transforming-growth-factor-beta and Diabetes-Mellitus--Type-1

Regulatory B cells (Bregs) have an anti-inflammatory role and can suppress autoimmunity, by employing both cytokine secretion and cell-contact mediated mechanisms. Numerous Breg subsets have been described and have overlapping phenotypes in terms of their immune expression markers or cytokine production. A hallmark feature of Bregs is the secretion of IL-10, although IL-35 and TGFβ-producing B cells have also been identified. To date, few reports have identified an impaired frequency or function of Bregs in individuals with type 1 diabetes; thus our understanding of the role played by these Breg subsets in the pathogenesis of this condition is limited. In this review we will focus on how regulatory B cells are altered in the development of type 1 diabetes, highlighting both frequency and function and discuss both human and animal studies.

Topics: Adolescent; Adult; Animals; Autoimmunity; B-Lymphocytes, Regulatory; Diabetes Mellitus, Type 1; Female; Humans; Immunophenotyping; Interleukin-10; Interleukins; Islets of Langerhans; Male; Mice; Transforming Growth Factor beta; Young Adult

Myeloid regulatory cell-based therapy has been shown to be a promising cell-based medicinal approach in organ transplantation and for the treatment of autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis, Crohn's disease and multiple sclerosis. Dendritic cells (DCs) are the most efficient antigen-presenting cells and can naturally acquire tolerogenic properties through a variety of differentiation signals and stimuli. Several subtypes of DCs have been generated using additional agents, including vitamin D3, rapamycin and dexamethasone, or immunosuppressive cytokines, such as interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β). These cells have been extensively studied in animals and humans to develop clinical-grade tolerogenic (tol)DCs. Regulatory macrophages (Mregs) are another type of protective myeloid cell that provide a tolerogenic environment, and have mainly been studied within the context of research on organ transplantation. This review aims to thoroughly describe the ex vivo generation of tolDCs and Mregs, their mechanism of action, as well as their therapeutic application and assessment in human clinical trials.

Topics: Animals; Arthritis, Rheumatoid; Cell- and Tissue-Based Therapy; Cholecalciferol; Dendritic Cells; Diabetes Mellitus, Type 1; Humans; Immune Tolerance; Interleukin-10; Macrophages; Transforming Growth Factor beta

Type 1 diabetes (T1D) is an autoimmune disease in which cells of the immune system destroy pancreatic β cells, which secrete insulin. The high prevalence of T1D in developed societies may be explained by environmental changes, including lower exposure to helminths. Indeed, infection by helminths such as Schistosoma, Filaria, and Heligmosomoides polygyrus and their by-products has been reported to ameliorate or prevent the development of T1D in human and animal models. Helminths can trigger distinct immune regulatory pathways, often involving adaptive immune cells that include T helper 2 (Th2) cells and regulatory T cells (Tregs) and innate immune cells that include dendritic cells, macrophages, and invariant natural killer T cells, which may act synergistically to induce Tregs in a Toll-like receptor-dependent manner. Cytokines such as interleukin (IL)-4, IL-10, and transforming growth factor (TGF)-β also play an important role in protection from T1D. Herein, we provide a comprehensive review of the effects and mechanisms underlying protection against T1D by helminths.

Topics: Animals; Dendritic Cells; Diabetes Mellitus, Type 1; Helminths; Humans; Insulin-Secreting Cells; Interleukin-10; Interleukin-4; Macrophages; Natural Killer T-Cells; T-Lymphocytes, Regulatory; Th2 Cells; Transforming Growth Factor beta

Transforming growth factor (TGF)-β has been implicated in regulation of the immune system, including autoimmunity. We have found that TGF-β is readily produced by T cells following immunization with self-peptide epitopes that downregulate autoimmune responses in type 1 diabetes (T1D) prone nonobese diabetic (NOD) mice. These include multiple peptide epitopes derived from the islet β-cell antigens GAD65 (GAD65 p202-221, GAD65 p217-236), GAD67 (GAD67 p210-229, GAD67 p225-244), IGRP (IGRP p123-145, IGRP p195-214) and insulin B-chain (Ins. B:9-23) that protected NOD mice from T1D. Immunization of NOD mice with the self-MHC class II I-A

Topics: Animals; Autoantigens; Autoimmunity; Diabetes Mellitus, Type 1; Humans; Immune Tolerance; Immunization; Mice; Mice, Inbred NOD; Peptides; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Cardiovascular diseases account for the major cause of morbidity and mortality among individuals with diabetes. The diabetic cardiomyopathy (DCM) is a type of diabetic cardiovascular disease, which further directs to the heart failure. The researchers found that diabetes induced cardiac fibrosis plays a vital role in several of the pathological changes that associated with DCM, causing left ventricular hypertrophy (LVH), diastolic dysfunction and systolic dysfunction. However, the mechanisms involved in the pathogenesis of DCM are still elusive. Many studies have demonstrated that the transforming growth factor beta (TGF-β) is one of the molecular mediators implicated in the progression of fibrogenesis. In diabetes, hyperglycemia causes the expression changes of microRNAs (miRNAs), long non-coding RNAs (lncRNAs), TGF-β genes, TGF-β proteins and their receptors. Activated TGF-β further leads to cardiac fibrosis, which in turn inducing DCM through the SMAD-dependent and independent pathways. Here, we reviewed the the molecular pathways that activate TGF-β then leading to cardiac fibrosis, which induced the pathological changes of DCM. Illustrating the pathways of TGF-ß would propose an efficient way for the management of diabetic cardiomyopathy (see Fig. 1).

Topics: Animals; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Cardiomyopathies; Fibrosis; Heart Failure; Humans; Myocardium; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor β (TGF-β) is a pleiotropic cytokine involved in both suppressive and inflammatory immune responses. After 30 years of intense study, we have only begun to elucidate how TGF-β alters immunity under various conditions. Under steady-state conditions, TGF-β regulates thymic T-cell selection and maintains homeostasis of the naïve T-cell pool. TGF-β inhibits cytotoxic T lymphocyte (CTL), Th1-, and Th2-cell differentiation while promoting peripheral (p)Treg-, Th17-, Th9-, and Tfh-cell generation, and T-cell tissue residence in response to immune challenges. Similarly, TGF-β controls the proliferation, survival, activation, and differentiation of B cells, as well as the development and functions of innate cells, including natural killer (NK) cells, macrophages, dendritic cells, and granulocytes. Collectively, TGF-β plays a pivotal role in maintaining peripheral tolerance against self- and innocuous antigens, such as food, commensal bacteria, and fetal alloantigens, and in controlling immune responses to pathogens.

Topics: Animals; Arthritis, Rheumatoid; Autoimmunity; B-Lymphocytes; Bacterial Infections; Cell Differentiation; Cell Lineage; Cell Proliferation; Cell Survival; Dendritic Cells; Diabetes Mellitus, Type 1; Granulocytes; Homeostasis; Humans; Immune Tolerance; Inflammatory Bowel Diseases; Isoantigens; Killer Cells, Natural; Lupus Erythematosus, Systemic; Lymphocyte Activation; Macrophages; Mast Cells; Mice; Monocytes; Parasitic Diseases; T-Lymphocytes; T-Lymphocytes, Regulatory; Thymus Gland; Transforming Growth Factor beta

Regulatory T lymphocyte cells (Treg) associated with interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) have implicated in the development of type 1 diabetes mellitus (T1DM), yet the existing evidence remains unclear. Hereby we performed a systematic review and meta-analysis to characterize the changes in T1DM patients. A total of 1407 T1DM patients and 1373 healthy controls from 40 case-control studies were eventually included in the pooling analysis. Compared with the controls, T1DM patients had decreased frequency of CD4(+)CD25(+)Treg (p=0.0003), CD4(+)CD25(+)Foxp3(+)Treg (p=0.020), and the level of TGF-β (p=0.030). Decrease in IL-10 (p=0.14) was not significant. All the changes remained significant when the studies with low NOS scores and publication bias were excluded. In conclusion, peripheral Treg and serum TGF-β are reduced in type 1 diabetes mellitus whereas changes in serum IL-10 are not significant.

Topics: Case-Control Studies; Diabetes Mellitus, Type 1; Female; Forkhead Transcription Factors; Humans; Interleukin-10; Interleukin-2 Receptor alpha Subunit; Male; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Chronic kidney disease (CKD) is becoming a worldwide epidemic, driven largely by the dramatic rise in the prevalence of diabetes and obesity. Novel targets and treatments for CKD are, therefore, desperately needed-to both mitigate the burden of this disease in the general population and reduce the necessity for renal replacement therapy in individual patients. This Review highlights new insights into the mechanisms that contribute to CKD, and approaches that might facilitate the development of disease-arresting therapies for CKD. Particular focus is given to therapeutic approaches using antifibrotic agents that target the transforming growth factor β superfamily. In addition, we discuss new insights regarding the roles of vascular calcification, the NADPH oxidase family, and inflammation in the pathogenesis of CKD. We also highlight a new understanding regarding kidney energy sensing pathways (AMPK, sirtuins, and mTOR) in a variety of kidney diseases and how they are linked to inflammation and fibrosis. Finally, exciting new insights have been made into the role of mitochondrial function and mitochondrial biogenesis in relation to progressive kidney disease. Prospective therapeutics based on these findings will hopefully renew hope for clinicians and patients in the near future.

Topics: AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Disease Progression; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibrosis; Glucuronidase; Humans; Klotho Proteins; Mitochondria; Models, Animal; NADPH Oxidases; NF-kappa B; Renal Insufficiency, Chronic; Transforming Growth Factor beta; Vascular Calcification

Following the discovery of interleukin (IL)-17 producing T helper (Th17) cells as a distinct lineage of CD4+ T helper cells it became clear that these cells play an important role in the host defense against extracellular fungal and bacterial pathogens and participate in the pathogenesis of multiple inflammatory and autoimmune disorders. Depending on the microenvironment, Th17 cells can alter their differentiation programme ultimately giving rise to either protective or pro-inflammatory pathogenic cells. We found that besides the conventional in vitro protocol for Th17 differentiation by transforming growth factor-beta (TGF-β) plus IL-6 cytokines, a combination of IL-23 plus IL-6 can also induce Th17 cells. The Th17 cells induced by IL-23 plus IL-6 (termed as effector Th17, Teff17 cells) are pathogenic upon adoptive transfer into non-obese diabetic (NOD) mice contributing to the development of type 1 diabetes (T1D) while cells induced by TGF-β plus IL-6 (termed as regulatory T cells, Treg17 cells) are non pathogenic and regulatory, and suppressed the pathogenic T cells in T1D. These cells differentially expressed a number of cytokines where Teff17 cells exhibited an increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-22 whereas Treg17 cells demonstrated increased expression of IL-21 and immunosuppressive cytokine IL-10. Differentiation of Th17 cells is controlled by a transcription factor, RORγT although these cells also express variable levels of T-bet and FoxP3 transcription factors. This points to a dual functional role of Th17 subsets in autoimmune diseases particularly T1D. We suggest that similar to conventional regulatory T cells (Treg), induction of regulatory Treg17 cells could play an important role in modulating and preventing certain autoimmune diseases.

Topics: Animals; Autoimmune Diseases; CD4-Positive T-Lymphocytes; Cell Differentiation; Cell Lineage; Diabetes Mellitus, Type 1; Humans; Interleukin-17; Mice; Th17 Cells; Transforming Growth Factor beta

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing β-cells in the pancreatic islets. There is an immediate need to restore both β-cell function and immune tolerance to control disease progression and ultimately cure T1D. Currently, there is no effective treatment strategy to restore glucose regulation in patients with T1D. FoxP3-expressing CD4(+) regulatory T cells (Tregs) are potential candidates to control autoimmunity because they play a central role in maintaining self-tolerance. However, deficiencies in either naturally occurring Tregs (nTregs) themselves and/or their ability to control pathogenic effector T cells have been associated with T1D. Here, we hypothesize that nTregs can be replaced by FoxP3(+) adaptive Tregs (aTregs), which are uniquely equipped to combat autoreactivity in T1D. Unlike nTregs, aTregs are stable and provide long-lived protection. In this review, we summarize the current understanding of aTregs and their potential for use as an immunological intervention to treat T1D.

Topics: Adoptive Transfer; Animals; Autoimmunity; Diabetes Mellitus, Type 1; Disease Progression; Forkhead Transcription Factors; Humans; Insulin-Secreting Cells; Integrin beta Chains; Interleukin-2; Mice; Self Tolerance; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Human fibroblasts in culture have been employed as an in vitro system to investigate some pathophysiological mechanisms of diabetes mellitus also associated with the development of diabetic nephropathy. In fact, there is increasing evidence that genetic factors either convey the risk of, or protect from, diabetic nephropathy and that the expression profiles and/or the behaviour of the cultured skin fibroblasts from type 1 diabetic patients could reflect these genetic influences. On the other hand, alterations could be attributable not only to changes in DNA sequence, but also to epigenetic factors. Our aim is to make a critical overview of the studies involving primary cultures of skin fibroblasts as tools to investigate the pathophysiology of diabetic nephropathy performed until now in this area. Cultured skin fibroblasts could be useful not only for the identification of patients at risk of developing diabetic renal disease, but also for a better understanding of the complex multifactorial mechanisms leading to the long-term complications in diabetes.

Topics: Cell Proliferation; Cells, Cultured; Collagen; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Fibroblasts; Humans; Hyperglycemia; Protein Kinase C; Proteomics; Risk Assessment; Skin; Sodium-Hydrogen Exchangers; Transforming Growth Factor beta

Type 1 diabetes mellitus (T1DM) is a T cell-mediated autoimmune disease resulting in islet β cell destruction, hypoinsulinemia, and severely altered glucose homeostasis. T1DM has classically been attributed to the pathogenic actions of auto-reactive effector T cells(Teffs) on the β cell. Recent literature now suggests that a failure of a second T cell subtype, known as regulatory T cells (Tregs), plays a critical role in the development of T1DM. During immune homeostasis, Tregs counterbalance the actions of autoreactive Teff cells, thereby participating in peripheral tolerance. An imbalance in the activity between Teff and Tregs may be crucial in the breakdown of peripheral tolerance, leading to the development of T1DM. In this review, we summarize our current understanding of Treg function in health and in T1DM, and examine the effect of experimental therapies for T1DM on Treg cell number and function in both mice and humans.

Topics: Animals; Antibodies, Monoclonal; Autoimmunity; CD3 Complex; Dendritic Cells; Diabetes Mellitus, Type 1; Immune Tolerance; Insulin-Secreting Cells; Interleukin-10; Mice; Recombinant Fusion Proteins; Sirolimus; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Podocyte loss is a common feature in human diabetes as well as in experimental diabetes in rodents. Almost all components of the diabetic milieu lead to serious podocyte stress, driving the cells towards cell cycle arrest and hypertrophy, detachment and apoptosis. Common pathway components induced by high glucose and advanced glycation end-products are reactive oxygen species, cyclin-dependent kinases (p27(Kip1)) and transforming growth factor-beta. In addition, mechanical stresses by stretch or shear forces, insulin deficiency or insulin resistance are independent components resulting in podocyte apoptosis and detachment. In this review, we discuss the common pathways leading to podocyte death as well as novel pathways and concepts of podocyte dedifferentiation and detachment that influence the progression of diabetic glomerulopathy.

Topics: Actins; Apoptosis; Chemokines; Cyclin-Dependent Kinase Inhibitor p27; Cytokines; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Glucose; Glycation End Products, Advanced; Humans; Hyperglycemia; Insulin; Insulin Resistance; Podocytes; Stress, Mechanical; Transforming Growth Factor beta

Topics: Antibodies; Autoimmunity; CD3 Complex; Diabetes Mellitus, Type 1; Humans; Self Tolerance; Transforming Growth Factor beta

The activation, expansion, and survival of regulatory T cells (Tregs) as well as the expression of their suppressive capacities result from distinct signaling pathways involving various membrane receptors and cytokines. Multiple studies have shown that thymus-derived naturally occurring Tregs constitutively express the forkhead/winged helix transcription factor FoxP3 in addition to high levels of CD25, the negative co-stimulatory molecule CTLA-4, and the glucocorticoid-induced TNF receptor-related protein GITR. At variance, adaptive or induced Tregs acquire these phenotypic markers as they differentiate in the periphery, following adequate stimulation in the appropriate environment, together with their capacity to produce immunomodulatory cytokines (mainly, IL-4, IL-10 and TGF-beta) and to display regulatory capacities. However, none of these molecules but FoxP3 are restricted to Tregs since they may also be expressed and upregulated on activated effector T cells. This explains why different hypotheses were proposed to interpret interesting reports showing that in vivo abrogation of CTLA-4 signaling using neutralizing CTLA-4 antibodies triggers different autoimmune or immune-mediated manifestations. Thus, an effect on pathogenic T cell effectors and/or Tregs has been proposed. Here we present and discuss recent results we obtained in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes, arguing for a key role of CTLA-4 in the functional activity of Tregs. Moreover, data are presented that simultaneous blockade of CTLA4 and TGF-beta further impairs immunoregulatory circuits that control disease progression.

Topics: Animals; Antigens, CD; Autoimmunity; CTLA-4 Antigen; Diabetes Mellitus, Type 1; Disease Progression; Homeostasis; Immune Tolerance; Mice; Mice, Inbred NOD; Signal Transduction; Systemic Inflammatory Response Syndrome; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Type 1 diabetes mellitus is an autoimmune disease characterized by progressive destruction of pancreatic beta cells by genetic and environmental factors which leads to an absolute dependence of insulin for survival and maintenance of health. Although the majority of mechanisms of beta cell destruction remain unclear, many molecules, including proinflammatory cytokines and chemokines such as tumor necrosis factor alpha and monocyte chemoattractant protein-1, are implicated in the development of beta cell damage. Furthermore, beta cell destruction is enhanced by the Th1 and Th17 subsets of CD4+ T cells. In contrast, there are mechanisms involved in the maintenance of peripheral tolerance by regulatory T cells, the function of which depends on the pleiotropic cytokine transforming growth factor beta. Development and progression of renal injuries in patients with diabetic nephropathy are also associated with several growth factors and proinflammatory cytokines, including tumor necrosis factor alpha, insulin-like growth factor-1, monocyte chemoattractant protein-1, vascular endothelial growth factor, and transforming growth factor beta. Although the pathogenic mechanisms underlying type 1 diabetes and diabetic nephropathy are principally different, i.e., autoimmunity and inflammation, some common factors, including susceptibility genes and proinflammatory cytokines, are involved in both mechanisms, including infiltrating cell recruitment, upregulation of other cytokines and chemokines, or apoptosis.

Topics: CD4-Positive T-Lymphocytes; Chemokine CCL2; Chemokines; Cytokines; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Genetic Predisposition to Disease; Humans; Insulin-Like Growth Factor I; Islets of Langerhans; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation; Vascular Endothelial Growth Factor A

It is now well-established that CD4+ regulatory T cells are instrumental in controlling immune responses both to self-antigens and to non-self-antigens. However, the precise modalities involved in their differentiation and survival, their mode of action and their antigen specificity are only partially understood. We have been particularly interested in the study of regulatory T cells controlling autoimmune insulin-dependent diabetes. Here, we provide evidence to support the phenotypic and functional diversity of regulatory T cells mediating transferable 'active' or 'dominant' peripheral tolerance in the non-obese diabetic mouse model (NOD). They include natural and adaptive regulatory T cells that are operational both in unmanipulated NOD mice and in animals undergoing treatments aimed at inducing/restoring tolerance to self-beta-cell antigens. At least in our hands, the differential cytokine-dependency appears as a major distinctive feature of regulatory T cells subsets. Among immunoregulatory cytokines, transforming growth factor-beta(TGF-beta) appeared to play a key role. Herein we discuss these results and the working hypothesis they evoke in the context of the present literature, where the role of TGF-beta-dependent T-cell-mediated immunoregulation is still debated.

Topics: Animals; Autoimmune Diseases; Diabetes Mellitus, Type 1; Mice; Mice, Inbred NOD; T-Lymphocytes, Regulatory; Toll-Like Receptors; Transforming Growth Factor beta

Islet transplantation has emerged as a viable long-term means of treating type I diabetes. This is largely due to the success of the "Edmonton protocol" which has produced insulin independence in 85% of patients 1 year after transplantation of allogeneic islets together with a non-steroid immunosuppressive regimen. While these data provide a clear and unequivocal demonstration that islet transplantation is a viable treatment strategy, the shortage of suitable donor tissue together with the debilitating consequences of life-long immunosuppression necessitate the development of novel means to enable transplantation of all type 1 diabetics including the young juvenile diabetics. One potential means of enabling islet transplantation takes advantage of the ability of Sertoli cells to provide local immunoprotection to co-grafted islets, including those from xenogeneic sources. Sertoli cells are normally found in the testes where one of their functions is to provide local immunologic protection to developing germ cells. In animal models, allogeneic and xenogeneic islets survive and function for extended periods of time when grafted into the testes. Moreover, isolated Sertoli cells protect co-grafted allogeneic and xenogeneic islets from immune destruction and reverse diabetes in immunocompetent and autoimmune animals. These benefits are discussed in the context of several potential underlying biological mechanisms.

Topics: Animals; Autoantigens; Diabetes Mellitus, Type 1; Fas Ligand Protein; fas Receptor; Graft Survival; Humans; Islets of Langerhans; Islets of Langerhans Transplantation; Major Histocompatibility Complex; Male; Membrane Glycoproteins; Models, Biological; Sertoli Cells; Testis; Transforming Growth Factor beta; Transplantation, Heterologous

The structural changes characterising diabetic microangiopathy, which may be referred to as 'abnormal growth' and 'impaired regeneration', strongly suggest a role for a number of aberrantly expressed growth factors, possibly acting in combination, in the development of these complications. This initial speculation has been supported by the detection of increased concentrations of several growth factors in the target tissues of diabetic long-term complications, and by enhanced expression of these growth factors consequent to the activation of the biochemical pathways linking hyperglycaemia to microvascular changes: the polyol pathway; non-enzymatic glycation of proteins; vasoactive hormones; oxidative stress, and hyperglycaemic pseudohypoxia. As to nephropathy, insulin-like growth factor I (IGF-I) seems to be implicated in the earlier stages of the disease, while transforming growth factor beta (TGF beta) is involved both in the early and later stages, being responsible, at least in part, for extracellular matrix (ECM) accumulation. Vascular endothelial growth factor (VEGF) plays a pivotal role both in non-proliferative and proliferative retinopathy. Finally, deficiency of several neurotrophic factors, namely nerve growth factor (NGF) and IGF-I has been related to the degeneration or impaired regeneration occurring in diabetic neuropathy. Knowledge of the involvement of growth factors in diabetic microangiopathy opens the way to new therapeutic interventions aimed at blocking the deleterious actions of several growth factors.

Topics: Diabetes Mellitus, Type 1; Diabetic Nephropathies; Diabetic Neuropathies; Diabetic Retinopathy; Endothelial Growth Factors; Growth Substances; Humans; Insulin-Like Growth Factor I; Lymphokines; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Patients with insulin-dependent diabetes mellitus (IDDM) and albuminuria are at high risk for severe micro- and macrovascular complications. Diabetic vascular complications are characterized by structural alterations of extracellular matrix (ECM) components in glomeruli and large vessel walls, namely, accumulation of collagen IV, collagen VI and fibronectin and relative decrease of heparan sulphate proteoglycan (HSPG). We hypothesize that the defect remodelling of ECM contributing to nephropathy and macrovascular disease is induced by overproduction of transforming growth factor-beta (TGF-beta). Recent reports indicate that hyperglycaemia, increased intraglomerular pressure, and glycated proteins potentially induce overproduction of TGF-beta in diabetes. TGF-beta stimulates production of ECM components such as collagen IV, fibronectin, proteoglycans (decorin and biglycan) without increasing HSPG. TGF-beta overproduction leads to glomerulosclerosis and TGF-beta is a causal factor in myointimal hyperplasia after balloon injury of carotid artery. It mediates angiotensin II modulator effect on smooth muscle cell growth. These findings may indicate TGF-beta overproduction to be a common pathogenetic step explaining the well-known association between micro- and macrovascular complications in diabetic patients. TGF-beta antagonists, such as decorin, betaglycan, and possibly also heparin, might be potential candidates for future therapy to prevent diabetic vascular disease.

Topics: Animals; Cytokines; Diabetes Mellitus, Type 1; Diabetic Angiopathies; Diabetic Nephropathies; Extracellular Matrix; Humans; Risk Factors; Transforming Growth Factor beta

Renal cells are a rich source of transforming growth factor (TGF)-beta, and they serve as targets for its actions. Our hypothesis that activation of the TGF-beta system in the kidney is implicated in the development of diabetic renal disease stems from the close similarity of actions of TGF-beta and high ambient glucose on renal cell growth and extracellular matrix metabolism. Proximal tubule cells and glomerular mesangial cells cultured in high glucose concentration express increased TGF-beta 1 mRNA and protein levels, and treatment with anti-TGF-beta antibodies results in prevention of the effects of high glucose to induce cellular hypertrophy and stimulate collagen biosynthesis. Several in vivo studies by different groups of investigators have reported overexpression of TGF-beta in the glomeruli in human and experimental diabetes. We have also observed that the development of renal hypertrophy in the insulin-dependent diabetic BB rat and NOD mouse is associated with increased expression of TGF-beta 1 in the kidney and that short-term administration of antibodies capable of neutralizing the activity of TGF-beta in the streptozotocin mouse model of diabetes results in attenuation of whole kidney and glomerular hypertrophy and overexpression of mRNAs encoding matrix components. Together, these findings are consistent with the hypothesis that the diabetic state stimulates TGF-beta expression in the kidney and that in turn this growth factor may mediate, in an autocrine/paracrine manner, some of the principal early manifestations of diabetic renal disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Extracellular Matrix; Gene Expression; Glomerular Mesangium; Humans; Hyperglycemia; Kidney; Kidney Tubules, Proximal; Mice; Mice, Inbred NOD; Rats; Rats, Inbred BB; Transforming Growth Factor beta

Topics: Animals; Anterior Chamber; Aqueous Humor; Autoimmune Diseases; Diabetes Mellitus, Type 1; Graft Rejection; Humans; Hypersensitivity, Delayed; Rats; Rats, Inbred BB; Transforming Growth Factor beta

Type 1 diabetes results from autoimmune destruction of insulin producing pancreatic β-cells. We have shown that treatment with alum-formulated glutamic acid decarboxylase 65 (GAD-alum) preserved residual insulin secretion and induced antigen-specific responses in children with recent onset type 1 diabetes. The aim of this study was to further investigate the immunomodulatory effect of GAD-alum, focusing on CD4(+)CD25(high) cells and their association to cytokine secretion. Samples obtained 21 and 30months after the initial injection of GAD-alum or placebo were included in the present study. GAD(65)-stimulation enhanced the percentage of CD4(+)CD25(high)FOXP3(+) cells, but reduced the percentage of CD4(+)CD25(+) cells, in samples from the GAD-alum treated group. Further, the GAD(65)-induced secretion of IL-5, -10, and -13 correlated with the expression of CD4(+)CD25(high)FOXP3(+) cells, but inversely with CD4(+)CD25(+) cells. These new data suggest that GAD-alum treatment induced GAD(65)-specific T cells with regulatory features.

Topics: Adolescent; Alum Compounds; Autoantibodies; CD4-Positive T-Lymphocytes; Cell Count; Child; Diabetes Mellitus, Type 1; Female; Forkhead Transcription Factors; Gene Expression; Glutamate Decarboxylase; Humans; Immunosuppression Therapy; Interferon-gamma; Interleukin-2 Receptor alpha Subunit; Interleukins; Leukocytes, Mononuclear; Lymphocyte Activation; Male; T-Lymphocytes, Regulatory; Th2 Cells; Transforming Growth Factor beta; Treatment Outcome; Tumor Necrosis Factor-alpha

Transforming growth factor ss (TGF-ss) is an immunosuppressor. It plays a role in regulating cell proliferation, and deletion of its gene in transgenic mice leads to an autoimmune-like disorder. A role of this cytokine has been proposed in the pathogenesis of type 1 diabetes and probably type 2 diabetes. Previous studies had shown an elevated serum level in type 2 diabetes and a reduced serum level in type 1 diabetes; however, these studies did not address the onset of the alterations of TGF-ss with regard to the duration of diabetes. In this study, we compared the levels of TGF-ss in the serum of groups of patients with type 1 and type 2 diabetes mellitus divided according to the duration of their disease. Twenty-six normoalbuminuric patients with type 1 diabetes and 25 normoalbuminuric patients with type 2 diabetes were divided into three groups according to the onset of their diabetes and were compared with 27 and 15 age-matched normal subjects, respectively. We conclude that in normoalbuminuric patients serum TGF-ss levels increased at the onset of type 2 diabetes and remained elevated throughout the disease; they did not change at the onset of type1 diabetes, however, they started to decrease around 2 yr after the onset of the disease.

Topics: Adolescent; Adult; Aging; Albuminuria; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Female; Glycated Hemoglobin; Humans; Male; Time Factors; Transforming Growth Factor beta

The renoprotective effect of captopril on progression of diabetic nephropathy was demonstrated by the Collaborative Study Group Captopril Trial and might be independent of blood pressure. Because angiotensin II is known to stimulate the prosclerotic cytokine, transforming growth factor-beta (TGF-beta), we postulated that the renoprotective effect may be due to inhibition of TGF-beta1 production. TGF-beta1 levels were measured in serum at baseline and 6 months from patients in the captopril trial. TGF-beta1 analyses were performed on all available patient sera. Analysis was performed between the percent change in TGF-beta1 levels during the first 6 months versus the percent change in glomerular filtration rate (GFR) in the subsequent 2 years. TGF-beta1 levels increased by 11% (P = 0. 003) in the placebo group (n = 24), whereas there was a decrease of 14% (P = 0.01) in the captopril group (n = 34). There was an inverse correlation between the percent change in TGF-beta1 levels during the first 6 months and the percent change in GFR over the ensuing 2-year period in patients from both the placebo (r = -0.55, P = 0. 005) and captopril groups (r = -0.45, P = 0.008). In patients with initial GFR below 75 mL/min, there was an even stronger correlation in percent change in TGF-beta1 levels and percent change in GFR in both placebo (n = 9, r = -0.69, P = 0.03) and captopril groups (n = 21, r = -0.73, P = 0.0001). Our data suggest that captopril decreases TGF-beta1 levels in diabetic nephropathy and that changes in TGF-beta1 levels may predict the course of diabetic nephropathy.

Topics: Adult; Angiotensin-Converting Enzyme Inhibitors; Antihypertensive Agents; Blood Pressure; Captopril; Diabetes Mellitus, Type 1; Diabetic Angiopathies; Female; Follow-Up Studies; Glomerular Filtration Rate; Glycated Hemoglobin; Humans; Hypertension, Renal; Kidney Function Tests; Male; Transforming Growth Factor beta

Nerve growth factor (NGF) and transforming growth factor-beta2 (TGF-beta2) are cytokines which have known immunological effects. An elevated level of NGF has been reported in certain autoimmune diseases, whereas TGF-beta2 is an immunosuppressor which is known to play a role in regulating cell proliferation. A role of this cytokine has been proposed in the pathogenesis of type-1 diabetes mellitus (IDDM), but no clinical studies have yet measured its serum level in this disease. In this study we measured the levels of NGF and TGF-beta2 in the sera of patients with IDDM (n = 26) and values were compared to those of age-matched normal subjects (n = 27) and also to patients with type-2 diabetes mellitus (NIDDM) (n = 26) with similar HbA1c levels and an equal duration of diabetes. Serum NGF levels were significantly elevated in IDDM patients compared to those of age-matched controls (p <.001) and NIDDM controls (p <.01). TGF-beta2 levels were lower in IDDM patients when compared with the healthy control (p <.001) and the NIDDM control (p <.05). There was no correlation between the levels of NGF and TGF-beta2. The duration of diabetes and the level of HbA1c did not affect the NGF and TGF-beta2 levels in the IDDM patients. We conclude that an increase in NGF and a suppression in TGF-beta2 levels are present in patients with type-1 diabetes mellitus and that both cytokines may play independent roles in the pathogenesis of this disease.

Topics: Adolescent; Adult; Aged; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Female; Humans; Male; Middle Aged; Nerve Growth Factor; Reference Values; Transforming Growth Factor beta

Urinary samples were concentrated rapidly and efficiently and were used to develop several protein assays that may be of value in monitoring individuals with progressive renal disorders. Transforming growth factor-beta1 (TGF-11) and retinol binding protein (RBP) were measured with modification of commercially available methods used to assay serum specimens; type 3 collagen (T3C) was measured with a new immunonephelometric assay. The precision characteristics of these assays are comparable with those reported for microalbuminuria. The clinical utility of measuring a panel of these markers was demonstrated in urine samples from 16 control subjects and from 46 individuals with insulin-dependent diabetes mellitus (IDDM) with various albumin excretion rates (AERs). TGF-beta1 and T3C were used as markers of cytokine expression and of the renal fibrogenic process, whereas RBP excretion served as a marker of tubular injury or dysfunction. Compared with controls, T3C excretion was significantly increased in 18 normoalbuminuric and further increased in 13 microalbuminuric (AER 20 < or = 200 microg/min) IDDM subjects. RBP excretion was increased in macroalbuminuric IDDM subjects (AER >200 microg/min, overt nephropathy). Significant correlations were also found between AER and RBP in all but macroalbuminuric individuals, whereas TGF-beta1 correlated with T3C excretion in controls and in normoalbuminuric diabetic subjects. Urinary RBP but not AER was an excellent predictor of diabetic nephropathy as defined by serum creatinine (P = 0.0001). This underscores the importance of an early tubulopathy in the subsequent development of glomerulopathy and overt nephropathy. The data suggest that longitudinal monitoring of a panel of urinary markers such as that used in the current study may better define their relevance in progressive glomerulosclerosis and may also provide greater insight into the mechanisms underlying such process.

Topics: Adult; Albuminuria; Biomarkers; Collagen; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Follow-Up Studies; Humans; Male; Middle Aged; Predictive Value of Tests; Regression Analysis; Retinol-Binding Proteins; Risk Factors; Transforming Growth Factor beta

The purpose of the study was to assess TGF-beta and IL-6 urinary excretion (measured with EIA) in 12 IDDM patients (7 F, 5 M, age 20-49 yrs, mean = 33.08) with albuminuria or microalbuminuria. Control group consists of 27 IDDM patients (12 F, 15 M, age 24-59 yrs. mean = 39.5) without albuminuria or microalbuminuria. Urinary excretion of IL-6 was significantly higher (p < 0.05) in IDDM patients with albuminuria (mean = 7.43 +/- 8.29 pg/mg creatinine) than in control group (mean = 3.74 +/- 2.64 pg/mg creatinine). Urinary excretion of TGF-beta was also higher (but not significantly in IDDM patients with albuminuria or microalbuminuria (mean = 42.0 +/- 30.0 pg/mg creatinine) than in control group (mean = 27.0 +/- 20.0 pg/mg creatinine). The data indicate that IL-6 and TGF-beta could be involved in the development of diabetic nephropathy.

