight Brucella proteins that induced lymphocyte proliferation were resolved by both procedures. Phenotypic analysis of the proliferating cell population demonstrated the presence of CD4+, CD8+, and immunoglobulin M+ lymphocytes. Two immunogenic proteins, 12 and 31 kDa, identified by two-dimensional cellular immunoblotting, were subjected to partial N-terminal amino acid analysis. The 12-kDa protein was within the area of greatest lymphocyte proliferation, while the 31-kDa protein was chosen for comparison with a 31-kDa protein previously reported by others. A search of the National Biomedical Research Foundation protein data bank showed that the sequences were not homologous with other known proteins. Identification of Brucella proteins immunogenic for bovine lymphocytes provides an important step in distinguishing the various proteins involved in pathogenicity and/or disease resistance.

One- and two-dimensional gel electrophoresis and cellular immunoblotting were used to compare the protein profiles and immunogenic capabilities of salt- and sodium dodecyl sulfate-extracted components isolated from different Brucella species. Cellular immunoblotting demonstrated that freshly isolated bovine peripheral blood mononuclear cells proliferated to similar molecular mass components from the various Brucella extractions. One- and two-dimensional gel electrophoresis showed similarities in protein profiles among the different Brucella species that correlated with lymphocyte reactivity. The results presented in this study provide preliminary evidence that common proteins that induce lymphocyte proliferation are present among different Brucella species, suggesting that a genuswide subunit vaccine may be feasible.