RESUMEN
The clinical efficacy of peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists in cell-mediated autoimmune diseases results from down-regulation of inflammatory cytokines and autoimmune effector cells. T cell islet autoimmunity has been demonstrated to be common in patients with phenotypic type 2 diabetes mellitus (T2DM) and islet-specific T cells (T(+) ) to be correlated positively with more severe beta cell dysfunction. We hypothesized that the beneficial effects of the PPAR-γ agonist, rosiglitazone, therapy in autoimmune T2DM patients is due, in part, to the immunosuppressive properties on the islet-specific T cell responses. Twenty-six phenotypic T2DM patients positive for T cell islet autoimmunity (T(+) ) were identified and randomized to rosiglitazone (n = 12) or glyburide (n = 14). Beta cell function, islet-specific T cell responses, interleukin (IL)-12 and interferon (IFN)-γ responses and islet autoantibodies were followed for 36 months. Patients treated with rosiglitazone demonstrated significant (P < 0·03) down-regulation of islet-specific T cell responses, although no change in response to tetanus, a significant decrease (P < 0·05) in IFN-γ production and significantly (P < 0·001) increased levels of adiponectin compared to glyburide-treated patients. Glucagon-stimulated beta cell function was observed to improve significantly (P < 0·05) in the rosiglitazone-treated T2DM patients coinciding with the down-regulation of the islet-specific T cell responses. In contrast, beta cell function in the glyburide-treated T2DM patients was observed to drop progressively throughout the study. Our results suggest that down-regulation of islet-specific T cell autoimmunity through anti-inflammatory therapy may help to improve beta cell function in autoimmune phenotypic T2DM patients.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/administración & dosificación , Islotes Pancreáticos/fisiología , PPAR gamma/agonistas , Linfocitos T/inmunología , Tiazolidinedionas/administración & dosificación , Adulto , Anciano , Autoanticuerpos/sangre , Autoinmunidad/efectos de los fármacos , Péptido C/sangre , Células Cultivadas , Diabetes Mellitus Tipo 2/inmunología , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Gliburida/administración & dosificación , Gliburida/efectos adversos , Gliburida/farmacología , Humanos , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/farmacología , Terapia de Inmunosupresión , Interferón gamma/inmunología , Interleucina-12/inmunología , Islotes Pancreáticos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Rosiglitazona , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Tiazolidinedionas/efectos adversos , Tiazolidinedionas/farmacologíaRESUMEN
Historically, type 2 diabetes (T2D) was considered a metabolic disease of ageing. However, recent discoveries have demonstrated the role of chronic systemic inflammation in the development of insulin resistance and subsequent progression to T2D. Over the years, investigations into the pathophysiology of T2D have identified the presence of islet-specific T cells and islet autoimmune disease in T2D patients. Moreover, the cell-mediated islet autoimmunity has also been correlated with the progressive loss of ß-cell function associated with T2D disease pathogenesis. In this manuscript, the involvement of cell-mediated islet autoimmune disease in the progression of T2D disease and the similarities in islet-specific T-cell reactivity between type 1 diabetes (T1D) and T2D are discussed.
Asunto(s)
Autoinmunidad/fisiología , Diabetes Mellitus Tipo 2/inmunología , Islotes Pancreáticos/inmunología , Autoanticuerpos/fisiología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 2/patología , Humanos , Islotes Pancreáticos/patología , Fenotipo , Linfocitos T/fisiologíaRESUMEN
Historically, the development of type 2 diabetes has been considered not to have an autoimmune component, in contrast to the autoimmune pathogenesis of type 1 diabetes. In this review we will discuss the accumulating data supporting the concept that islet autoreactivity and inflammation is present in type 2 diabetes pathogenesis, and the islet autoimmunity appears to be one of the factors associated with the progressive nature of the type 2 diabetes disease process.
Asunto(s)
Enfermedades Autoinmunes/etiología , Diabetes Mellitus Tipo 2/inmunología , Inflamación/inmunología , Islotes Pancreáticos/inmunología , Reacción de Fase Aguda/inmunología , Animales , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedad Crónica , Citocinas/biosíntesis , Diabetes Mellitus Tipo 2/complicaciones , Progresión de la Enfermedad , Humanos , Inflamación/complicaciones , Mediadores de Inflamación/inmunología , Resistencia a la Insulina , Grasa Intraabdominal/inmunología , Grasa Intraabdominal/patología , Modelos Inmunológicos , Obesidad/etiología , Obesidad/inmunología , Ratas , Autotolerancia/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
Autoimmune T cell responses directed against insulin-producing ß cells are central to the pathogenesis of type 1 diabetes (T1D). Detection of such responses is therefore critical to provide novel biomarkers for T1D 'immune staging' and to understand the mechanisms underlying the disease. While different T cell assays are being developed for these purposes, it is important to optimize and standardize methods for processing human blood samples for these assays. To this end, we review data relevant to critical parameters in peripheral blood mononuclear cell (PBMC) isolation, (cryo)preservation, distribution and usage for detecting antigen-specific T cell responses. Based on these data, we propose recommendations on processing blood samples for T cell assays and identify gaps in knowledge that need to be addressed. These recommendations may be relevant not only for the analysis of T cell responses in autoimmune disease, but also in cancer and infectious disease, particularly in the context of clinical trials.
