ABSTRACT
Type 1 diabetes (T1D) is caused by the selective destruction of the insulin-producing beta cells of the pancreas by an autoimmune response. Due to ethical and practical difficulties, the features of the destructive process are known from a small number of observations, and transcriptomic data are remarkably missing. Here we report whole genome transcript analysis validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and correlated with immunohistological observations for four T1D pancreases (collected 5 days, 9 months, 8 and 10 years after diagnosis) and for purified islets from two of them. Collectively, the expression profile of immune response and inflammatory genes confirmed the current views on the immunopathogenesis of diabetes and showed similarities with other autoimmune diseases; for example, an interferon signature was detected. The data also supported the concept that the autoimmune process is maintained and balanced partially by regeneration and regulatory pathway activation, e.g. non-classical class I human leucocyte antigen and leucocyte immunoglobulin-like receptor, subfamily B1 (LILRB1). Changes in gene expression in islets were confined mainly to endocrine and neural genes, some of which are T1D autoantigens. By contrast, these islets showed only a few overexpressed immune system genes, among which bioinformatic analysis pointed to chemokine (C-C motif) receptor 5 (CCR5) and chemokine (CXC motif) receptor 4) (CXCR4) chemokine pathway activation. Remarkably, the expression of genes of innate immunity, complement, chemokines, immunoglobulin and regeneration genes was maintained or even increased in the long-standing cases. Transcriptomic data favour the view that T1D is caused by a chronic inflammatory process with a strong participation of innate immunity that progresses in spite of the regulatory and regenerative mechanisms.
Subject(s)
Diabetes Mellitus, Type 1/metabolism , Diabetes Mellitus, Type 1/pathology , Gene Expression Profiling , Islets of Langerhans/metabolism , Pancreas/metabolism , Pancreas/pathology , Adolescent , Adult , Antigens, CD/analysis , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , C-Reactive Protein/genetics , C-Reactive Protein/metabolism , Cell Count , Diabetes Mellitus, Type 1/immunology , Down-Regulation/genetics , Female , Gene Expression/genetics , Glucagon-Secreting Cells/metabolism , HLA Antigens/genetics , HLA Antigens/metabolism , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Humans , Immunity, Innate/genetics , Inflammation/genetics , Insulin-Secreting Cells/metabolism , Islets of Langerhans/pathology , Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Leukocytes/metabolism , Male , Middle Aged , Pancreatitis-Associated Proteins , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation/genetics , Young Adult , HLA-E AntigensABSTRACT
Dendritic cells (DCs) are powerful antigen-presenting cells capable of maintaining peripheral tolerance. The possibility to generate tolerogenic DCs opens new therapeutic approaches in the prevention or remission of autoimmunity. There is currently no treatment inducing long-term tolerance and remission in type 1 diabetes (T1D), a disease caused by autoimmunity towards beta cells. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow islet regeneration. Apoptotic cells--a source of autoantigens--are cleared rapidly by macrophages and DCs through an immunologically silent process that contributes to maintaining tolerance. Our aims were to prevent T1D and to evaluate the re-establishment of peripheral tolerance using autologous DCs pulsed in vitro with apoptotic bodies from beta cells. Immature DCs derived from bone marrow of non-obese diabetic (NOD) mice were obtained and pulsed with antigen-specific apoptotic bodies from the beta cell line NIT-1. Those DCs that phagocytosed apoptotic cells diminished the expression of co-stimulatory molecules CD40 and CD86 and reduced secretion of proinflammatory cytokines. Moreover, these cells were resistant to increase the expression of co-stimulatory molecules after lipopolysaccharide activation. The administration of these cells to NOD transgenic mice expressing interferon-beta in their insulin-producing cells, a model of accelerated autoimmune diabetes, decreased diabetes incidence significantly and correlated positively with insulitis reduction. DCs pulsed with apoptotic cells that express disease-associated antigens constitutes a promising strategy to prevent T1D.