Topics: Adult; Albuminuria; Biomarkers; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Humans; Interleukin-6; Male; Middle Aged; Transforming Growth Factor beta

Topics: Cytokines; Diabetes Mellitus, Type 1; Disease Progression; Forkhead Transcription Factors; GATA3 Transcription Factor; Humans; Interleukin-10; Leukocytes, Mononuclear; Nuclear Receptor Subfamily 1, Group F, Member 3; RNA, Messenger; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

MSCs have been demonstrated to have a great benefit for type 1 diabetes mellitus (T1DM) due to their strong immunosuppressive and regenerative capacity. However, the comprehensive mechanism is still unclear. Our previous study indicated that transforming growth factor beta induced (TGFBI) is highly expressed in human umbilical cord-derived mesenchymal stem or stromal cells (hUC-MSCs), which are also implicated in T1DM. In this study, we found that infusion of TGFBI knockdown hUC-MSCs displayed impaired therapeutic effects in T1DM mice and decreased immunosuppressive capability. TGFBI knockdown hUC-MSCs could increase the proportion of T-cell infiltration while increasing the expression of IFN-gamma and interleukin-17A in the spleen. In addition, we also revealed that hUC-MSC-derived TGFBI could repress activated T-cell proliferation by interfering with G1/S checkpoint CyclinD2 expression. Our results demonstrate that TGFBI plays a critical role in MSC immunologic regulation. TGFBI could be a new immunoregulatory molecule controlling MSC function for new treatments of T1DM. Schematic Representation of the Immunosuppression capacity of hUC-MSC by TGFBI.

Topics: Animals; Cell Proliferation; Diabetes Mellitus, Type 1; Humans; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Streptozocin; Transforming Growth Factor beta; Umbilical Cord

Anti-CD20 therapy delays type 1 diabetes mellitus (T1DM) progression in both nonobese diabetic (NOD) mice and new-onset patients. The mechanism is not completely defined. This study aimed to investigate the effects of anti-CD20 therapy on T helper 17 (Th17) cells and regulatory T cells (Tregs) in NOD mice. The role of B cell depletion in T1DM development was also examined.. NOD mice were randomly divided into two groups. The mice in the experimental group were treated with an anti-CD20 antibody, while the control mice were treated with an isotype-matched control antibody. After treatment, islet morphology and inflammation, Th17 and Treg cell frequencies in the pancreas and spleen, serum cytokine and anti-glutamic acid decarboxylase (GAD) antibody levels, interleukin (IL)-17A levels in the pancreas and spleen, insulin expression in islet cells and islet β cell function were measured.. Decreased blood glucose and increased insulin secretion were found in the exprimental group compared with the CON group. A lower islet inflammation score was also found in the experimental group. Decreased Th17 cell and IL-17A levels and augmented Treg cell levels were found in the spleen and pancreas after anti-CD20 treatment. The serum levels of B cell activating factor (BAFF), IL-17A, IL-17F, IL-23 and anti-GAD autoantibodies were decreased in the experimental group, while higher serum levels of IL-10 and transforming growth factor (TGF)-β were found.. Anti-CD20 therapy might have some beneficial effects that improve β cell function by relieving islet inflammation through regulation of Th17/Treg cells and the proinflammatory/anti-inflammatory balance.

Topics: Animals; Diabetes Mellitus, Type 1; Humans; Inflammation; Interleukin-17; Mice; Mice, Inbred NOD; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta

Type 1 diabetes mellitus (T1D) is a chronic autoimmune condition in which the immune system destroys insulin-producing pancreatic β cells. In addition to well-established pathogenic effector T cells, regulatory T cells (Tregs) have also been shown to be defective in T1D. Thus, an increasing number of therapeutic approaches are being developed to target Tregs. However, the role and mechanisms of TGF-β-induced Tregs (iTregs) in T1D remain poorly understood. Here, using a streptozotocin (STZ)-induced preclinical T1D mouse model, we found that iTregs could ameliorate the development of T1D and preserve β cell function. The preventive effect was associated with the inhibition of type 1 cytotoxic T (Tc1) cell function and rebalancing the Treg/Tc1 cell ratio in recipients. Furthermore, we showed that the underlying mechanisms were due to the TGF-β-mediated combinatorial actions of mTOR and TCF1. In addition to the preventive role, the therapeutic effects of iTregs on the established STZ-T1D and nonobese diabetic (NOD) mouse models were tested, which revealed improved β cell function. Our findings therefore provide key new insights into the basic mechanisms involved in the therapeutic role of iTregs in T1D.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Female; Gene Expression Regulation; Hepatocyte Nuclear Factor 1-alpha; Insulin-Secreting Cells; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Colonization factor antigen I (CFA/I) fimbria, an adhesin from enterotoxigenic Escherichia coli, confers protection in murine autoimmune models for type 1 diabetes (T1D), multiple sclerosis, and rheumatoid arthritis. Although CFA/I fimbriae's initial mode of action is in a bystander or in an antigen (Ag)-independent fashion, protection is ultimately dependent upon the induction and/or activation of auto-Ag-specific regulatory T cells (Tregs). However, little is known about how protection transitions from bystander suppression to Ag-specific Tregs. Since dendritic cells (DCs) play an integral role in fate decisions for T cells becoming inflammatory or tolerogenic, the described study tests the hypothesis that Lactococcus lactis expressing CFA/I (LL-CFA/I) stimulates DCs to establish a regulatory microenvironment. To this end, bone marrow-derived dendritic cells (BMDCs) were infected in vitro with LL-CFA/I. Results revealed increased production of IL-10, TGF-β, and indoleamine 2,3-deoxygenase (IDO). Although co-culture of LL-CFA/I infected BMDCs with naïve T cells did not promote Foxp3 expression, TNF-α and IFN-γ production was suppressed. NOD mice orally dosed with LL-CFA/I showed an increase in regulatory plasmacytoid DCs (pDCs) expressing IDO and TGF-β in pancreatic lymph nodes (PaLNs) and spleen three days post-treatment. However, Tregs did not appear in the mucosal inductive sites until much later. These findings show that LL-CFA/I influences specific DC populations to establish tolerance.

Topics: Administration, Oral; Animals; Antigens, Bacterial; Blood Glucose; CD4-Positive T-Lymphocytes; Cells, Cultured; Coculture Techniques; Dendritic Cells; Diabetes Mellitus, Type 1; Disease Models, Animal; Female; Fimbriae Proteins; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Lactococcus lactis; Lymph Nodes; Mice; Mice, Transgenic; Primary Cell Culture; Probiotics; Spleen; Transforming Growth Factor beta

Epithelial-to-mesenchymal transition (EMT) is required for renal fibrosis, which is a characteristic of diabetic nephropathy (DN). Our previous study demonstrated that fibroblast growth factor 21 (FGF21) prevented DN associated with the suppressing renal connective tissue growth factor expression, a key marker of renal fibrosis. Therefore, the effects of FGF21 on renal fibrosis in a DN mouse model and the underlying mechanisms were investigated in this study.. Type 1 diabetes mellitus was induced in C57BL/6J mice by intraperitoneal injections of multiple low doses of streptozotocin. Then, diabetic and non-diabetic mice were treated with or without FGF21 in the presence of pifithrin-α (p53 inhibitor) or 10-[4'-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride (10-DEBC) hydrochloride (Akt inhibitor) for 4 months.. DN was diagnosed by renal dysfunction, hypertrophy, tubulointerstitial lesions, and glomerulosclerosis associated with severe fibrosis, all of which were prevented by FGF21. FGF21 also suppressed the diabetes-induced renal EMT in DN mice by negatively regulating transforming growth factor beta (TGF-β)-induced nuclear translocation of Smad2/3, which is required for the transcription of multiple fibrotic genes. The mechanistic studies showed that FGF21 attenuated nuclear translocation of Smad2/3 by inhibiting renal activity of its conjugated protein p53, which carries Smad2/3 into the nucleus. Moreover pifithrin-α inhibited the FGF21-induced preventive effects on the renal EMT and subsequent renal fibrosis in DN mice. In addition, 10-DEBC also blocked FGF21-induced inhibition of renal p53 activity by phosphorylation of mouse double minute-2 homolog (MDM2).. FGF21 prevents renal fibrosis via negative regulation of the TGF-β/Smad2/3-mediated EMT process by activation of the Akt/MDM2/p53 signaling pathway.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Epithelial-Mesenchymal Transition; Fibroblast Growth Factors; Fibrosis; Kidney Tubules; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad2 Protein; Streptozocin; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Neutrophils releasing neutrophil extracellular traps (NETs) infiltrate the pancreas prior to type 1 diabetes (T1D) onset; however, the precise nature of their contribution to disease remains poorly defined. To examine how NETs affect immune functions in T1D, we investigated NET composition and their effect on dendritic cells (DCs) and T lymphocytes in T1D children. We showed that T1D patient NET composition differs substantially from that of healthy donors and that the presence of T1D-NETs in a mixed peripheral blood mononuclear cell culture caused a strong shift toward IFNγ-producing T lymphocytes, mediated through activation of innate immunity cells in T1D samples. Importantly, in a monocyte-derived DC (moDC) culture, NETs induced cytokine production, phenotypic change and IFNγ-producing T cells only in samples from T1D patients but not in those from healthy donors. RNA-seq analysis revealed that T1D-NETs presence causes TGFβ downregulation and IFNα upregulation and creates pro-T1D signature in healthy moDCs.

Topics: Adolescent; Adult; Cell Differentiation; Cells, Cultured; Child; Dendritic Cells; Diabetes Mellitus, Type 1; Extracellular Traps; Female; Gene Expression Regulation; Humans; Immunity, Innate; Interferon-gamma; Male; Neutrophils; Th1 Cells; Th1-Th2 Balance; Transforming Growth Factor beta; Young Adult

To explore the role and molecular mechanism of regulatory T (Treg) cells in type 1 diabetes (T1D).. Patients with T1D and the healthy volunteers were selected and a number of CD3. Treg content in patients with T1D was significantly decreased compared with the control volunteers. Treg content in rats was markedly decreased after injection with STZ to induce T1D rat model, while Treg infusion weakened the decrease. The change scope of weight and blood glucose in the model and Treg group was bigger than the control group, and the change in the infusion group was lighter than the model group. T1D decreased the expressions of IL-10, IL-4, TGF-β, and IL-2, while Treg infusion weakened the decrease. The expressions of IL-17 and IFN-γ in the T1D group was increased, while Treg infusion weakened the increase.. Autologous Treg infusion can strengthen the immunologic and islet function to treat T1D which may be via regulating the expression of inflammatory factors.

Topics: Animals; Cells, Cultured; Cytokines; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Interferon-gamma; Interleukin-10; Interleukin-2; Interleukin-4; Male; Rats; Real-Time Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The International Diabetic Federation estimated that 415 million adults currently have diabetes and 318 million adults had impaired glucose tolerance, putting them at high risk of developing diabetes in the future. In Type 1 Diabetes (T1D), the β cells are lost because of autoimmune reactions. Although islet transplantation has been a promising therapy for T1D, it is greatly limited by pancreatic donors. Here, we describe a protocol to generate glucose- responsive pancreatic β-like (GRPβ-L) cells from human-induced pluripotent stem (iPS) cells. We recapitulate in vivo pancreas development by in vitro induction of differentiating human (iPS) cells with stage-specific signaling molecules and proteins. Inhibition of Tyrosine Kinase receptor AXL, TGF-β, and Notch signaling pathways in the final stage of the five-stage protocol could efficiently generate GRPβ-L from the endocrine progenitor. Differentiation of human iPS cells through the protocol could result in functional GRPβ-L cells, which could be used in pharmaceutical and β cell biology studies. © 2018 by John Wiley & Sons, Inc.

Topics: Axl Receptor Tyrosine Kinase; Cell Culture Techniques; Cell Differentiation; Cell Lineage; Diabetes Mellitus, Type 1; Glucose; Humans; Induced Pluripotent Stem Cells; Insulin-Secreting Cells; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta

To identify single-cell transcriptional signatures of dendritic cells (DCs) that are associated with autoimmunity, and determine whether those DC signatures are correlated with the clinical heterogeneity of autoimmune disease.. Blood-derived DCs were single-cell sorted from the peripheral blood of patients with rheumatoid arthritis, systemic lupus erythematosus, or type 1 diabetes as well as healthy individuals. DCs were analyzed using single-cell gene expression assays, performed immediately after isolation or after in vitro stimulation of the cells. In addition, protein expression was measured using fluorescence-activated cell sorting.. CD1c+ conventional DCs and plasmacytoid DCs from healthy individuals exhibited diverse transcriptional signatures, while the DC transcriptional signatures in patients with autoimmune disease were altered. In particular, distinct DC clusters, characterized by up-regulation of TAP1, IRF7, and IFNAR1, were abundant in patients with systemic autoimmune disease, whereas DCs from patients with type 1 diabetes had decreased expression of the regulatory genes PTPN6, TGFB, and TYROBP. The frequency of CD1c+ conventional DCs that expressed a systemic autoimmune profile directly correlated with the extent of disease activity in patients with rheumatoid arthritis (Spearman's r = 0.60, P = 0.03).. DC transcriptional signatures are altered in patients with autoimmune disease and are associated with the level of disease activity, suggesting that immune cell transcriptional profiling could improve our ability to detect and understand the heterogeneity of these diseases, and could guide treatment choices in patients with a complex autoimmune disease.

Topics: Adaptor Proteins, Signal Transducing; Arthritis, Rheumatoid; ATP Binding Cassette Transporter, Subfamily B, Member 2; Autoimmune Diseases; Case-Control Studies; Dendritic Cells; Diabetes Mellitus, Type 1; Flow Cytometry; Gene Expression Profiling; Humans; Inflammation; Interferon Regulatory Factor-7; Lupus Erythematosus, Systemic; Membrane Proteins; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Receptor, Interferon alpha-beta; Severity of Illness Index; Single-Cell Analysis; Transforming Growth Factor beta; Up-Regulation

Type 1 diabetes (T1D) is a chronic metabolic disease of unknown etiology that results from β-cell destruction. The onset of the disease, which arises after a long asymptomatic period of autoimmune attack, may be followed by a relapsing and remitting progression, a phenomenon that is most evident during the partial remission phase (PR). This stage lasts for a few months, shows minor requirements of exogenous insulin and could be explained by a recovery of immunological tolerance. This study aims to identify new biomarkers at early stages of pediatric T1D that reflect immunoregulatory changes. To that end, pediatric patients with T1D (n = 52) and age-related control subjects (n = 30) were recruited. Immune response-related molecules and lymphocyte subsets were determined starting at T1D onset and until the second year of progression. Results showed that circulating TGF-β levels decreased during PR, and that betatrophin concentration was increased in all the considered stages without differing among studied checkpoints. Moreover, an increase of regulatory T, B and NK subsets was found during T1D progression, probably reflecting an attempt to restore self-tolerance. By contrast, a reduction in monocyte levels was observed at the early stages of diabetes. The results reveal significant changes in immunological parameters during the different early stages of T1D in children, which could ultimately serve as potential biomarkers to characterize the progression of T1D.

Topics: Angiopoietin-Like Protein 8; Angiopoietin-like Proteins; Biomarkers; Body Mass Index; Case-Control Studies; Child; Diabetes Mellitus, Type 1; Disease Progression; Female; Humans; Immunologic Memory; Lymphocyte Subsets; Male; Monocytes; Peptide Hormones; Pilot Projects; Remission Induction; Transforming Growth Factor beta

Polyclonal T regulatory cells (Treg - CD4

Topics: Animals; Antigens, CD; Apyrase; Cell Proliferation; Cell Separation; Coculture Techniques; Dendritic Cells; Diabetes Mellitus, Type 1; Disease Models, Animal; Gene Expression; Humans; Insulin; Interleukin-10; Interleukin-2; Mice; Mice, Inbred NOD; Primary Cell Culture; Programmed Cell Death 1 Receptor; Receptors, Antigen, T-Cell; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Autoimmune diabetes is a disorder of immune homeostasis that leads to targeted insulin-secreting islet β cell destruction characterized by insulitis. Human amylin (hA) is an important neuroendocrine hormone co-secreted with insulin by pancreatic β cells. Here, we report hA immune-modulatory action through inducing regulatory T cells. We ex vivo-treated human peripheral blood mononuclear cells (hPBMCs) with hA for 24 h and counted CD4+Foxp3+ regulatory T cells (Treg) using flow cytometry. Diabetic status was monitored and splenic Treg were measured in non-obese diabetic (NOD) male mice. NOD mice were intraperitoneally injected once daily with hA (n = 25) or solvent for control (n = 25) for 7 months continuously. Spleen tissues were collected at the end of intervention and processed for flow cytometry and Western blot. We found a 2.9-fold (p < 0.05) increase of CD4+Foxp3+ Treg in hPBMCs treated with 10 nmol/L hA compared with negative control. Incidence of diabetes in hA-treated NOD mice decreased 44% (p = 0.045) in the 6th month and 57% (p = 0.0002) in the 7th month. Meanwhile, the hA treatment induced a 1.5-fold increase of CD4+Foxp3+ Treg from mouse splenocytes (p = 0.0013). Expression of transforming growth factor-β (TGF-β) and toll-like receptor-4 (TLR-4) were upregulated in hA-treated mice. Human amylin might protect against autoimmune diabetes via the induction of CD4+Foxp3+ Treg, which suggests a novel approach to improve autoimmune conditions.

Topics: Animals; CD4 Antigens; Cells, Cultured; Diabetes Mellitus, Type 1; Forkhead Transcription Factors; Humans; Immunomodulation; Insulin-Secreting Cells; Islet Amyloid Polypeptide; Male; Mice; Mice, Inbred NOD; T-Lymphocytes, Regulatory; Toll-Like Receptor 4; Transforming Growth Factor beta

Sugars have pathogenic roles in diabetes. In Nature Medicine, Zhang et al. revisit this dogma and show that mannose induces the generation of regulatory T (Treg) cells. The immunoregulatory effect of mannose not only prevented the onset of autoimmune diabetes but also blocked disease progression in new-onset diabetic mice.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Mice; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Antigen-specific regulatory T cells (Tregs) have proven to be effective in reversing established autoimmunity in type 1 diabetes (T1D). Cord blood (CB) can serve as an efficient and safe source for Tregs for antigen-specific immunomodulation in T1D, a strategy that is yet to be explored. Therefore, we assessed the potential of CB in generation of proinsulin (PI)-specific Tregs by using HLA class II tetramers.. We analyzed the frequency of PI-specific natural Tregs (nTregs) and induced Tregs (iTregs) derived from the CB as well as peripheral blood (PB) of patients with T1D and healthy control subjects. For this, CD4+CD25+CD127. Following stimulation, we observed that CB harbors a significantly higher frequency of PI-specific Tregs than PB of subjects with T1D (P = 0.0003). Further, the proportion of PI-specific Tregs was significantly higher in both the nTreg (P = 0.01) and iTreg (P = 0.0003) compartments of CB as compared with PB of subjects with T1D. In co-culture experiments, the PI-specific Tregs suppressed the proliferation of effector T cells significantly (P = 0.0006). The expanded nTregs were able to retain hypomethylation status at their Tregs-specific demethylated region (TSDR), whereas iTregs were unable to acquire the characteristic demethylation pattern.. Our study demonstrates that CB can serve as an excellent source for generation of functional antigen-specific Tregs for immunotherapeutic approaches in subjects with T1D.

Topics: Adult; CD4-Positive T-Lymphocytes; Cell- and Tissue-Based Therapy; Cells, Cultured; Diabetes Mellitus, Type 1; Female; Fetal Blood; Forkhead Transcription Factors; Humans; Immunomodulation; Infant, Newborn; Interleukin-2 Receptor alpha Subunit; Proinsulin; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Umbilical Cord

We previously reported the development of an oral vaccine for diabetes based on live attenuated Salmonella-expressing preproinsulin (PPI) as the autoantigen. When combined with host cell-expressed TGFβ, the vaccine prevented the onset of diabetes in non-obese diabetic (NOD) mice. Herein, we investigated factors that could affect vaccine efficacy including vaccination number, optimization of the autoantigen codon sequence, Salmonella SPI2-TTSS promoter/effector combinations, concurrent short-course low-dose anti-CD3. We also evaluated autoantigen GAD65 and cytokine IL10 treatment upon vaccine efficacy. T-cells we employed to elucidate the mechanism of the vaccine action. Our results showed that GAD65+TGFβ or PPI+TGFβ+IL10 prevented the onset of diabetes in the NOD mice and maintained glucose tolerance. However, increasing the number of vaccine doses, codon-optimization of the autoantigen(s) or use of other Salmonella promoter/effector combinations had no in vivo effect. Interestingly, two doses of vaccine (PPI+TGFβ+IL10) combined with a sub-therapeutic dose of anti-CD3 prevented diabetes and decreased hyperglycemia in mice. The combined therapy also increased splenic Tregs and local Tregs in pancreatic lymph nodes (PLN) and increased regulatory (IL10 and IL2) but reduced inflammatory (IFNγ and TNFα) cytokines. Together, these results indicate that the combination of low vaccine dose number, less vaccine autoantigen expression and short-course low-dose anti-CD3 can increase regulatory mechanisms and suppress autoimmunity.

Topics: Animals; Autoantigens; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Drug Therapy, Combination; Female; Immunotherapy; Insulin; Interleukin-10; Mice; Mice, Inbred NOD; Protein Precursors; Salmonella; Spleen; T-Lymphocytes; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Aberrant expression of microRNAs (miRs) contributes to diabetic renal complications, including renal hypertrophy and matrix protein accumulation. Reduced expression of phosphatase and tensin homolog (PTEN) by hyperglycemia contributes to these processes. We considered involvement of miR in the downregulation of PTEN. In the renal cortex of type 1 diabetic mice, we detected increased expression of miR-214 in association with decreased levels of PTEN and enhanced Akt phosphorylation and fibronectin expression. Mesangial and proximal tubular epithelial cells exposed to high glucose showed augmented expression of miR-214. Mutagenesis studies using 3'-UTR of PTEN in a reporter construct revealed PTEN as a direct target of miR-214, which controls its expression in both of these cells. Overexpression of miR-214 decreased the levels of PTEN and increased Akt activity similar to high glucose and lead to phosphorylation of its substrates glycogen synthase kinase-3β, PRAS40, and tuberin. In contrast, quenching of miR-214 inhibited high-glucose-induced Akt activation and its substrate phosphorylation; these changes were reversed by small interfering RNAs against PTEN. Importantly, respective expression of miR-214 or anti-miR-214 increased or decreased the mammalian target of rapamycin complex 1 (mTORC1) activity induced by high glucose. Furthermore, mTORC1 activity was controlled by miR-214-targeted PTEN via Akt activation. In addition, neutralization of high-glucose-stimulated miR-214 expression significantly inhibited cell hypertrophy and expression of the matrix protein fibronectin. Finally, the anti-miR-214-induced inhibition of these processes was reversed by the expression of constitutively active Akt kinase and hyperactive mTORC1. These results uncover a significant role of miR-214 in the activation of mTORC1 that contributes to high-glucose-induced mesangial and proximal tubular cell hypertrophy and fibronectin expression.

Topics: 3' Untranslated Regions; Animals; Blood Glucose; Cell Proliferation; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disease Models, Animal; Epithelial Cells; Fibronectins; Gene Expression Regulation, Enzymologic; Glomerular Mesangium; Hypertrophy; Kidney Glomerulus; Kidney Tubules, Proximal; Mechanistic Target of Rapamycin Complex 1; Mice; MicroRNAs; Multiprotein Complexes; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Rats; RNA Interference; Signal Transduction; TOR Serine-Threonine Kinases; Transfection; Transforming Growth Factor beta

D-mannose, a C-2 epimer of glucose, exists naturally in many plants and fruits, and is found in human blood at concentrations less than one-fiftieth of that of glucose. However, although the roles of glucose in T cell metabolism, diabetes and obesity are well characterized, the function of D-mannose in T cell immune responses remains unknown. Here we show that supraphysiological levels of D-mannose safely achievable by drinking-water supplementation suppressed immunopathology in mouse models of autoimmune diabetes and airway inflammation, and increased the proportion of Foxp3

Topics: Adoptive Transfer; Animals; Colitis; Colon; Diabetes Mellitus, Type 1; Dietary Supplements; Disease Models, Animal; Fatty Acids; Flow Cytometry; Forkhead Transcription Factors; Humans; In Vitro Techniques; Inflammation; Integrins; Lipid Metabolism; Lung; Lung Diseases; Mannose; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Ovalbumin; Oxidation-Reduction; Pancreas; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Respiratory Hypersensitivity; Reverse Transcriptase Polymerase Chain Reaction; Spleen; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Up-Regulation

Decreased soluble guanylate cyclase activity and cGMP levels in diabetic kidneys were shown to influence the progression of nephropathy. The regulatory effects of soluble guanylate cyclase activators on renal signaling pathways are still unknown, we therefore investigated the renal molecular effects of the soluble guanylate cyclase activator cinaciguat in type-1 diabetic (T1DM) rats. Male adult Sprague-Dawley rats were divided into 2 groups after induction of T1DM with 60 mg/kg streptozotocin: DM, untreated (DM, n = 8) and 2) DM + cinaciguat (10 mg/kg per os daily, DM-Cin, n = 8). Non-diabetic untreated and cinaciguat treated rats served as controls (Co (n = 10) and Co-Cin (n = 10), respectively). Rats were treated for eight weeks, when renal functional and molecular analyses were performed. Cinaciguat attenuated the diabetes induced proteinuria, glomerulosclerosis and renal collagen-IV expression accompanied by 50% reduction of TIMP-1 expression. Cinaciguat treatment restored the glomerular cGMP content and soluble guanylate cyclase expression, and ameliorated the glomerular apoptosis (TUNEL positive cell number) and podocyte injury. These effects were accompanied by significantly reduced TGF-ß overexpression and ERK1/2 phosphorylation in cinaciguat treated diabetic kidneys. We conclude that the soluble guanylate cyclase activator cinaciguat ameliorated diabetes induced glomerular damage, apoptosis, podocyte injury and TIMP-1 overexpression by suppressing TGF-ß and ERK1/2 signaling.

Topics: Animals; Benzoates; Cyclic GMP; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disease Models, Animal; Enzyme Activators; Kidney; Male; MAP Kinase Signaling System; Rats, Sprague-Dawley; Transforming Growth Factor beta; Treatment Outcome

CD3-specific monoclonal antibody (mAb) treats autoimmune disease in animal models and has shown promise in clinical trials of type 1 diabetes. Whereas intravenous administration of CD3-specific mAb acts primarily by transient depletion of activated effector T cells, oral CD3-specific mAb acts primarily by the induction Tregs. We investigated whether oral CD3-specific mAb inhibits disease in non obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, closely resembling human type 1 diabetes. We found that oral CD3-specific mAb treatment delayed onset and reduced incidence of diabetes in NOD mice, inducing changes in both effector and regulatory T cell compartments. The therapeutic effect was associated with decreased T cell proliferation, decreased IFNγ and IL-17 production, and increased TGF-β and IL-10 production in vitro. In vivo transfer experiments demonstrated that oral CD3-specific mAb decreased diabetogenicity of effector T cells and increased the function of regulatory T cells. Oral OKT3, a monoclonal antibody specific for human CD3 had equivalent effects in transgenic NOD mice expressing the human CD3 epsilon chain which serves as a preclinical model for testing human CD3-specific mAb. These results suggest that oral CD3-specific mAb has the potential for treating autoimmune diabetes in humans.

Topics: Administration, Mucosal; Animals; Antibodies, Monoclonal; CD3 Complex; Diabetes Mellitus, Type 1; Disease Models, Animal; Female; Immune Tolerance; Immunity, Mucosal; Interleukin-10; Lymphocyte Activation; Mice; Mice, Inbred NOD; Mice, Transgenic; Protective Agents; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The role of transforming growth factor-β (TGF-β) has recently gained much attention in diabetic nephropathy and kidney fibrosis. In this study, we extend this to an assessment of transcriptional regulation of the entire TGF-β superfamily in kidneys from diabetic vs. healthy mice. In order to study the translation between mouse model and patients, we evaluated the signature of phosphorylated Sma- and Mad-related protein 2 (pSmad2), as molecular marker of TGF-β/activin activity, in the kidneys of streptozotocin (STZ)-treated mice compared to that of type 1 diabetes (T1D) patients.. Patterns of pSmad2 were determined in kidneys from T1D patients with progressed diabetic nephropathy (DN), defined by hyperglycemia, microalbuminuria, and increased levels of serum creatinine. They were compared to changes seen in the STZ-induced DN mouse model. This was studied by immunohistochemistry (IHC) with an antibody specific for pSmad2. Diabetic mice were also characterized by pSmad1/5/8 (IHC), pSmad2/3 (flow cytometry), and TGF-β family members including bone morphogenetic protein (BMP)-like proteins (quantitative real-time polymerase chain reaction [qPCR]).. Renal tubules in DN patients and in STZ mice showed upregulation of pSmad2 concomitant with significantly enlarged distal tubule lumens (p < 0.0001). Renal-derived CD11b+ cells from STZ mice showed elevated pSmad2/3, while endothelial cells had reduced pSmad2/3 levels. No pSmad1/5/8 was observed in the tubule compartment of STZ-treated mice. On total kidney mRNA level, a signature favoring activation of the TGF-β/activin pathway and inhibition of the BMP pathway was demonstrated by qPCR.. Although the pre-clinical DN model lacks the features of fibrosis present in human DN, both species show induction of a local milieu favoring pSmad2 signaling, which may be useful as a disease biomarker in pre-clinical models.

Topics: Activins; Adult; Aged; Aged, 80 and over; Animals; Bone Morphogenetic Proteins; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Epithelial Cells; Female; Humans; Kidney Tubules; Male; Mice; Mice, 129 Strain; Middle Aged; Models, Biological; Phosphorylation; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation

Mesenchymal stem cells (MSCs) are advantageous candidates for cell therapy of Type 1 diabetes (T1D). Considering immunomodulatory effect of MSC, in this study, we engineered MSCs with TGF-β gene to increase MSC potency for T1D therapy in mouse model.. Two plans were designed for prevention and treatment of diabetes, respectively. In both of them, MSCs were injected i.v. and then, the diabetes features including serum insulin, blood glucose, glucose tolerance, splenocytes proliferation, and IL-4/IFN-γ production were evaluated.. TGF-β/MSCs treatment program resulted in the restoration of serum glucose after 3weeks, while prevention program could delay diabetes progression for two weeks. TGF-β/MSCs treatment elevated the levels of serum insulin and Th2 cytokine shift on 5th week after start of treatment. TGF-β/MSCs (and MSCs alone) could also diminish body weight and enhance mice survival comparing to untreated diabetic mice.. Engineered TGF-β/MSCs could restore some T1D features, including the regulation of adverse immune responses and could be potent tools for cell therapy of T1D comparing MSCs alone.

Topics: Animals; Blood Glucose; Cells, Cultured; Cytokines; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Genetic Engineering; Humans; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Th1-Th2 Balance; Transforming Growth Factor beta

In addition to lowering blood glucose in patients with type 2 diabetes mellitus, dipeptidyl peptidase 4 (DPP4) inhibitors have been shown to be antifibrotic and anti-inflammatory. We have previously shown that DPP4 inhibition in human kidney proximal tubular cells exposed to high glucose reduced fibrotic and inflammatory markers. Hence, we wanted to demonstrate renoprotection in an in vivo model.. We used a type 1 diabetic animal model to explore the renoprotective potential of saxagliptin independent of glucose lowering. We induced diabetes in enos -/- mice using streptozotocin and matched glucose levels using insulin. Diabetic mice were treated with saxagliptin and outcomes compared with untreated diabetic mice.. We provide novel data that saxagliptin limits renal hypertrophy, transforming growth factor beta-related fibrosis and NF-κBp65-mediated macrophage infiltration. Overall, there was a reduction in histological markers of tubulointerstitial fibrosis. There was no reduction in albuminuria or glomerulosclerosis.. Our findings highlight the potential of DPP4 inhibition as additional therapy in addressing the multiple pathways to achieve renoprotection in diabetic nephropathy.

Topics: Adamantane; Albuminuria; Animals; Blood Glucose; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Dipeptides; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Fibronectins; Fibrosis; Glomerulonephritis; Hypertrophy; Insulin; Kidney; Male; Mice, Knockout; Nephritis, Interstitial; Nitric Oxide Synthase Type III; Phosphorylation; Signal Transduction; Smad2 Protein; Smad3 Protein; Streptozocin; Transcription Factor RelA; Transforming Growth Factor beta

The inflammatory state associated with Crohn's disease (CD) and ulcerative colitis (UC) remains incompletely defined. To understand more clearly the extracellular milieu associated with inflammatory bowel disease (IBD), we employed a bioassay whereby plasma of treatment naive paediatric IBD patients (n = 22 CD, n = 15 UC) and unrelated healthy controls (uHC, n = 10) were used to induce transcriptional responses in a healthy leucocyte population. After culture, gene expression was measured comprehensively with microarrays and analysed. Relative to uHC, plasma of CD and UC patients induced distinct responses consisting, respectively, of 985 and 895 regulated transcripts [|log2 ratio| ≥ 0·5 (1·4-fold); false discovery rates (FDR) ≤ 0·01]. The CD:uHC and UC:uHC signatures shared a non-random, commonly regulated, intersection of 656 transcripts (χ(2)  = P < 0·001) and were highly correlative [Pearson's correlation coefficient = 0·96, 95% confidence interval (CI) = 0.96, 0.97]. Despite sharing common genetic susceptibility loci, the IBD signature correlated negatively with that driven by plasma of type 1 diabetes (T1D) patients (Pearson's correlation coefficient = -0·51). Ontological analyses revealed the presence of an immunoregulatory plasma milieu in IBD, as transcripts for cytokines/chemokines, receptors and signalling molecules consistent with immune activation were under-expressed relative to uHC and T1D plasma. Multiplex enzyme-linked immunosorbent assay (ELISA) and receptor blockade studies confirmed transforming growth factor (TGF)-β and interleukin (IL)-10 as contributors to the IBD signature. Analysis of CD patient signatures detected a subset of transcripts associated with responsiveness to 6-mercaptopurine treatment. Through plasma-induced signature analysis, we have defined a unique, partially TGF-β/IL-10-dependent immunoregulatory signature associated with IBD that may prove useful in predicting therapeutic responsiveness.

Topics: Adolescent; Blood Proteins; Child; Child, Preschool; Colitis, Ulcerative; Crohn Disease; Diabetes Mellitus, Type 1; Female; Healthy Volunteers; Humans; Immunologic Factors; Interleukin-10; Leukocytes, Mononuclear; Male; Primary Cell Culture; Protein Array Analysis; RNA, Messenger; Transcriptome; Transforming Growth Factor beta

Human umbilical cord Wharton jelly-derived mesenchymal stem cells (hUCMS) might apply to treating chronic autoimmune disorders, as already shown for Sjögren's syndrome, including type 1 diabetes mellitus (T1D). Since naked hUCMS grafts encountered restraints, we enveloped hUCMS, within immunoisolatory microcapsules (CpS-hUCMS), made of our endotoxin-free, clinical grade alginate. We then examined the vitro effects of interferon (IFN)-γ-pretreated CpS-hUCMS on Th17 and Treg of T1D patients (n=15) and healthy controls (n=10). Peripheral blood mononuclear cells (PBMCs) were co-cultured with PBMC/CpS-hUCMS: lymphocyte proliferation was assessed by carboxyfluorescein succinimidyl esther (CFSE) dilution assay, and phenotypic analysis of regulatory and effector Tc was also performed. Cytokine expression was performed by bead array and qPCR on IFN-γ-pretreated hUCMS before PBMCs co-culture. CpS-hUCMS restored a correct Treg/Th17 ratio, relevant to the T1D disease process. In summary, we have preliminarily developed a new biohybrid system, associated with immunoregulatory properties, that is ready for in vivo application.