Asunto(s)
Conservación de la Sangre/métodos , Separación Celular/métodos , Criopreservación/métodos , Diabetes Mellitus Tipo 1/inmunología , Células Secretoras de Insulina/inmunología , Monocitos/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Superficie/inmunología , Enfermedades Autoinmunes/inmunología , Conservación de la Sangre/normas , Separación Celular/normas , Ensayos Clínicos como Asunto , Criopreservación/normas , Humanos , Infecciones/inmunología , Ratones , Neoplasias/inmunologíaRESUMEN
BACKGROUND: Islet-antigen-specific CD4+ T cells are known to promote auto-immune destruction in T1D. Measuring T-cell number and function provides an important biomarker. In response to this need, we evaluated responses to proinsulin and GAD epitopes in a multicentre study. METHODS: A tetramer-based assay was used in five participating centres to measure T-cell reactivities to DR0401-restricted epitopes. Three participating centres concurrently performed ELISPOT or immunoblot assays. Each centre used blind-coded, centrally distributed peptide and tetramer reagents. RESULTS: All participating centres detected responses to auto-antigens and the positive control antigen, and in some cases cloned the corresponding T cells. However, response rates varied among centres. In total, 74% of patients were positive for at least one islet epitope. The most commonly recognized epitope was GAD270-285. Only a minority of the patients tested by tetramer and ELISPOT were concordant for both assays. CONCLUSIONS: This study successfully detected GAD and proinsulin responses using centrally distributed blind-coded reagents. Centres with little previous experience using class II tetramer reagents implemented the assay. The variability in response rates observed for different centres suggests technical difficulties and/or heterogeneity within the local patient populations tested. Dual analysis by tetramer and ELISPOT or immunoblot assays was frequently discordant, suggesting that these assays detect distinct cell populations. Future efforts should investigate shared blood samples to evaluate assay reproducibility and longitudinal samples to identify changes in T-cell phenotype that correlate with changes in disease course.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Epítopos de Linfocito T/inmunología , Antígenos HLA-DR/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Adulto , Ensayo de Immunospot Ligado a Enzimas , Humanos , Proinsulina/inmunologíaRESUMEN
BACKGROUND: Type 1 diabetes (T1D) is a cell-mediated autoimmune disease characterized by destruction of the pancreatic islet cells. The use of cryopreserved cells is preferable to the use of freshly isolated cells to monitor clinical trials to decrease assay and laboratory variability. METHODS: The T-Cell Workshop Committee of the Immunology of Diabetes Society compared two widely accepted T-cell freezing protocols (warm and cold) to freshly isolated peripheral blood mononuclear cells from patients with T1D and controls in terms of recovery, viability, cell subset composition, and performance in functional assays currently in use in T1D-related research. Nine laboratories participated in the study with four different functional assays included. RESULTS: The cold freezing method yielded higher recovery and viability compared with the warm freezing method. Irrespective of freezing protocol, B cells and CD8+ T cells were enriched, monocyte fraction decreased, and islet antigen-reactive responses were lower in frozen versus fresh cells. However, these results need to take in to account that the overall response to islet autoantigens was low in some assays. CONCLUSIONS: In the current study, none of the tested T-cell functional assays performed well using frozen samples. More research is required to identify a freezing method and a T-cell functional assay that will produce responses in patients with T1D comparable to responses using fresh peripheral blood mononuclear cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Criopreservación/métodos , Diabetes Mellitus Tipo 1/inmunología , Islotes Pancreáticos/inmunología , Supervivencia Celular , Humanos , Proinsulina/inmunologíaRESUMEN
Type 1 diabetes (T1D) is an autoimmune disease caused by the T cell-mediated destruction of the pancreatic insulin-producing beta cells. Currently there are no widely accepted and standardized assays available to analyse the function of autoreactive T cells involved in T1D. The development of such an assay would greatly aid efforts to understand the pathogenesis of T1D and is also urgently required to guide the development of antigen-based therapies intended to prevent, or cure, T1D. Here we describe some of the assays used currently to detect autoreactive T cells in human blood and review critically their strengths and weaknesses. The challenges and future prospects for the T cell assays are discussed.