Subject(s)
Apoptosis/immunology , Autoantigens/immunology , Dendritic Cells , Diabetes Mellitus, Type 1/prevention & control , Immune Tolerance/immunology , Immunotherapy/methods , Insulin-Secreting Cells/immunology , Animals , Autoantigens/administration & dosage , Cells, Cultured , Cytoplasmic Vesicles/immunology , Dendritic Cells/immunology , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/immunology , Endocytosis , Epitopes , Female , Insulin-Secreting Cells/pathology , Interferon-beta/genetics , Lipopolysaccharides/pharmacology , Lymphocyte Activation/drug effects , Mice , Mice, Inbred NOD , Mice, Transgenic , Recombinant Fusion Proteins/genetics , Specific Pathogen-Free OrganismsABSTRACT
The destruction of beta cells by the islet infiltrating lymphocytes causes type 1 diabetes. Transgenic mice models expressing interferon (IFN)-beta in beta cells, in the non-obese diabetic (NOD) strain and in a diabetes-free, major histocompatibility complex-matched, homologous strain, the non-obese resistant (NOR) mice, developed accelerated type 1 diabetes after 3 weeks of age. Our aim was to determine if natural killer (NK) cells could affect the acceleration of the disease. We determined the amount of NK cells in the pancreas, spleen and lymph nodes from NOD rat insulin promoter (RIP)-IFN-beta mice. Pancreatic cytokines were assessed by quantitative real-time polymerase chain reaction and protein arrays. To confirm the relevance of NK cells in the acceleration of autoimmune diabetes this subset was depleted with anti-asialo GM1 antibodies. An increase of intrapancreatic NK cells characterized the accelerated onset of diabetes both in NOD and NOR RIP-IFN-beta transgenic models. Cytokines involved in NK function and migration were found to be hyperexpressed in the pancreas from accelerated diabetic mice. Interestingly, the depletion of NK cells in vivo abolished completely the acceleration of diabetes. NK cells connect innate to adaptive immunity and might play a role in autoimmunity. We report here that NK cells are required critically in the pancreas for accelerated diabetes. This model links inflammation to acceleration of beta cell-specific autoimmunity mediated by NK cells.
Subject(s)
Diabetes Mellitus, Type 1/immunology , Interferon-beta/immunology , Killer Cells, Natural/immunology , Animals , B-Lymphocytes/immunology , Cytokines/metabolism , Disease Models, Animal , Disease Progression , G(M1) Ganglioside/immunology , Islets of Langerhans/immunology , Lymph Nodes/immunology , Lymphocyte Subsets/immunology , Mice , Mice, Inbred NODABSTRACT
AIMS/HYPOTHESIS: The expression of IFNbeta in beta cells results in accelerated type 1 diabetes. The REG family of beta cell proliferation factors have been described as autoantigens in autoimmune diabetes. The aim of this study was to determine the effect of IFNbeta on Reg expression, and the implications of this in terms of autoimmunity. METHODS: Reg gene expression was determined in islets from non-obese diabetic (NOD) RIP-HuIFNbeta mice by cDNA microarray, quantitative real-time PCR and immunohistochemistry. The effect of IFNbeta on Reg1 and Reg2 expression was assessed in the NOD insulinoma cell line NIT-1. IL-6, known to induce Reg expression, was measured in the insulitis microenvironment. Morphological studies were carried out to determine islet enlargement in this model. RESULTS: Reg2 was upregulated in islets from the NOD RIP-HuIFNbeta mice at the onset of the autoimmune attack. IFNbeta upregulates Reg1 and Reg2 genes in NIT-1 cells. The expression of Il6 was increased in islets from transgenic mice and in NIT-1 cells exposed to HuIFNbeta. Moreover, islets from transgenic mice were enlarged compared with those from wild-type mice. CONCLUSIONS/INTERPRETATION: Reg overexpression correlates well with the acceleration of diabetes in this model. The upregulation of Reg suggests that islets try to improve hyperglycaemia by regenerating the cells lost in the autoimmune attack. Reg expression is regulated by several factors such as inflammation. Therefore, the overexpression of an IFNbeta-induced autoantigen (REG) in the islets during inflammation might contribute to the premature onset of diabetes.