Topics: Antiviral Agents; Blotting, Western; Capsules; Case-Control Studies; Cells, Cultured; Diabetes Mellitus, Type 1; Flow Cytometry; Forkhead Transcription Factors; Gene Expression; Gene Expression Profiling; HLA-G Antigens; Humans; Hypoxanthine Phosphoribosyltransferase; Immunohistochemistry; In Vitro Techniques; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interferon-gamma; Interleukin-10; Interleukin-17; Interleukin-2; Interleukin-6; Interleukins; Mesenchymal Stem Cells; Nitric Oxide Synthase Type II; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocyte Subsets; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Umbilical Cord; Wharton Jelly

Eucommia bark, Eucommia ulmoides Oliver barks (Du-Zhong in Mandarin), is an herb used for renal dysfunction in Chinese traditional medicine. In an attempt to develop this herb as a treatment for diabetic nephropathy (DN), we investigated the effects of Du-Zhong on renal dysfunction in type 1-like diabetic rats.. Streptozotocin (STZ) was used to induce type 1-like diabetes in rats (STZ-diabetic rats). In addition to hyperglycemia, STZ-diabetic rats showed significant nephropathy, including higher plasma levels of blood urea nitrogen, creatinine, and renal fibrosis. Western blot analysis of renal cortical tissue was applied to characterize the changes in potential signals related to nephropathy.. Oral administration of Du-Zhong (1 g/kg/day) to STZ-diabetic rats for 20 days not only decreased the plasma levels of blood urea nitrogen and creatinine but also improved renal fibrosis, whereas the plasma glucose level was not changed. The higher expressions of protein levels of transforming growth factor-beta (TGF-β) and connective tissue growth factor in diabetic rats were markedly attenuated by Du-Zhong. The increased phosphorylation of Smad2/3 in STZ-diabetic rats was also reduced by Du-Zhong. However, Du-Zhong cannot reverse the hyperglycemia-induced overproduction of signal transducers and activators of transcription 3 in the diabetic kidney.. Oral administration of Du-Zhong improves STZ-induced DN in rats by inhibiting TGF-β/Smad signaling and suppressing TGF-β/connective tissue growth factor expression. Therefore, active principle from Du-Zhong is suitable to develop as new agent for DN in the future.

Topics: Administration, Oral; Animals; Blood Glucose; Connective Tissue Growth Factor; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Eucommiaceae; Male; Medicine, Chinese Traditional; Rats; Rats, Wistar; Signal Transduction; Streptozocin; Structure-Activity Relationship; Transforming Growth Factor beta

Human type 1 diabetes results from a destructive auto-reactive immune response in which CD8(+) T lymphocytes play a critical role. Given the intense ongoing efforts to develop immune intervention to prevent and/or cure the disease, biomarkers suitable for prediction of disease risk and progress, as well as for monitoring of immunotherapy are required. We undertook separate multi-parameter analyses of single naïve and activated/memory CD8(+) T lymphocytes from pediatric and adult patients, with the objective of identifying cellular profiles associated with onset of type 1 diabetes. We observe global perturbations in gene and protein expression and in the abundance of T cell populations characterizing pediatric but not adult patients, relative to age-matched healthy individuals. Pediatric diabetes is associated with a unique population of CD8(+) T lymphocytes co-expressing effector (perforin, granzyme B) and regulatory (transforming growth factor β, interleukin-10 receptor) molecules. This population persists after metabolic normalization and is especially abundant in children with high titers of auto-antibodies to glutamic acid decarboxylase and with elevated HbA1c values. These findings highlight striking differences between pediatric and adult type 1 diabetes, indicate prolonged large-scale perturbations in the CD8(+) T cell compartment in the former, and suggest that CD8(+)CD45RA(-) T cells co-expressing effector and regulatory factors are of interest as biomarkers in pediatric type 1 diabetes.

Topics: Adolescent; Adult; Autoantibodies; Biomarkers; CD8-Positive T-Lymphocytes; Child; Child, Preschool; Diabetes Mellitus, Type 1; Female; Glutamate Decarboxylase; Glycated Hemoglobin; Granzymes; Humans; Leukocyte Common Antigens; Lymphocyte Activation; Male; Middle Aged; Perforin; Receptors, Interleukin-10; Transcriptome; Transforming Growth Factor beta; Young Adult

Type 1 diabetes (T1D) is thought to involve chronic inflammation, which is manifested by the activation and expression of different inflammatory mediators. Th1- and Th17-associated cytokines are factors that have been shown to exert profound pro-inflammatory activities and have been implicated in the pathogenesis of T1D in mice and humans.. Therefore, the aim of this case control study was to determine the serum level of IL-17, IL-21, IL-27, transforming growth factor beta (TGF-β), and IFN-γ and their reciprocal relationship in Iranian T1D patients.. Blood samples were collected from 48 T1D patients and 49 healthy individuals with no history of malignancies or autoimmune disorders based on simple sampling. The serum levels of IL-17, IL-21, IL-27, TGF-β, and IFN-γ were measured by the enzyme linked immunosorbent assay (ELISA).. The serum levels of IL-17 and IL-21 were significantly higher in T1D patients compared to the healthy individuals (p = 0.005 and 0.01, respectively), but interestingly, the opposite was the case for IL-27 (p < 0.0001). However, there were no significant differences in TGF-β and IFN-γ between both groups. In addition, IL-17/IFN-γ and IL-17/IL-27 ratios were higher in patients compared to the control group.. Our results indicated dominant Th17-associated IL-17, suggesting a shift from the Treg and Th1 phenotypes toward the Th17 phenotype. Therefore, it can promote inflammation in β cells in T1D. In addition, it suggests the role of Th17 and Th17/Th1 ratios as a potential contributor to β cells destruction and the Th17/Th1 response ratio may provide a novel biomarker for rapid T1D diagnosis before the destruction of β cells and progression of the disease to the clinical end stages.

Topics: Adolescent; Animals; Case-Control Studies; Child; Child, Preschool; Diabetes Mellitus, Type 1; Female; Humans; Inflammation; Interferon-gamma; Interleukin-17; Interleukins; Iran; Male; Mice; Th1 Cells; Th17 Cells; Transforming Growth Factor beta; Young Adult

Upregulation of the intermediate filament protein nestin was identified in a subpopulation of fibroblasts during reactive and reparative fibrosis and directly contributed to the enhanced proliferative phenotype. The present study tested the hypothesis that nestin was expressed in lung fibroblasts and the pattern of expression represented a distinct marker of pulmonary remodeling secondary to myocardial infarction and type I diabetes. Nestin((+)) fibroblasts were detected in rat lungs and a subpopulation exhibited a myofibroblast phenotype delineated by the co-expression of smooth muscle α-actin. In the lungs of myocardial infarcted rats, interstitial collagen content and nestin mRNA/protein levels were significantly increased despite the absence of secondary pulmonary hypertension, whereas smooth muscle α-actin protein expression was unchanged. Exposure of rat pulmonary fibroblasts to pro-fibrotic stimuli angiotensin II and transforming growth factor-β significantly increased nestin protein levels. In the lungs of type I diabetic rats, the absence of a reactive fibrotic response was associated with a significant downregulation of nestin mRNA/protein expression. Nestin was reported a target of miR-125b, albeit miR-125b levels were unchanged in pulmonary fibroblasts treated with pro-fibrotic stimuli. Nestin((+)) cells lacking smooth muscle α-actin/collagen staining were also identified in rodent lungs and a transgenic approach revealed that expression of the intermediate filament protein was driven by intron 2 of the nestin gene. The disparate regulation of nestin characterized a distinct pattern of pulmonary remodeling secondary to myocardial infarction and type I diabetes and upregulation of the intermediate filament protein in lung fibroblasts may have facilitated in part the reactive fibrotic response.

Topics: Actins; Airway Remodeling; Angiotensin II; Animals; Biomarkers; Cell Differentiation; Collagen Type I; Diabetes Mellitus, Type 1; Fibroblasts; Heart Failure; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Lung; Male; MicroRNAs; Myocardial Contraction; Myocardial Infarction; Nestin; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; RNA, Messenger; Streptozocin; Transforming Growth Factor beta

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is particularly effective in improving osteogenesis in patients with diminished bone healing capabilities, such as individuals with type 1 diabetes mellitus (T1DM) who have impaired bone healing capabilities and increased risk of developing osteoporosis. This study measured the effects of rhBMP-2 treatment on osteogenesis by observing the dose-dependent effect of localized delivery of rhBMP-2 on biomechanical parameters of bone using a hydroxyapatite/tri-calcium phosphate (HA/TCP) carrier in a T1DM-related osteoporosis animal model.. Two different doses of rhBMP-2 (LD low dose, HD high dose) with a HA/TCP carrier were injected into the femoral intramedullary canal of rats with T1DM-related osteoporosis. Two more diabetic rat groups were injected with saline alone and with HA/TCP carrier alone. Radiographs and micro-computed tomography were utilized for qualitative assessment of bone mineral density (BMD). Biomechanical testing occurred at 4- and 8-week time points; parameters tested included torque to failure, torsional rigidity, shear stress, and shear modulus.. At the 4-week time point, the LD and HD groups both exhibited significantly higher BMD than controls; at the 8-week time point, the HD group exhibited significantly higher BMD than controls. Biomechanical testing revealed dose-dependent, higher trends in all parameters tested at the 4- and 8-week time points, with minimal significant differences.. Groups treated with rhBMP-2 demonstrated improved bone mineral density at both 4 and 8 weeks compared to control saline groups, in addition to strong trends towards improvement of intrinsic and extrinsic biomechanical properties when compared to control groups. Data revealed trends toward dose-dependent increases in peak torque, torsional rigidity, shear stress, and shear modulus 4 weeks after rhBMP-2 treatment.. Not applicable.

Topics: Animals; Biomechanical Phenomena; Bone Density; Bone Morphogenetic Protein 2; Calcium Phosphates; Diabetes Mellitus, Type 1; Disease Models, Animal; Durapatite; Femur; Male; Osteogenesis; Osteoporosis; Rats, Wistar; Recombinant Proteins; Transforming Growth Factor beta; X-Ray Microtomography

Curing type 1 diabetes (T1D) will require lasting control of the autoimmune response that destroys insulin-producing islet β-cells. Re-establishing tolerance by restoring/replacing Tregs has significant potential for treatment of T1D but will require strategies to augment and maintain their efficacy. We previously showed that polyclonal in vitro-induced Tregs can reverse recent onset of T1D in ∼ 50% of NOD mice. Here we report that treatment of newly hyperglycemic animals with a short course of anti-CD44 at the time of Treg transfer improved diabetes reversal to >90%. Anti-CD44 treatment alone delayed diabetes onset and increased the frequencies of pancreatic CD4(+) T cells producing IL-2 or TGF-β, cytokines that support Treg function and survival, without altering production of IFN-γ. These anti-CD44 effects on endogenous T cells were also observed in the context of polyclonal Treg transfer, and the combination treatment also reduced pancreatic infiltrates. The results provide compelling evidence that approaches to modulate the pancreatic milieu to support Treg function and counteract inflammation in the pancreas can greatly enhance the efficacy of adoptively transferred Tregs, and suggest that approaches achieving these outcomes hold promise for long-term control of autoimmunity in T1D.

Topics: Adoptive Transfer; Animals; Antibodies, Blocking; CD4-Positive T-Lymphocytes; Combined Modality Therapy; Diabetes Mellitus, Type 1; Female; Flow Cytometry; Humans; Hyaluronan Receptors; Interferon-gamma; Interleukin-2; Male; Mice, Inbred NOD; Mice, SCID; Pancreas; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Hyperlipidemia is thought to be a major risk factor for the progression of renal diseases in diabetes. Recent studies have shown that lipid profiles are commonly abnormal early on type 2 diabetes mellitus (T2DM) with diabetic nephropathy. However, the early effects of triglyceride and cholesterol abnormalities on renal injury in type 1 diabetes mellitus (T1DM) are not fully understood and require reliable animal models for exploration of the underlying mechanisms. Hamster models are important tools for studying lipid metabolism because of their similarity to humans in terms of lipid utilization and high susceptibility to dietary cholesterol and fat.. Twenty-four male Golden Syrian hamsters (100-110 g) were rendered diabetes by intraperitoneal injections of streptozotocin (STZ) on consecutive 3 days at dose of 30 mg/kg, Ten days after STZ injections, hamsters with a plasma Glu concentration more than 12 mmol/L were selected as insulin deficient ones and divided into four groups (D-C, D-HF, D-HC, and D-HFHC), and fed with commercially available standard rodent chow, high-fat diet, high-cholesterol diet, high-fat and cholesterol diet respectively, for a period of four weeks.. After an induction phase, a stable model of renal injury was established with the aspects of early T1DM kidney disease, These aspects were severe hypertriglyceridemia, hypercholesterolemia, proteinuria with mesangial matrix accumulation, upgraded creatinine clearance, significant cholesterol and triglyceride deposition, and increasing glomerular surface area, thickness of basement membrane and mesangial expansion. The mRNA levels of sterol regulatory element binding protein-1c, transforming growth factors-β, plasminogen activator inhibitor-1, tumor necrosis factor-α and interleukin-6 in the D-HFHC group were significantly up-regulated compared with control groups.. This study presents a novel, non-transgenic, non-surgical method for induction of renal injury in hamsters, which is an important complement to existing diabetic models for pathophysiological studies in early acute and chronic kidney disease, especially hyperlipidemia. These data suggest that both severe hypertriglyceridemia and hypercholesterolemia can accelerate renal injury in the early development of T1DM.

Topics: Animals; Blood Glucose; Cholesterol, Dietary; Creatinine; Cricetinae; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Diet, High-Fat; Disease Models, Animal; Hypercholesterolemia; Hypertriglyceridemia; Interleukin-6; Kidney; Male; Mesocricetus; Plasminogen Activator Inhibitor 1; Proteinuria; RNA, Messenger; Sterol Regulatory Element Binding Protein 1; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

To investigate the effects of ibuprofen on cardiac fibrosis in a rat model of type 1 diabetes.. The diabetic model was established by injecting streptozotocin into the rats. Then, ibuprofen or pioglitazone was given by gavage for 8 weeks. The cardiac fibrosis was assessed, and the major components of the renin-angiotensin system, the transforming growth factor β1 (TGF-β1) and the mammalian target of rapamycin (mTOR), were evaluated by histopathological, immunohistochemical, Western blot analysis or ELISA assay.. Obvious cardiac fibrosis was detected in the diabetic group and was alleviated by ibuprofen treatment. Angiotensin-converting enzyme (ACE), angiotensin (Ang) II and AngII type 1 receptor (AT1-R) levels were higher, and ACE2, Ang(1-7) and Mas receptor (Mas-R) were lower in the diabetic group. The ratio of ACE to ACE2 was raised in the diabetic group. All these changes were ameliorated by ibuprofen. TGF-β1 and mTOR were raised in the hearts of the diabetic group and were attenuated by ibuprofen treatment. There was no significant difference between the ibuprofen and the pioglitazone groups.. Ibuprofen could ameliorate the cardiac fibrosis in diabetic rats by reduction of the ACE/AngII/AT1-R axis and enhancement of the ACE2/Ang(1-7)/Mas-R axis, leading to a decrease in TGF-β1 and mTOR.

Topics: Angiotensin-Converting Enzyme 2; Animals; Cardiotonic Agents; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Angiopathies; Down-Regulation; Fibrosis; Ibuprofen; Male; Myocardium; Peptidyl-Dipeptidase A; Rats, Sprague-Dawley; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Up-Regulation

Toll-like receptor 4 (TLR4) activation has been proposed to be important for islet cell inflammation and eventually β cell loss in the course of type 1 diabetes (T1D) development. However, according to the "hygiene hypothesis", bacterial endotoxin lipopolysaccharide (LPS), an agonist on TLR4, inhibits T1D progression. Here we investigated possible mechanisms for the protective effect of LPS on T1D development in non-obese diabetic (NOD) mice. We found that LPS administration to NOD mice during the prediabetic state neither prevented nor reversed insulitis, but delayed the onset and decreased the incidence of diabetes, and that a multiple-injection protocol is more effective than a single LPS intervention. Further, LPS administration suppressed spleen T lymphocyte proliferation, increased the generation of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), reduced the synthesis of strong Th1 proinflammatory cytokines, and downregulated TLR4 and its downstream MyD88-dependent signaling pathway. Most importantly, multiple injections of LPS induced a potential tolerogenic dendritic cell (DC) subset with low TLR4 expression without influencing the DC phenotype. Explanting DCs from repeated LPS-treated NOD mice into NOD/SCID diabetic mice conferred sustained protective effects against the progression of diabetes in the recipients. Overall, these results suggest that multiple mechanisms are involved in the protective effects of LPS against the development of diabetes in NOD diabetic mice. These include Treg induction, down-regulation of TLR4 and its downstream MyD88-dependent signaling pathway, and the emergence of a potential tolerogenic DC subset.

Topics: Animals; Cell Differentiation; Cell Proliferation; Dendritic Cells; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Down-Regulation; Female; Insulin-Secreting Cells; Interferon-gamma; Interleukin-10; Interleukin-2; Lipopolysaccharides; Lymphocyte Activation; Mice; Mice, Inbred NOD; Myeloid Differentiation Factor 88; Spleen; T-Lymphocytes, Regulatory; Toll-Like Receptor 4; Transforming Growth Factor beta

Type 1 diabetes is one of the most extensively studied autoimmune diseases, but the cellular and molecular mechanisms leading to T cell-mediated destruction of insulin-producing β cells are still not well understood. In this study, we show that regulatory T cells (T(regs)) in NOD mice undergo age-dependent loss of suppressor functions exacerbated by the decreased ability of activated effector T cells to upregulate Foxp3 and generate T(regs) in the peripheral organs. This age-dependent loss is associated with reduced intercellular communication mediated by gap junctions, which is caused by impaired upregulation and decreased expression of connexin 43. Regulatory functions can be corrected, even in T cells isolated from aged, diabetic mice, by a synergistic activity of retinoic acid, TGF-β, and IL-2, which enhance connexin 43 and Foxp3 expression in T(regs) and restore the ability of conventional CD4(+) T cells to upregulate Foxp3 and generate peripherally derived T(regs). Moreover, we demonstrate that suppression mediated by T(regs) from diabetic mice is enhanced by a novel reagent, which facilitates gap junction aggregation. In summary, our report identifies gap junction-mediated intercellular communication as an important component of the T(reg) suppression mechanism compromised in NOD mice and suggests how T(reg) mediated immune regulation can be improved.

Topics: Age Factors; Animals; Cell Communication; Cell Differentiation; Connexin 43; Diabetes Mellitus, Type 1; Female; Forkhead Transcription Factors; Gap Junctions; Immunosuppressive Agents; Interleukin-2; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Smad2 Protein; Smad3 Protein; STAT5 Transcription Factor; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tretinoin; Up-Regulation

The anti-inflammatory cytokine IL-35 is produced by regulatory T (Treg) cells to suppress autoimmune and inflammatory responses. The role of IL-35 in type 1 diabetes (T1D) remains to be answered. To elucidate this, we investigated the kinetics of Treg cell response in the multiple low dose streptozotocin induced (MLDSTZ) T1D model and measured the levels of IL-35 in human T1D patients. We found that Treg cells were increased in MLDSTZ mice. However, the Treg cells showed a decreased production of anti-inflammatory (IL-10, IL-35, TGF-β) and increased pro-inflammatory (IFN-γ, IL-2, IL-17) cytokines, indicating a phenotypic shift of Treg cells under T1D condition. IL-35 administration effectively both prevented development of, and counteracted established MLDSTZ T1D, seemingly by induction of Eos expression and IL-35 production in Treg cells, thus reversing the phenotypic shift of the Treg cells. IL-35 administration reversed established hyperglycemia in NOD mouse model of T1D. Moreover, circulating IL-35 levels were decreased in human T1D patients compared to healthy controls. These findings suggest that insufficient IL-35 levels play a pivotal role in the development of T1D and that treatment with IL-35 should be investigated in treatment of T1D and other autoimmune diseases.

Topics: Animals; Cytokines; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Forkhead Transcription Factors; Humans; Hyperglycemia; Interleukin-10; Interleukin-2; Interleukins; Leukocytes, Mononuclear; Male; Mice; Mice, Inbred NOD; Minor Histocompatibility Antigens; Phenotype; Receptors, Cytokine; Recombinant Proteins; Spleen; Streptozocin; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Thymus Gland; Transforming Growth Factor beta

To investigate the therapeutic effects and potential mechanisms of icariside II (ICA II) on reversing diabetic nephropathy in streptozotocin (STZ)-induced type I diabetic rats.. Newborn male Sprague Dawley rats were labeled with thymidine analog 5-ethynyl-2-deoxyuridine (EdU) for tracking endogenous label retaining progenitor cells (LRCs). At age of 8 weeks, 48 rats were randomly divided into three groups: normal control group (n=16), diabetes mellitus group (DM; n=16), and diabetes mellitus plus ICA II therapy group (DM+ICA II, n=16). Eight weeks induced for diabetes with STZ, rats in DM group and DM+ICA II group were treated with vehicle or ICA II (5 mg/kg/day) for another 8 weeks, respectively. Then, blood creatinine, 24-hour urine protein, blood urea nitrogen, and glycosylated hemoglobin were measured, as well as the expression of von Willebrand factor, malondialdehyde, transforming growth factor-β/drosophila mothers against decapentaplegic protein/connective tissue growth factor (TGF-β/Smad/CTGF) signaling, marker of proliferation Ki-67, and EdU+ LRCs in renal tissues.. Increased levels of creatinine, 24-hour urine protein, and blood urea nitrogen and remarkably decreased proportion of normal glomeruli and increased proportions of I, IIa, IIb, and III glomeruli were observed in diabetic rats, while ICA II could reverse these changes. Interestingly, ICA II could significantly downregulate the levels of malondialdehyde and TGF-β/Smad/CTGF signaling and increase the expression of von Willebrand factor, Ki-67, and EdU+ LRCs in the kidney.. ICA II treatment could ameliorate diabetic nephropathy in STZ-induced diabetic rats by increasing endothelial cell contents, downregulating TGF-β/Smad/CTGF signaling pathway and oxidative stress level, and promoting cell proliferation both in kidney cortex and medulla. These beneficial effects appear to be mediated by its antioxidant capacity and recruitment of endogenous EdU+ progenitor cells into the kidney tissue.

Topics: Animals; Biomarkers; Blood Urea Nitrogen; Cell Proliferation; Cell Tracking; Connective Tissue Growth Factor; Creatinine; Cytoprotection; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Endothelial Progenitor Cells; Flavonoids; Glycated Hemoglobin; Kidney; Male; Oxidative Stress; Proteinuria; Rats, Sprague-Dawley; Signal Transduction; Smad Proteins; Time Factors; Transforming Growth Factor beta

Impaired wound healing is considered to be one of the most serious complications associated with diabetes as it significantly increases the susceptibility of patients to infection. Propolis is a natural bee product used extensively in foods and beverages that has significant benefits to human health. In particular, propolis has antioxidant, anti-inflammatory and analgesic effects that could be useful for improving wound healing. In this study, we investigated the effects of topical application of propolis on the healing and closure of diabetic wounds in a streptozotocin (STZ)-induced type I diabetic mouse model.. Sixty male mice were distributed equally into 3 experimental groups: group 1, non-diabetic control mice; group 2, diabetic mice; and group 3, diabetic mice treated daily with a topical application of propolis.. We found that diabetic mice exhibited delayed wound closure characterized by a significant decrease in the levels of TGF-β1 and a prolonged elevation of the levels of inflammatory cytokines (IL-1β, IL-6 and TNF-α) and MMP9 in wound tissues compared with control non-diabetic mice. Moreover, the wound tissues of diabetic mice showed a marked reduction in the phosphorylation of Smad2 and Smad3 as well as a marked reduction in collagen production. Interestingly, compared with untreated diabetic mice, topical application of propolis significantly enhanced the closure of diabetic wounds and decreased the levels of IL-1β, IL-6, TNF-α and MMP9 to near normal levels. Most importantly, compared with untreated diabetic mice, the treatment of diabetic mice with propolis significantly enhanced the production of collagen via the TGF-β1/Smad2,3 signaling axis in wounded tissues.. Our findings reveal the molecular mechanisms underlying the improved healing and closure of diabetic wounds following topical propolis application.

Topics: Administration, Topical; Animals; Collagen; Cytokines; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Drug Administration Schedule; Gene Expression Regulation; Humans; Male; Matrix Metalloproteinase 9; Mice; Propolis; Smad Proteins; Streptozocin; Transforming Growth Factor beta; Wound Healing

βig-h3/TGF-βi is a secreted protein capable of binding to both extracellular matrix and cells. Human genetic studies recently revealed that in the tgfbi gene encoding for βig-h3, three single nucleotide polymorphisms were significantly associated with type 1 diabetes (T1D) risk. Pancreatic islets express βig-h3 in physiological conditions, but this expression is reduced in β-cell insult in T1D. Since the integrity of islets is destroyed by autoimmune T lymphocytes, we thought to investigate the impact of βig-h3 on T-cell activation. We show here that βig-h3 inhibits T-cell activation markers as well as cytotoxic molecule production as granzyme B and IFN-γ. Furthermore, βig-h3 inhibits early T-cell receptor signaling by repressing the activation of the early kinase protein Lck. Moreover, βig-h3-treated T cells are unable to induce T1D upon transfer in Rag2 knockout mice. Our study demonstrates for the first time that T-cell activation is modulated by βig-h3, an islet extracellular protein, in order to efficiently avoid autoimmune response.

Topics: Animals; Autoimmunity; Biomarkers; Cadaver; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Extracellular Matrix Proteins; Female; Humans; Hypoglycemic Agents; Lymph Nodes; Lymphocyte Activation; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Protein Kinase Inhibitors; Receptors, Antigen, T-Cell; Recombinant Proteins; Signal Transduction; Specific Pathogen-Free Organisms; T-Lymphocytes; Transforming Growth Factor beta

CD26 is a T cell activation marker consisting in a type II transmembrane glycoprotein with dipeptidyl peptidase IV (DPPIV) activity in its extracellular domain. It has been described that DPPIV inhibition delays the onset of type 1 diabetes and reverses the disease in non-obese diabetic (NOD) mice. The aim of the present study was to assess the effect of MK626, a DPPIV inhibitor, in type 1 diabetes incidence and in T lymphocyte subsets at central and peripheral compartments. Pre-diabetic NOD mice were treated with MK626. Diabetes incidence, insulitis score, and phenotyping of T lymphocytes in the thymus, spleen and pancreatic lymph nodes were determined after 4 and 6 weeks of treatment, as well as alterations in the expression of genes encoding β-cell autoantigens in the islets. The effect of MK626 was also assessed in two in vitro assays to determine proliferative and immunosuppressive effects. Results show that MK626 treatment reduces type 1 diabetes incidence and after 6 weeks of treatment reduces insulitis. No differences were observed in the percentage of T lymphocyte subsets from central and peripheral compartments between treated and control mice. MK626 increased the expression of CD26 in CD8+ T effector memory (TEM) from spleen and pancreatic lymph nodes and in CD8+ T cells from islet infiltration. CD8+TEM cells showed an increased proliferation rate and cytokine secretion in the presence of MK626. Moreover, the combination of CD8+ TEM cells and MK626 induces an immunosuppressive response. In conclusion, treatment with the DPPIV inhibitor MK626 prevents experimental type 1 diabetes in association to increase expression of CD26 in the CD8+ TEM lymphocyte subset. In vitro assays suggest an immunoregulatory role of CD8+ TEM cells that may be involved in the protection against autoimmunity to β pancreatic islets associated to DPPIV inhibitor treatment.

Topics: Animals; Autoantigens; CD8-Positive T-Lymphocytes; Diabetes Mellitus, Type 1; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Islets of Langerhans; Lymphocyte Activation; Mice; Mice, Inbred NOD; Sitagliptin Phosphate; Transforming Growth Factor beta

Fibroblast growth factor 21 (FGF21) is an important regulator in glucose and lipid metabolism, and has been considered as a potential therapy for diabetes. The effect of FGF21 on the development and progression of diabetes-induced pathogenic changes in the aorta has not currently been addressed. To characterize these effects, type 1 diabetes was induced in both FGF21 knockout (FGF21KO) and C57BL/6 J wild type (WT) mice via multiple-dose streptozotocin injection. FGF21KO diabetic mice showed both earlier and more severe aortic remodeling indicated by aortic thickening, collagen accumulation and fibrotic mediator connective tissue growth factor expression. This was accompanied by significant aortic cell apoptosis than in WT diabetic mice. Further investigation found that FGF21 deletion exacerbated aortic inflammation and oxidative stress reflected by elevated expression of tumor necrosis factor α and transforming growth factor β, and the accumulation of 3-nitrotyrocine and 4-Hydroxynonenal. FGF21 administration can reverse the pathologic changes in FGF21KO diabetic mice. These findings demonstrate that FGF21 deletion aggravates aortic remodeling and cell death probably via exacerbation of aortic inflammation and oxidative stress. This marks FGF21 as a potential therapy for the treatment of aortic damage due to diabetes.

Topics: Aldehydes; Animals; Aorta; Aortic Diseases; Apoptosis; Collagen; Connective Tissue Growth Factor; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Angiopathies; Fibroblast Growth Factors; Fibrosis; Gene Deletion; Genetic Predisposition to Disease; Male; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase Type III; Oxidative Stress; Phenotype; Signal Transduction; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Tyrosine; Vascular Remodeling

There are contradictory results about the effect of angiotensin-converting enzyme inhibitors (ACEIs) on bone. This study was performed to address the skeletal renin-angiotensin system (RAS) activity and the effects of the ACEI, captopril, on the bone of streptozotocin-induced type 1 diabetic mice. Histochemical assessment on bone paraffin sections was conducted by Safranin O staining and tartrate-resistant acid phosphatase staining. Micro-computed tomography was performed to analyze bone biological parameters. Gene and protein expression were determined by real-time polymerase chain reaction and immunoblotting, respectively. Type 1 diabetic mice displayed osteopenia phenotype and captopril treatment showed no osteoprotective effects in diabetic mice as shown by the reduction of bone mineral density, trabecular thickness and bone volume/total volume. The mRNA expression of ACE and renin receptor, and the protein expression of renin and angiotensin II were markedly up-regulated in the bone of vehicle-treated diabetic mice compared to those of non-diabetic mice, and these molecular changes of skeletal RAS components were effectively inhibited by treatment with captopril. However, treatment with captopril significantly elevated serum tartrate-resistant acid phosphatase 5b levels, reduced the ratio of osteoprotegerin/receptor activator of nuclear factor-κB ligand expression, increased carbonic anhydrase II mRNA expression and the number of matured osteoclasts and decreased transforming growth factor-β and osteocalcin mRNA expression in the tibia compared to those of diabetic mice. The present study demonstrated that the use of the ACEI, captopril, has no beneficial effect on the skeletal biological properties of diabetic mice. However, this could be attributed, at least partially, to its suppression of osteogenesis and stimulation of osteoclastogenesis, even though it could effectively inhibit high activity of local RAS in the bone of diabetic mice.

Topics: Acid Phosphatase; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Bone and Bones; Bone Density; Bone Diseases, Metabolic; Captopril; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Isoenzymes; Male; Mice; Mice, Inbred DBA; NF-kappa B; Osteocalcin; Osteoclasts; Random Allocation; Renin; Renin-Angiotensin System; RNA, Messenger; Streptozocin; Tartrate-Resistant Acid Phosphatase; Tibia; Transforming Growth Factor beta; Up-Regulation

Infections with helminth parasites prevent/attenuate auto-inflammatory disease. Here we show that molecules secreted by a helminth parasite could prevent Type 1 Diabetes (T1D) in nonobese diabetic (NOD) mice. When delivered at 4 weeks of age (coincident with the initiation of autoimmunity), the excretory/secretory products of Fasciola hepatica (FhES) prevented the onset of T1D, with 84% of mice remaining normoglycaemic and insulitis-free at 30 weeks of age. Disease protection was associated with suppression of IFN-γ secretion from autoreactive T cells and a switch to the production of a regulatory isotype (from IgG2a to IgG1) of autoantibody. Following FhES injection, peritoneal macrophages converted to a regulatory M2 phenotype, characterised by increased expression levels of Ym1, Arg-1, TGFβ and PD-L1. Expression of these M2 genetic markers increased in the pancreatic lymph nodes and the pancreas of FhES-treated mice. In vitro, FhES-stimulated M2 macrophages induced the differentiation of Tregs from splenocytes isolated from naïve NOD mice. Collectively, our data shows that FhES contains immune-modulatory molecules that mediate protection from autoimmune diabetes via the induction and maintenance of a regulatory immune environment.

Topics: Animals; Autoantibodies; Autoimmunity; B7-H1 Antigen; beta-N-Acetylhexosaminidases; Cell Differentiation; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Fasciola hepatica; Female; Helminths; Interferon-gamma; Lectins; Lymph Nodes; Macrophages, Peritoneal; Mice; Mice, Inbred NOD; Pancreas; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Type 1 diabetes (T1D) is a metabolic disease that is initiated by the autoimmune destruction of pancreatic insulin-producing beta cells that is accompanied by the development of antigen-specific antibodies and cytotoxic T lymphocytes (CTLs). Several studies have shown that vaccination with diabetic autoantigens provides some protection against this process. In this report we describe a new oral vaccine that utilizes live attenuated Salmonella for simultaneous delivery of autoantigens in conjunction with immunomodulatory cytokine genes to immune cells in the gut mucosa. Recent data showed that live attenuated Salmonella is a safe, simple and effective vector for expression of antigens and cytokines by antigen-presenting cells (APCs) of gut-associated lymphatic tissue (GALT). This novel strategy was tested by fusion of the diabetic autoantigen preproinsulin with Salmonella secretory effector protein (SseF) of pathogenicity island-2 (SPI2). In this way the autoantigen is only expressed inside the host immune cells and translocated to the host cell cytosol. In addition Salmonella was used to deliver the gene for the immunomodulatory cytokine transforming growth factor beta (TGFβ) for host cell expression. Oral co-vaccination of 8 week-old non-obese diabetic (NOD) mice with three weekly doses of both the autoantigen and cytokine significantly reduced the development of diabetes, improved the response to glucose challenge, preserved beta cell mass, and reduced the severity of insulitis compared with controls and autoantigen alone. Combination therapy also resulted in increased circulating levels of IL10 four weeks post-vaccination and IL2 for 12 weeks post-vaccination, but without effect on proinflammatory cytokines IL6, IL12(p70), IL17 and IFNγ. However, in non-responders there was a significant rise in IL12 compared with responders. Future studies will examine the mechanism of this vaccination strategy in more detail. In conclusion, Salmonella-based oral vaccines expressing autoantigens combined with imunomodulatory cytokines appears to be a promising therapy for prevention of T1D.

Topics: Administration, Oral; Animals; Autoantigens; Bacterial Proteins; Cell Line; Diabetes Mellitus, Type 1; Female; Genetic Vectors; Insulin; Interleukins; Membrane Proteins; Mice, Inbred NOD; Protein Precursors; Salmonella; Transforming Growth Factor beta; Vaccines

Transforming growth factor beta-induced (TGFBI/βIG-H3), also known as βig-H3, is a protein inducible by TGFβ1 and secreted by many cell types. It binds to collagen, forms part of the extracellular matrix and interacts with integrins on the cell surface. Recombinant TGFBI and transgenic TGFBI overexpression can promote both islet survival and function. In this study, we generated TGFBI KO mice and further assessed TGFBI function and signaling pathways in islets. Islets from KO mice were of normal size and quantity, and these animals were normoglycemic. However, KO islet survival and function was compromised in vitro. In vivo, KO donor islets became inferior to wild-type donor islets in achieving normoglycemia when transplanted into KO diabetic recipients. TGFBI KO mice were more prone to straptozotocin-induced diabetes than the wild-type counterpart. Phosphoprotein array analysis established that AKT1S1, a molecule linking the AKT and mTORC1 signaling pathways, was modulated by TGFBI in islets. Phosphorylation of four molecules in the AKT and mTORC1 signaling pathway, i.e. AKT, AKT1S1, RPS6 and EIF4EBP1, was upregulated in islets upon TGFBI stimulation. Suppression of AKT activity by a chemical inhibitor, or knockdown of AKT1S1, RPS6 and EIF4EBP1 expression by small interfering RNA, modulated islet survival, proving the relevance of these molecules in TGFBI-triggered signaling. Human genetic studies revealed that in the TGFBI gene and its vicinity, three single-nucleotide polymorphisms were significantly associated with type 1 diabetes risks, and one with type 2 diabetes risks. Our study suggests that TGFBI is a potential risk gene for human diabetes.