Asunto(s)
Autoantígenos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Pruebas Inmunológicas/métodos , Islotes Pancreáticos/inmunología , Linfocitos T/inmunología , Humanos , Activación de Linfocitos , Linfocitos T/citologíaRESUMEN
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.
Asunto(s)
Autoanticuerpos/sangre , Autoantígenos/inmunología , Diabetes Mellitus Tipo 2/inmunología , Inmunidad Celular , Islotes Pancreáticos/inmunología , Adolescente , Adulto , Anciano , Autoantígenos/química , Índice de Masa Corporal , Niño , Femenino , Humanos , Immunoblotting , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Peso MolecularRESUMEN
BACKGROUND: Insulin-dependent (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 transplantation and at 3-month intervals after transplantation. Our study population included 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 transplantation, all nine subjects tested negative for islet cell autoantibody (ICA), and seven of nine subjects tested positive for glutamic acid decarboxylase autoantibody (GADAb). After transplantation, all subjects became ICA(+), and the two patients who were GADAb(-) before transplantation, became GADAb(+) after transplantation. Maximum PBMC reactivity to separated human fetal pancreatic proteins was observed in four patients at 3 months, in one patient at 6 months, in two patients at 9 months, and in one patient at 12 months after transplantation. One subject, who had PBMC reactivity to multiple islet proteins before transplantation, 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 transplant recipients is most consistent with reactivation of the type 1 diabetes disease process.
Asunto(s)
Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 1/cirugía , Trasplante de Tejido Fetal , Trasplante de Páncreas , Adulto , Anciano , Autoanticuerpos/análisis , División Celular/fisiología , Diabetes Mellitus Tipo 1/inmunología , Femenino , Proteínas Fetales/fisiología , Glutamato Descarboxilasa/inmunología , Humanos , Terapia de Inmunosupresión , Masculino , Persona de Mediana Edad , Monocitos/patología , Páncreas/citología , Páncreas/embriologíaRESUMEN
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.
Asunto(s)
Diabetes Mellitus Tipo 1/cirugía , Trasplante de Tejido Fetal/inmunología , Terapia de Inmunosupresión , Trasplante de Islotes Pancreáticos/inmunología , Adulto , Anciano , Autoanticuerpos/biosíntesis , Diabetes Mellitus Tipo 1/inmunología , Femenino , Glutamato Descarboxilasa/inmunología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Feto/inmunología , Leucocitos Mononucleares/inmunología , Trasplante de Páncreas/inmunología , Páncreas/inmunología , Adulto , Humanos , Peso Molecular , RiesgoRESUMEN
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)
Asunto(s)
Insulina/metabolismo , Interleucina-1/farmacología , Islotes Pancreáticos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Técnicas de Cultivo , Citotoxicidad Inmunológica , Interacciones Farmacológicas , Secreción de Insulina , Interleucina-1/toxicidad , Islotes Pancreáticos/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
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.
Asunto(s)
Autoanticuerpos/sangre , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 2/diagnóstico , Glutamato Descarboxilasa/inmunología , Isoenzimas/inmunología , Adulto , Factores de Edad , Anciano , Pueblo Asiatico , Autoanticuerpos/clasificación , Población Negra , Índice de Masa Corporal , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 2/inmunología , Diagnóstico Diferencial , Femenino , Humanos , Hiperglucemia/etiología , Insulina/inmunología , Masculino , Persona de Mediana Edad , Fenotipo , Poliuria/etiología , Valor Predictivo de las Pruebas , Población BlancaRESUMEN
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.