Subject(s)
Diabetes Mellitus, Type 1/genetics , Gene Expression Regulation , Interferon-beta/physiology , Islets of Langerhans/physiopathology , Lithostathine/genetics , Animals , Cell Line , Crosses, Genetic , Female , Humans , Insulin/genetics , Islets of Langerhans/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD/immunology , Mice, Transgenic , Promoter Regions, Genetic , Rats , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Los grupos internacionales más relevantes en tolerancia inmunológica y autoinmunidad participaron en el Simposio Keystone Tolerancia, Autoinmunidad y Regulación Inmunológica en Breckenridge, Colorado, entre el 21 y el 26 de marzo de 2006. Las ponencias describieron los avances en la investigación en el campo de la tolerancia y la autoinmunidad. En este resumen se ha organizado la información en cinco áreas: 1) Genes y señalización en la regulación del sistema inmune, 2) Desencadenantes extrínsecos y aspectos intrínsecos de la autoinmunidad, 3) Supresión y regulación del sistema inmune, 4) Ensayos clínicos de inmunotolerancia, 5) Debate y reconocimientos a la trayectoria científica. Los resultados presentados en el simposio demuestran los continuos avances en el conocimiento de la tolerancia y ponen de manifiesto la necesidad de continuar investigando en esta línea. Esto contribuiría al desarrollo de ensayos clínicos de prevención de enfermedades autoinmunes en sujetos de riesgo
The most relevant international groups working in immunological tolerance and autoimmunity participated in the Keystone Symposia Tolerance, Autoimmunity and Immune Regulation in Breckenridge, Colorado, between the 21st and 26th of March 2006. Lectures and abstracts described new research work and advances relevant to tolerance and autoimmunity. In this report, the information is organized in five areas: 1) Genes and signals in immune regulation, 2) Extrinsic triggers and intrinsic drivers of autoimmunity, 3) Suppression and regulation of the immune system, 4) Translating tolerance to the clinic, 5) Debate and awards. The results presented in the meeting demonstrate the continuous advances in the knowledge of tolerance and prove the need to pursue research on this field. This may help to develop clinical trials to prevent autoimmune diseases in at risk subjects
Subject(s)
Humans , Autoimmunity/immunology , Immune Tolerance/immunology , Congress , Biomedical ResearchABSTRACT
Pancreatic islet transplantation has been proposed as an attractive option for the treatment of type I diabetes. Transplantation into different sites has been investigated, among them those that are immuno-logically privileged (e.g., thymus, uterus, brain, anterior eye chamber, and testicle). Because of their characteristics, seminal vesicles could be considered as immunologically privileged organs, but there is no worldwide experience that can confirm it. The purpose of the present study is to assess the viability and functionality of islet transplantation into seminal vesicles of diabetic rats. One hundred ninety inbred adult male syngeneic Lewis rats were used as donors (n = 72), receptors (n = 36), and controls(n = 11). Diabetes was chemically induced through a single intraperitoneal injection of streptozotocin. Groups of 1200 purified islets were introduced in the right seminal vesicle of diabetic rats. Diabetic control rats were sham transplanted. Body weight and glycemia were monitored every 2 d. Of transplanted rats, 16.7% achieved a good function due to islet engraftment, while 30.6% achieved a partially good response, and 52.7% were considered as nonresponding. This is the first report about islet transplantation into seminal vesicles of diabetic animals. Our results indicate that islet transplantation into rat seminal vesicles is technically possible, and that islets can function normally after engraftment into the wall of the seminal vesicle.
Subject(s)
Diabetes Mellitus, Experimental/surgery , Graft Survival , Islets of Langerhans Transplantation/methods , Seminal Vesicles/surgery , Animals , Blood Glucose , Body Weight , Diabetes Mellitus, Experimental/blood , Hyperglycemia/blood , Hyperglycemia/surgery , Male , Rats , Rats, Inbred LewABSTRACT