Topics: Animals; Diabetes Mellitus; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Extracellular Matrix Proteins; Genetic Predisposition to Disease; Humans; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphoproteins; Phosphorylation; Polymorphism, Single Nucleotide; Risk Factors; Signal Transduction; Tissue Survival; Transforming Growth Factor beta; Transforming Growth Factor beta1

Type 1 diabetes is recognized as an autoimmune inflammatory disease and low grade inflammation is also observed in type 2 diabetic patients. Interleukin 17 (IL-17) is a new player in inflammation. Th17 cells, as the main source of IL-17, require transforming growth factor β (TGF-β) and interleukin 23 (IL-23). The aim of this study was to investigate serum IL-17, IL-23 and TGF-β levels in diabetic patients and controls. In this case-control study, serum levels of IL-17, IL-23, and TGF-β were measured in 24 type 1 diabetic patients and 30 healthy controls using the ELISA method. Simultaneously, the same methodology was used to compare serum concentration of these three cytokines in 38 type 2 diabetic patients and 40 healthy controls. There was no significant difference between serum levels of IL-17 and IL-23 cytokines between cases and controls. However, TGF-β was significantly lower in type 1 diabetic patients (P < 0.001). Serum IL-17 and IL-23 levels demonstrate no association with type 1 and type 2 diabetes, but, in line with previous studies, TGF-β levels were lower in type 1 diabetic patients.

Topics: Adolescent; Adult; Case-Control Studies; Child; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Female; Healthy Volunteers; Humans; Interleukin-17; Interleukin-23; Male; Transforming Growth Factor beta

Gut microbiota (GM) and diet both appear to be important in the pathogenesis of type 1 diabetes. Fermentable fibres (FFs), of which there is an ample supply in natural, diabetes-promoting diets, are used by GM as a source of energy. Our aim was to determine whether FFs modify GM and diabetes incidence in the NOD mouse.. Female NOD mice were weaned to a semisynthetic diet and the effects of FF supplementation on diabetes incidence and insulitis were evaluated. Real-time quantitative PCR was employed to determine the effects imposed to gene transcripts in the colon and lymph nodes. Changes to GM were analysed by next-generation sequencing.. NOD mice fed semisynthetic diets free from FFs were largely protected from diabetes while semisynthetic diets supplemented with the FFs pectin and xylan (PX) resulted in higher diabetes incidence. Semisynthetic diet free from FFs altered GM composition significantly; addition of PX changed the composition of the GM towards that found in natural-diet-fed mice and increased production of FF-derived short-chain fatty acid metabolites in the colon. The highly diabetogenic natural diet was associated with expression of proinflammatory and stress-related genes in the colon, while the semisynthetic diet free from FFs promoted Il4, Il22, Tgfβ and Foxp3 transcripts in the colon and/or pancreatic lymph node. PX in the same diet counteracted these effects and promoted stress-related IL-18 activation in gut epithelial cells. 16S RNA sequencing revealed each diet to give rise to its particular GM composition, with different Firmicutes to Bacteroidetes ratios, and enrichment of mucin-degrading Ruminococcaceae following diabetes-protective FF-free diet.. FFs condition microbiota, affect colon homeostasis and are important components of natural, diabetes-promoting diets in NOD mice.

Topics: Animals; Colon; Diabetes Mellitus, Type 1; Female; Gastrointestinal Tract; Hepatocyte Nuclear Factor 3-gamma; Interleukin-18; Interleukin-22; Interleukin-4; Interleukins; Lymph Nodes; Mice; Mice, Inbred NOD; Microbiota; Pectins; Transforming Growth Factor beta; Xylans

TGF-β signaling in T cells is critical for peripheral T-cell tolerance by regulating effector CD4(+) T helper (Th) cell differentiation. However, it is still controversial to what extent TGF-β signaling in Foxp3(+) regulatory T (Treg) cells contributes to immune homeostasis. Here we showed that abrogation of TGF-β signaling in thymic T cells led to rapid type 1 diabetes (T1D) development in NOD mice transgenic for the BDC2.5 T-cell receptor. Disease development in these mice was associated with increased peripheral Th1 cells, whereas Th17 cells and Foxp3(+) Treg cells were reduced. Blocking of IFN-γ signaling alone completely suppressed diabetes development in these mice, indicating a critical role of Th1 cells in this model. Furthermore, deletion of TGF-β signaling in peripheral effector CD4(+) T cells, but not Treg cells, also resulted in rapid T1D development, suggesting that conventional CD4(+) T cells are the main targets of TGF-β to suppress T1D. TGF-β signaling was dispensable for Treg cell function, development, and maintenance, but excessive IFN-γ production due to the absence of TGF-β signaling in naive CD4(+) T cells indirectly caused dysregulated Treg cell homeostasis. We further showed that T cell-derived TGF-β1 was critical for suppression of Th1 cell differentiation and T1D development. These results indicate that autocrine/paracrine TGF-β signaling in diabetogenic CD4(+) T cells, but not Treg cells, is essential for controlling T1D development.

Topics: Animals; Cell Communication; Diabetes Mellitus, Type 1; DNA Primers; Flow Cytometry; Homeostasis; Interferon-gamma; Mice; Mice, Inbred NOD; Mice, Transgenic; Real-Time Polymerase Chain Reaction; Receptors, Antigen, T-Cell; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; T-Lymphocytes, Regulatory; Th1 Cells; Transforming Growth Factor beta

The dilute plasma cytokine milieu associated with type 1 diabetes (T1D), while difficult to measure directly, is sufficient to drive transcription in a bioassay that uses healthy leukocytes as reporters. Previously, we reported disease-associated, partially IL-1 dependent, transcriptional signatures in both T1D patients and the BioBreeding (BB) rat model. Here, we examine temporal signatures in congenic BBDR.lyp/lyp rats that develop spontaneous T1D, and BBDR rats where T1D progresses only after immunological perturbation in young animals. After weaning, the BBDR temporal signature showed early coincident induction of transcription related to innate inflammation as well as IL-10- and TGF-β-mediated regulation. BBDR plasma cytokine levels mirrored the signatures showing early inflammation, followed by induction of a regulated state that correlated with failure of virus to induce T1D in older rats. In contrast, the BBDR.lyp/lyp temporal signature exhibited asynchronous dynamics, with delayed induction of inflammatory transcription and later, weaker induction of regulatory transcription, consistent with their deficiency in regulatory T cells. Through longitudinal analyses of plasma-induced signatures in BB rats and a human T1D progressor, we have identified changes in immunoregulatory processes that attenuate a preexisting innate inflammatory state in BBDR rats, suggesting a mechanism underlying the decline in T1D susceptibility with age.

Topics: Age Factors; Animals; Diabetes Mellitus, Type 1; Disease Resistance; Humans; Interleukin-10; Parvovirus; Rats; Rats, Inbred Strains; Transcriptome; Transforming Growth Factor beta

NADPH oxidase (Nox) isoforms have been implicated in contributing to diabetic microvascular complications, but the functional role of individual isoforms in diabetic kidney are unclear. Nox2, in particular, is highly expressed in phagocytes and may play a key inflammatory role in diabetic kidney disease. To determine the role of Nox2, we evaluated kidney function and pathology in wild-type (WT; C57BL/6) and Nox2 knockout (KO) mice with type 1 diabetes. Diabetes was induced in male Nox2 KO and WT mice with a multiple low-dose streptozotocin protocol. Groups were studied for kidney disease after 8 and 20 wk of diabetes. Hyperglycemia and body weights were similar in WT and Nox2 KO diabetic mice. All functional and structural features of early and later stage diabetic kidney disease (albuminuria, mesangial matrix, tubulointerstitial disease, and gene expression of matrix and transforming growth factor-β) were similar in both diabetic groups compared with their respective nondiabetic groups, except for reduction of macrophage infiltration and monocyte chemoattractant protein-1 in the diabetic Nox2 KO mice. Systolic blood pressure by telemetry was surprisingly increased in Nox2 KO mice; however, the systolic blood pressure was reduced in the diabetic WT and Nox2 KO mice by tail-cuff. Interestingly, diabetic Nox2 KO mice had marked upregulation of renal Nox4 at both the glomerular and cortical levels. The present results demonstrate that lack of Nox2 does not protect against diabetic kidney disease in type 1 diabetes, despite a reduction in macrophage infiltration. The lack of renoprotection may be due to upregulation of renal Nox4.

Topics: Albuminuria; Animals; Blood Pressure; Chemokine CCL2; Collagen Type IV; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Fibronectins; Macrophages; Male; Membrane Glycoproteins; Mice; Mice, Knockout; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidases; p38 Mitogen-Activated Protein Kinases; Transforming Growth Factor beta; Up-Regulation

Leading hypotheses to explain helminth-mediated protection against autoimmunity postulate that type 2 or regulatory immune responses induced by helminth infections in the host limit pathogenic Th1-driven autoimmune responses. We tested these hypotheses by investigating whether infection with the filarial nematode Litomosoides sigmodontis prevents diabetes onset in IL-4-deficient NOD mice and whether depletion or absence of regulatory T cells, IL-10, or TGF-β alters helminth-mediated protection. In contrast to IL-4-competent NOD mice, IL-4-deficient NOD mice failed to develop a type 2 shift in either cytokine or Ab production during L. sigmodontis infection. Despite the absence of a type 2 immune shift, infection of IL-4-deficient NOD mice with L. sigmodontis prevented diabetes onset in all mice studied. Infections in immunocompetent and IL-4-deficient NOD mice were accompanied by increases in CD4(+)CD25(+)Foxp3(+) regulatory T cell frequencies and numbers, respectively, and helminth infection increased the proliferation of CD4(+)Foxp3(+) cells. However, depletion of CD25(+) cells in NOD mice or Foxp3(+) T cells from splenocytes transferred into NOD.scid mice did not decrease helminth-mediated protection against diabetes onset. Continuous depletion of the anti-inflammatory cytokine TGF-β, but not blockade of IL-10 signaling, prevented the beneficial effect of helminth infection on diabetes. Changes in Th17 responses did not seem to play an important role in helminth-mediated protection against autoimmunity, because helminth infection was not associated with a decreased Th17 immune response. This study demonstrates that L. sigmodontis-mediated protection against diabetes in NOD mice is not dependent on the induction of a type 2 immune shift but does require TGF-β.

Topics: Animals; Diabetes Mellitus, Type 1; Female; Filariasis; Filarioidea; Interleukin-10; Interleukin-4; Mice; Mice, 129 Strain; Mice, Inbred NOD; Mice, Knockout; Mice, Transgenic; T-Lymphocytes, Regulatory; Th1 Cells; Transforming Growth Factor beta

The protective effects of TGF-β have been documented in various autoimmune diseases, mostly in organ-specific autoimmunity including type 1 diabetes mellitus (T1DM). However, TGF-β also plays a role as a pro-inflammatory mediator by induction of Th17 cytokine production. IL-23 also plays a key role in differentiation of Th17 cells, which are implicated in pathogenesis of autoimmune conditions including T1DM. The aim of this study was to investigate and compare the difference in the level of TGF-β1 and IL-23 gene expression in unstimulated peripheral blood mononuclear cells (PBMCs) of patients with different forms of diabetes compared with normal healthy controls subjects. Patients with T1DM were grouped as early-onset T1DM (N = 20) with age at diagnosis <18 years and late-onset T1DM (N = 20) with the age at onset >18 years. Patients with T2DM (N = 20) and normal healthy controls (N = 20) were recruited from the same area. TGF-β1 and IL-23 gene expression in fresh unstimulated PBMCs was determined in each group using quantitative real-time PCR. The results confirmed that a significant difference in TGF-β1 and IL-23 gene expression was observed in both forms of juvenile-onset T1DM and adult-onset T1DM compared to the controls and T2DM patients. There was no significant difference for TGF-β gene expression in patients with T2DM and controls. We therefore conclude that our results support the previous data on TGF-β gene down-regulation in T1DM. Also up-regulation of IL-23 has been observed in T1DM whilst it was down-regulated in T2DM. We also found no significant difference between juvenile-onset and adult-onset T1DM indicating same mechanism might be involved in the pathogenesis of both types. More studies on different cytokines in Th17 pathways are required to further confirm our finding.

Topics: Adolescent; Adult; Age of Onset; Aged; Child; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Female; Gene Expression Regulation; Humans; Interleukin-23; Iran; Leukocytes, Mononuclear; Male; Middle Aged; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Young Adult

High levels of soluble cytotoxic T-lymphocyte antigen 4 (soluble CTLA-4), an alternative splice form of the regulatory T-cell (Treg) associated CTLA-4 gene, have been associated with type 1 diabetes (T1D) and other autoimmune diseases, such as Grave's disease and myasthenia gravis. At the same time, studies have shown soluble CTLA-4 to inhibit T-cell activation through B7 binding. This study aimed to investigate the role of soluble CTLA-4 in relation to full-length CTLA-4 and other Treg-associated markers in T1D children and in individuals with high or low risk of developing the disease.. T1D children were studied at 4 days, 1 and 2 years after diagnosis in comparison to individuals with high or low risk of developing the disease. Isolated peripheral blood mononuclear cells were stimulated with the T1D-associated glutamic acid decarboxylase 65 and phytohaemagglutinin. Subsequently, soluble CTLA-4, full-length CTLA-4, FOXP3 and TGF-β mRNA transcription were quantified and protein concentrations of soluble CTLA-4 were measured in culture supernatant and sera.. Low protein concentrations of circulating soluble CTLA-4 and a positive correlation between soluble CTLA-4 mRNA and protein were seen in T1D, in parallel with a negative correlation in healthy subjects. Further, low levels of mitogen-induced soluble CTLA-4 were accompanied by low C-peptide levels. Interestingly, low mitogen-induced soluble CTLA-4 mRNA and low TGF-β mRNA expression were seen in high risk individuals, suggesting an alteration in activation and down-regulating immune mechanisms during the pre-diabetic phase.

Topics: Adolescent; Autoimmunity; Biomarkers; Blood Cells; C-Peptide; Child; Child, Preschool; CTLA-4 Antigen; Diabetes Mellitus, Type 1; Female; Forkhead Transcription Factors; Gene Expression; Humans; Longitudinal Studies; Male; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes, Regulatory; Transcription, Genetic; Transforming Growth Factor beta

TGF-β1 upregulates microRNA-192 (miR-192) in cultured glomerular mesangial cells and in glomeruli from diabetic mice. miR-192 not only increases collagen expression by targeting the E-box repressors Zeb1/2 but also modulates other renal miRNAs, suggesting that it may be a therapeutic target for diabetic nephropathy. We evaluated the efficacy of a locked nucleic acid (LNA)-modified inhibitor of miR-192, designated LNA-anti-miR-192, in mouse models of diabetic nephropathy. LNA-anti-miR-192 significantly reduced levels of miR-192, but not miR-194, in kidneys of both normal and streptozotocin-induced diabetic mice. In the kidneys of diabetic mice, inhibition of miR-192 significantly increased Zeb1/2 and decreased gene expression of collagen, TGF-β, and fibronectin; immunostaining confirmed the downregulation of these mediators of renal fibrosis. Furthermore, LNA-anti-miR-192 attenuated proteinuria in these diabetic mice. In summary, the specific reduction of renal miR-192 decreases renal fibrosis and improves proteinuria, lending support for the possibility of an anti-miRNA-based translational approach to the treatment of diabetic nephropathy.

Topics: Albuminuria; Animals; Collagen; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disease Models, Animal; Fibronectins; Fibrosis; Homeodomain Proteins; Kidney; Kruppel-Like Transcription Factors; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; MicroRNAs; Oligonucleotides; Proteinuria; Repressor Proteins; Streptozocin; Transforming Growth Factor beta; Zinc Finger E-box Binding Homeobox 2; Zinc Finger E-box-Binding Homeobox 1

Clinical studies suggest metabolic memory to hyperglycemia. We tested whether diabetes leads to persistent systematic in vitro gene expression alterations in patients with type 1 diabetes (T1D) compared with their monozygotic, nondiabetic twins. Microarray gene expression was determined in skin fibroblasts (SFs) of five twin pairs cultured in high glucose (HG) for ∼6 weeks. The Exploratory Visual Analysis System tested group differences in gene expression levels within KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. An overabundance of differentially expressed genes was found in eight pathways: arachidonic acid metabolism (P = 0.003849), transforming growth factor-β signaling (P = 0.009167), glutathione metabolism (P = 0.01281), glycosylphosphatidylinositol anchor (P = 0.01949), adherens junction (P = 0.03134), dorsal-ventral axis formation (P = 0.03695), proteasome (P = 0.04327), and complement and coagulation cascade (P = 0.04666). Several genes involved in epigenetic mechanisms were also differentially expressed. All differentially expressed pathways and all the epigenetically relevant differentially expressed genes have previously been related to HG in vitro or to diabetes and its complications in animal and human studies. However, this is the first in vitro study demonstrating diabetes-relevant gene expression differences between T1D-discordant identical twins. These SF gene expression differences, persistent despite the HG in vitro conditions, likely reflect "metabolic memory", and discordant identical twins thus represent an excellent model for studying diabetic epigenetic processes in humans.

Topics: Adult; Arachidonic Acid; Capillary Permeability; Cells, Cultured; Diabetes Mellitus, Type 1; Diseases in Twins; Female; Fibroblasts; Gene Expression Profiling; Glutathione; Humans; Male; Middle Aged; Signal Transduction; Skin; Transforming Growth Factor beta; Twins, Monozygotic

Tregs play an important role in controlling immune responses, particularly autoimmunity. In NOD mouse model, an excellent model for autoimmune diabetes, transfer of Tregs was shown to prevent diabetes, whereas depletion of Tregs in vivo enhanced disease progression, suggesting that Treg dysfunction contributes to the pathogenesis of diabetes. However, the mechanisms leading to Treg dysfunction and their role in diabetes progression has remained unclear. In this study we assessed quantitative and qualitative changes in Tregs during the development of autoimmune diabetes in NOD. We compared female NOD with males that have similar predisposition to but a lower incidence of diabetes and found that Treg numbers remained unchanged between 6 to 16 weeks of age in both groups. Although female Tregs produced lower TGF-β compared to male, regulatory function of female Tregs was only marginally inferior to male upon GAD65 autoantigen stimulation. GAD65-reactive female Teffectors were more responsive and progressively became refractory to regulation compared to male effectors, in part due to lower expression of TGF-β RII, accounting for reduced sensitivity to Tregs. Moreover, we unexpectedly found that TGF-β suppressed IFN-γ production to GAD65 antigen in male, not in female responders. These data suggest that TGF-β plays a major role in Teff resistance to regulation and Treg dysfunction, and may account for autoimmune diabetes. Our study implies that development of a successful supplemental Treg therapy for halting autoimmunity may require further understanding of Teff responses to regulation in order to generate highly effective Tregs.

Topics: Animals; Autoantigens; Autoimmunity; Cells, Cultured; Diabetes Mellitus, Type 1; Disease Progression; Female; Glutamate Decarboxylase; Interferon-gamma; Male; Mice; Mice, Inbred NOD; Receptors, Transforming Growth Factor beta; T-Lymphocytes, Regulatory; Th1 Cells; Transforming Growth Factor beta

Regulatory T (Treg) cells, driven by the Foxp3 transcription factor, are responsible for limiting autoimmunity and chronic inflammation. We showed that a well-characterized Foxp3(gfp) reporter mouse, which expresses an N-terminal GFP-Foxp3 fusion protein, is a hypomorph that causes profoundly accelerated autoimmune diabetes on a NOD background. Although natural Treg cell development and in vitro function are not markedly altered in Foxp3(gfp) NOD and C57BL/6 mice, Treg cell function in inflammatory environments was perturbed and TGF-β-induced Treg cell development was reduced. Foxp3(gfp) was unable to interact with the histone acetyltransferase Tip60, the histone deacetylase HDAC7, and the Ikaros family zinc finger 4, Eos, which led to reduced Foxp3 acetylation and enhanced K48-linked polyubiquitylation. Collectively this results in an altered transcriptional landscape and reduced Foxp3-mediated gene repression, notably at the hallmark IL-2 promoter. Loss of controlled Foxp3-driven epigenetic modification leads to Treg cell insufficiency that enables autoimmunity in susceptible environments.

Topics: Acetylation; Animals; Autoimmunity; Carrier Proteins; Diabetes Mellitus, Type 1; DNA-Binding Proteins; Epigenesis, Genetic; Forkhead Transcription Factors; Histone Acetyltransferases; Histone Deacetylases; Interleukin-2; Lysine Acetyltransferase 5; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Nerve Tissue Proteins; Promoter Regions, Genetic; T-Lymphocytes, Regulatory; Trans-Activators; Transcription Factors; Transforming Growth Factor beta

MicroRNAs (miRNAs) are crucial for regulatory T cell (Treg) stability and function. We report that microRNA-10a (miR-10a) is expressed in Tregs but not in other T cells including individual thymocyte subsets. Expression profiling in inbred mouse strains demonstrated that non-obese diabetic (NOD) mice with a genetic susceptibility for autoimmune diabetes have lower Treg-specific miR-10a expression than C57BL/6J autoimmune resistant mice. Inhibition of miR-10a expression in vitro leads to reduced FoxP3 expression levels and miR-10a expression is lower in unstable "exFoxP3" T cells. Unstable in vitro TGF-ß-induced, iTregs do not express miR-10a unless cultured in the presence of retinoic acid (RA) which has been associated with increased stability of iTreg, suggesting that miR-10a might play a role in stabilizing Treg. However, genetic ablation of miR-10a neither affected the number and phenotype of natural Treg nor the capacity of conventional T cells to induce FoxP3 in response to TGFβ, RA, or a combination of the two. Thus, miR-10a is selectively expressed in Treg but inhibition by antagomiRs or genetic ablation resulted in discordant effects on FoxP3.

Topics: Animals; Cells, Cultured; Diabetes Mellitus, Type 1; Gene Expression; Mice; Mice, Inbred NOD; MicroRNAs; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tretinoin

The highest annual increase in the incidence of type 1 diabetes (T1D) in children under the age of 5 years and aggressive process of β-cell destruction in this age group indicate the need to assess the immune system. The aim of this study was to evaluate regulatory T cells (Tregs) frequency in the peripheral blood of children <5 years of age with newly diagnosed T1D in comparison with diabetic children diagnosed at a later age and healthy controls. 40 children with newly diagnosed T1D (20 children <5 years of age and 20 older patients) and 40 age-matched controls were included in this study. Flow cytometric analysis of Tregs was performed using the following markers: CD4, CD25, CD127, FoxP3, IL-10, and TGF-β. Apoptosis was measured using anti-active caspase 3 monoclonal antibody. Fasting C-peptide and HbA1c were monitored as well. We showed that T1D children <5 years had lower C-peptide concentration than diabetic children ≥5 years of age (0.32 vs. 0.80 ng/ml, respectively, p = 0.0005). There was lower frequency of CD4(+)CD25(high)CD127(low)FoxP3(+) Tregs in T1D children <5 years than ≥5 years of age (0.87 vs. 1.56 %, respectively, p = 0.017). Diabetic children <5 years had lower CD4(+)CD25(high)CD127(low)FoxP3(+), CD4(+)CD25(high)IL-10, and CD4(+)CD25(high)TGF-β Tregs compared to age-matched controls. There was no difference in Tregs apoptosis between the examined groups. This study highlights the distinctiveness of diabetes in children <5 years of age. Understanding the differences of immune system activity in the young diabetic children would open the way to identify children at risk for T1D and enables the use of novel forms of intervention.

Topics: Adolescent; Antigens, CD; C-Peptide; Cell Separation; Child; Child, Preschool; Diabetes Mellitus, Type 1; Female; Flow Cytometry; Forkhead Transcription Factors; Glycated Hemoglobin; Humans; Infant; Interleukin-10; Male; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Continuous diabetes-associated complications are a major source of immune system exhaustion and an increased incidence of infection. Diabetes can cause poor circulation in the feet, increasing the likelihood of ulcers forming when the skin is damaged and slowing the healing of the ulcers. Whey proteins (WPs) enhance immunity during childhood and have a protective effect on some immune disorders. Therefore, in this study, we investigated the effects of camel WP on the healing and closure of diabetic wounds in a streptozotocin (STZ)-induced type I diabetic mouse model.. Diabetic mice exhibited delayed wound closure characterized by a significant decrease in an anti-inflammatory cytokine (namely, IL-10) and a prolonged elevation of the levels of inflammatory cytokines (TNF-α, IL-1β and IL-6) in wound tissue. Moreover, aberrant expression of chemokines that regulate wound healing (MIP-1α, MIP-2, KC and CX3CL1) and growth factors (TGF-β) were observed in the wound tissue of diabetic mice compared with control nondiabetic mice. Interestingly, compared with untreated diabetic mice, supplementation with WP significantly accelerated the closure of diabetic wounds by limiting inflammatory stimuli via the restoration of normal IL-10, TNF-α, IL-1β and IL-6 levels. Most importantly, the supplementation of diabetic mice with WP significantly modulated the expression of MIP-1α, MIP-2, KC, CX3CL1 and TGF-β in wound tissue compared with untreated diabetic mice.. Our data demonstrate the benefits of WP supplementation for improving the healing and closure of diabetic wounds and restoring the immune response in diabetic mice.

Topics: Animals; Biomarkers; Chemokine CCL3; Chemokine CX3CL1; Chemokine CXCL1; Chemokine CXCL2; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Dietary Supplements; Male; Mice; Milk Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Treatment Outcome; Whey Proteins; Wound Healing

Regulatory T cells (Tregs) are critical for the peripheral maintenance of the autoreactive T cells in autoimmune disorders such as type 1 diabetes (T1D). Pharmacological inhibition of Janus tyrosine kinase 3 (JAK3) has been proposed as a basis for new treatment modalities against autoimmunity and allogeneic responses. Targeting JAK3 with an inhibitor has previously been shown to exhibit protective action against the development of T1D in non-obese diabetic (NOD) mice. As the mechanism of such preventative action has been unknown, we hypothesized that JAK3 inhibition induces generation of Tregs. Here, we show that the JAK3 inhibitor 4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131) suppresses proliferation of short-term cultured NOD CD4(+) T cells through induction of apoptosis, while promoting survival of a particular population of long-term cultured cells. It was found that the surviving cells were not of the CD4(+)CD25(+)FoxP3(+) phenotype. They secreted decreased amounts of IL-10, IL-4 and interferon (IFN)-γ compared to the cells not exposed to the optimal concentrations of JAK3 inhibitor. However, an elevated transforming growth factor (TGF)-β secretion was detected in their supernatants. In vivo treatment of prediabetic NOD mice with WHI-P131 did not affect the frequency and number of splenic and pancreatic lymph node CD4(+)FoxP3(+) Tregs, while generating an elevated numbers of CD4(+)FoxP3(-) TGF-β-secreting T cells. In conclusion, our data suggest an induction of TGF-β-secreting CD4(+) T cells as the underlying mechanism for antidiabetogenic effects obtained by the treatment with a JAK3 inhibitor. To our knowledge, this is the first report of the JAK3 inhibitor activity in the context of the murine Tregs.

Topics: Animals; Apoptosis; CD4 Lymphocyte Count; Cells, Cultured; Cytoprotection; Diabetes Mellitus, Type 1; Female; Humans; Janus Kinase 3; Mice; Mice, Inbred NOD; Quinazolines; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Up-Regulation

Macrophages are multifunctional immune cells that may either drive or modulate disease pathogenesis depending on their activation phenotype. Autoimmune type 1 diabetes (T1D) is a chronic proinflammatory condition characterized by unresolved destruction of pancreatic islets. Adoptive cell transfer of macrophages with immunosuppressive properties represents a novel immunotherapy for treatment of such chronic autoimmune diseases. We used a panel of cytokines and other stimuli to discern the most effective regimen for in vitro induction of immunosuppressive macrophages (M2r) and determined interleukin (IL)-4/IL-10/transforming growth factor-β (TGF-β) to be optimal. M2r cells expressed programmed cell death 1 ligand-2, fragment crystallizable region γ receptor IIb, IL-10, and TGF-β, had a potent deactivating effect on proinflammatory lipopolysaccharide/interferon-γ-stimulated macrophages, and significantly suppressed T-cell proliferation. Clinical therapeutic efficacy was assessed after adoptive transfer in NOD T1D mice, and after a single transfer of M2r macrophages, >80% of treated NOD mice were protected against T1D for at least 3 months, even when transfer was conducted just prior to clinical onset. Fluorescent imaging analyses revealed that adoptively transferred M2r macrophages specifically homed to the inflamed pancreas, promoting β-cell survival. We suggest that M2r macrophage therapy represents a novel intervention that stops ongoing autoimmune T1D and may have relevance in a clinical setting.

Topics: Animals; Cell Proliferation; Cell Tracking; Cells, Cultured; Diabetes Mellitus, Type 1; Female; Gene Expression Regulation; Immunotherapy, Adoptive; Insulin-Secreting Cells; Interferon-gamma Release Tests; Interleukin-10; Interleukin-4; Lymph Nodes; Macrophage Activation; Macrophages; Mice; Mice, Inbred NOD; Mice, Transgenic; Pancreas; Prediabetic State; RNA, Messenger; Transforming Growth Factor beta

The cartography of β-cell epitopes targeted by CD8(+) T cells in type 1 diabetic (T1D) patients remains largely confined to the common HLA-A2 restriction. We aimed to identify β-cell epitopes restricted by the HLA-B7 (B*07:02) molecule, which is associated with mild T1D protection. Using DNA immunization on HLA-B7-transgenic mice and prediction algorithms, we identified GAD and preproinsulin candidate epitopes. Interferon-γ (IFN-γ) enzyme-linked immunospot assays on peripheral blood mononuclear cells showed that most candidates were recognized by new-onset T1D patients, but not by type 2 diabetic and healthy subjects. Some epitopes were highly immunodominant and specific to either T1D children (GAD(530-538); 44% T cell-positive patients) or adults (GAD(311-320); 38%). All epitopes displayed weak binding affinity and stability for HLA-B7 compared with HLA-A2-restricted ones, a general feature of HLA-B7. Single-cell PCR analysis on β-cell-specific (HLA-B7 tetramer-positive) T cells revealed uniform IFN-γ and transforming growth factor-β (TGF-β) mRNA expression, different from HLA-A2-restricted T cells. We conclude that HLA-B7-restricted islet epitopes display weak HLA-binding profiles, are different in T1D children and adults, and are recognized by IFN-γ(+)TGF-β(+)CD8(+) T cells. These features may explain the T1D-protective effect of HLA-B7. The novel epitopes identified should find valuable applications for immune staging of HLA-B7(+) individuals.

Topics: Adolescent; Adult; Aged; Animals; CD8-Positive T-Lymphocytes; Child; Child, Preschool; Diabetes Mellitus, Type 1; Epitopes; Female; HLA-B7 Antigen; Humans; Insulin-Secreting Cells; Interferon-gamma; Leukocytes, Mononuclear; Male; Mice; Middle Aged; Transforming Growth Factor beta

Transforming growth factor (TGF-β1) is a pleiotropic cytokine, secreted by immune and nonhematopoietic cells. TGF-β is involved in many different critical processes, such as embryonal development, cellular maturation and differentiation, wound healing, and immune regulation. It maintains immune homeostasis by acting as a potent immune suppressor through inhibition of proliferation, differentiation, activation, and effector function of immune cells. Paradoxically, depending on the context, it displays proinflammatory properties by being a potent chemoattractant for neutrophils and promoting inflammation. In addition, it does not only induce differentiation into the anti-inflammatory Treg cells, but also into the proinflammatory Th17 and Th9 cells and inhibits Th22 differentiation. TGF-β has been demonstrated to be involved in multiple pathologies. In infections, it protects against collateral damages caused by the immune system, but it also promotes immune evasion and chronic infections. In autoimmune diseases, a TGF-β dysfunction leads to the loss of tolerance to self-antigens. In cancer, TGF-β is a potent inhibitor of cell proliferation and acts as a tumor suppressor at the beginning of tumorogenesis. However, once the cells become resistant to TGF-β, it mainly supports tumor growth and metastasis by promoting immune evasion and angiogenesis. In asthma, it is assumed to promote allergen tolerance, but plays a detrimental role in irreversible remodeling of the airways. Despite the high numbers of TGF-β-targeted pathways, it is a promising drug target for treatment of autoimmunity, cancer, fibrosis, if cell specificity can be achieved.This review summarizes the progresses that have been accomplished on the understanding of TGF-β's signaling in the immune homeostasis and its role in pathogenesis.

Topics: Arthritis, Rheumatoid; Base Sequence; CD8 Antigens; Cells, Cultured; Cytokines; Diabetes Mellitus, Type 1; Forkhead Transcription Factors; Gene Expression Regulation; Humans; Immune Tolerance; Interleukin-2; Interleukin-4; Malaria; Molecular Sequence Data; Multiple Sclerosis; Promoter Regions, Genetic; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

Effective therapies that prevent chronic inflammation from developing into type 1 diabetes remain elusive. In this study, we show that expression of TGF-β for just 1 wk in inflamed islets of NOD mice significantly delays diabetes development. Time course studies demonstrated that the brief TGF-β pulse protects only if administered when extensive β cell destruction has occurred. Surprisingly, TGF-β-mediated protection is not linked to enhanced Foxp3(+) regulatory T cell activity or to decreased intrapancreatic presentation of islet Ags. Instead, TGF-β disables the transition of primed autoreactive CD8(+) T cells to cytotoxic effectors and decreases generation, or maintenance, of CD8(+) memory T cells within the pancreas, significantly impairing their diabetogenic capacity.

Topics: Animals; Cell Survival; Cytotoxicity Tests, Immunologic; Diabetes Mellitus, Type 1; Disease Models, Animal; Disease Progression; Epitopes, T-Lymphocyte; Forkhead Transcription Factors; Immunologic Memory; Inflammation Mediators; Islets of Langerhans; Mice; Mice, Inbred NOD; Mice, SCID; Mice, Transgenic; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta

Type 1 diabetes (T1D) is an autoimmune disease resulting from T cell-mediated destruction of insulin-producing β cells, and viral infections can prevent the onset of disease. Invariant natural killer T cells (iNKT cells) exert a regulatory role in T1D by inhibiting autoimmune T cell responses. As iNKT cell-plasmacytoid dendritic cell (pDC) cooperation controls viral replication in the pancreatic islets, we investigated whether this cellular cross talk could interfere with T1D development during viral infection. Using both virus-induced and spontaneous mouse models of T1D, we show that upon viral infection, iNKT cells induce TGF-β-producing pDCs in the pancreatic lymph nodes (LNs). These tolerogenic pDCs convert naive anti-islet T cells into Foxp3(+) CD4(+) regulatory T cells (T reg cells) in pancreatic LNs. T reg cells are then recruited into the pancreatic islets where they produce TGF-β, which dampens the activity of viral- and islet-specific CD8(+) T cells, thereby preventing T1D development in both T1D models. These findings reveal a crucial cooperation between iNKT cells, pDCs, and T reg cells for prevention of T1D by viral infection.