Asunto(s)
Diabetes Mellitus Experimental/cirugía , Hipoglucemiantes/farmacología , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/efectos de los fármacos , Tiazolidinedionas/farmacología , Animales , Biomarcadores/sangre , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/metabolismo , Supervivencia de Injerto/efectos de los fármacos , Humanos , Hipoglucemiantes/sangre , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Rosiglitazona , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Tiazolidinedionas/sangre , Factores de TiempoRESUMEN
CONTEXT: The previously reported absence of 65-kDa glutamate decarboxylase antibody (GAD65Ab)-specific antiidiotypic antibodies (anti-Id) in type 1 diabetes (T1D) patients at clinical onset could be due to an inability to mount an antibody response to GAD65Ab or a longitudinal decline in anti-Id levels. OBJECTIVE AND DESIGN: We investigated anti-Id levels in longitudinal samples obtained from T1D patients (n = 41) (clinical diagnosis - 12 months), and latent autoimmune diabetes in adults (LADA) patients (n = 32) who received alum-formulated human recombinant GAD65 (baseline - 12 months). We also determined anti-Id levels in a small cohort of Type 2 diabetes patients during their development of autoimmune T cell responses. RESULTS: At clinical onset T1D patients presented no or low anti-Id levels. However, 22/41 T1D patients showed ≥50% increase in GAD65Ab-specific anti-Id levels during follow-up; peaking at 3 (n = 1), 6 (n = 10), 9 (n = 10), or 12 (n = 1) months. Increasing anti-Id levels marked patients who experienced a temporary increase in C-peptide levels. Anti-Id levels correlated significantly with glycated hemoglobin and C-peptide levels at 6 and 9 months (P values ranged from <0.001 to <0.05). In LADA patients receiving placebo, anti-Id levels declined in seven of nine patients, whereas four of five patients receiving 20 µg alum-formulated human recombinant GAD65 showed increasing anti-Id levels. Changes in anti-Id and C-peptide levels closely correlated (P < 0.0001). The significant decline in anti-Id levels (P = 0.03) in T2D patients developing T cell autoimmune responses supports our hypothesis that declining anti-Id levels are associated with developing islet autoimmunity. CONCLUSIONS: The close association between GAD65Ab-specific anti-Id levels and ß-cell function may provide a novel marker for the progression of autoimmune diabetes.
Asunto(s)
Anticuerpos Antiidiotipos/sangre , Autoinmunidad/inmunología , Péptido C/sangre , Diabetes Mellitus Tipo 1/sangre , Progresión de la Enfermedad , Glutamato Descarboxilasa/sangre , Islotes Pancreáticos/inmunología , Adolescente , Análisis de Varianza , Anticuerpos Antiidiotipos/inmunología , Péptido C/inmunología , Niño , Preescolar , Diabetes Mellitus Tipo 1/inmunología , Femenino , Glutamato Descarboxilasa/inmunología , Humanos , Lactante , Masculino , Estudios ProspectivosRESUMEN
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.
Asunto(s)
Anticuerpos/inmunología , Especificidad de Anticuerpos , Autoanticuerpos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Insulina/inmunología , Afinidad de Anticuerpos , Unión Competitiva , Humanos , Epítopos Inmunodominantes/inmunologíaRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/inmunología , Brucella/inmunología , Activación de Linfocitos , Animales , Bovinos , Técnicas In Vitro , Cloruro de Sodio/farmacología , Dodecil Sulfato de Sodio/farmacologíaRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/análisis , Proteínas Bacterianas/análisis , Brucella abortus/inmunología , Activación de Linfocitos , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Bovinos , Electroforesis en Gel de Poliacrilamida , Femenino , Citometría de Flujo , Immunoblotting , Masculino , Datos de Secuencia Molecular , Peso MolecularRESUMEN
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.
Asunto(s)
Insulina/metabolismo , Interleucina-1/toxicidad , Islotes Pancreáticos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Radioisótopos de Cromo , Relación Dosis-Respuesta a Droga , Glucosa/farmacología , Humanos , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Cinética , Ratas , Proteínas Recombinantes/toxicidadRESUMEN
Intra- and intermolecular spreading of T cell responses to autoantigens has been implicated in the pathogenesis of autoimmune diseases. Therefore, we questioned whether T cell responses from subjects identified as at-risk (positive for autoantibody reactivity to islet proteins) for the development of type 1 diabetes, a cell-mediated autoimmune disease, would demonstrate intermolecular Ag spreading of T cell responses to islet cell proteins. Previously, we have demonstrated that by the time subjects develop type 1 diabetes, they have T cell responses to numerous islet proteins, whereas T cells from normal controls respond to a limited number of islet proteins. Initial testing of PBMC responses from 25 nondiabetic at-risk subjects demonstrated that 16 of the 25 subjects have PBMC responses to islet proteins similar to controls. Fourteen of these 16 subjects were available for follow-up. Eleven of the 14 developed T cell responses to increasing numbers of islet proteins, and 6 of these subjects developed type 1 diabetes. In the nine subjects who already demonstrated T cell Ag spreading at the initial visit, four were available for follow-up. Of these four, two had increases in T cell reactivity to islet proteins, while two maintained their initial levels of T cell reactivity. We also observed Ag spreading in autoantibody reactivity to islet proteins in nine of the 18 at-risk subjects available for follow-up. Our data strongly support the conclusion that intermolecular spreading of T cell and Ab responses to islet proteins occurs during the preclinical period of type 1 diabetes.