La diabetes tipo 1 (DT1) es una enfermedad autoinmunitaria causada por la destrucción de las células beta pancreáticas productoras de insulina. La patogénesis de DT1 es compleja, interviniendo factores genéticos, inmunológicos y ambientales. Las infecciones víricas serían uno de los factores ambientales que, juntamente con la susceptibilidad genética, podrían estar implicados en la patogénesis de la enfermedad. En la presente revisión, resumimos las hipótesis de la contribución de los virus en las enfermedades autoinmunes en general, centrándonos en la DT1: Mimetismo molecular, aumento del procesamiento y presentación antigénica de autoantígenos durante la infección o diseminación de epítopos (epitope spreading), efectos colaterales, reconocimiento de epitopos crípticos por parte de linfocitos T autoreactivos, activación de linfocitos T con dos receptores (dual TCRs), reactivación de células T de memoria por mecanismos antígeno inespecíficos,anticuerpos anti-idiotípicos y superantígenos. Resultadosde estudios epidemiológicos, serológicos y experimentales sugieren la asociación entre varios virus y el desarrollo de DT1. Los datos más convincentes son las variaciones estacionales de la incidencia de la enfermedad, el aumento de la frecuencia de diabetes en pacientes con rubeola congénita o infección por enterovirus y la detección de secuencias de DNA de citomegalovirus en los linfocitos de pacientes con DT1. Sin embargo, los efectos causales directos son muy dificiles de demostrar. El objetivo de la presente revisión es resumir los datos sobre el papel de los virus en el desarrollo de la DT1 (AU)
Type 1 diabetes (T1D) is an autoimmune disease that results from the destruction of insulin producing pancreatic is let betacells. The pathogenesis of T1D is complex and results from a combination of genetic, immunologic and environmental factors. Viruses seem to play a role among the many environmental factors that, together with the genetic susceptibility, have been implicatedin the pathogenesis of T1D. Here we review the various hypotheses of the contribution of viruses to autoimmune diseases in general, focusing on T1D: Molecular mimicry, increased processing and presentation of autoantigens during infection or epitopespreading, direct bystander effects, recognition of cryptic epitopesby autoreactive T cells, activation of T cells with dual TCRs, reactivation of memory T cells by nonantigen-specific mechanisms,anti-idiotypic antibodies and superantigens. Epidemiological,serological and experimental studies suggest the association of several viruses to the development of autoimmune diabetes.The most convincing findings are the seasonal variations in the incidence of the disease, the increased frequency of T1D in patients with congenital rubella syndrome or enterovirus infection and the detection of CMV DNA sequences in lymphocytes from T1D patients. However, direct causative effects are difficult to verify. The aim of the present review is to summarize the findings regarding the role of viruses in the development of T1D (AU)
Subject(s)
Humans , Virus Diseases/complications , Diabetes Mellitus, Type 1/etiology , Autoimmune Diseases/complications , Cytomegalovirus/pathogenicity , Enterovirus/pathogenicity , Rubella virus/pathogenicityABSTRACT
La diabetes tipo 1 (DT1) es una enfermedad autoinmunitariacausada por la destrucción de las células beta pancreáticas productorasde insulina. La patogénesis de DT1 es compleja, interviniendofactores genéticos, inmunológicos y ambientales. Lasinfecciones víricas serían uno de los factores ambientales que, juntamentecon la susceptibilidad genética, podrían estar implicadosen la patogénesis de la enfermedad. En la presente revisión, resumimoslas hipótesis de la contribución de los virus en las enfermedadesautoinmunes en general, centrándonos en la DT1: Mimetismomolecular, aumento del procesamiento y presentación antigénicade autoantígenos durante la infección o diseminación deepítopos (epitope spreading), efectos colaterales, reconocimientode epitopos crípticos por parte de linfocitos T autoreactivos, activaciónde linfocitos T con dos receptores (dual TCRs), reactivaciónde células T de memoria por mecanismos antígeno inespecíficos,anticuerpos anti-idiotípicos y superantígenos. Resultadosde estudios epidemiológicos, serológicos y experimentales sugierenla asociación entre varios virus y el desarrollo de DT1. Losdatos más convincentes son las variaciones estacionales de la incidenciade la enfermedad, el aumento de la frecuencia de diabetesen pacientes con rubeola congénita o infección por enterovirusy la detección de secuencias de DNA de citomegalovirus enlos linfocitos de pacientes con DT1. Sin embargo, los efectos causalesdirectos son muy dificiles de demostrar. El objetivo de lapresente revisión es resumir los datos sobre el papel de los virusen el desarrollo de la DT1