Topics: Animals; Antigens, Surface; Apoptosis Regulatory Proteins; B7-1 Antigen; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Cell Communication; Dendritic Cells; Diabetes Mellitus, Type 1; Interleukin-10; Islets of Langerhans; Lymph Nodes; Lymphocyte Activation; Membrane Glycoproteins; Mice; Natural Killer T-Cells; Peptides; Programmed Cell Death 1 Receptor; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Virus Diseases

Signaling through TLR2 promotes inflammation and modulates CD4(+) CD25(+) Tregs. We assessed mechanistically how this molecule would alter immunoregulation in type 1 diabetes (T1D). We also asked whether TLR2 may be involved in our recent discovery that viral infection can protect from autoimmune diabetes by expanding and invigorating Tregs. Treatment of prediabetic mice with a synthetic TLR2 agonist diminished T1D and increased the number and function of CD4(+) CD25(+) Tregs, also conferring DCs with tolerogenic properties. TLR2 ligation also promoted the expansion of Tregs upon culture with DCs and ameliorated their capacity to prevent the disease. Protection from T1D by lymphocytic choriomeningitis virus (LCMV) infection depended on TLR2. LCMV increased the frequency of CD4(+) CD25(+) Tregs and their production of TGF-β more significantly in WT than TLR2-deficient mice. Furthermore, LCMV infection in vivo or LCMV-infected DCs in vitro rendered, via TLR2, CD4(+) CD25(+) Tregs capable of diminishing T1D. We identify novel mechanisms by which TLR2 promotes immunoregulation and controls autoimmune diabetes in naïve or infected hosts. This work should help understand T1D etiology and develop novel immune-based therapeutic interventions.

Topics: Animals; Arenaviridae Infections; CD4 Antigens; Cells, Cultured; Dendritic Cells; Diabetes Mellitus, Type 1; Flow Cytometry; Immunity, Innate; Inflammation; Interleukin-2 Receptor alpha Subunit; Lymphocytic choriomeningitis virus; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Prediabetic State; Signal Transduction; T-Lymphocytes, Regulatory; Toll-Like Receptor 2; Transforming Growth Factor beta

The predominant transcription factors regulating key genes in diabetic kidney disease have not been established. The transcription factor upstream stimulatory factor 1 (USF1) is an important regulator of glucose-mediated transforming growth factor (TGF)-β1 expression in mesangial cells; however, its role in the development of diabetic kidney disease has not been evaluated. In the present study, wild-type (WT; USF1 +/+), heterozygous (USF1 +/-), and homozygous (USF1 -/-) knockout mice were intercrossed with Akita mice (Ins2/Akita) to induce type 1 diabetes. Mice were studied up to 36 wk of age. The degree of hyperglycemia and kidney hypertrophy were similar in all groups of diabetic mice; however, the USF1 -/- diabetic mice had significantly less albuminuria and mesangial matrix expansion than the WT diabetic mice. TGF-β1 and renin gene expression and protein were substantially increased in the WT diabetic mice but not in USF1 -/- diabetic mice. The underlying pathway by which USF1 is regulated by high glucose was investigated in mesangial cell culture. High glucose inhibited AMP-activated protein kinase (AMPK) activity and increased USF1 nuclear translocation. Activation of AMPK with AICAR stimulated AMPK activity and reduced nuclear accumulation of USF1. We thus conclude that USF1 is a critical transcription factor regulating diabetic kidney disease and plays a critical role in albuminuria, mesangial matrix accumulation, and TGF-β1 and renin stimulation in diabetic kidney disease. AMPK activity may play a key role in high glucose-induced regulation of USF1.

Topics: Albuminuria; Alleles; AMP-Activated Protein Kinases; Animals; Cell Line; Cell Nucleus; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disease Progression; Extracellular Matrix; Female; Hyperglycemia; Hypertrophy; Kidney; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Renin-Angiotensin System; RNA, Messenger; Transforming Growth Factor beta; Upstream Stimulatory Factors

The pre-ligand assembly domain (PLAD) of tumor necrosis factor receptors mediates specific ligand-independent receptor assembly and subsequent signaling. However, the physiological role of PLAD in the regulation of TNFR-mediated immune responses in autoimmunity is still unclear. By using the recombinant PLAD.Fc protein to block TNFR1 assembly, we demonstrated that PLAD.Fc treatment significantly reduced the TNFR1-driving proinflammatory cytokines and protected NOD mice from diabetes. Strikingly, Th17 differentiation was significantly inhibited in PLAD.Fc-treated NOD and TNFR1-deficient mice, indicating a TNFR1-dependent Th17 development. PLAD.Fc-modulated effects on DCs, in terms of the downregulation of Th17-inducing cytokines, IL-6 and TGF-β, explained the potential mechanism for Th17 suppression. Finally, we provided an additional result that PLAD.Fc administration diminished the infiltration of Th17 cells in the central nervous system and ameliorated the experimental autoimmune encephalomyelitis in mice. Collectively, these data demonstrated that targeting PLAD of TNFR1 provides protection from autoimmune diseases through the downregulation of Th17 and suggested a therapeutic potential of PLAD-modulation in TNF-involved inflammatory diseases.

Topics: Animals; Autoimmunity; Cell Differentiation; Cell Movement; Central Nervous System; Diabetes Mellitus, Type 1; Down-Regulation; Encephalomyelitis, Autoimmune, Experimental; Female; Humans; Interleukin-6; Jurkat Cells; Mice; Mice, Inbred NOD; Molecular Targeted Therapy; Plasmids; Protein Structure, Tertiary; Receptors, Tumor Necrosis Factor, Type I; Recombinant Fusion Proteins; Signal Transduction; Th17 Cells; Transfection; Transforming Growth Factor beta

Immunization with Schistosoma mansoni soluble antigen preparations protects non-obese diabetic (NOD) mice against the development of type 1 diabetes. These preparations have long been known to induce Th2 responses in vitro and in vivo. Recently, two separate groups have reported that ω-1, a well-characterized glycoprotein in S. mansoni soluble egg antigens (SEA), which with IL-4 inducing principle of S. mansoni eggs (IPSE/α-1) is one of the two major glycoproteins secreted by live eggs, is a major SEA component responsible for this effect. We found that ω-1 induces Foxp3 as well as IL-4 expression when injected in vivo. We confirmed that ω-1 conditions DCs to drive Th2 responses and further demonstrated that ω-1 induces Foxp3(+) T cells from NOD mouse naïve T cells. In contrast, IPSE/α-1 did not drive Foxp3 responses. The in vitro development of Foxp3-expressing T cells by ω-1 was TGF-β- and retinoic acid-dependent. Our work, therefore, identifies ω-1 as an important factor for the induction of Foxp3(+) T cells by SEA in NOD mice.

Topics: Animals; CD4-Positive T-Lymphocytes; Cell Differentiation; Cells, Cultured; Dendritic Cells; Diabetes Mellitus, Type 1; Egg Proteins; Forkhead Transcription Factors; Helminth Proteins; Immunization; Interleukin-4; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Schistosoma mansoni; Th2 Cells; Transforming Growth Factor beta; Tretinoin

In spite of intensive research the pathogenesis of type 1 diabetes mellitus is not thoroughly understood. One of the ideas which currently has a great number of supporters is the theory of the participation of T regulatory cells in the mechanism of insufficient suppression of the immune response against pancreatic self -antigens. According to some authors, the infusion of T regulatory cells in autoimmune diseases could lead to long -term remission or even a complete cure.. The aim of our present study was to achieve T regulatory cells from conventional T lymphocytes isolated from a small amount of peripheral blood in children with type 1 diabetes mellitus and their comparison to the Tregs generated from the blood of control children. Additionally, we assessed the changes in the expression of selected genes essential for the function of these cells during Tregs generation.. The examined group consisted of 20 children with type 1 diabetes mellitus, the control group consisted of 20 non -diabetic children. From the peripheral blood CD4+CD25 - cells were separated and cultured with T -reg expander and interleukin (IL) 2. Before and after the culture the cells were analysed according to the expression of transcription factor FoxP3 and other molecules/cytokines: OX40, 4 -1BB, GITR, ICOS -1, CTLA -4 and IFN -γ, IL -10 and TGF -β.. We observed a significantly higher percentage of T regulatory cells after the culture (with no difference between diabetic and control children). Moreover, we observed a lower expression of mRNA for GITR molecule and a higher IL -10 expression in the cultures of diabetic children compared to the control ones. The cells cultured from the blood of control children were characterised by a higher increase in the expression of mRNA for ICOS -1 and a lower expression of mRNA for TGF -β in comparison to the cultures from diabetic children.. The results of our investigations confirm the possibility of generating T regulatory cells from conventional T lymphocytes from peripheral blood of children with newly recognised type 1 diabetes mellitus.

Topics: Adolescent; Antigens, CD; Cells, Cultured; Child; Diabetes Mellitus, Type 1; Female; Glucocorticoid-Induced TNFR-Related Protein; Humans; Inducible T-Cell Co-Stimulator Ligand; Interleukin-10; Male; Pancreas; RNA, Messenger; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Type 1 diabetes (T1D) develops as a consequence of a progressive autoimmune response that destroys insulin-producing β-cells in pancreatic islets. Because of their role(s) in controlling immune responses, considerable effort has been directed toward resolving whether regulatory T cells (Tregs) offer a clinical treatment to restore tolerance in T1D. We previously reported that in vitro-induced adaptive Treg cells (aTregs) can reverse T1D and persist as protective memory cells in the NOD mouse model. In the current study, we investigated mechanisms that regulate aTregs. We found that these FoxP3(+) aTregs expressed high levels of the IL-7 receptor, IL-7Rα, without the high affinity receptor for IL-2, CD25, which is found on natural Treg cells (nTregs). IL-7Rα expression was mirrored by the dependency of aTregs on IL-7 for persistence. IL-10 and TGF-β, effector cytokines of aTregs, were not essential for their maintenance at the level of systemic antibody blocking. Nevertheless, IL-10 modulated cytokine production by aTregs and TGF-β was critical for protection. aTregs were found to infiltrate islets and the expression of integrin-β7 was required for their localization in the pancreas. Furthermore, blocking aTreg entry into the pancreas prevented their control of diabetogenic effector T cells, implying the need for local control of the autoimmune response. The distinct homeostatic regulation of aTregs independently of a response to IL-2, which is defective in T1D patients, suggests that these cells represent a translatable candidate to control the autoimmune response.

Topics: Adaptive Immunity; Adoptive Transfer; Animals; Autoimmunity; Cell Differentiation; Cell Movement; Diabetes Mellitus, Type 1; Female; Humans; Integrin beta Chains; Interleukin-10; Interleukin-2; Interleukin-2 Receptor alpha Subunit; Interleukin-7; Mice; Mice, Inbred NOD; Mice, Knockout; Pancreas; Receptors, Interleukin-7; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Recent studies have demonstrated that complement contributes to the development of autoimmune diabetes. However, the mechanisms remain unknown. Herein, using a model of streptozotocin (STZ)-induced diabetes, we found the presence of immune tolerance to self islet in complement C3-deficient mice after STZ. Higher number of CD4+CD25+ regulatory T cells (Tregs) with characteristics of expressing Foxp3 was observed in C3-/- mice. These C3-/- Tregs exhibited enhanced suppressive capacity to effector cell proliferation. The central role of Tregs was further evidenced by that depleting these cells using anti-CD25 antibody dramatically abrogated the preventive effects of C3 deficiency on STZ-induced diabetes. Importantly, transforming growth factor-β (TGF-β) was a key factor for Treg-mediated immune suppression as blocking TGF-β activity reversed suppressive capacity of Tregs in vitro and diabetes-resistant effects of C3 deficiency in vivo. These findings suggest that resistance to overt diabetes in STZ-treated C3-/- mice involves a population of Tregs in TGF-β-dependent manner.

Topics: Animals; Complement C3; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Forkhead Transcription Factors; Immune Tolerance; Interleukin-2 Receptor alpha Subunit; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Type 1 diabetes (T1D) is expected to cause significant changes in the serum proteome; however, few studies have systematically assessed the proteomic profile change associated with the disease. In this study, a semiquantitative spectral counting-based two dimensional liquid chromatography mass spectrometry platform was used to analyze serum samples from T1D patients and controls. In this discovery phase, significant differences were found for 21 serum proteins implicated in inflammation, oxidation, metabolic regulation, and autoimmunity. To assess the validity of these findings, six candidate proteins including adiponectin, insulin-like growth factor binding protein 2, serum amyloid protein A, C-reactive protein, myeloperoxidase, and transforming growth factor beta induced were selected for subsequent immune assays for 1139 T1D patients and 848 controls. A series of statistical analyses using cases and controls matched for age, sex, and genetic risk confirmed that T1D patients have significantly higher serum levels for four of the six proteins: adiponectin (odds ratio (OR) = 1.95, p = 10(-27)), insulin-like growth factor binding protein 2 (OR = 2.02, p < 10(-20)), C-reactive protein (OR = 1.13, p = 0.007), serum amyloid protein A (OR = 1.51, p < 10(-16)); whereas the serum levels were significantly lower in patients than controls for the two other proteins: transforming growth factor beta induced (OR = 0.74, p < 10(-5)) and myeloperoxidase (OR = 0.51, p < 10(-41)). Compared with subjects in the bottom quartile, subjects in the top quartile for adiponectin (OR = 6.29, p < 10(-37)), insulin-like growth factor binding protein 2 (OR = 7.95, p < 10(-46)), C-reactive protein (OR = 1.38, p = 0.025), serum amyloid protein A (OR = 3.36, p < 10(-16)) had the highest risk of T1D, whereas subjects in the top quartile of transforming growth factor beta induced (OR = 0.41, p < 10(-11)) and myeloperoxidase (OR = 0.10, p < 10(-43)) had the lowest risk of T1D. These findings provided valuable information on the proteomic changes in the sera of T1D patients.

Topics: Adiponectin; Adolescent; Adult; Age Factors; Aged; Aged, 80 and over; Biomarkers; C-Reactive Protein; Case-Control Studies; Child; Child, Preschool; Chromatography, Liquid; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix Proteins; Female; Humans; Infant; Infant, Newborn; Insulin-Like Growth Factor Binding Protein 2; Male; Middle Aged; Multivariate Analysis; Peroxidase; Serum Amyloid A Protein; Sex Factors; Tandem Mass Spectrometry; Transforming Growth Factor beta; Young Adult

Congenic NOD.ABH(D18Mit8-D18Mit214) mice, which contain greater than 12.8 Mb of DNA encompassing Idd21.1 from diabetes-resistant Biozzi/ABH mice, have a lower frequency of diabetes compared with the parental nonobese diabetic (NOD) strain, possibly due to reduced pathogenicity of β-islet-infiltrating immune cells.. The objective of the study was to identify an Idd21.1 candidate gene.. The methods used in the study were adoptive transfer into scid mice lacking an adaptive immune system; dendritic cell phenotyping and gene expression analysis; and fine-mapping Idd21.1 by congenic mapping.. Diabetes incidences of NOD.scid.ABH(D18Mit8-D18Mit214) mice receiving splenocytes from NOD and NOD.ABH(D18Mit8-D18Mit214) were similar to that previously observed in NOD.scid recipients, suggesting that the diabetes resistance in NOD.ABH(D18Mit8-D18Mit214) is primarily mediated by the adaptive immune system, findings supported by adoptive transfer of CD4(+) T cells. In activated dendritic cells, there were no conclusive differences in cytokine profiles and activation marker expression. However, microarray analysis comparing gene expression between activated dendritic cells from NOD and NOD.ABH (D18Mit8-D18Mit214) revealed that Smad2, in a maximal 6.5-Mb region to which Idd21.1 was further resolved by congenic mapping, was differentially expressed (increased in NOD). Quantitative real-time PCR confirmed the differential expression of Smad2, and other genes in the TGF-β signaling pathway, in activated dendritic cells.. These results implicate Smad2 as an Idd21.1 candidate and Smad2 and the TGF-β signaling pathway in activated dendritic cells in diabetogenesis. With suggestive evidence from human genome-wide association studies supporting a role for SMAD7 in human type 1 diabetes, a comprehensive genetic investigation of the SMAD genes in type 1 diabetes is warranted.

Topics: Animals; Diabetes Mellitus, Type 1; Female; Genetic Loci; Genetic Predisposition to Disease; Mice; Mice, Inbred NOD; Pancreas; Signal Transduction; Smad2 Protein; Spleen; Transforming Growth Factor beta

Type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice is a T cell-mediated autoimmune disease characterized by lymphocytic infiltration of pancreatic islets with subsequent destruction of the insulin-producing cells. The T regulatory (Treg) cell has been suggested to play an important role in controlling T cell-mediated inflammatory T1D. We previously demonstrated that induction of antigen-specific Treg cells in vivo by co-immunization with a DNA vaccine and its encoded protein can effectively inhibit T cell-mediated inflammatory diseases. To further demonstrate the potential of this strategy, we show here that co-immunization of NOD mice twice with DNA encoding proinsulin plus insulin protein prevents the onset of T1D and induces the impairment of antigen-specific T cell responses in a dose-dependent manner. We further show that the inhibitory function is due to the induction of TGF-beta-producing CD4(+)CD25(-) islet-specific iTreg cells against the onset of T1D in NOD mice. Induced iTreg cells were observed only in the co-immunization group, but derived neither from the DNA vaccine nor the protein alone, suggesting that a biased helper T cell type 1 response plays no inhibitory role. A strategy based on co-immunization to induce a protective response against the onset of diabetes in NOD mice may lead to the development of an immunotherapeutic/preventive protocol against T1D in humans.

Topics: Animals; Diabetes Mellitus, Type 1; Female; Humans; Immunization; Insulin; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Proinsulin; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Treatment Outcome; Vaccines, DNA

Whether interleukin (IL)-17 promotes a diabetogenic response remains unclear. Here we examined the effects of neutralization of IL-17 on the progress of adoptively transferred diabetes. IL-17-producing cells in non-obese diabetic (NOD) mice were identified and their role in the pathogenesis of diabetes examined using transfer and co-transfer assays. Unexpectedly, we found that in vivo neutralization of IL-17 did not protect NOD-severe combined immunodeficiency (SCID) mice against diabetes transferred by diabetic splenocytes. In NOD mice, gammadelta(+) T cells were dominated by IL-17-producing cells and were found to be the major source of IL-17. Interestingly, these IL-17-producing gammadelta T cells did not exacerbate diabetes in an adoptive transfer model, but had a regulatory effect, protecting NOD mice from diabetes by up-regulating transforming growth factor (TGF)-beta production. Our data suggest that the presence of IL-17 did not increase the chance of the development of diabetes; gammadelta T cells protected NOD mice from diabetes in a TGF-beta-dependent manner, irrespective of their role as major IL-17 producers.

Topics: Adoptive Transfer; Animals; Antibodies, Monoclonal; Blood Glucose; Cells, Cultured; Diabetes Mellitus, Type 1; Female; Interferon-gamma; Interleukin-17; Mice; Mice, Inbred NOD; Mice, SCID; Receptors, Antigen, T-Cell, gamma-delta; Spleen; T-Lymphocytes; Transforming Growth Factor beta

The aim of this study was to define novel mediators of tubule injury in diabetic kidney disease. For this, we used state-of-the-art proteomic methods combined with a label-free quantitative strategy to define protein expression differences in kidney tubules from transgenic OVE26 type 1 diabetic and control mice. The analysis was performed with diabetic samples that displayed a pro-fibrotic phenotype. We have identified 476 differentially expressed proteins. Bioinformatic analysis indicated several clusters of regulated proteins in relevant functional groups such as TGF-beta signaling, tight junction maintenance, oxidative stress, and glucose metabolism. Mass spectrometry detected expression changes of four physiologically relevant proteins were confirmed by immunoblot analysis. Of these, the Grb2-related adaptor protein (GRAP) was up-regulated in kidney tubules from diabetic mice and fibrotic kidneys from diabetic patients, and subsequently confirmed as a novel component of TGF-beta signaling in cultured human renal tubule cells. Thus, indicating a potential novel role for GRAP in TGF-beta-induced tubule injury in diabetic kidney disease. Although we targeted a specific disease, this approach offers a robust, high-sensitivity methodology that can be applied to the discovery of novel mediators for any experimental or disease condition.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Humans; Kidney Tubules; Mice; Models, Biological; Proteomics; Recombinant Proteins; Signal Transduction; Tandem Mass Spectrometry; Transforming Growth Factor beta; Up-Regulation

We have shown that Schistosoma mansoni egg soluble antigen (SEA) prevents diabetes in the nonobese diabetic (NOD) mouse inducing functional changes in antigen presenting cells (APCs) and expanding T helper (Th) 2 and regulatory T cell (Treg) responses. A Th2 response to S. mansoni infection or its antigens is key to both the establishment of tolerance and successfully reproduction in the host. More recently we demonstrated that SEA treatment upregulates bioactive TGFbeta on T cells with consequent expansion of Foxp3+ Tregs, and these cells might be important in SEA-mediated diabetes prevention together with Th2 cells. In this study we profile further the phenotypic changes that SEA induces on APCs, with particular attention to cytokine expression and markers of macrophage alternative activation. Our studies suggest that TGFbeta from T cells is important not just for Treg expansion but also for the successful Th2 response to SEA, and therefore, for diabetes prevention in the NOD mouse.

Topics: Adaptive Immunity; Animals; Antigens, Helminth; Cell Proliferation; Cytokines; Dendritic Cells; Diabetes Mellitus, Type 1; Female; Forkhead Transcription Factors; Host-Parasite Interactions; Immunity, Innate; Macrophages; Mice; Mice, Inbred NOD; Models, Immunological; Phenotype; Schistosoma mansoni; Schistosomiasis mansoni; Statistics, Nonparametric; T-Lymphocytes, Regulatory; Th2 Cells; Transforming Growth Factor beta

Antigen-specific regulatory CD4(+) T cells have been described but there are few reports on regulatory CD8(+) T cells. We generated islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific regulatory CD8(+) T cells from 8.3-NOD transgenic mice. CD8(+) T cells from 8.3-NOD splenocytes were cultured with IGRP, splenic dendritic cells (SpDCs), TGF-beta, and all-trans retinoic acid (ATRA) for 5days. CD8(+) T cells cultured with either IGRP alone or IGRP and SpDCs in the absence of TGF-beta and ATRA had low Foxp3(+) expression (1.7+/-0.9% and 3.2+/-4.5%, respectively). In contrast, CD8(+) T cells induced by exposure to IGRP, SpDCs, TGF-beta, and ATRA showed the highest expression of Foxp3(+) in IGRP-reactive CD8(+) T cells (36.1+/-10.6%), which was approximately 40-fold increase compared with that before induction culture. CD25 expression on CD8(+) T cells cultured with IGRP, SpDCs, TGF-beta, and ATRA was only 7.42%, whereas CD103 expression was greater than 90%. These CD8(+) T cells suppressed the proliferation of diabetogenic CD8(+) T cells from 8.3-NOD splenocytes in vitro and completely prevented diabetes onset in NOD-scid mice in cotransfer experiments with diabetogenic splenocytes from NOD mice in vivo. Here we show that exposure to ATRA and TGF-beta induces CD8(+)Foxp3(+) T cells ex vivo, which suppress diabetogenic T cells in vitro and in vivo.

Topics: Animals; CD8-Positive T-Lymphocytes; Diabetes Mellitus, Type 1; Forkhead Transcription Factors; Glucose-6-Phosphatase; Mice; Mice, Inbred NOD; Mice, Transgenic; Proteins; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tretinoin

Diabetic nephropathy (DN) is the most common indication for the development of end stage renal diseases. Inflammation is increasingly seen as the core process in the development of diabetes. Inflammatory markers e.g. NFkappaB (p65 levels), TNFalpha, COX-2 and TGFbeta-smad signaling are the key elements in the development of DN. Renin-angiotensin system suppressors like telmisartan have been used to treat DN, but they are not able to prevent completely because of development of resistance against them. Anti-inflammatory agents like, aspirin acts through both COX dependent and COX independent pathways. Hence, we thought that combining aspirin with telmisartan will be better therapeutic option in preventing the progression of nephropathy in diabetes. In the present study we studied the effect of this combination on inflammatory markers [COX-2, NFkappaB (p65 levels), TNFalpha], TGFbeta-smad expression in preventing the progression of streptozotocin-induced type I diabetic nephropathy. Treatment of aspirin significantly prevented the progression of nephropathy and inhibited the augmented COX-2, NFkappaB (p65 levels), TNFalpha, and TGFbeta-smad expression. Combination of aspirin with telmisartan resulted in a further decrease in the development of nephropathy and inflammatory markers in comparison to aspirin alone treatment. This is the first report which shows that aspirin in combination with telmisartan is more proficient in the treatment of diabetic nephropathy than any single drug therapy and involves the change in expression of inflammatory markers and TGFbeta-smad signaling.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Benzimidazoles; Benzoates; Cyclooxygenase 2; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Dose-Response Relationship, Drug; Drug Therapy, Combination; Gene Expression Regulation; Inflammation; Male; NF-kappa B; Oxidative Stress; Rats; Rats, Sprague-Dawley; Signal Transduction; Smad Proteins; Streptozocin; Telmisartan; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Antigen-specific immunotherapy is expected to be an ideal strategy for treating type 1 diabetes (T1D). We investigated the therapeutic efficacy of a peptide in the leader sequence of preproinsulin, which was selected because of its binding affinity to the MHC I-A(g7) molecule. Preproinsulin-1 L7-24 peptide (L7-24) emulsified in Freund's incomplete adjuvant was administered subcutaneously to NOD mice. Administration of L7-24 increased the proportion of regulatory T cells in the spleen. Splenocytes of NOD mice immunized with this peptide secreted IL-4 and IL-10 in response to L7-24. This peptide also significantly prevented the development of diabetes and cured some newly diabetic NOD mice without recurrence. L7-24 peptide, which has a high affinity for pockets of I-A(g7), induced regulatory T cells and showed therapeutic effects. This peptide may provide a new approach for developing antigen-specific immunotherapy for autoimmune diabetes.

Topics: Amino Acid Sequence; Animals; Autoimmunity; Blood Glucose; Cell Count; Concanavalin A; Diabetes Mellitus, Type 1; Female; Forkhead Transcription Factors; Histocompatibility Antigens Class II; Immunotherapy, Active; Insulin; Interferon-gamma; Interleukin-10; Interleukin-4; Islets of Langerhans; Lymphocyte Activation; Mice; Mice, Inbred NOD; Mice, Knockout; Peptide Fragments; Protein Binding; Protein Precursors; Spleen; T-Lymphocytes; T-Lymphocytes, Regulatory; Th2 Cells; Transforming Growth Factor beta; Vaccination

Type 1 diabetes results from an insufficiency of insulin production as a result of autoimmune destruction of the insulin-secreting pancreatic beta-cells. It can be treated by transplantation of islets of Langerhans from human donors, but widespread application of this therapy is restricted by the scarcity of donor tissue. Generation of functional beta-cells from embryonic stem (ES) cells in vitro could provide a source of an alternative graft material. Several ES cell differentiation protocols have reported the production of insulin-producing cells by mimicking the in vivo developmental stages of pancreatic organogenesis in which cells are transitioned through mesendoderm, definitive endoderm, foregut endoderm, pancreatic endoderm, and the endocrine precursor stage, until mature beta-cells are obtained. These studies provide proof of concept that recapitulating pancreatic development in vitro offers a useful strategy for generating beta-cells, but current differentiation protocols employ a bewildering variety of growth factors, mitogens, and pharmacological agents. In this review, we will attempt to clarify the functions of these agents in in vitro differentiation strategies by focusing on the intracellular signaling pathways through which they operate - phosphatidylinositol 3-kinase, transforming growth factor beta, Wnt/beta-catenin, Hedgehog, and Notch.

Topics: Animals; beta Catenin; Cell Differentiation; Diabetes Mellitus, Type 1; Embryonic Stem Cells; Hedgehog Proteins; Humans; Insulin; Insulin Secretion; Insulin-Secreting Cells; Islets of Langerhans Transplantation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Diabetogenic autoreactive T cells with effector/memory characteristics are described in type 1 diabetes patients (T1D). Alternatively activated dendritic cells (aaDCs) have been regarded as promising tools for clinical application in autoimmune diseases (ADs), although their ability to induce antigen-specific tolerance in T cells derived from ADs has yet to be determined.. Monocyte-derived dendritic cells (DCs) were produced utilizing GM-CSF and IL-4, and aaDCs by adding IL-10 and TGF-beta (10/TGF-DC) during differentiation. Both cell groups were insulin-loaded, maturated with lipopolysaccharide, and cocultured with autologous effector/memory T cells derived from T1D individuals, in order to evaluate the induction of insulin-specific tolerance.. In five of eight T1D patients analyzed in vitro, 10/TGF-DC were able to induce insulin-specific tolerance in effector/memory CD4+ T cells (50.4% +/- 13.2 less proliferation), without affecting the proliferative response to an unrelated antigen (candidin). Tolerance induction was dependent on the current activation state of CD4+ T cells in each patient. 10/TGF-DC-stimulated T cells acquired an IL-2(low)IFN-gamma(low)IL-10(high) cytokine profile, and their hyporesponsiveness could be reverted upon exposure to IL-2. This study shows a perspective about the in vitro ability of monocyte-derived 10/TGF-DC to induce antigen-specific tolerance in effector/memory T cells generated during the course of an autoimmune disease.

Topics: Adult; Antigen Presentation; Antigens, Differentiation; CD4-Positive T-Lymphocytes; Cell Differentiation; Dendritic Cells; Diabetes Mellitus, Type 1; Female; Humans; Immune Tolerance; Immunologic Memory; Immunotherapy; Insulin; Interleukin-10; Lymphocyte Activation; Male; T-Lymphocyte Subsets; Transforming Growth Factor beta

Diabetic nephropathy is characterized as the progressive development of renal insufficiency in a setting of hyperglycemia. Previous studies indicate that reactive oxygen species (ROS) play an important role in high glucose-induced renal injury. Cilostazol was reported to lower the production of superoxide significantly in situ. We hypothesized that cilostazol administration in streptozotocin-induced diabetic rats exerts effects via improving oxidative stress. Male Sprague-Dawley rats were fed with cilostazol (5 mg/kg or 25 mg/kg) for 12 weeks after streptozotocin-induced diabetes mellitus. The results showed that cilostazol decreased reactive oxygen species activity significantly in the kidneys of diabetic rats and improved the urine albumin/creatinine ratio. Cilostazol can also improve the levels of serum cholesterol, triglyceride, and LDL-cholesterol. Additionally, diabetes-caused increased glomerular size, TGF-beta, and NF-kappaB decreased under treatment with cilostazol in diabetic rats. Our results indicate that cilostazol has beneficial effects in early diabetic nephropathy.

Topics: Animals; Blood Glucose; Body Weight; Cilostazol; Creatinine; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Gene Expression Regulation; Kidney; Lipid Metabolism; Male; NF-kappa B; Organ Size; Oxidative Stress; Rats; Rats, Sprague-Dawley; Serum Albumin; Tetrazoles; Transforming Growth Factor beta

Zymosan is a complex fungal component shown to be capable of both promoting and suppressing the development of autoimmune disorders in mice. In this study, we show that a single injection of zymosan just prior to diabetes onset can significantly delay the progression of disease in NOD mice. Zymosan treatment of NOD mice induced the production of biologically active TGF-beta from cells infiltrating the pancreas and was associated with expansion of programmed cell death 1 ligand 1(+)TGF-beta(+) macrophages and Foxp3(+) regulatory T cells in vivo. Neutralization of either TGF-beta or programmed cell death 1 ligand 1 abrogated the protective effects of zymosan. Zymosan acted through TLR2 as well as ERK and p38 MAPK to induce macrophage secretion of TGF-beta and promotion of Foxp3(+) regulatory T cells in vitro and in vivo.

Topics: Animals; B7-1 Antigen; B7-H1 Antigen; Cell Differentiation; Cell Line; Cell Proliferation; Cells, Cultured; Diabetes Mellitus, Type 1; Dose-Response Relationship, Immunologic; Female; Immunity, Innate; Membrane Glycoproteins; Mice; Mice, Inbred NOD; Mink; Peptides; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Zymosan

Numerous cytokines have been shown to participate in the pathogenesis of type 1 diabetes (T1D). As gene polymorphisms can influence cytokine production or function, they may potentially contribute to genetic predisposition to the disease. The aim of this study was therefore to investigate the role of 22 single nucleotide polymorphisms (SNPs) in 13 cytokine and cytokine receptor genes in genetic susceptibility to T1D. Polymerase chain reaction with sequence-specific primers was used to genotype cytokine SNPs and HLA-DRB1 alleles in 151 diabetics and 140 healthy individuals of Slovak origin. Univariate analysis showed that transforming growth factor (TGF)-beta1 codon 10 TT homozygotes were significantly more susceptible to developing T1D than C allele carriers (P (c) = 0.0066, OR = 2.46). Furthermore, tumor necrosis factor (TNF)-alpha -308 A allele carriers were also significantly overrepresented among the diabetics (P (c) = 0.0031, OR = 2.62); however, the association of the -308 A allele with T1D might be due to its strong linkage disequilibrium with the susceptibility allele HLA-DRB1*0301. An association was also found with interleukin (IL)-6 -174 G/C and nt565 G/A SNPs; however, its significance was lost when statistical correction was applied. These data suggest that the TGF-beta1 codon 10 SNP is among numerous genetic variations with small individual effects on T1D development. Moreover, a possible role of TNF-alpha and IL-6 SNPs cannot be ruled out, although their association with T1D was due to strong LD with the HLA class II susceptibility allele or did not withstand statistical correction, respectively.

Topics: Diabetes Mellitus, Type 1; DNA Mutational Analysis; Genetic Association Studies; Genetic Predisposition to Disease; Genetic Testing; Genotype; HLA-DR Antigens; HLA-DRB1 Chains; Humans; Inflammation Mediators; Interleukin-6; Linkage Disequilibrium; Polymorphism, Single Nucleotide; Slovakia; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

We have previously shown that two injections of 20 µg alum-formulated glutamic acid decarboxylase 65 (GAD(65)) (GAD-alum; Diamyd(®)) in children with recent-onset type 1 diabetes lead to preservation of residual insulin secretion. In vitro cytokine production at the 15 months' follow-up indicated immunomodulation. In the present study, we took advantage of peripheral blood mononuclear cells, cryopreserved during early follow-ups, to investigate whether the immunomodulatory effect of GAD-alum was apparent earlier after treatment, preceding the changes previously reported at 15 months.. Peripheral blood mononuclear cells from 70 type 1 diabetic children, randomly assigned GAD-alum (n = 35) or placebo (n = 35), that had been frozen at baseline (n = 27) and after 1 (n = 58), 3 (n = 67) and 9 (n = 66) months, were stimulated in vitro with GAD(65), tyrosine phosphatase-like protein IA-2 peptide, insulin peptide, GAD-alum, alum formulation or phytohaemagglutinin. Interleukin (IL)-5, -6, -10, -12, -13, -17, tumour necrosis factor and interferon-γ were measured in cell supernatants and serum samples using Luminex. Expression of FOXP3 and transforming growth factor-β was determined by real-time reverse transcription polymerase chain reaction.. Already 1 month after the first injection, GAD(65)-induced IL-5 and IL-13 together with FOXP3 were enhanced in GAD-alum-treated patients compared to those with placebo. The in vitro response at 3 and 9 months was characterized by a broader range of cytokines in the treated group. Notably, only the T-helper 2-associated cytokines IL-5 and IL-13 together with FOXP3 increased continuously over time.. Treatment with GAD-alum in type 1 diabetic children induced an early T-helper 2 immune enhanced response to GAD(65), followed by a wider spectrum of cytokines at 3 and 9 months.

Topics: Adolescent; Alum Compounds; Autoantibodies; Child; Diabetes Mellitus, Type 1; Follow-Up Studies; Forkhead Transcription Factors; Glutamate Decarboxylase; Humans; Immunologic Factors; Insulin; Interferon-gamma; Interleukins; Monocytes; Multicenter Studies as Topic; Protein Tyrosine Phosphatases; Randomized Controlled Trials as Topic; Th2 Cells; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Diabetic patients experience exaggerated intimal hyperplasia after endovascular procedures. Recently it has been shown that circulating smooth muscle progenitor cells (SPC) contribute to intimal hyperplasia. We hypothesized that SPC differentiation would be increased in diabetes and focused on modulation of TGF-β/BMP-6 signaling as potential underlying mechanism.. We isolated SPC from C57Bl/6 mice with streptozotocin-induced diabetes and controls. SPC differentiation was evaluated by immunofluorescent staining for αSMA and collagen Type I. SPC mRNA expression of TGF-β and BMP-6 was quantified using real-time PCR. Intima formation was assessed in cuffed femoral arteries. Homing of bone marrow derived cells to cuffed arterial segments was evaluated in animals transplanted with bone marrow from GFP-transgenic mice.. We observed that SPC differentiation was accelerated and numeric outgrowth increased in diabetic animals (24.6 ± 8.8 vs 8.3 ± 1.9 per HPF after 10 days, p < 0.05). Quantitative real-time PCR showed increased expression of TGF-β and decreased expression of the BMP-6 in diabetic SPC. SPC were MAC-3 positive, indicative of monocytic lineage. Intima formation in cuffed arterial segments was increased in diabetic mice (intima/media ratio 0.68 ± 0.15 vs 0.29 ± 0.06, p < 0.05). In GFP-chimeric mice, bone marrow derived cells were observed in the neointima (4.4 ± 3.3 cells per section) and particularly in the adventitia (43.6 ± 9.3 cells per section). GFP-positive cells were in part MAC-3 positive, but rarely expressed α-SMA.. In conclusion, in a diabetic mouse model, SPC levels are increased and SPC TGF-β/BMP-6 expression is modulated. Altered TGF-β/BMP-6 expression is known to regulate smooth muscle cell differentiation and may facilitate SPC differentiation. This may contribute to exaggerated intimal hyperplasia in diabetes as bone marrow derived cells home to sites of neointima formation.

Topics: Animals; Bone Marrow Cells; Bone Morphogenetic Protein 6; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; Femoral Artery; Green Fluorescent Proteins; Hyperplasia; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Smooth, Vascular; RNA, Messenger; Signal Transduction; Stem Cells; Transforming Growth Factor beta; Tunica Intima

This study investigated whether metabolic derangement at diagnosis of diabetes mellitus affects the function of the basement membrane and the excretion of several components and whether insulin treatment can normalize this. It was designed to evaluate urinary excretion rates of transforming growth factor-beta(1) (TGF-beta(1)), the carboxy-terminal domain of collagen IV (NC1) and albumin in children during the first 20 days of treatment after diagnosis of type 1 diabetes.. Thirty-four newly diagnosed diabetic children between 4 and 16 years of age and 26 healthy children of matching age were studied with timed overnight urine collections. Urine was collected during the first 20 days of treatment.. Urinary excretion of albumin and TGF-beta(1) in diabetic children were significantly increased at entry but normalized during 20 days of treatment with insulin compared with control children. In contrast, the non-significant high NC1 excretion at diagnosis did not change but became significantly increased after 20 days of insulin treatment. Overall, the kidney size was within normal limits and unaffected by treatment. The largest kidneys had less NC1 excretion (R= - 0.67, p<0.05, n=13) and a lower glomerular filtration rate (R= - 0.77, p<0.01, n=10) than the smallest kidneys. After 20 days of treatment TGF-beta(1) excretion had decreased in children with kidney size > 8.5 cm.. Correction of the metabolic derangement with insulin decreased excretion of albumin and TGF-beta(1), but had no effect on kidney size and urine NC1 excretion, presumably because the observation period was too short.

Topics: Adolescent; Basement Membrane; Child; Child, Preschool; Collagen Type IV; Diabetes Mellitus, Type 1; Female; Glomerular Filtration Rate; Humans; Insulin; Male; Transforming Growth Factor beta

Tubulointerstitial fibrosis (TIF) is a prominent feature of progressive diabetic nephropathy. The goal of this study was to determine if hallmarks of TIF occur in the transgenic OVE26 type 1 diabetic mouse and define signaling events associated with TIF.. The expression patterns of several phenotypic markers of TIF were determined in kidneys of OVE26 diabetic and control mice by immunohistochemistry and immunoblot analysis.. Pathological signatures of TIF are an accumulation of myofibroblasts and excessive deposition of extracellular matrix in the tubulointerstitium. Kidneys from OVE26 diabetic animals exhibited an increase in tubulointerstitial myofibroblast marker (alpha-smooth muscle actin), fibronectin and collagen I staining. Abundance of the pro-fibrotic cytokine TGF-beta was also enhanced in diabetic tubules. As injury involving loss of epithelial cell-cell contact promotes tissue fibrosis, we examined expression of the adhesion protein, E-cadherin. The percent of E-cadherin-stained tubules was decreased in diabetic kidneys. Prominent regulators of TGF-beta signaling, glycogen synthase kinase-3 (GSK-3) alpha and beta, were also differentially expressed.. These results indicate that TGF-beta-induced TIF occurs in OVE26 diabetic mice, providing a practical in vivo model for defining novel regulatory events and treatment strategies for diabetes-induced TIF.

Topics: Actins; Animals; Cadherins; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disease Models, Animal; Extracellular Matrix; Female; Fibrosis; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Kidney Tubules; Mice; Mice, Transgenic; Nephritis, Interstitial; Transforming Growth Factor beta

Abnormalities in DC function are implicated in defective immune regulation that leads to type-1 diabetes (T1D) in NOD mice and humans. In this study, we used GM-CSF and Flt3-L to modulate DC function in NOD mice and observed the effects on T1D development. Treatment with either ligand at earlier stages of insulitis suppressed the development of T1D. Unlike Flt3-L, GM-CSF was more effective in suppressing T1D, even when administered at later stages of insulitis. In vitro studies and in vivo adoptive transfer experiments revealed that CD4+CD25+ T cells from GM-CSF-treated mice could suppress effector T cell response and T1D. This suppression is likely mediated through enhanced IL-10 and TGF-beta1 production. Adoptive transfer of GM-CSF exposed DCs to naive mice resulted in an expansion of Foxp3+ T cells and a significant delay in T1D onset. Our results indicate that GM-CSF acted primarily on DCs and caused an expansion of Foxp3+ Tregs which delayed the onset of T1D in NOD mice.

Topics: Age of Onset; Animals; Dendritic Cells; Diabetes Mellitus, Type 1; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Immunologic Factors; Interleukin-10; Mice; Mice, Inbred NOD; Recombinant Proteins; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Up-Regulation

Schistosoma mansoni soluble egg antigens (SEA) profoundly regulate the infected host's immune system. We previously showed that SEA prevents type 1 diabetes in NOD mice and that splenocytes from SEA-treated mice have reduced ability to transfer diabetes to NOD.scid recipients. To further characterize the mechanism of diabetes prevention we examined the cell types involved and showed that CD25(+) T-cell depletion of splenocytes from SEA-treated donors restored their ability to transfer diabetes. Furthermore, SEA treatment increased the number and proportional representation of Foxp3(+) T cells in the pancreas of NOD mice. We have used in vitro systems to analyze the effect of SEA on the development of NOD Foxp3(+) T cells. We find that SEA can induce Foxp3 expression in naïve T cells in a TGF-beta-dependent manner. Foxp3 induction requires the presence of DC, which we also show are modified by SEA to upregulate C-type lectins, IL-10 and IL-2. Our studies show that SEA can have a direct effect on CD4(+) T cells increasing expression of TGF-beta, integrin beta8 and galectins. These effects of SEA on DC and T cells may act in synergy to induce Foxp3(+) Treg in the NOD mouse.

Topics: Animals; Antigens, Helminth; Dendritic Cells; Diabetes Mellitus, Type 1; Disease Models, Animal; Forkhead Transcription Factors; Interleukin-10; Lectins, C-Type; Mice; Mice, Inbred NOD; Ovum; Pancreas; Schistosoma mansoni; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Topics: Activins; Androstadienes; Cell Differentiation; Diabetes Mellitus, Type 1; Embryonic Stem Cells; Epidermal Growth Factor; Humans; Insulin-Secreting Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Pluripotent Stem Cells; Transforming Growth Factor beta; Wortmannin

Treg can suppress autoimmune diseases such as type 1 diabetes, but their in vivo activity during suppression remains poorly characterized. In type 1 diabetes, Treg activity has been demonstrated in the pancreatic lymph node, but little has been studied in the pancreas, the site of autoimmune islet destruction. In this study we induced islet-specific Treg from the BDC-6.9 TCR transgenic mouse by activation of T cells in the presence of TGF-beta. These Treg can suppress spontaneous diabetes as well as transfer of diabetes into NOD.scid mice by diabetic NOD spleen cells or activated BDC-2.5 TCR transgenic Th1 effector T cells. In the latter transfer model, we observed infiltration of the pancreas by both effector T cells and Treg, suggesting that Treg are active in the inflammatory site and are not just restricted to the draining lymph node. Within the pancreas, we demonstrate that Treg transfer causes a reduction in the number of effector Th1 T cells and macrophages, and also inhibits effector T-cell cytokine and chemokine production. Although we found no role for TGF-beta in vitro, transfection of effector T cells with a dominant-negative TGF-beta receptor demonstrated that in vivo suppression of diabetes by TGF-beta-induced Treg is TGF-beta-dependent.

Topics: Adoptive Transfer; Animals; Cytokines; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Female; Macrophages; Male; Mice; Mice, Inbred NOD; Mice, SCID; Mice, Transgenic; Pancreas; Receptors, Transforming Growth Factor beta; Specific Pathogen-Free Organisms; T-Lymphocytes, Regulatory; Th1 Cells; Transforming Growth Factor beta

Type 1 diabetes (T1D) is an autoimmune disease that is caused by the destruction of insulin-producing beta cells. Viral infections induce immune responses that can damage beta cells and promote T1D or on the other hand prevent the development of the disease. However, the opposing roles of viral infections in T1D are not understood mechanistically. We report here that viruses that do not inflict damage on beta cells provided protection from T1D by triggering immunoregulatory mechanisms. Infection of prediabetic NOD mice with Coxsackie virus B3 or lymphocytic choriomeningitis virus (LCMV) delayed diabetes onset and reduced disease incidence. Delayed T1D onset was due to transient upregulation of programmed cell death-1 ligand 1 (PD-L1) on lymphoid cells, which prevented the expansion of diabetogenic CD8+ T cells expressing programmed cell death-1 (PD-1). Reduced T1D incidence was caused by increased numbers of invigorated CD4+CD25+ Tregs, which produced TGF-beta and maintained long-term tolerance. Full protection from T1D resulted from synergy between PD-L1 and CD4+CD25+ Tregs. Our results provide what we believe to be novel mechanistic insight into the role of viruses in T1D and should be valuable for prospective studies in humans.

Topics: Acute Disease; Animals; B7-1 Antigen; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Coxsackievirus Infections; Cytomegalovirus Infections; Diabetes Mellitus, Type 1; Female; Interferon-alpha; Interleukin-2 Receptor alpha Subunit; Membrane Glycoproteins; Mice; Mice, Inbred NOD; Peptides; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Up-Regulation

While many candidate type 1 diabetes (T1D) susceptibility genes have been identified, evidence suggests that environmental stimuli, such as viral infections, may also be involved in T1D pathogenesis. However, how viral infections may prevent or trigger the diabetogenic process remains unclear. In this issue of the JCI, Filippi et al. show that infection of NOD mice with Coxsackie virus B3 or lymphocytic choriomeningitis virus, neither of which directly destroys insulin-secreting pancreatic beta cells, triggers the activation of two distinct immunoregulatory mechanisms, involving both the innate and adaptive immune system, that protect against the development of T1D in these animals (see the related article beginning on page 1515).

Topics: Animals; Autoimmunity; Coxsackievirus Infections; Cytomegalovirus Infections; Diabetes Mellitus, Type 1; Insulin-Secreting Cells; Interferon-beta; T-Lymphocytes; Transforming Growth Factor beta

Recently, the anti-CD3 antibody has been shown to be a promising candidate for the efficient treatment of overt autoimmunity. However, the mechanisms underlying this effect remain unclear. Our previous studies demonstrated that natural killer (NK)T cells and transforming growth factor (TGF)-beta were key elements in anti-CD3 F(ab')(2)-mediated re-establishment of glucose homeostasis and restoration of self tolerance to islets in type 1 diabetes. In this report, we further investigate the regulatory pathways involved, especially the cellular source of TGF-beta production. The treatment of new-onset nonobese diabetic mice with anti-CD3 F(ab')(2) resulted in a significant increase in the numbers of NK cells in spleen and pancreatic lymph nodes that secrete TGF-beta. Depletion of this cell population with a specific anti-AsGM1 antibody abrogated anti-CD3 F(ab')(2) therapeutic effects and splenic TGF-beta production. When fractionated from recovered mice after CD3 antibody therapy, these NK cells actively suppressed diabetogenic cell proliferation and prevented the cotransfer of diabetes into nonobese diabetic-severe combined immunodeficient mice in a TGF-beta-dependent manner. In addition, the regulatory NKT cells from remitting mice were capable of causing NK cells to exhibit a TGF-beta-producing phenotype by the secretion of the T helper 2 cytokines interleukins 4 and 10. Overall, these data indicate that NK cells are the main source of TGF-beta production after anti-CD3 F(ab')(2) treatment, which are controlled by a population of T helper 2-like NKT cells.

Topics: Animals; Antibodies, Monoclonal; Autoimmunity; CD3 Complex; Diabetes Mellitus, Type 1; Immunoglobulin Fab Fragments; Interleukin-10; Interleukin-4; Killer Cells, Natural; Lymph Nodes; Mice; Mice, Inbred NOD; Pancreas; Spleen; Transforming Growth Factor beta

Type I diabetes mellitus is caused by autoimmune destruction of pancreatic beta cells, and effective treatment of the disease might require rescuing beta cell function in a context of reinstalled immune tolerance. Sertoli cells (SCs) are found in the testes, where their main task is to provide local immunological protection and nourishment to developing germ cells. SCs engraft, self-protect, and coprotect allogeneic and xenogeneic grafts from immune destruction in different experimental settings. SCs have also been successfully implanted into the central nervous system to create a regulatory environment to the surrounding tissue which is trophic and counter-inflammatory. We report that isolated neonatal porcine SC, administered alone in highly biocompatible microcapsules, led to diabetes prevention and reversion in the respective 88 and 81% of overtly diabetic (nonobese diabetic [NOD]) mice, with no need for additional beta cell or insulin therapy. The effect was associated with restoration of systemic immune tolerance and detection of functional pancreatic islets that consisted of glucose-responsive and insulin-secreting cells. Curative effects by SC were strictly dependent on efficient tryptophan metabolism in the xenografts, leading to TGF-beta-dependent emergence of autoantigen-specific regulatory T cells and recovery of beta cell function in the diabetic recipients.

Topics: Adoptive Transfer; Animals; Cell Separation; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Progression; Forkhead Transcription Factors; GATA3 Transcription Factor; Gene Expression Regulation; Indoleamine-Pyrrole 2,3,-Dioxygenase; Insulin; Islets of Langerhans; Male; Mice; Mice, Inbred NOD; Nuclear Receptor Subfamily 1, Group F, Member 3; Receptors, Retinoic Acid; Receptors, Thyroid Hormone; Sertoli Cells; Sus scrofa; T-Box Domain Proteins; Transforming Growth Factor beta; Transplantation, Heterologous

Originally predicated on the recognition of an increasing prevalence of allergy, the hygiene hypothesis was later found to accommodate the contrasting epidemiologic trends in developed countries for infectious vs autoimmune diseases. Experimentally, reduced exposure to infections will increase the risk of disease in several models of experimental autoimmunity. Although TLRs were initially considered as stimulatory molecules capable of activating early defense mechanisms against invading pathogens, emerging data suggest that they can also exert a regulatory function. In the present study, we evaluated whether TLR3 and TLR9, recognizing microbial dsDNA and CpG-containing DNA sequences, respectively, play a role in the protection from experimental autoimmune diabetes induced in C57BL/6 mice by streptozotocin. In wild-type animals, the disease was accompanied by up-regulation of IDO in pancreatic lymph nodes and would be greatly exacerbated by in vivo administration of an IDO inhibitor. Conversely, administration of a CpG-containing oligodeoxynucleotide greatly attenuated the disease in an IDO-dependent fashion. TLR9-, but not TLR3-deficient mice developed a more robust disease, an event accompanied by lack of IDO induction in pancreatic lymph nodes. Thus, our data suggest that the TLR9-IDO axis may represent a valuable target in the prevention/therapy of type 1 diabetes.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Female; Forkhead Transcription Factors; Indoleamine-Pyrrole 2,3,-Dioxygenase; Insulin-Secreting Cells; Interleukin-17; Interleukin-6; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Oligodeoxyribonucleotides; T-Lymphocytes, Regulatory; Toll-Like Receptor 3; Toll-Like Receptor 9; Transforming Growth Factor beta

Extracorporeal photochemotherapy (ECP) has demonstrated immunological effects. The proposed cytotoxic lymphocyte antigen 4 (CTLA-4) involvement, together with forkhead box P3 (FoxP3) and transforming growth factor (TGF)-beta are associated with regulatory T cell activity. The aim of the study was to evaluate the regulatory T cell-associated effect of ECP in recent onset type 1 diabetic (T1D) children. Children (n = 20) with T1D received photopheresis 8-methoxypsoralen + ECP or placebo + shampheresis. Peripheral blood mononuclear cells (PBMC) collected pretreatment (day 1) and post-treatment (day 90) were stimulated with phytohaemagglutinin (PHA) and T1D-associated glutamic acid decarboxylase 65 (GAD(65)) peptide a.a. 247-279. CTLA-4, sCTLA-4, FoxP3 and TGF-beta mRNA transcription was quantified. Photopheresis-treated individuals' relative mRNA expression was generally maintained during the course of the study. Placebo individuals increased in spontaneous CTLA-4 mRNA (P < 0.05) but decreased in expression after stimulation with GAD(65)-peptide (P < 0.05) and PHA (P < 0.05). Spontaneous TGF-beta (P < 0.05) increased whereas PHA- (P < 0.01) and GAD(65)-peptide (P < 0.01)-induced TGF-beta expression decreased in the placebo group, whereas it was maintained in the treated group. Without intervention, expression of CTLA-4 and TGF-beta, stimulated with PHA and GAD(65) peptide, decreased with time, with a parallel reduction of GAD(65)-peptide and PHA-stimulated TGF-beta expression. These parameters were counteracted by ECP. In conclusion, our results indicate that ECP maintains regulatory T cell-associated activity in recent-onset T1D.

Topics: Adolescent; Antigens, CD; Biomarkers; Case-Control Studies; Child; CTLA-4 Antigen; Diabetes Mellitus, Type 1; Female; Forkhead Transcription Factors; Gene Expression Regulation; Glutamate Decarboxylase; Humans; Immune Tolerance; Lymphocyte Activation; Male; Photopheresis; Phytohemagglutinins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Cryopreserved peripheral blood mononuclear cells (PBMC) are commonly used when assessing immune responses in clinical trials, both for practical reasons and to minimize interassay variation, as samples are often collected and studied over time. This study investigated the effect of cryopreservation on cytokine and chemokine secretion, and on expression of regulatory T-cell associated markers, in samples from children with type 1 diabetes. PBMC were cultured before and after cryopreservation either with GAD(65) or PHA. Secretion of cytokines (IL-5, -6, -10, -12, -13 -17, IFN-gamma and TNF-alpha) and chemokines (IP-10, MCP-1, MIP-1alpha, MIP-1beta and RANTES) was analysed in cell supernatants using multiplex fluorochrome technique (Luminex). Expression of FOXP3 and TGF-beta mRNA was detected by multiplex real-time RT-PCR. Increased spontaneous secretion of IL-6, -10, -12, -13, IFN-gamma and MCP-1, and mRNA expression of FOXP3 and TGF-beta, was detected after cryopreservation. Stimulation with GAD(65) induced higher levels of IL-6, IFN-gamma, TNF-alpha and MIP-1alpha, whereas lower secretion was found for IL-10 and IL-13 in cryopreserved PBMC. Stimulation with PHA induced lower secretion of IP-10, MCP-1 and RANTES and FOXP3 mRNA expression after cryopreservation. Thus, cryopreserved PBMC were suitable to assess the immunological markers included in this study, even though their expression could differ from freshly handled cells.

Topics: Adolescent; Cells, Cultured; Chemokines; Child; Cryopreservation; Cytokines; Diabetes Mellitus, Type 1; Female; Forkhead Transcription Factors; Humans; Leukocytes, Mononuclear; Male; RNA, Messenger; Transforming Growth Factor beta

Studies of animals with spontaneous autoimmune diabetes have revealed that autoreactive T cells that mediate islet beta cell destruction can be manipulated by the administration of Th(2) cytokines. Using gene delivery to express the targeted protein, we can overcome the need for frequent administration of cytokines on account of their short half-lives. In this study, the effect of hTGFbeta gene delivery was evaluated both in vitro and in vivo using an adenovirus vector (Ad) constructed with an hTGFbeta cDNA. In vitro transfection assays of the construct in HepG2, beta cell lines, and islets showed good expression levels of hTGFbeta and activation of smad3. Ad-hTGFbeta enhanced differentiation and proliferation in the beta cell line or islets without causing apoptosis. Of interest, Ad-hTGFbeta transduction in CD4(+)CD25(-) T cells resulted in a significant enhanced expression of CD25 and a regulatory T cell-specific transcription factor, Foxp3. To evaluate in vivo efficacy, Ad-hTGFbeta was intravenously injected into 7-week-old NOD mice and compared to the transduction using the vector only. The Ad-hTGFbeta group had persistent gene expression for longer than 5 weeks, and high TGFbeta serum level was secreted. There was no difference in the degree of insulitis between the Ad-hTGFbeta group and controls. Although we found favorable in vitro results, such as decrease in islet apoptosis, enhanced proliferation and differentiation, and increase in the level of CD4(+)CD25(+) regulatory T cells, there was no difference in reduction of the development of T1D between controls and Ad-hTGFbeta-injected mice. Nevertheless, if we find the appropriate mode and timing of TGFbeta gene transduction, Ad-hTGFbeta gene therapy might be useful in therapeutic cytokine delivery for the treatment of T1D.

Topics: Adenoviridae; Animals; Cells, Cultured; Cloning, Molecular; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Female; Gene Transfer Techniques; Genetic Therapy; Humans; Mice; Mice, Inbred NOD; Plasmids; Smad3 Protein; Smad7 Protein; Transforming Growth Factor beta

The role of regulatory T cells (Tregs) in type 1 diabetes has been studied extensively. The most prevalent way to define Tregs has been by their surface expression of CD4 and CD25. As currently the transcription factor FoxP3 and the low expression of CD127 are regarded to be the most specific markers of Tregs, we analysed the number of Tregs defined by these molecules in peripheral blood mononuclear cells of diabetic patients and healthy controls. The gene expression of transforming growth factor beta and two isoforms of FoxP3 was measured as well. There were no significant differences between diabetic patients and healthy controls regarding the number of Tregs, or the expression of FoxP3 isoforms and TGFbeta in peripheral blood mononuclear cells. However, we found significantly higher expression of both full-length and Delta2FoxP3 in study subjects, positive for either GAD65 or IA-2 autoantibodies. The ratio of the expression of different isoforms was not changed. This study shows the possible role of FoxP3 in the development of tissue characteristic humoral immunity in type 1 diabetes.

Topics: Adult; Autoantibodies; CD4 Lymphocyte Count; Diabetes Mellitus, Type 1; Female; Flow Cytometry; Forkhead Transcription Factors; Gene Expression; Glutamate Decarboxylase; Humans; Leukocytes, Mononuclear; Male; Receptor-Like Protein Tyrosine Phosphatases, Class 8; RNA, Messenger; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Type 1 diabetes mellitus is associated with a number of disorders of skeletal health, conditions that rely, in part, on dynamic bone formation. A mouse model of distraction osteogenesis was used to study the consequences of streptozotocin-induced diabetes and insulin treatment on bone formation and osteoblastogenesis. In diabetic mice compared with control mice, new bone formation was decreased, and adipogenesis was increased in and around, respectively, the distraction gaps. Although insulin treatment restored bone formation to levels observed in nondiabetic control mice, it failed to significantly decrease adipogenesis. Molecular events altered during de novo bone formation in untreated type 1 diabetes mellitus, yet restored with insulin treatment were examined so as to clarify specific osteogenic genes that may contribute to diabetic bone disease. RNA from distraction gaps was analyzed by gene microarray and quantitative RT-PCR for osteogenic genes of interest. Runt-related transcription factor 2 (RUNX2), and several RUNX2 target genes, including matrix metalloproteinase-9, Akp2, integrin binding sialoprotein, Dmp1, Col1a2, Phex, Vdr, osteocalcin, and osterix, were all significantly down-regulated in the insulin-deficient, hyperglycemic diabetic animals; however, insulin treatment of diabetic animals significantly restored their expression. Expression of bone morphogenic protein-2, transcriptional coactivator with PDZ-binding motif, and TWIST2, all important regulators of RUNX2, were not impacted by the diabetic condition, suggesting that the defect in osteogenesis resides at the level of RUNX2 expression and its activity. Together, these data demonstrate that insulin and/or glycemic status can regulate osteogenesis in vivo, and systemic insulin therapy can, in large part, rescue the diabetic bone phenotype at the tissue and molecular level.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Core Binding Factor Alpha 1 Subunit; Diabetes Mellitus, Type 1; Down-Regulation; Female; Insulin; Matrix Metalloproteinase 9; Mice; Osteogenesis; Osteogenesis, Distraction; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

Diabetic kidney disease is associated with monocyte chemoattractant CC chemokine ligand 2 (CCL2)-dependent glomerular and interstitial macrophage recruitment. In addition, nephropathy is delayed in Ccl2 mutant diabetic mice. However, whether the late onset of therapeutic Ccl2 blockade modulates the progression of advanced diabetic nephropathy remains unknown. We addressed this question by antagonizing Ccl2 with mNOX-E36-3'PEG, an anti-Ccl2 L-enantiomeric RNA aptamer (ie, a Spiegelmer), which binds murine Ccl2 and blocks the recruitment of ex vivo-labeled macrophages to the kidneys of db/db mice with type 2 diabetes. We injected mNOX-E36-3'PEG subcutaneously at a dose of 50 mg/kg three times per week into uninephrectomized (1K) db/db mice with advanced glomerulopathy from 4 to 6 months of age. mNOX-E36-3'PEG reduced the number of glomerular macrophages by 40% compared with nonfunctional (control) Spiegelmer-treated 1K db/db mice. This result was associated with protection from diffuse glomerulosclerosis and significantly improved the glomerular filtration rate. mNOX-E36-3'PEG also reduced renal Ccl2 mRNA and protein expression compared with control Spiegelmer-treated 1K db/db mice of the same age. Together, the late onset of therapeutic Ccl2 blockade, eg, with specific Spiegelmers, offers protection from diffuse glomerulosclerosis in type 2 diabetic db/db mice and, thus, may represent a novel therapeutic strategy for advanced glomerulosclerosis.

Topics: Animals; Aptamers, Nucleotide; Cell Count; Cell Movement; Cell Proliferation; Cells, Cultured; Chemokine CCL2; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Glomerular Filtration Rate; Kidney; Macrophages; Male; Mice; Mice, Inbred NOD; Nephrectomy; Nephritis, Interstitial; Receptors, CCR2; RNA, Messenger; Time Factors; Transforming Growth Factor beta

Coxsackievirus infections have long been associated with the induction of type 1 diabetes. Infection with coxsackievirus B4 (CB4) enhances type 1 diabetes onset in NOD mice by accelerating the presentation of beta-cell antigen to autoreactive T-cells. It has been reported that a progressive defect in regulatory T-cell (Treg) function is, in part, responsible for type 1 diabetes onset in NOD mice. This defect may contribute to susceptibility to viral-induced type 1 diabetes. We asked whether the immune response after CB4 infection could be manipulated to reestablish peripheral tolerance while maintaining the immune response to virus.. NOD mice expressing transforming growth factor-beta (TGF-beta) specifically in the beta-cells were infected with CB4, and the functional role of Tregs in disease protection was measured. Systemic treatments with TGF-beta were used to assess its therapeutic potential.. Here, we report that Tregs induced after CB4 infection in the presence of TGF-beta prevented type 1 diabetes. The capacity to directly infect pancreatic beta-cells correlated with increased numbers of pancreatic Tregs, suggesting that presentation of beta-cell antigen is integral to induction of diabetogenic protective Tregs. Furthermore, the presence of these viral induced Tregs correlated with protection from type 1 diabetes without altering the antiviral response. Finally, when TGF-beta was administered systemically to NOD mice after infection, the incidence of type 1 diabetes was reduced, thereby signifying a potential therapeutic role for TGF-beta.. We demonstrate manipulations of the immune response that result in Treg-mediated protection from type 1 diabetes without concomitant loss of the capacity to control viral infection.

Topics: Animals; Coxsackievirus Infections; Diabetes Mellitus, Type 1; Disease Models, Animal; Flow Cytometry; Humans; Mice; Mice, Inbred NOD; Mice, Transgenic; Recombinant Proteins; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Costimulatory ligands CD80 and CD86 have different binding preferences and affinities to their receptors, CD28 and CTLA-4. Earlier, we demonstrated that CD80 binds to CTLA-4 with higher affinity and has a role in suppressing T cell response. The current study demonstrates that not only did blockade of CD86 upon Ag presentation by bone marrow-derived dendritic cells (DC) to OVA-specific T cells result in induction of hyporesponsive T cells but also that these T cells could suppress the proliferative response of effector T cells. These T cells showed TGF-beta1 on their surface and secreted TGF-beta1 and IL-10 upon restimulation. Although blockade of CTLA-4 and neutralization of IL-10 profoundly inhibited the induction of these TGF-beta1(+) T cells, their ability to suppress the effector T cell proliferation was abrogated by neutralization of TGF-beta1 alone. Induction of TGF-beta1(+) and IL-10(+) T cells was found to be independent of natural CD4(+)CD25(+) regulatory T cells, demonstrating that preferential ligation of CTLA-4 by CD80 induced IL-10 production by effector T cells, which in turn promoted the secretion of TGF-beta1. Treatment of prediabetic NOD mice with islet beta cell Ag-pulsed CD86(-/-) DCs, but not CD80(-/-) DCs, resulted in the induction of TGF-beta1- and IL-10-producing cells, significant suppression of insulitis, and delay of the onset of hyperglycemia. These observations demonstrate not only that CD80 preferentially binds to CTLA-4 but also that interaction during Ag presentation can result in IL-10-dependent TGF-beta1(+) regulatory T cell induction, reinstating the potential of approaches to preferentially engage CTLA-4 through CD80 during self-Ag presentation in suppressing autoimmunity.

Topics: Animals; Antigen Presentation; Antigens, CD; B7-1 Antigen; B7-2 Antigen; CTLA-4 Antigen; Dendritic Cells; Diabetes Mellitus, Type 1; Female; Flow Cytometry; Interleukin-10; Lymphocyte Activation; Mice; Mice, Inbred NOD; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

We tested the hypothesis that AMP-activated protein kinase (AMPK), an energy sensor, regulates diabetes-induced renal hypertrophy. In kidney glomerular epithelial cells, high glucose (30 mM), but not equimolar mannitol, stimulated de novo protein synthesis and induced hypertrophy in association with increased phosphorylation of eukaryotic initiation factor 4E binding protein 1 and decreased phosphorylation of eukaryotic elongation factor 2, regulatory events in mRNA translation. These high-glucose-induced changes in protein synthesis were phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin (mTOR) dependent and transforming growth factor-beta independent. High glucose reduced AMPK alpha-subunit theronine (Thr) 172 phosphorylation, which required Akt activation. Changes in AMP and ATP content could not fully account for high-glucose-induced reductions in AMPK phosphorylation. Metformin and 5-aminoimidazole-4-carboxamide-1beta-riboside (AICAR) increased AMPK phosphorylation, inhibited high-glucose stimulation of protein synthesis, and prevented high-glucose-induced changes in phosphorylation of 4E binding protein 1 and eukaryotic elongation factor 2. Expression of kinase-inactive AMPK further increased high-glucose-induced protein synthesis. Renal hypertrophy in rats with Type 1 diabetes was associated with reduction in AMPK phosphorylation and increased mTOR activity. In diabetic rats, metformin and AICAR increased renal AMPK phosphorylation, reversed mTOR activation, and inhibited renal hypertrophy, without affecting hyperglycemia. AMPK is a newly identified regulator of renal hypertrophy in diabetes.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Carrier Proteins; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Glucose; Hypertrophy; Intracellular Signaling Peptides and Proteins; Kidney; Metformin; Multienzyme Complexes; Phosphoproteins; Protein Serine-Threonine Kinases; Rats; Ribonucleosides; Transforming Growth Factor beta

Proteomics combined with cell fractionation was used to identify proteins regulated by high glucose (HG) in human mesangial cells (HMC). Total membrane and cytosolic fraction proteins derived from HMC after 7 days of HG exposure were resolved by a two-dimensional gel electrophoresis approach. DeCyder software was used to analyze the HG-induced protein spot dysregulation. In the membrane subproteome, of the 92 spots that were matched across all gels, HG induced significant downregulation of only 4 protein spots. The dysregulated spots from the membrane subproteome included binding protein (BiP), calreticulin precursor protein, a 63-kDa transmembrane protein from a ER/Golgi intermediate, and beta-subunit of collagen proline 4-hydroxylase. In the cytosolic subproteome, of the 122 spots that were matched across all gels, HG induced downregulation of 3 protein spots and upregulation of 2 protein spots significantly. Enolase 1, annexin VI, and gamma(2)-actin were decreased, whereas heat shock protein-70 kDa and calmodulin (CaM) were increased. Further confocal microscopy and Western immunoblotting of mesangial cells validated the increase in CaM. Immunoblotting of diabetic mouse and rat kidneys exhibited a marked increase in CaM at both early and late stages of diabetes, reflecting the potential physiological relevance of CaM upregulation. CaM-specific inhibitors blocked glucose transport stimulated by transforming growth factor-beta and insulin in mesangial cells. In conclusion, using a combination of cell fractionation and protein expression profiling, we identified a cohort of HG-dysregulated proteins in the HMC and identified a critical and as yet unrecognized role for CaM in glucose transport in mesangial cells.