Type 1 diabetes (T1D) is an autoimmune disease that resultsfrom the destruction of insulin producing pancreatic islet betacells. The pathogenesis of T1D is complex and results from a combinationof genetic, immunologic and environmental factors. Virusesseem to play a role among the many environmental factorsthat, together with the genetic susceptibility, have been implicatedin the pathogenesis of T1D. Here we review the various hypothesesof the contribution of viruses to autoimmune diseases ingeneral, focusing on T1D: Molecular mimicry, increased processingand presentation of autoantigens during infection or epitopespreading, direct bystander effects, recognition of cryptic epitopesby autoreactive T cells, activation of T cells with dual TCRs,reactivation of memory T cells by nonantigen-specific mechanisms,anti-idiotypic antibodies and superantigens. Epidemiological,serological and experimental studies suggest the associationof several viruses to the development of autoimmune diabetes.The most convincing findings are the seasonal variationsin the incidence of the disease, the increased frequency of T1D inpatients with congenital rubella syndrome or enterovirus infectionand the detection of CMV DNA sequences in lymphocytesfrom T1D patients. However, direct causative effects are difficultto verify. The aim of the present review is to summarize the findingsregarding the role of viruses in the development of T1D
Subject(s)
Humans , Virus Diseases/complications , Diabetes Mellitus, Type 1/immunology , Genetic Predisposition to Disease , Epitopes/immunology , Autoimmunity/immunology , Enterovirus/pathogenicity , Retroviridae/pathogenicity , Reoviridae/pathogenicity , Mumps virus/pathogenicity , Encephalomyocarditis virus/pathogenicity , Cytomegalovirus/pathogenicity , Parvovirus/pathogenicity , Mengovirus/pathogenicityABSTRACT
Gran parte de lo que sabemos sobre el desarrollo de la diabetes mellitus tipo 1 (T1D), se lo debemos a los estudios realizados en el ratón NOD, actualmente uno de los mejores modelos experimentales de esta enfermedad. Estudios realizados en esta cepa murina han demostrado que los linfocitos T son los principales efectores de la destrucción de las células beta pancreáticas. Sin embargo, también se ha observado que otras células del sistema inmune están implicadas en el desarrollo de la enfermedad. Entre ellas, las células dendríticas, los macrófagos y los linfocitos B, son imprescindibles tanto en el inicio como en fases mas avanzadas de la enfermedad. El objetivo de la presente revisión es sintetizar los recientes conocimientos sobre el papel de estas poblaciones celulares como células efectoras en el desarrollo de la T1D (AU)
Subject(s)
Humans , B-Lymphocytes/immunology , Autoimmunity , Diabetes Mellitus, Type 1/immunology , Dendritic Cells/immunology , Autoantibodies/immunology , Macrophages/immunology , CD8-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , T-Lymphocytes, Regulatory/immunology , Islets of Langerhans/immunologyABSTRACT
El 7º Congreso Internacional de la Immunology of Diabetes Society (IDS), tuvo lugar en Cambridge (UK) entre el 28 y el 31de Marzo de 2004, con la participación de los grupos más relevantes que trabajan en la inmunología de la diabetes, la fisiología de la célula beta y el transplante de islotes. Se presentaron muy diversas novedades científicas en el campo de la diabetes tipo 1. A modo de compendio en este resumen, los trabajos se agruparon en 8 áreas: 1) Talleres de autoanticuerpos y linfocitos T, 2) utilidad de los modelos animales en diabetes tipo 1, 3) transplante de islotes en pacientes diabéticos, 4) hipótesis de la higiene, 5) contribución de la célula beta y los linfocitos T en el ataque autoinmune ("la víctima y el asesino"), 6) variaciones en la diabetes tipo 1 (forma fulminante en Japón), 7) nuevo modelo animal para el estudio de la diabetes tipo 1 y 8) simposio satélite de transplante. Los datos presentados reflejan los lentos pero constantes avances en el conocimiento de la diabetes tipo 1 y evidencian la necesidad de seguir investigando hasta llegar a comprender la etiopatogenia de esta enfermedad y poder desarrollar tanto ensayos de prevención en sujetos de riesgo como tratamientos eficaces y seguros para los pacientes (AU)
Subject(s)
Animals , Humans , Autoimmunity/immunology , Diabetes Mellitus, Type 1/immunology , Islets of Langerhans/immunology , Autoantigens/immunology , Disease Models, Animal , B-Lymphocytes , T-Lymphocytes , Islets of Langerhans Transplantation/trendsABSTRACT