Topics: Animals; Calmodulin; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Down-Regulation; Gene Expression Profiling; Glucose; Humans; Insulin; Mesangial Cells; Mice; Proteome; Rats; Transforming Growth Factor beta

Antigen-specific immunotherapy, an approach to selectively block autoimmune diabetes, generally declines in nonobese diabetic (NOD) mice as disease progresses. To define the parameters influencing the efficacy of antigen-specific immunotherapy once diabetes is established, plasmid DNA (pDNA) vaccination was used to suppress autoimmune-mediated destruction of syngeneic islet grafts in diabetic NOD recipients. pDNAs encoding a glutamic acid decarboxylase 65 (GAD65)-Ig molecule (pGAD65), interleukin (IL)-4 (pIL4), and IL-10 (pIL10) significantly delayed the onset of recurrent diabetes compared with pGAD65+pIL10-vaccinated recipients. Despite differences in efficacy, a similar frequency of GAD65-specific CD4(+) T-cells secreting IL-4, IL-10, or interferon-gamma were detected in mice treated with pGAD65+pIL4+pIL10 and pGAD65+pIL10. However, the frequency of FoxP3-expressing CD4(+)CD25(+)CD62L(hi) T-cells was increased in the renal and pancreatic lymph nodes of diabetic recipients vaccinated with pGAD65+pIL4+pIL10. These immunoregulatory CD4(+)CD25(+) T-cells (CD4(+)CD25(+) Treg) exhibited enhanced in vivo and in vitro suppressor activity that partially was transforming growth factor-beta dependent. Furthermore, duration of islet graft protection in pGAD65+pIL4+pIL10-vaccinated diabetic recipients correlated with the persistence of CD4(+)CD25(+) Treg. These data demonstrate that the frequency and maintenance of FoxP3-expressing CD4(+)CD25(+) Treg influence antigen-induced suppression of ongoing beta-cell autoimmunity in diabetic recipients.

Topics: Animals; Antigens; CD4 Lymphocyte Count; CD4-Positive T-Lymphocytes; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Glutamate Decarboxylase; Immunotherapy; Insulin-Secreting Cells; Interleukin-2 Receptor alpha Subunit; Interleukin-4; Isoenzymes; Mice; Mice, Inbred NOD; Muramidase; Polymerase Chain Reaction; Transforming Growth Factor beta

Previous results have shown that CD4(+)CD25(+) regulatory T cells (Tregs) control autoimmunity in a spontaneous model of type 1 diabetes, the nonobese diabetic (NOD) mouse. Moreover, anti-CD3 reverses diabetes in this setting by promoting Tregs that function in a TGF-beta-dependent manner. This finding contrasts with a large body of work suggesting that CD4(+)CD25(high) Tregs act in a cytokine-independent manner, thus suggesting that another type of Treg is operational in this setting. We sought to determine the basis of suppression both in untreated NOD mice and in those treated with anti-CD3. Our present results show that a subset of foxP3(+) cells present within a CD4(+)CD25(low) lymphocyte subset suppresses T cell immunity in spontaneously diabetic NOD mice in a TGF-beta-dependent manner, a functional property typical of "adaptive" regulatory T cells. This distinct Treg subset is evident in NOD, but not normal, mice, suggesting that the NOD mice may generate these adaptive Tregs in an attempt to regulate ongoing autoimmunity. Importantly, in two distinct in vivo models, these TGF-beta-dependent adaptive CD4(+)CD25(low) T cells can be induced from peripheral CD4(+)CD25(-) T lymphocytes by anti-CD3 immunotherapy which correlates with the restoration of self-tolerance.

Topics: Adoptive Transfer; Animals; Antibodies; CD28 Antigens; CD3 Complex; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Forkhead Transcription Factors; Immunoglobulin Fab Fragments; Immunotherapy; Mice; Mice, Inbred NOD; Mice, Knockout; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Immuno-regulatory defects, including a reduction in the number and function of regulatory T cells, play an important role in the development of autoimmune diabetes in both humans and non-obese diabetic (NOD) mice. In this study we tested the effect of introduction of FoxP3 into antigen non-specific polyclonal and antigen-specific monoclonal T cells on diabetes development in NOD mice. Transduction of FoxP3 into antigen-specific monoclonal (insulin or BDC2.5 mimotope specific) or antigen non-specific polyclonal T cells using retroviral transduction delayed or prevented diabetes development. However, transduced antigen-specific monoclonal T cells were considerably more effective than polyclonal T cells. Regulatory activity was not limited to CD4 T cells as potent diabetogenic CD8 T cells specific for insulin, were also reduced in pathogenicity by FoxP3 induction. The disease suppressive effect, in both CD4 and CD8 cells, was more evident in spontaneously diabetes-prone NOD hosts (non-lymphopenic) than in lymphopenic NOD.scid hosts. We suggest that this strategy of transducing antigen-specific CD4 or CD8 T cells may be a useful therapeutic approach in the prevention of autoimmune diabetes.

Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Forkhead Transcription Factors; Genetic Therapy; Insulin; Mice; Mice, Inbred NOD; Mice, SCID; Retroviridae; Transduction, Genetic; Transforming Growth Factor beta

Anti-CD3 monoclonal antibody (mAb) has been shown to induce tolerance and to be an effective treatment for diabetes both in animal models and in human trials. We have shown that anti-CD3 mAb given orally is biologically active in the gut and suppresses experimental autoimmune encephalitis by the induction of a regulatory T-cell that expresses latency-associated peptide (LAP) on its surface. In the present study, we investigated the effect of oral anti-CD3 mAb on the prevention of autoimmune diabetes in AKR mice in which the low-dose streptozocin (STZ) model induces autoimmunity to the beta-cells of the islets. We found that oral anti-CD3 mAb given at doses of 50 and 250 microg/feeding suppressed the incidence of diabetes in this model with the best effects seen at the 50 microg/dose. Associated with suppression, we observed decreased cell proliferation in the spleen and conversion of T-helper (Th)1 responses into Th2/Th3 responses in the periphery, including the pancreatic lymph nodes. Oral anti-CD3 mAb increased the expression of LAP on CD4(+) T-cells, and these cells could adoptively transfer protection. Protection by oral anti-CD3 was transforming growth factor-beta dependent. Our results demonstrate that oral anti-CD3 is effective in the model of STZ-induced diabetes and may be a useful form of therapy for type 1 diabetes in humans.

Topics: Administration, Oral; Adoptive Transfer; Animals; Antibodies, Monoclonal; CD3 Complex; CD4 Antigens; CD4-Positive T-Lymphocytes; Cell Proliferation; Cells, Cultured; Cytokines; Diabetes Mellitus, Type 1; Immunotherapy; Insulin; Intestinal Absorption; Intestines; Male; Mice; Mice, Inbred AKR; Pancreas; Spleen; Streptozocin; Transforming Growth Factor beta

Several strategies are being designed to test the therapeutic potential of Ag-specific regulatory T cells to prevent or treat autoimmune diseases. In this study, we demonstrate that naive CD4+ Foxp3- T cells specific for a naturally expressed autoantigen (H+/K+ ATPase) can be converted to Foxp3+ T regulatory cells (Tregs) when stimulated in presence of TGFbeta. TGFbeta-induced Tregs (iTregs) have all the characteristics of naturally generated regulatory T cells in vitro, and more importantly, are effective at preventing organ-specific autoimmunity in a murine model of autoimmune gastritis. H+/K+ ATPase specific iTregs were able to inhibit the initial priming and proliferation of autoreactive T cells, and appear to do so by acting on H+/K+ ATPase presenting dendritic cells (DC). DC exposed to iTregs in vivo were reduced in their ability to stimulate proliferation and cytokine production by H+/K+ ATPase specific T cells. iTregs specifically reduced CD80 and CD86 expression on the surface of H+/K+ ATPase presenting DC in vitro. These studies reveal the therapeutic potential of Ag specific iTregs to prevent autoimmunity, and provide a mechanism by which this population of regulatory T cells, and perhaps others, mediate their suppressive effects in vivo.

Topics: Animals; Autoantigens; Autoimmunity; B7-1 Antigen; B7-2 Antigen; CD4 Lymphocyte Count; Cells, Cultured; Dendritic Cells; Diabetes Mellitus, Type 1; Female; Forkhead Transcription Factors; Gastritis; Lymph Nodes; Lymphocyte Activation; Mice; Organ Specificity; Phenotype; T-Lymphocytes, Regulatory; Time Factors; Transforming Growth Factor beta

Diabetic nephropathy is the most common cause of end-stage renal failure in the United States. Hyperglycemia is an important factor in the pathogenesis of diabetic nephropathy. Hyperglycemia upregulates the expression of transforming growth factor-beta (TGF-beta), which stimulates extracellular matrix deposition in the kidney, contributing to the development of diabetic nephropathy. Our previous studies demonstrated that the transcription factor, upstream stimulatory factor 2 (USF2), was upregulated by high glucose, which bound to an 18-bp sequence in the thrombospondin 1 (TSP1) gene promoter and regulated high glucose-induced TSP1 expression and TGF-beta activity in mesangial cells, suggesting that USF2 might play a role in the development of diabetic nephropathy. In the present studies, we examined the effect of overexpression of USF2 on the development of diabetic nephropathy. Type 1 diabetes was induced in USF2 transgenic mice [USF2 (Tg)] and their wild-type littermates (WT) by injection of streptozotocin. Four groups of mice were studied: control WT, control USF2 (Tg), diabetic WT, and diabetic USF2 (Tg). Mice were killed after 15 wk of diabetes onset. At the end of studies, control USF2 (Tg) mice ( approximately 6 mo old) exhibited increased urinary albumin excretion. These mice also exhibited glomerular hypertrophy, accompanied by increased TSP1, active TGF-beta, fibronectin accumulation in the glomeruli compared with control WT littermates. Type 1 diabetes onset further augmented the urinary albumin excretion and glomerular hypertrophy in the USF2 (Tg) mice. These findings suggest that overexpression of USF2 accelerates the development of diabetic nephropathy.

Topics: Albuminuria; Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Electrophoretic Mobility Shift Assay; Fibronectins; Hypertrophy; Kidney Glomerulus; Male; Mice; Mice, Transgenic; Promoter Regions, Genetic; Thrombospondin 1; Time Factors; Transforming Growth Factor beta; Upstream Stimulatory Factors

Our previous studies have demonstrated that splenocytes, transduced with glutamate decarboxylate 65 (GAD) and IgG fusion construct, protect non-obese diabetes (NOD) mice from diabetes. However, the mechanism by which this strategy prevents diabetes is not well understood. Here, we found that CD4(+)Foxp3(+)Treg cells, in vitro induced by GAD-IgG-transduced splenocytes, after transfer, were responsible for prevention of diabetes in NOD mice. Further studies suggested that GAD-IgG-transduced B cells could secrete high level of TGF-beta and stimulated CD4(+)T cells to secrete high level of IFN-gamma. Finally, we found that when TGF-beta and/or IFN-gamma were blocked, CD4(+)Foxp3(-)T cells were not converted into CD4(+)Foxp3(+)Treg cells. The results suggest that GAD-IgG-transduced B cells via TGF-beta and IFN-gamma in vitro induce the CD4(+)Foxp3(+)Treg cells which are responsible for prevention of diabetes in NOD mice by GAD-IgG-gene transfer.

Topics: Animals; Antigens; B-Lymphocytes; Cell Transplantation; Diabetes Mellitus, Type 1; Female; Forkhead Transcription Factors; Genetic Therapy; Glutamate Decarboxylase; Immune Tolerance; Immunoglobulin G; Interferon-gamma; Mice; Mice, Inbred NOD; Recombinant Fusion Proteins; Spleen; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transduction, Genetic; Transforming Growth Factor beta

Decorin, a proteoglycan that inhibits active transforming growth factor-beta, is increased in diabetic nephropathy; however, its functional significance is unclear. In this study, we used low-dose streptozotocin to induce type 1 diabetes in wild-type (C57BL/6J Dcn(+/+)), Dcn(-/-), and Dcn(+/-) mice and studied the mice for up to 1 year of diabetes. Decorin gene dose had no effect on severity of diabetes; however, the Dcn(-/-) diabetic mice died significantly earlier than nondiabetic controls (57 versus 7.3% mortality). In contrast to wild-type diabetic mice, which failed to develop significant nephropathy, the Dcn(-/-) diabetic mice developed a significant increase in albuminuria and plasma creatinine and a concurrent decrease in circulating adiponectin levels. Interestingly, adiponectin levels at 6 months of diabetes were predictive of mortality in diabetic mice. Dcn(-/-) diabetic mice exhibited advanced glomerular lesions, including diffuse mesangial matrix accumulation and fibrin cap formation. By immunohistochemistry, Dcn(-/-) diabetic mice exhibited significant increases in glomerular transforming growth factor-beta, type I collagen, macrophage infiltration, and Nox4. We conclude that decorin is a natural protective factor against diabetic nephropathy and that the Dcn(-/-) diabetic mouse is a useful new model of progressive diabetic nephropathy.

Topics: Animals; Collagen Type I; Decorin; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Extracellular Matrix Proteins; Kidney Glomerulus; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; NADPH Oxidase 4; NADPH Oxidases; Proteoglycans; Streptozocin; Transforming Growth Factor beta

The onset of type 1 diabetes in NOD mice is delayed by oral administration of a bacterial extract (OM-85) and can be completely prevented by its intraperitoneal administration. Optimal prevention is observed when starting treatment at 3 or 6 weeks of age, and some effect is still observed with treatment at 10 weeks of age. Using genetically deficient mice and cytokine-neutralizing monoclonal antibodies, we demonstrate here that the therapeutic effect does not involve T-helper type 2 cytokines (interleukin [IL]-4 and -10) but is tightly dependent on transforming growth factor (TGF)-beta. Natural killer T-cells also participate in the therapeutic effect because CD1d(-/-) NOD mice are partially resistant to the protective effect of OM-85. The question remains of the specificity of the protective effect of OM-85, which may include proinflammatory components. It will thus be important to further characterize the molecular components that afford protection from type 1 diabetes. Lipopolysaccharide is excluded, but other Toll-like receptor (TLR) agonists could be involved because OM-85 stimulated dendritic cells and induced TGF-beta production by splenocytes in a TLR-2-, TLR-4-, and MyD88-dependent fashion.

Topics: Adaptor Proteins, Signal Transducing; Aging; Animals; Antigens, CD1; Bacteria; Cell Extracts; Diabetes Mellitus, Type 1; Dose-Response Relationship, Drug; Female; Gene Deletion; Interleukin-4; Killer Cells, Natural; Ligands; Mice; Mice, Inbred NOD; Myeloid Differentiation Factor 88; Toll-Like Receptors; Transforming Growth Factor beta

Adaptive regulatory T cells that develop from naive CD4 cells in response to exposure to Ag can act as immunotherapeutic agents to control immune responses. We show that effectors generated from murine islet-specific CD4 cells by TCR stimulation with IL-2 and TGF-beta1 have potent suppressive activity. They prevent spontaneous development of type 1 diabetes in NOD mice and inhibit development of pancreatic infiltrates and disease onset orchestrated by Th1 effectors. These regulatory T cells do not require innate CD25+ regulatory cells for generation or function, nor do they share some characteristics typically associated with them, including expression of CD25. However, the adaptive population does acquire the X-linked forkhead/winged helix transcription factor, FoxP3, which is associated with regulatory T cell function and maintains expression in vivo. One mechanism by which they may inhibit Th1 cells is via FasL-dependent cytotoxicity, which occurs in vitro. In vivo, they eliminate Th1 cells in lymphoid tissues, where Fas/FasL interactions potentially play a role because Th1 cells persist when this pathway is blocked. The results suggest that adaptive regulatory CD4 cells may control diabetes in part by impairing the survival of islet-specific Th1 cells, and thereby inhibiting the localization and response of autoaggressive T cells in the pancreatic islets.

Topics: Adoptive Transfer; Animals; Antigens; CD4-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Diabetes Mellitus, Type 1; Fas Ligand Protein; Female; In Vitro Techniques; Interleukin-2; Islets of Langerhans; Membrane Glycoproteins; Mice; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Mice, Transgenic; Th1 Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factors

We have shown previously that incorporation of a cDNA coding for the pro-apoptotic protein BAX into plasmid DNA coding for a secreted form of the pancreatic beta-cell antigen glutamic acid decarboxylase (GAD) promotes prevention of type 1 diabetes in non-obese diabetic (NOD) mice. Here we present evidence indicating that injection of the same vaccine at time of early diabetes onset could ameliorate the disease with efficacy, with 42% of mice overtly diabetic by 40 weeks of age compared to 92% in control groups. In addition, immunological analysis revealed that the DNA vaccine induced CD4(+)CD25(+) T cells cultured from draining lymph nodes that had immunosuppressive function in vitro. The induced regulatory T cells (Tregs) expressed the foxp3 gene and showed cell-contact-dependent as well as TGF-beta- and IL-10-independent immunosuppressive activity. Data also revealed that CD4(+)CD25(-) T cells from mice immunized with the DNA vaccine yielded a cell population that was foxp3(+), showed increased expression of CD25 compared to control, and had immunosuppressive function in vitro, indicating that Tregs could have developed from antigen-induced, peripheral T lymphocytes. In contrast, injection of DNA coding for SGAD55 or BAX alone did not induce Tregs. Altogether, our data confirm that pro-apoptotic DNA vaccination can be used as an immunosuppressive strategy and demonstrate its potential for therapy of pathological autoimmunity.

Topics: Aging; Animals; Apoptosis; bcl-2-Associated X Protein; CD4-Positive T-Lymphocytes; Cell Proliferation; Diabetes Mellitus, Type 1; Forkhead Transcription Factors; Interleukin-10; Mice; Mice, Inbred NOD; Receptors, Interleukin-2; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Vaccination; Vaccines, DNA

Safe induction of autoantigen-specific long-term tolerance is the "holy grail" for the treatment of autoimmune diseases. In animal models of type 1 diabetes, oral or i.n. immunization with islet antigens induces Tregs that are capable of bystander suppression. However, such interventions are only effective early in the prediabetic phase. Here, we demonstrate that a novel combination treatment with anti-CD3epsilon-specific antibody and i.n. proinsulin peptide can reverse recent-onset diabetes in 2 murine diabetes models with much higher efficacy than with monotherapy with anti-CD3 or antigen alone. In vivo, expansion of CD25(+)Foxp3(+) and insulin-specific Tregs producing IL-10, TGF-beta, and IL-4 was strongly enhanced. These cells could transfer dominant tolerance to immunocompetent recent-onset diabetic recipients and suppressed heterologous autoaggressive CD8 responses. Thus, combining a systemic immune modulator with antigen-specific Treg induction is more efficacious in reverting diabetes. Since Tregs act site-specifically, this strategy should also be expected to reduce the potential for systemic side effects.

Topics: Administration, Intranasal; Animals; Autoantigens; CD3 Complex; Diabetes Mellitus, Type 1; Interleukin-10; Interleukin-4; Mice; Mice, Transgenic; Proinsulin; Remission Induction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Rotavirus infections have been implicated as a possible trigger of type 1 diabetes. We elucidated this connection by comparing peripheral blood T cell responses to rotavirus between children with newly diagnosed type 1 diabetes (n = 43), healthy children with multiple diabetes-associated autoantibodies (n = 36) and control children carrying human leukocyte antigen (HLA)-conferred susceptibility to type 1 diabetes but without autoantibodies (n = 104). Lymphocyte proliferation assays based on stimulation with an antigen were performed using freshly isolated peripheral blood mononuclear cells (PBMC) and IgG and IgA class rotavirus antibodies were measured using plasma samples collected from the children. The expression of interferon (IFN)-gamma, interleukin (IL)-4, IL-10 and transforming growth factor (TGF)-beta in PBMC was studied with real-time polymerase chain reaction (PCR) in a subgroup of 38 children. No differences were observed in the strength or frequency of positive T cell responses to rotavirus between children with overt diabetes, children with multiple autoantibodies and control children. Children with diabetes-associated autoantibodies had, instead, stronger T cell responses to purified coxsackie B4 virus than control children. Rotavirus-stimulated lymphocytes from autoantibody-positive children produced more IL-4 and phytohaemagglutinin (PHA)-stimulated lymphocytes more IL-4 and IFN-gamma than lymphocytes from control children. PHA-stimulated lymphocytes from children with diabetes also produced more IL-4 and purified protein derivative (PPD)-stimulated lymphocytes less TGF-beta than lymphocytes from autoantibody-negative control children. In conclusion, our lymphocyte proliferation studies did not provide evidence supporting an association between rotavirus infections and the development of type 1 diabetes or diabetes-associated autoantibodies in young children.

Topics: Antigens, Viral; Autoantibodies; Case-Control Studies; Cell Proliferation; Child; Child, Preschool; Cytokines; Diabetes Mellitus, Type 1; Female; Humans; Insulin-Secreting Cells; Interferon-gamma; Interleukin-10; Interleukin-4; Linear Models; Lymphocyte Activation; Male; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Rotavirus; T-Lymphocytes; Transforming Growth Factor beta

We hypothesize that transforming growth factor-beta (TGF-beta), a multifunctional growth factor which plays a key role in the development of tissue fibrosis, may be involved in the pathophysiology of diabetic nephropathy. Our aim was to examine three polymorphisms within the TGF-beta 1 gene, in codons 10, 25 and 263, for association with nephropathy in Type 1 diabetes.. We conducted a large case-control study using cases with Type 1 diabetes and clinical nephropathy. Controls were Type 1 diabetic subjects who have been injecting insulin for at least 50 years and have extremely low risk of nephropathy. Genotyping was by polymerase chain reaction with sequence-specific primers.. There was a significant difference in the frequency of the TGF-beta 1 codon 10 genotypes in the diabetic nephropathy group (n = 420) when compared with the controls (n = 410, P = 0.007). There were no significant differences when the frequencies of the TGF-beta1 codons 25 and 263 genotypes in the diabetic nephropathy group were compared with the control group.. In our study the TGF-beta 1 codon 10 polymorphism is associated with nephropathy in Type 1 diabetes and variation in this gene may contribute to the genetic predisposition to this complication in Type 1 diabetes.

Topics: Adult; Case-Control Studies; Codon; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Genetic Predisposition to Disease; Genotype; Humans; Male; Polymorphism, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1

We generated the homozygous transgenic mice with expression of the active form of TGF-beta1 by the glucagon promoter (homozygous NOD-TGF-beta1). The homozygous NOD-TGF-beta1 showed severe diabetes in 84.6%, impaired glucose tolerance, and low serum insulin levels. The final size of endocrine and whole pancreas decreased, respectively, to 6 and 34%, compared to wild-type mice. The homozygous N(2) backcross to C57BL/6 (B6-TGF-beta1) showed no diabetes, but impaired glucose tolerance and low serum insulin levels. In homozygous NOD-TGF-beta1, the expression of p15(INK4b) was induced by 3.4-fold in pancreatic islets than that in wild-type mice. Based on these, we conclude first that excessive paracrine TGF-beta1 signaling in islets results in endocrine and exocrine pancreatic hypoplasia, second that TGF-beta1decrease the final size of endocrine and exocrine pancreas presumably through regulating cell cycle via p15(INK4b) at least in endocrine pancreas, and third that hypoplastic action of TGF-beta1 of pancreatic islets is independent of the genetic background.

Topics: Animals; Cell Cycle; Cell Cycle Proteins; Crosses, Genetic; Cyclin-Dependent Kinase Inhibitor p15; Diabetes Mellitus, Type 1; Female; Glucagon; Glucose Tolerance Test; Homozygote; Hyperglycemia; Insulin; Islets of Langerhans; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Transgenic; Pancreas, Exocrine; Promoter Regions, Genetic; Swine; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Suppressor Proteins

Accumulating evidence that granulocyte colony-stimulating factor (G-CSF), the key hematopoietic growth factor of the myeloid lineage, not only represents a major component of the endogenous response to infections, but also affects adaptive immune responses, prompted us to investigate the therapeutic potential of G-CSF in autoimmune type 1 diabetes. Treatment with G-CSF protected NOD mice from developing spontaneous diabetes. G-CSF triggered marked recruitment of dendritic cells (DCs), particularly immature CD11c(lo)B220(+) plasmacytoid DCs, with reduced costimulatory signal expression and higher interferon-alpha but lower interleukin-12p70 release capacity than DCs in excipient-treated mice. G-CSF recipients further displayed accumulation of functional CD4(+)CD25(+) regulatory T-cells that produce transforming growth factor-beta1 (TGF-beta1) and actively suppressed diabetes transfer by diabetogenic effector cells in secondary NOD-SCID recipients. G-CSF's ability to promote key tolerogenic interactions between DCs and regulatory T-cells was demonstrated by enhanced recruitment of TGF-beta1-expressing CD4(+)CD25(+) cells after adoptive transfer of DCs isolated from G-CSF- relative to vehicle-treated mice into naive NOD recipients. The present results suggest that G-CSF, a promoter of tolerogenic DCs, may be evaluated for the treatment of human type 1 diabetes, possibly in association with direct inhibitors of T-cell activation. They also provide a rationale for a protective role of the endogenous G-CSF produced during infections in early diabetes.

Topics: Aging; Animals; CD4 Antigens; CD4-Positive T-Lymphocytes; Cytokines; Dendritic Cells; Diabetes Mellitus, Type 1; Female; Flow Cytometry; Granulocyte Colony-Stimulating Factor; Mice; Mice, Inbred NOD; Receptors, Interleukin-2; Recombinant Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

The nature of the T-cell response to antigen is governed by the activation state of the antigen-presenting dendritic cell (DC). Immature or resting DCs have been shown to induce T-cell responses that may protect against the development of autoimmune disease. Effectively harnessing this "tolerogenic" effect of resting DCs requires that it be disease-specific and that activation of DCs by manipulation ex vivo is avoided. We reasoned that this could be achieved by transferring in vivo partially differentiated myeloid progenitor cells encoding a disease-specific autoantigen. With the aim of preventing autoimmune diabetes, we transferred myeloid progenitor cells encoding proinsulin into NOD mice. Bone marrow (BM) was cultured in granulocyte macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta1, a cytokine combination that expands myeloid cells but inhibits terminal DC differentiation, to yield Gr-1(+)/CD11b(+)/CD11c(-) myeloid progenitor cells and a minor population of CD11c(+)/CD11b(+)/CD86(lo) immature DCs. After transfer, Gr-1(+) myeloid cells acquired the characteristics of resting DCs (CD11c(+)/MHC classII(int)/CD86(lo)/CD40(lo)). Gr-1(+) myeloid cells generated from transgenic NOD mice that expressed proinsulin controlled by a major histocompatibility complex (MHC) class II promoter, but not from wild-type NOD mice, transferred into 4-week-old female NOD mice significantly suppressed diabetes development. The transfer of DC progenitors encoding a disease-specific autoantigen is, therefore, an effective immunotherapeutic strategy that could be applied to humans.

Topics: Animals; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Dendritic Cells; Diabetes Mellitus, Type 1; Granulocyte-Macrophage Colony-Stimulating Factor; Immune Tolerance; Mice; Mice, Inbred NOD; Myeloid Cells; Proinsulin; Stem Cells; T-Lymphocytes; Transforming Growth Factor beta

Natural CD4(+)CD25(+) regulatory T (CD4(+)CD25(+) T reg) cells play a key role in the immunoregulation of autoimmunity. However, little is known about the interactions between CD4(+)CD25(+) T reg cells and autoreactive T cells. This is due, in part, to the difficulty of using cell surface markers to identify CD4(+)CD25(+) T reg cells accurately. Using a novel real-time PCR assay, mRNA copy number of FoxP3, TGFbeta1, and interleukin (IL)-10 was measured in single cells to characterize and quantify CD4(+)CD25(+) T reg cells in the nonobese diabetic (NOD) mouse, a murine model for type 1 diabetes (T1D). The suppressor function of CD4(+)CD25(+)CD62L(hi) T cells, mediated by TGFbeta, declined in an age-dependent manner. This loss of function coincided with a temporal decrease in the percentage of FoxP3 and TGFbeta1 coexpressing T cells within pancreatic lymph node and islet infiltrating CD4(+)CD25(+)CD62L(hi) T cells, and was detected in female NOD mice but not in NOD male mice, or NOR or C57BL/6 female mice. These results demonstrate that the majority of FoxP3-positive CD4(+)CD25(+) T reg cells in NOD mice express TGFbeta1 but not IL-10, and that a defect in the maintenance and/or expansion of this pool of immunoregulatory effectors is associated with the progression of T1D.

Topics: Age Factors; Animals; CD4 Lymphocyte Count; CD4-Positive T-Lymphocytes; Diabetes Mellitus, Type 1; DNA-Binding Proteins; Female; Forkhead Transcription Factors; Gene Expression; Interleukin-10; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; RNA, Messenger; Sex Factors; T-Lymphocyte Subsets; Transforming Growth Factor beta; Transforming Growth Factor beta1

Type 1 diabetes results from auto-aggressive T-cell-mediated destruction of beta cells of the pancreas. Recent data suggest that restoration of self-tolerance may facilitate islet-cell regeneration/recovery. In view of the immunoregulatory activity of transforming growth factor (TGF)-beta1, we investigated whether systemic TGF-beta1 gene therapy blocks islet destructive autoimmunity and facilitates regeneration of beta-cell function in overtly diabetic nonobese diabetic (NOD) mice.. We used site-directed mutagenesis to create cysteine to serine mutation at sites 224 and 226 and constructed a replication deficient adenovirus (Ad) vector encoding active form of human TGF-beta1 (Ad-hTGF-beta1). Overtly diabetic NOD mice received intravenous injection of Ad-hTGF-beta1. Seven to 14 days after the injection, the mice received transplants with 500 syngeneic islets under the kidney capsule. Islet-graft survival and regeneration of endogenous beta-cell function were examined.. Syngeneic islet grafts failed by day 17 in all untreated mice, whereas Ad-hTGF-beta1 therapy prolonged survival of islet grafts. Islet grafts from treated mice showed well-preserved islets with a peri-islet infiltrate primarily of CD4+ T cells and expression of CD25 and Foxp3. Systemic TGF-beta1 gene therapy was associated with islet regeneration in the native pancreas. Native pancreas of treated mice revealed islets staining strongly for insulin. Similar to what was found in the syngeneic islet graft, there were well-demarcated peri-islet infiltrates that were positive for CD4, TGF-beta1, and Foxp3.. Our data demonstrate that systemic TGF-beta1 gene therapy blocks islet destructive autoimmunity, facilitates islet regeneration, and cures diabetes in diabetic NOD mice.

Topics: Animals; Diabetes Mellitus, Type 1; Disease Models, Animal; DNA-Binding Proteins; Extracellular Matrix Proteins; Forkhead Transcription Factors; Genetic Therapy; Graft Survival; Humans; Islets of Langerhans; Islets of Langerhans Transplantation; Mice; Mice, Inbred NOD; Mice, Transgenic; Nephrectomy; Regeneration; Transforming Growth Factor beta

Diabetic nephropathy is one of the major microvascular complications in diabetes and is the leading cause of end-stage renal disease worldwide. Among various factors, angiogenesis-associated factors such as vascular endothelial growth factor (VEGF)-A and angiopoietin (Ang)-2 are involved in the development of diabetic nephropathy. We previously reported the therapeutic efficacy of antiangiogenic tumstatin peptide in the early diabetic nephropathy model. Here, we examine the effect of endostatin peptide, a potent inhibitor of angiogenesis derived from type XVIII collagen, in preventing progression in the type 1 diabetic nephropathy mouse model. Endostatin peptide did not affect hyperglycemia induced by streptozotocin (STZ). Glomerular hypertrophy, hyperfiltration, and albuminuria were significantly suppressed by endostatin peptide (5 mg/kg) in STZ-induced diabetic mice. Glomerular mesangial matrix expansion, the increase of glomerular type IV collagen, endothelial area (CD31(+)), and F4/80(+) monocyte/macrophage accumulation were significantly inhibited by endostatin peptide. Increase in the renal expression of VEGF-A, flk-1, Ang-2, an antagonist of angiopoietin-1, transforming growth factor-beta1, interleukin-6, and monocyte chemoattractant protein-1 was inhibited by endostatin peptide in diabetic mice. Decrease of nephrin mRNA and protein in diabetic mice was suppressed by treatment with endostatin peptide. The level of endostatin in the renal cortex and sera was increased in diabetic mice. Endogenous renal levels of endostatin were decreased in endostatin peptide-treated groups in parallel with VEGF-A. Although serum levels of endostatin were decreased in the low-dose endostatin-peptide group, high-dose administration resulted in elevated serum levels of endostatin. These results demonstrate the potential use of antiangiogenic endostatin peptide as a novel therapeutic agent in diabetic nephropathy.

Topics: Albuminuria; Amino Acid Sequence; Animals; Blood Glucose; Body Weight; Collagen Type IV; Creatinine; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Endostatins; Female; Hyperglycemia; Hypertrophy; Immunohistochemistry; Integrin alpha5beta1; Kidney; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Organ Size; Peptide Fragments; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

We quantified the glomerular expression of thrombospondin-1 (THBS1, also known as TSP-1), transforming growth factor beta 1 (TGFB1, also known as TGF-beta1) and connective tissue growth factor (CTGF) at each stage of diabetic nephropathy. We also examined the roles of THBS1 and CTGF in mediating high-glucose- and glycated-albumin-induced synthesis of the matrix protein, fibronectin, by mesangial cells.. THBS1, latent and active TGFB1, and CTGF, were detected by immunohistochemistry and in situ hybridisation in biopsies from 19 insulin-dependent diabetic patients with incipient, manifest and advanced diabetic nephropathy, and in 11 control kidneys. Findings were quantified by image analysis. Human mesangial cells were cultured with normal or high glucose, albumin or glycated albumin (Amadori product), +/-THBS1 or CTGF antisense oligonucleotides, or with peptide W, an inhibitor of TGFB1 bioactivation by THBS1. Proteins were measured by western blot analysis or ELISA.. In glomeruli of normal kidneys, mRNA and protein levels for THBS1, latent-TGFB1 and CTGF were low. They were increased in the incipient stage of diabetic nephropathy, predominantly in mesangial areas, with further increases at later stages of the disease. Little or no active TGFB1 immunostaining was detected prior to manifest diabetic nephropathy. In contrast to high-glucose conditions, increases in fibronectin synthesis that were stimulated by glycated albumin were not dependent on THBS1 activation of latent TGFB1. However, increased fibronectin synthesis in both conditions required CTGF.. Increased glomerular expression of all three factors occurs from the earliest stage of diabetic nephropathy. In contrast to THBS1, CTGF is required for mesangial synthesis of fibronectin stimulated by high glucose or glycated albumin, and is thus a potential therapeutic target.

Topics: Adult; Aged; Blotting, Western; Case-Control Studies; Connective Tissue Growth Factor; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Extracellular Matrix Proteins; Female; Fibronectins; Gene Expression Regulation; Glomerular Mesangium; Glucose; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Hyperglycemia; Immediate-Early Proteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; RNA, Messenger; Serum Albumin; Thrombospondin 1; Transforming Growth Factor beta

Rats fed a high fat diet and given a low dose of streptozotocin (STZ) (35 mg/kg) develop type 2 diabetes with insulin resistance, hyperinsulinemia, moderate hyperglycemia, hyperlipidemia, and salt-sensitive hypertension. We postulated that rats with noninsulinopenic (type 2) diabetes develop lesions of diabetic nephropathy significantly more prominent than those seen in classic insulinopenic (type 1) diabetic rats.. Rats were fed regular chow or high fat diet (60% calories from fat and 70% animal fat). After 5 weeks, rats fed regular chow received vehicle (controls) or 55 mg/kg STZ (type 1 diabetes mellitus). Rats fed high fat diet received vehicle (high fat) or low dose STZ, 35 mg/kg (type 2 diabetes mellitus). Rats were sacrificed 14 weeks after STZ/vehicle injection.. Blood glucose, systolic blood pressure, and urinary protein excretion were significantly higher in both diabetes groups than in controls. Serum insulin levels (ng/mL) were higher in type 2 diabetes than in type 1 diabetes groups (0.49 +/- 0.12 vs. 0.07 +/- 0.07) (P= 0.01). Percentage of sclerosed glomeruli was significantly higher in type 2 diabetes group than in control and type 1 diabetes groups. Fibronectin expression was significantly increased in high fat, type 1 and type 2 diabetes groups compared to controls. The expression of type IV collagen, connective tissue growth factor (CTGF), and transforming growth factor-beta (TGF-beta) was significantly increased in high fat and type 2 diabetes groups compared to controls.. Rats fed a high fat diet and given a low dose of STZ developed diabetes (with normal/high insulin levels), hypertension, and proteinuria. Kidney lesions in this type 2 model appear to be more pronounced than in type 1 diabetic rats despite lower blood glucose levels and proteinuria. We present a nongenetic rat model of type 2 diabetes mellitus and nephropathy.