La diabetes mellitus tipo 1 es una enfermedad de etiología desconocida causada por la destrucción de las células productoras de insulina por parte del sistema inmunitario. Se conocen factores genéticos y desencadenantes ambientales que confieren susceptibilidad de desarrollar la enfermedad, así como alteraciones inmunológicas asociadas al proceso inflamatorio de los islotes que se detallan en esta revisión. Estos datos, junto con los modelos animales desarrollados -espontáneos o genéticamente manipulados- y los ensayos clínicos para evitar la manifestación de la enfermedad, han generado conocimiento y multitud de hipótesis, pero hasta el momento no existe prevención efectiva frente a la diabetes autoinmune. Es de esperar que las investigaciones presentes y futuras sobre la diabetes tipo 1 consigan determinar la etiología de la enfermedad y lograr la prevención o tratamientos de tolerancia inmunológica eficaces (AU)
Subject(s)
Animals , Humans , Autoimmunity/physiology , Diabetes Mellitus, Type 1/immunology , Risk Factors , Autoantibodies/immunology , Disease Models, AnimalABSTRACT
CD14, a GPI-linked membrane protein, is a component of the lipopolysaccharide (LPS) receptor complex, one of the pattern-recognizing receptors (PRR) expressed by myeloid lineage cells. Here we report that CD14, the functionally linked toll-like receptor molecules, TLR2 and TLR4, and the associated molecule MD-2 are expressed in endocrine cells of the human pancreatic islets. CD14 expression in human pancreatic islets was determined by immunofluorescence staining of tissue sections and primary cultures, and confirmed by flow cytometry of dispersed normal islets and SV40-transformed islet cells (HP62). The latter cells synthesized and secreted CD14 in response to lipopolysaccharide (LPS) in a time- and dose-dependent manner. Reverse transcription polymerase chain reaction (RT-PCR)-Southern was positive for CD14, TLR2, TLR4 and MD-2 in human pancreas, purified islets and HP62 cells. In vitro experiments using rat islets (also positive for CD14 by RT-PCR) and HP62 cells showed that LPS regulates glucose-dependent insulin secretion and induces inflammatory cytokines [interleukin (IL)-1alpha, IL-6 and tumour necrosis factor (TNF)-alpha]. The functional expression of CD14 and associated molecules in islet beta cells adds a new pathway that islet cells may follow to adjust their function to endotoxaemia situations and become vulnerable to the inflammatory events that occur during diabetogenic insulitis.
Subject(s)
Islets of Langerhans/metabolism , Lipopolysaccharide Receptors/metabolism , Membrane Glycoproteins/metabolism , Receptors, Cell Surface/metabolism , Adolescent , Adult , Antigens, Surface/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Female , Glucose/antagonists & inhibitors , Glucose/pharmacology , Humans , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/drug effects , Lipopolysaccharide Receptors/genetics , Lipopolysaccharides/pharmacology , Lymphocyte Antigen 96 , Male , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Species Specificity , Toll-Like Receptor 2 , Toll-Like Receptor 4 , Toll-Like Receptors , Tumor Cells, CulturedSubject(s)
Blood Glucose/metabolism , Diabetes Mellitus, Experimental/surgery , Islets of Langerhans Transplantation/methods , Seminal Vesicles , Animals , Diabetes Mellitus, Experimental/blood , Disease Models, Animal , Islets of Langerhans Transplantation/pathology , Islets of Langerhans Transplantation/physiology , Male , Rats , Rats, Inbred Lew , Seminal Vesicles/pathology , Time Factors , Transplantation, HeterologousABSTRACT
INTRODUCTION: Islet transplantation is an attractive solution for type I diabetes, but the results are at the present discouraging. Collagenase, the enzyme used to obtain islets for transplantation, presents interbatch variability and endotoxin contamination that induces inflammatory cytokine production. Liberase (Roche, Basel, Switzerland), a new mixture of purified enzymes, has the same composition in all batches and is endotoxin-free. AIMS: To compare the engraftment of islets obtained using either enzyme in streptozotocin-induced diabetic rats. METHODOLOGY: Collagenase- or Liberase-isolated islets were transplanted under the kidney capsule of diabetic rats. Collagenase islets restored glycemia and insulinemia in all animals at 24 hours, and both parameters were maintained in 45% of rats over 90 days; however, Liberase islets failed to reverse diabetes in all subjects. RESULTS: In vitro experiments showed that Liberase islets did not maintain active insulin secretion. Cytotoxicity assays showed toxicity of Liberase to islets; both enzymes induced inflammatory cytokine production by macrophages. CONCLUSION: In summary, in our model, Liberase is not a good substitute for collagenase as an islet-isolating reagent. A major effort and investment in developing enzymes for tissue dispersion is needed to improve the outcome of islet transplantation.
Subject(s)
Collagenases , Diabetes Mellitus, Experimental/surgery , Graft Survival , Islets of Langerhans Transplantation , Thermolysin , Animals , Blood Glucose/analysis , Cell Death/drug effects , Collagenases/metabolism , Collagenases/pharmacology , Diabetes Mellitus, Experimental/blood , Endotoxins/analysis , In Vitro Techniques , Insulin/metabolism , Insulin Secretion , Interleukin-1/genetics , Interleukin-6/genetics , Islets of Langerhans/physiology , Macrophages/drug effects , Macrophages/metabolism , Male , RNA, Messenger/biosynthesis , Rats , Rats, Inbred Lew , Reverse Transcriptase Polymerase Chain Reaction , Specimen Handling , Thermolysin/metabolism , Thermolysin/pharmacology , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Thyroid follicular cells (TFC) in Graves' disease (GD) hyperexpress HLA class I and express ectopic HLA class II molecules, probably as a consequence of cytokines produced by infiltrating T cells. This finding led us to postulate that TFC could act as antigen-presenting cells, and in this way be responsible for the induction and/or maintenance of the in situ autoimmune T cell response. Invariant chain (li) and HLA-DM molecules are implicated in the antigen processing and presentation by HLA class II molecules. We have investigated the expression of these molecules by TFC from GD glands. The results demonstrate that class II+ TFC from GD patients also express li and HLA-DM, and this expression is increased after IFN-gamma stimulation. The level of HLA-DM expression by TFC was low but sufficient to catalyze peptide loading into the HLA class II molecules and form stable HLA class II-peptide complexes expressed at the surface of TFC. These results have implications for the understanding of the possible role of HLA class II+ TFC in thyroid autoimmune disease.
Subject(s)
Antigens, Differentiation, B-Lymphocyte/analysis , Graves Disease/immunology , HLA-D Antigens/analysis , HLA-DR Antigens/analysis , Histocompatibility Antigens Class II/analysis , Thyroid Gland/immunology , Antigen Presentation/immunology , Antigens, Differentiation, B-Lymphocyte/genetics , Blotting, Northern , Flow Cytometry , Fluorescent Antibody Technique , Graves Disease/physiopathology , HLA-D Antigens/genetics , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Humans , Interferon-gamma/pharmacology , Peptide Fragments/immunology , Peptide Fragments/metabolism , Precipitin Tests , Thyroid Gland/cytology , Thyroid Gland/drug effectsABSTRACT
Clinical transplantation of human islets has a disappointingly low rate of success. We report here the identification of a possible causative factor: endotoxin present in the collagenase preparations used to disperse the pancreatic tissue before islet purification and transplantation. Supporting evidence includes (1) detection of unexpectedly high levels of endotoxin in most collagenase solutions currently used to digest human pancreases; (2) demonstration that supernatants generated during islet separation are able to induce the inflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha) in macrophages; and (3) induction of IL-1, IL-6, and TNF-alpha in the islets during the separation procedure. Cytokine expression was assessed by reverse transcription-polymerase chain reaction and, for TNF-alpha, confirmed by enzyme-linked immunoabsorbent assay. It is proposed that endotoxin and locally induced cytokines carried over with the graft activate the endothelium and promote lymphomonocytic infiltration of grafted islets and surrounding liver tissue favoring primary nonfunction and early rejection. These results also have implications for the numerous experimental procedures that use collagenase, and they point to possible ways to improve islet preparation and transplantation protocols.
Subject(s)
Endotoxins/analysis , Islets of Langerhans Transplantation/methods , Adolescent , Adult , Cell Separation/methods , Collagenases/chemistry , Cytokines/metabolism , Female , Humans , Macrophages/immunology , Male , Middle Aged , Tissue DonorsABSTRACT
Autoimmune thyroid diseases (AITD) and insulin-dependent diabetes mellitus (IDDM) are two autoimmune syndromes of unknown etiology with common immune features. One is that the target cells, thyrocytes and pancreatic islet beta cells respectively, hyperexpress several proteins encoded in the HLA region: HLA class I, HLA class II and transporter associated with antigen processing (TAP-1): the clinical course and many aspects of the immunopathology are, however, quite different. Low-molecular-mass polypeptides 2 and 7 (LMP2 and LMP7) are proteasome subunits that increase the efficiency of endogenous antigen processing and are encoded in close vicinity to the TAP genes. We investigated whether LMP2 and LMP7 are hyperexpressed in thyrocytes and islet cells in AITD and IDDM. Thyroid tissue from Graves' disease patients (GD, n = 8) and Hashimoto thyroiditis (HT, n = 1) and pancreatic tissue from IDDM patients (n = 4) as well as control tissues were examined by the two-color indirect immunofluorescence technique. The results demonstrate that, in normal glands, thyrocytes and pancreatic islet cells express comparable moderate to low levels of LMP2 and LMP7. In AITD and IDDM, expression of LMP2/7 in the endocrine cells was disparate: while in AITD glands there was hyperexpression of LMP2 and 7 parallel to that of HLA class I and TAP-1, in the islet cells of recent onset diabetic pancreases (n = 2) the level of LMP2 and 7 expression was totally normal, including islets that were infiltrated by lymphocytes and hyperexpressed HLA class I and TAP-1. These observations suggest different mechanisms of endogenous peptides generation at the target cells in AITD from IDDM. Since this is a key step for the maintenance of peripheral tolerance, it may help to understand some of the different clinical features of the two autoimmune diseases.
Subject(s)
Autoimmunity , Cysteine Endopeptidases , Diabetes Mellitus, Type 1/metabolism , Graves Disease/metabolism , Multienzyme Complexes , Proteins/metabolism , Thyroiditis, Autoimmune/metabolism , Adolescent , Adult , Aged , Diabetes Mellitus, Type 1/immunology , Female , Fluorescent Antibody Technique, Indirect , Graves Disease/immunology , Humans , Male , Middle Aged , Pancreas/metabolism , Proteasome Endopeptidase Complex , Thyroid Gland/metabolism , Thyroiditis, Autoimmune/immunologyABSTRACT
A possible role of transporter associated with antigen processing (TAP)-1 in the pathogenesis of IDDM has been investigated by examining the level of TAP-1 expression in the islets of IDDM pancreas and by studying in vitro the effect of interferon (IFN)-gamma, IFN-alpha, and tumor necrosis factor-alpha in TAP-1 expression by cultured islet cells. A remarkable hyperexpression of TAP-1 has been found in the endocrine cells (beta and non-beta) of IDDM islets, which constitutes first evidence of hyperexpression of this molecule in the target organ of an autoimmune disease. TAP-1 hyperexpression correlated clearly with HLA class I hyperexpression but only very partially with HLA class II ectopic expression. IFN-gamma and IFN-alpha, both cytokines putatively implicated in IDDM pathogenesis, were capable of inducing TAP-1 protein (as assessed by immunofluorescence flow cytometry) and message (by Northern blot analysis and reverse transcription polymerase chain reaction). These findings suggest that under the influence of cytokines (most probably IFN-alpha) beta-cells may express in their surface a high density of HLA class I-peptide complexes that may facilitate their recognition and lysis by low-affinity CD8+ T-cells.
Subject(s)
ATP-Binding Cassette Transporters , Cytokines/pharmacology , Diabetes Mellitus, Type 1/metabolism , Extracellular Matrix Proteins/biosynthesis , Gene Expression/drug effects , Islets of Langerhans/metabolism , Nerve Tissue Proteins/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 2 , Adolescent , Adult , Aged , Base Sequence , Cell Line , Cells, Cultured , DNA Primers , Diabetes Mellitus, Type 1/immunology , Female , Flow Cytometry , Gene Expression/immunology , Humans , Interferon-alpha/pharmacology , Interferon-gamma/pharmacology , Islets of Langerhans/drug effects , Islets of Langerhans/immunology , Male , Middle Aged , Molecular Sequence Data , Polymerase Chain Reaction , Proteoglycans/biosynthesis , Recombinant Proteins/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Human islet transplantation has a high rate of failure, often due to primary nonfunction, which suggests that islets are damaged during the processing of the pancreas. The preparation of human islets for transplantation is still a complex process that requires large teams of surgical and laboratory personnel. To overcome this problem, we have adopted the use of the IBM 2991 COBE cell separator and a metrizamide/Ficoll density medium that is easy to prepare. Twenty-seven pancreatic glands have been processed using the COBE cell separator, 23 of which were purified in metrizamide/Ficoll gradients and 4 in bovine serum albumin gradients. The results show an improvement of recovery and viability in these preparations when compared retrospectively with manual gradients. More importantly, the time required for purification was shortened to one fourth the usual time and total processing time is about half as long. Moreover, a team of two laboratory staff was regularly able to prepare islets for transplantation, reducing the separation time from 7 hr to 3.5 hr. We conclude that the automatic cell separator and metrizamide-based separation medium are useful modifications of current islet purification methods.