Topics: Animals; Blood Glucose; Blood Pressure; Body Weight; Collagen Type IV; Connective Tissue Growth Factor; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Fibronectins; Glycated Hemoglobin; Immediate-Early Proteins; Insulin; Intercellular Signaling Peptides and Proteins; Kidney; Lipids; Male; Organ Size; Proteinuria; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

Transforming growth factor-beta1 (TGF-beta1) is a potent multifunctional polypeptide that is involved in normal renal function and in the development of glomerular sclerosis. It is also an important mediator of the immune and anti-inflammatory responses. The purpose of this study was to examine whether the measurement of urinary TGF-beta1 excretion in patients with different types of renal diseases and in newly diagnosed type 1 diabetes mellitus represents a non-invasive tool to evaluate disease activity and to monitor response to therapy. We studied the urinary excretion of TGF-beta1 in 57 nephropathic patients divided in different groups according to the underlying disease: 15 had mesangial glomerulonephritis (IgAGN), 9 membranous glomerulonephritis (MGN), 7 rapidly progressive glomerulonephritis (RPGN), 8 systemic lupus erythematosus (SLE), 9 interstitial nephritis (IN), 9 chronic renal failure (CRF). TGF-beta1 was also measured in 38 patients with type 1 (insulin-dependent) diabetes mellitus (12 with newly diagnosed diabetes, 26 long-standing diabetes) and 31 healthy controls. Total urinary TGF-beta1 concentration was assayed by enzyme-linked immunoassay (ELISA), and expressed as a ratio to urinary creatinine concentration. The urinary TGF-beta1 levels were compared with the findings of biopsy and clinical parameters. Urinary TGF-beta1 excretion was significantly increased in all groups except MGN, IN and CRF. In non-diabetic patients, urinary TGF-beta1 levels correlated with crescent formation, floccular adhesion and mesangial proliferation, but not with the degree of tubulo-interstitial fibrosis. Urinary TGF-beta1 levels did not correlate with indices of renal function (serum creatinine, glomerular filtration rate (GFR), albumin excretion rate [AER]). Among diabetic patients, HbA(1C) significantly correlated with TGF-beta1 urinary excretion. Urinary TGF-beta1 levels may represent a valid indicator of acute glomerular flogosis associated with mesangial proliferation in glomerulonephrities. In newly diagnosed diabetic patients, hyperglycaemia seems to represent the principal factor leading to TGF-beta1 overproduction. Follow-up studies of urinary TGF-beta1 levels measured during optimal glycaemic control are necessary to clarify the relationship between hyperglycaemia and TGF-beta1 excretion.

Topics: Adolescent; Adult; Aged; Chi-Square Distribution; Diabetes Mellitus, Type 1; Female; Humans; Kidney Diseases; Male; Middle Aged; Statistics, Nonparametric; Transforming Growth Factor beta; Transforming Growth Factor beta1

Oxidative stress and transforming growth factor-beta 1 (TGF-beta1) are associated with diabetic complications, and smoking is a risk factor.. This study aimed (i) to compare urinary 8-epi-PGF2alpha and plasma and urinary TGF-beta1 levels obtained in heavy smokers with Type 1 diabetes with those observed in age-matched non-smoker patients with Type 1 diabetes and controls, and (ii) to investigate the effects of smoking cessation (SC) on the above-mentioned parameters in patients with Type 1 diabetes.. Compared with control subjects (n = 12), non-smoker diabetic patients (n = 12) presented higher values of urinary 8-epi-PGF2alpha (74.2 +/- 29.6 vs. 29.6 +/- 11.1 pg/mg urinary creatinine, P = 0.01), plasma TGF-beta1 (7.7 +/- 4.7 vs. 3.6 +/- 1.7 ng/ml, P = 0.001) and urinary TGF-beta1 (15.3 +/- 6.3 vs. 8.1 +/- 4.4 ng/mg urinary creatinine, P = 0.02). Compared with non-smoker diabetic patients, smoker diabetic patients (n = 16) showed higher levels of urinary 8-epi-PGF2alpha (107.8 +/- 40.2 vs. 74.2 +/- 29.6 pg/mg urinary creatinine, P = 0.0001), plasma TGF-beta1 (12.6 +/- 4.9 vs. 7.7 +/- 4.7 ng/ml, P = 0.001) and urinary TGF-beta1 (27.5 +/- 16.0 vs. 15.3 +/- 6.3 ng/mg urinary creatinine, P = 0.01). Smoker patients were included in a smoking cessation programme. In the 10 patients that gave up smoking there was a reduction of urinary 8-epi-PGF2alpha (basal: 110.47 +/- 47.0 vs. week 12: 73.2 +/- 25.6; P < 0.001), plasma TGF-beta1 (basal: 11.2 +/- 5.9 vs. week 12: 4.89 +/- 2.25; P < 0.01) and urinary TGF-beta1 (basal: 18.12 +/- 9.27 vs. week 12: 10.32 +/- 2.0; P < 0.01) levels.. In patients with Type 1 diabetes, smoking increased oxidative stress, evaluated by lipid peroxidation, and TGF-beta1 production. Smoking cessation decreased these parameters, providing additional support to encourage diabetic patients to give up smoking.

Topics: Adult; Cotinine; Cross-Sectional Studies; Diabetes Mellitus, Type 1; Dinoprost; Female; Gas Chromatography-Mass Spectrometry; Humans; Immunoassay; Male; Smoking; Smoking Cessation; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vasoconstrictor Agents

Connective tissue growth factor (CTGF) is strongly upregulated in fibrotic disorders and has been hypothesized to play a role in the development and progression of diabetes complications. The aim of the present study was to investigate the possible association of plasma CTGF levels in type 1 diabetic patients with markers relevant to development of diabetes complications.. Plasma CTGF levels (full-length and NH2-terminal fragments) were determined in 62 well-characterized patients with type 1 diabetes and in 21 healthy control subjects. Correlations of these plasma CTGF levels with markers of glycemic control, platelet activation, endothelial activation, nephropathy, and retinopathy were investigated.. -Elevated plasma NH2-terminal fragment of CTGF (CTGF-N) levels were detected in a subpopulation of type 1 diabetic patients and were associated with diabetic nephropathy. Stepwise regression analysis revealed contribution of albuminuria, creatinine clearance, and duration of diabetes as predictors of plasma CTGF-N level. Elevation of plasma CTGF-N levels in patients with retinopathy was probably due to renal comorbidity.. Plasma CTGF-N levels are elevated in type 1 diabetic patients with nephropathy and appear to be correlated with proteinuria and creatinine clearance. Further studies will be needed to determine the relevance of plasma CTGF as a clinical marker and/or pathogenic factor in diabetic nephropathy.

Topics: Adult; Biomarkers; Connective Tissue Growth Factor; Creatinine; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Proteinuria; Reference Values; Regression Analysis; Transforming Growth Factor beta

The aims of the study were to determine whether transforming growth factor beta1 TGF-beta1 levels are raised at diagnosis of Type 1 diabetes mellitus and are related to blood glucose.. Fourteen patients (mean age 24.3 +/- 4.9 years) admitted to hospital for onset of Type 1 diabetes were studied. On the first day of hospitalization, before insulin therapy, and at 1, 4 and 16 weeks, fasting blood glucose, HbA(1c), lipid profile and TGF-beta1 levels and TGF-beta1 levels in 24-h urine were determined. The control group included 14 non-diabetic subjects with similar characteristics to those of the diabetic group.. Plasma and urinary TGF-beta1 levels were significantly lower in controls (4.7 (1.6-6.8) ng/ml P < 0.001; 5.7 (1.5-8.5) ng/mg urinary creatinine, P < 0.01) than in patients with Type 1 diabetes mellitus [10.5 (1.8-24.9) ng/ml; 10.1 (4.2-29.8) ng/mg urinary creatinine]. On study completion, HbA(1c) fell from 11.6 +/- 2.0 to 5.4 +/- 0.6% (P < 0.001). Improved metabolic control was not associated with changes in plasma (9.4 (2.6-19.5)/5.9 (1.6-21.5)/7.0 (2.3-30.2)/10.5 (1.8-24.9) ng/ml at baseline, 1, 4 and 16 weeks, respectively) or urinary (12.0 (4.7-29.5)/10.9 (1.5-20.5)/8.7 (4.3-16.9)/10.1 (4.2-29.8) ng/mg urinary creatinine) TGF-beta1 levels. A statistically significant correlation was observed between plasma TGF-beta1 and insulin dosage (U/kg/day) (r = 0.52, P = 0.037).. The increased TGF-beta1 production observed herein was not modulated by glycaemic reduction and could be a response to immuno-inflammatory activation present at the onset of Type 1 diabetes.

Topics: Adult; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Humans; Hypoglycemic Agents; Insulin; Male; Pilot Projects; Transforming Growth Factor beta; Transforming Growth Factor beta1

Previous studies have demonstrated that antigen-specific tolerance could be induced by lipopolysaccharide (LPS)-stimulated B cells retrovirally transduced with an immunoglobulin-antigen (or epitope-containing peptide) fusion construct. To investigate the mechanism of this gene therapy system, we now adapted this approach to immunotherapy of spontaneous diabetes in nonobese diabetic (NOD) mice, a T-cell-mediated autoimmune disease triggered, in part, by a pathogenic response to glutamate decarboxylase (GAD) 65. We demonstrate that LPS-stimulated splenocytes, retrovirally transfected with GAD-IgG fusion construct, induce a significant antigen-specific hyporesponsiveness at both cellular and humoral levels and reduce the incidence of diabetes in female NOD mice. Parallel with disease protection, we observed a prolonged increase of the numbers of CD4+CD25+ T cells in the periphery of GAD-IgG-treated mice, compared to those treated with a control IgG vector, both in the prediabetic period and persisting even 8 months after gene therapy. This increase appeared to be induced by the repeated stimulation of the antigen in the periphery instead of a result of differentiation of T-cell precursor in the thymus. Moreover, CD4+CD25+ T cells induced by GAD-IgG fusion construct were capable of suppressing the proliferative response of CD4+CD25- T cells in vitro; and ablation of the activity of CD4+CD25+ T cells by blocking antibody against CD25 could reverse GAD-specific T-cell hyporesponsiveness. These results suggested that CD4+CD25+ T-cell subset induced in GAD-IgG-treated NOD mice represented the regulatory or suppressive CD4+CD25+ T cells (Treg) and might play an important role in the induction and maintenance of tolerance in NOD mice. Furthermore, the numbers of splenic CD4+CD62L+ regulatory T cells in GAD-IgG-treated mice during the prediabetic period and serum TGF-beta levels in 34-38-week-old GAD-IgG-protected mice were also increased, compared to control IgG-treated ones. Therefore, we propose that the induction of tolerance and the prevention of diabetes incidence in NOD female mice induced by the GAD-IgG fusion construct may require CD4+ regulatory T cells, and the possible mediation of TGF-beta.

Topics: Animals; CD4-Positive T-Lymphocytes; Coculture Techniques; Diabetes Mellitus, Type 1; Female; Flow Cytometry; Genetic Therapy; Genetic Vectors; Glutamate Decarboxylase; Immune Tolerance; Immunoglobulin G; Interleukin-10; Lipopolysaccharides; Mice; Mice, Inbred NOD; Recombinant Fusion Proteins; Retroviridae; Transforming Growth Factor beta

Activation-induced cell death (AICD) plays a key role in the homeostasis of the immune system. Autoreactive T cells are eliminated through AICD both from the thymus and periphery. In this study, we show that NOD peripheral T cells, especially CD8(+) T cells, display a decreased susceptibility to anti-CD3-induced AICD in vivo compared with T cells from diabetes-resistant B6, nonobese diabetes-resistant, and NOD.B6Idd4 mice. The susceptibility of NOD CD8(+) T cells to AICD varies in an age- and dose-dependent manner upon stimulation in vivo with either a mitogenic or nonmitogenic anti-CD3. NOD T cells preactivated by anti-CD3 in vivo are less susceptible than B6 T cells to TCR-induced AICD. Treatment of NOD mice with a mitogenic anti-CD3 depletes CD4(+)CD25(-)CD62L(+) but not CD4(+)CD25(+)CD62L(+) T cells, thereby resulting in an increase of the latter subset in the spleen. Treatment with a nonmitogenic anti-CD3 mAb delays the onset of T1D in 8.3 TCR transgenic NOD mice. These results demonstrate that the capacity of anti-CD3 to protect NOD mice from T1D correlates with its ability to perturb T cell homeostasis by inducing CD8(+) T cell AICD and increasing the number of CD4(+)CD25(+)CD62L(+) T cells in the periphery.

Topics: Aging; Animals; Antibodies, Monoclonal; Apoptosis; CD3 Complex; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Death; Diabetes Mellitus, Type 1; Female; Genetic Predisposition to Disease; Homeostasis; Humans; Interferon-gamma; Interleukin-10; Interleukin-4; L-Selectin; Lymphocyte Activation; Lymphocyte Depletion; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Transgenic; Prediabetic State; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Interleukin-2; Spleen; T-Lymphocyte Subsets; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Autoimmunity presumably manifests as a consequence of a shortfall in the maintenance of peripheral tolerance by CD4(+)CD25(+) T regulatory cells (Tregs). However, the mechanism underlying the functional impairment of Tregs remains largely undefined. In this study a glutamic acid decarboxylase (GAD) diabetogenic epitope was expressed on an Ig to enhance tolerogenic function, and the resulting Ig-GAD expanded Tregs in both young and older insulitis-positive, nonobese diabetic (NOD) mice, but delayed autoimmune diabetes only in the former. Interestingly, Tregs induced at 4 wk of age had significant active membrane-bound TGF-beta (mTGF-beta) and sustained protection against diabetes, whereas Tregs expanded during insulitis had minimal mTGF-beta and could not protect against diabetes. The Tregs probably operate suppressive function through mTGF-beta, because Ab blockade of mTGF-beta nullifies protection against diabetes. Surprisingly, young Tregs that modulated pathogenic T cells maintained stable frequency over time in the protected animals, but decreased their mTGF-beta at the age of 8 wk. More strikingly, these 8-wk-old mTGF-beta-negative Tregs, which were previously protective, became unable to confer resistance against diabetes. Thus, a developmental decline in active mTGF-beta nullifies Treg function, leading to a break in tolerance and the onset of diabetes.

Topics: Aging; Amino Acid Sequence; Animals; Cell Differentiation; Clone Cells; Diabetes Mellitus, Type 1; Down-Regulation; Epitopes, T-Lymphocyte; Female; Glutamate Decarboxylase; Membrane Proteins; Mice; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Molecular Sequence Data; Peptides; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Abundant evidence suggests that cytokines involve in the pathogenesis of latent autoimmune diabetes of adults (LADA). This is a slowly progressive form of type 1 diabetes, which is initially diagnosed as type 2 diabetes. In this study, healthy individuals LADA and type 2 diabetic patients were genotyped for IL-6-174G/C, TNF-alpha-308A/G, TGF-beta1-codon10T/C, TGF-beta1-codon25G/C, IL-10-1082A/G, IL-10-819T/C, IL-10-592A/C gene polymorphisms, by sequence-specific-primer polymerase chain reaction methodology. A significant difference in the frequencies of -1082A/G IL-10 alleles was observed, with the -1082*A allele (known to be associated with low IL-10 production), predominating in LADA diabetics than type 2 diabetics (p=0.036). No significant differences of genotypes, phenotypes, or haplotype frequencies in the remaining cytokine polymorphisms were observed. Analysis of allele combinations revealed a significant involvement of the low and high in vitro production IL-10 alleles in the development of LADA and type 2 diabetes, respectively. These results suggest that the G/A mutation at position -1082 of IL-10 promoter gene region might be one of the factors participating to the pathogenesis of LADA diabetes and that identification of cytokine gene polymorphisms might contribute to the characterization of the different types of diabetes mellitus.

Topics: Adult; Cytokines; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Genotype; Haplotypes; Humans; Interleukin-10; Interleukin-6; Phenotype; Polymerase Chain Reaction; Polymorphism, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Transforming growth factor-beta1 (TGF-beta1) is a profibrotic cytokine suspected to be a crucial factor underlying glomerulosclerosis in advanced diabetic nephropathy. However, its potential role as a susceptibility gene for the development of this microvascular complication is unresolved.. We examined whether DNA sequence variants in the TGF-beta1 gene are associated with advanced diabetic nephropathy among Caucasians with type 1 diabetes mellitus. These variants included three coding (Leu10Pro, Arg25Pro, and Thr263Ile) and two noncoding single-nucleotide polymorphisms (-800 and -509), as well as an insertion/deletion of a cytosine residue in intron 4. A large case-control study design was used in which cases were patients with type 1 diabetes with advanced diabetic nephropathy (presence of persistent proteinuria or end-stage renal disease [ESRD]; n = 298) and controls were patients who remained normoalbuminuric despite greater than 15 years of type 1 diabetes (n = 263).. Genotype frequencies for all polymorphisms were in Hardy-Weinberg equilibrium. Genotype distributions of all six DNA sequence variants were very similar between cases and controls (P = not significant). There was no significant difference in genotype distributions among cases regardless of whether these individuals with diabetes were proteinuric at the time of examination or had already developed ESRD secondary to diabetic nephropathy. Stratified analyses according to diabetes duration and glycemic control likewise did not detect an association between DNA sequence variants and advanced diabetic nephropathy.. Genetic variation at the TGF-beta1 locus is unlikely to confer significant susceptibility to advanced diabetic nephropathy in patients with type 1 diabetes mellitus.

Topics: Adolescent; Adult; Albuminuria; Case-Control Studies; Creatinine; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Genetic Markers; Genetic Predisposition to Disease; Genetic Variation; Genotype; Humans; Insulin; Male; Polymorphism, Genetic; Serum Albumin; Transforming Growth Factor beta; Transforming Growth Factor beta1; White People

The imbalance of Th1/Th2 subsets is an important pathogenic mechanism for insulin-dependent diabetes mellitus (IDDM). Calcitonin gene-related peptide (CGRP) has been found to play important roles in the regulation of T lymphocytes. We hypothesize that exogenous CGRP administration during insulitis may modulate the balance of Th lymphocytes, thereby providing a therapeutic intervention for IDDM. We established CGRP gene transfer by naked plasmid injection into the skeletal muscles with electroporation enhancement. The effect of CGRP gene transfer on pathogenesis of IDDM was observedin autoimmune diabetic C57BL mice induced by multiple low-dose streptozotocin (MLDS) administration. The treatment significantly decreased morbidity of diabetes, ameliorated hyperglycemia and insulin deficiency, and inhibited lymphocyte infiltration into the islets, indicating the protection of beta cells against autoimmune destruction. CGRP gene transfer significantly inhibited T cell proliferation and secretion of the Th1 cytokine IFN-gamma, increased the level of the Th2 cytokine IL-10, but had no effect on IL-4 and TGF-beta1 secretion. CGRP gene transfer also decreased IL-12 and IFN-gamma levels in peritoneal effusion. Our results demonstrate that CGRP gene transfer selectively suppresses the pro-inflammatory Th1 subsets and promote anti-inflammatory Th2 subsets, resulting in amelioration of beta cell destruction and reduction of IDDM occurrence in mice with MLDS-induced diabetes.

Topics: Animals; Aorta; Ascitic Fluid; Calcitonin Gene-Related Peptide; Cell Division; Diabetes Mellitus, Type 1; Gene Expression; Humans; Injections, Intramuscular; Interferon-gamma; Interleukin-12; Interleukin-4; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Smooth, Vascular; Rabbits; RNA, Messenger; Streptozocin; T-Lymphocytes; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transgenes

We have recently described an impaired proliferative response of CD4(+) T-cells to primary antigens in patients with insulin-dependent diabetes mellitus (IDDM) [Clin. Immunol. 103 (2002) 249]. In order to further investigate possible mechanisms underlying this impairment, several factors known to be involved in the down-regulation of the immune response both at the level of APCs and CD4(+) T-cells were investigated: Monocyte-derived dendritic cells (MDDC) from IDDM patients were shown to express elevated amounts of CD86 (B7.2) (p=0.003) and reduced amounts of the adhesion molecule CD54 (ICAM-1) (p=0.03) on their cell surface compared to age-matched healthy controls and patients with non-insulin-dependent diabetes mellitus (NIDDM) as well as decreased SDS-PAGE stability of HLA-DQ and -DR peptide complexes directly isolated from the IDDM patients' peripheral blood mononuclear cells (PBMCs). Expression of CTLA-4 (CD152), known to be involved in the down-regulation of the immune response, was shown to be increased on CD4(+) T-cells from IDDM patients after exposure to the primary antigen KLH (keyhole limpet hemocyanin) presented by MDDC (p=0.0047). Likewise, purified CD4(+) T-cells from IDDM patients produced elevated levels of the cytokine TGF-beta1 after stimulation with immobilized monoclonal antibodies directed against CD3 and CD28 (p=0.014). When monocytes from IDDM patients were stimulated with lipopolysaccharide (LPS), an increased tendency to produce the inhibitory cytokine interleukin (IL)-10 (p=0.007) and the acute phase cytokine IL-6 (p=0.044) was observed, whereas the concentrations of tumor necrosis factor (TNF)-alpha, IL-1beta, and IL-12 were comparable to controls. Taken together, our data suggest that a deviation in the expression of certain molecules known to be involved in the peripheral control of the immune response is present in IDDM patients and is underlying the observed impairment of the primary immune response.

Topics: Abatacept; Adult; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation; B7-2 Antigen; CD28 Antigens; CD3 Complex; CD4-Positive T-Lymphocytes; Cells, Cultured; CTLA-4 Antigen; Cytokines; Dendritic Cells; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Electrophoresis, Polyacrylamide Gel; Female; HLA-DQ Antigens; HLA-DR Antigens; Humans; Immunoconjugates; Intercellular Adhesion Molecule-1; Lipopolysaccharides; Lymphocyte Activation; Male; Membrane Glycoproteins; Middle Aged; Monocytes; Transforming Growth Factor beta

Activation-induced cell death (AICD) represents a major means of peripheral tolerance induction, eliminating effector cells. NOD mice, a widely used model for autoimmune diabetes, are characterized by high levels of circulating T lymphocytes and by resistance to several apoptosis-inducing signals. The aim of this study was to analyse AICD in peripheral NOD T lymphocytes. First, we demonstrated in an in vitro AICD model that NOD T lymphocytes are more resistant to AICD (64+/-2%) compared to non-autoimmune C57BL/6 T lymphocytes (73+/-2%), but also diabetes-resistant NOR T lymphocytes (76+/-3%, P<0.05). Moreover, both CD4(+)and CD8(+)subsets were affected. Analysis of the cellular and molecular pathways revealed lower caspase 8 levels, a central caspase proximally involved in the AICD-pathway (fluorescence of 258+/-47 in NOD vs. 441+/-16 in NOR and 414+/-61 in C57BL/6 T lymphocytes, P<0.05). Gene expression analysis using real-time RT-PCR additionally revealed low expression of Fas and FasL, the death receptor system activating caspase 8 and contributing to AICD. Additionally, low IL-2 levels, together with high TGFbeta and Bclx-L levels, confirm the presence of a NOD-specific AICD-resistance profile. In conclusion, we present cellular and molecular evidence for disturbed AICD mechanisms in NOD T lymphocytes. This resistance in AICD may contribute to defective tolerance induction to autoantigens in NOD mice.

Topics: Animals; Apoptosis; bcl-X Protein; Caspase 8; Caspases; Cell Survival; Diabetes Mellitus, Type 1; Fas Ligand Protein; fas Receptor; Female; Gene Expression; In Vitro Techniques; Interleukin-2; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Prediabetic State; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; T-Lymphocytes; Transforming Growth Factor beta

The present study was undertaken to analyze the regulatory T cells generated in response to class I derived self-I-A beta(g7) (54-76) peptide. It was observed T cells from young unprimed type 1 diabetes (T1D) prone NOD mice did not respond to self-I-A beta(g7) (54-76) peptide although T cells from primed young NOD mice showed a strong response. T cells from young unprimed BALB/c mice responded to self-I-A beta(d) (62-78) peptide. However, a breakdown of tolerance to these peptides was observed with age in both the strains. Culture supernatant from I-A beta(g7) (54-76) peptide-primed cells secreted large amounts of TGF-beta and inhibited T cell responses in allogeneic-MLR. Further, I-A beta(g7) (54-76) peptide specific T cell lines from young (I-A.Y) and diabetic (I-A.D) NOD mice were established. I-A.Y secreted IL-4, TGF-beta and IL-10 while I-A.D T cell line secreted IL-10 and IFN-gamma. We found that I-A.D T cell line induced diabetes when transferred in NOD/SCID mice but I-A.Y T cell line did not induce disease. These results show that immunization of NOD mice with I-A beta(g7) (54-76) peptide at a younger age induces a regulatory T cell response suggesting that correcting the defects in immunoregulatory mechanisms using self-MHC peptides may be one of the approaches to prevent autoimmune diseases like T1D.

Topics: Animals; Autoantigens; Cytokines; Diabetes Mellitus, Type 1; Female; Histocompatibility Antigens Class II; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, SCID; Peptides; Spleen; T-Lymphocytes; Transforming Growth Factor beta

CD3-specific antibodies have the unique capacity to restore self-tolerance in established autoimmunity. They induce long-term remission of overt diabetes in nonobese diabetic (NOD) mice and in human type I diabetes. The underlying mechanisms had been unclear until now. Here we report that treatment with CD3epsilon-specific antibodies induces transferable T-cell-mediated tolerance involving CD4+CD25+ cells. However, these CD4+CD25+ T cells are distinct from naturally occurring regulatory T cells that control physiological autoreactivity. CD3-specific antibody treatment induced remission in NOD Cd28-/- mice that were devoid of such regulatory cells. Remission of diabetes was abrogated by coadministration of a neutralizing transforming growth factor (TGF)-beta-specific antibody. The central role of TGF-beta was further suggested by its increased, long-lasting production by CD4+ T cells from tolerant mice. These data explain the intriguing tolerogenic effect of CD3-specific antibodies and position them as the first clinically applicable pharmacological stimulant of TGF-beta-producing regulatory CD4+ T cells.

Topics: Animals; Antibodies; Antigens, CD; Antigens, Differentiation; CD3 Complex; CD4-Positive T-Lymphocytes; CTLA-4 Antigen; Diabetes Mellitus, Type 1; Immune Tolerance; Mice; Mice, Inbred NOD; Mice, SCID; Receptors, Interleukin-2; Spleen; T-Lymphocytes; Transforming Growth Factor beta

Excretion of growth factors in the urine has been implicated in the pathogenesis of tubulointerstitial disease that characterizes proteinuric renal disease. In this cross-sectional study, we sought to examine the urinary excretion of the profibrotic cytokine connective tissue growth factor (CTGF) in type 1 diabetic patients with incipient and overt diabetic nephropathy.. We recruited 31 subjects with type 1 diabetes from a hospital diabetes outpatient clinic. Of these, 10 subjects were normoalbuminuric, 8 were microalbuminuric and not receiving ACE inhibitor treatment, and 13 were macroalbuminuric, 8 of whom were receiving ACE inhibitor treatment. Urinary CTGF NH(2)-terminal fragment (CTGF-N) was determined by enzyme-linked immunosorbent assay and expressed relative to urinary creatinine.. Urinary CTGF-N was closely correlated with the degree of albuminuria (r = 0.76, P < 0.001). In comparison with normoalbuminuric subjects, urinary CTGF-N was increased 10- and 100-fold in micro- and untreated macroalbuminuric subjects, respectively (CTGF-N-to-creatinine ratio: normoalbuminuria 0.23 x// 1.3 ng/mg, microalbuminuria 2.1 x// 1.7 ng/mg, untreated macroalbuminuria 203 x// 3.8 ng/mg, and geometric mean x// tolerance factor; P < 0.05 for normoalbuminuria versus microalbuminuria, P < 0.001 for microalbuminuria versus macroalbuminuria). Urinary CTGF-N was lower (<30-fold) in macroalbuminuric subjects treated with ACE inhibitors (6.5 x// 1.7 ng/mg; P < 0.01 vs. untreated macroalbuminuria) compared with their untreated counterparts.. In this cross-sectional study, the magnitude of urinary CTGF-N excretion was related to the severity of diabetic nephropathy. In the context of its known profibrotic actions, these findings suggest that CTGF may contribute to the chronic tubulointerstitial fibrosis that accompanies proteinuric renal disease. Prospective and interventional studies will be needed to determine whether urinary CTGF-N may provide a reliable surrogate marker of renal injury and a meaningful indicator of response to therapy.

Topics: Adult; Albuminuria; Biomarkers; Blood Pressure; Creatinine; Cross-Sectional Studies; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Glycated Hemoglobin; Humans; Male; Middle Aged; Transforming Growth Factor beta

Pancreatic lymph node-derived CD4+CD25+ T regulatory (Treg) cells inhibit in situ differentiation of islet-reactive CD8+ T cells into cytotoxic T lymphocytes, thereby preventing diabetes progression. The mechanism by which these Treg cells suppress anti-islet CD8+ T cells is unknown. Here, we show by using a CD8+ T cell-mediated model of type 1 diabetes that transforming growth factor (TGF)-beta-TGF-beta receptor signals are critical for CD4+CD25+ Treg cell regulation of autoreactive islet-specific cytotoxic T lymphocytes. Transgenic expression of tumor necrosis factor alpha from birth to 25 days of age in the islets of B6 mice that constitutively express CD80 on their beta cells results in accumulation of CD4+CD25+TGF-beta+ cells exclusively in the islets and pancreatic lymph nodes, which delays diabetes progression. In contrast, expression of tumor necrosis factor alpha until 28 days of age prevents islet accumulation of CD4+CD25+TGF-beta+ Treg cells, resulting in acceleration to diabetes. Furthermore, adoptive transfer experiments demonstrated that CD4+CD25+ Treg cells could not control naïve or activated islet-reactive CD8+ T cells bearing a dominant negative TGF-beta receptor type II. Our data demonstrate that, in vivo, TGF-beta signaling in CD8+ T cells is critical for CD4+CD25+ Treg cell suppression of islet-reactive CD8+ T cells in type 1 diabetes.

Topics: Adoptive Transfer; Animals; CD4 Antigens; CD8-Positive T-Lymphocytes; Diabetes Mellitus, Type 1; Islets of Langerhans; Mice; Mice, Inbred C57BL; Pancreas; Receptors, Interleukin-2; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

NOD mice have a relative deficiency of CD4+CD25+ regulatory T cells that could result in an inability to maintain peripheral tolerance. The aim of this study was to induce the generation of CD4+CD25+ regulatory T cells in response to autoantigens to prevent type 1 diabetes (T1D). We found that immunization of NOD mice with insulin B-chain peptide B:9-23 followed by 72 h in vitro culture with B:9-23 peptide induces generation of CD4+CD25+ regulatory T cells. Route of immunization has a critical role in the generation of these cells. Non-autoimmune mice BALB/c, C57BL/6 and NOR did not show up regulation of CD4+CD25+ regulatory T cells. These cells secreted large amounts of TGF-beta and TNF-alpha with little or no IFN-gamma and IL-10. Adoptive transfer of these CD4+CD25+ regulatory T cells into NOD-SCID mice completely prevented the adoptive transfer of disease by diabetogenic T cells. Although, non-self antigenic OVA (323-339) peptide immunization and in vitro culture with OVA (323-339) peptide does result in up regulation of CD4+CD25+ T cells, these cells did not prevent transfer of diabetes. Our study for the first time identified the generation of antigen-specific CD4+CD25+ regulatory T cells specifically in response to immunization with B:9-23 peptide in NOD mice that are capable of blocking adoptive transfer of diabetes. Our results suggest the possibility of using autoantigens to induce antigen-specific regulatory T cells to prevent and regulate autoimmune diabetes.

Topics: Adoptive Transfer; Animals; Antibodies, Monoclonal; Antigen Presentation; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; CD3 Complex; CD4 Antigens; Coculture Techniques; Diabetes Mellitus, Type 1; Drug Administration Routes; Female; Glycosuria; Immune Tolerance; Insulin; Interferon-gamma; Interleukin-10; Islets of Langerhans; L-Selectin; Lectins, C-Type; Leukocyte Common Antigens; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; Ovalbumin; Peptide Fragments; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Receptors, Interleukin-2; Spleen; T-Lymphocytes; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vaccination

Transforming growth factor-beta (TGF-beta) may be critical in the development of diabetic nephropathy (DN), and genetic predisposition is an important determinant of DN risk. We evaluated mRNA expression levels of TGF-beta system components in cultured skin fibroblasts (SFs) from type 1 diabetic patients with fast versus slow development of DN. A total of 125 long-standing type 1 diabetic patients were ranked by renal mesangial expansion score (MES) based on renal biopsy findings and diabetes duration. Patients in the highest quintile of MES who were also microalbuminuric or proteinuric (n = 16) were classified as "fast-track" for DN, while those in the lowest quintile who were also normoalbuminuric (n = 23) were classsified as "slow-track" for DN. Twenty-five normal subjects served as control subjects. SFs were cultured in medium with 25 mmol/l glucose for 36 h. SF mRNA expression levels for TGF-beta1, TGF-beta type II receptor (TGF-beta RII), thrombospondin-1, and latent TGF-beta binding protein-1 (LTBP-1) were measured by real-time RT-PCR. LTBP-1 mRNA expression was reduced in slow-track (0.99 +/- 0.38) versus fast-track patients (1.65 +/- 0.52, P = 0.001) and control subjects (1.41 +/- 0.7, P = 0.025). mRNA levels for TGF-beta1, TGF-beta RII, and thrombospondin-1 were similar in the three groups. Reduced LTBP-1 mRNA expression in SFs from slow-track patients may reflect genetically determined DN protection and suggests that LTBP-1 may be involved in the pathogenesis of DN through the regulation of TGF-beta activity.

Topics: Adult; Albuminuria; Carrier Proteins; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disease Progression; Female; Fibroblasts; Glomerular Mesangium; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Latent TGF-beta Binding Proteins; Male; Middle Aged; Protein Serine-Threonine Kinases; Proteinuria; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reference Values; RNA, Messenger; Skin; Thrombospondin 1; Time Factors; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta1 is overexpressed in diabetes as a consequence of hyperglycemia and the creation of early glycated end products and may be responsible for the characteristic structural renal changes associated with diabetes. We sought to examine the role of both urinary and circulating TGF-beta1 and its promoter Amadori albumin in the vascular complications of type 1 diabetes.. The present article reports on a nested case-control study from the EURODIAB Prospective Complications Study of Europeans with type 1 diabetes. Case subjects (n = 356) were all individuals with one or more complications of diabetes; control subjects (n = 185) were all individuals with no evidence of complications.. Urinary TGF-beta1 and Amadori albumin were elevated in patients with micro- or macroalbuminuria. Standardized regression effects (SREs) for macroalbuminuria versus normoalbuminuria were 2.45 (95% CI 1.88-3.18, P = 0.0001 for urinary TGF-beta1) and 1.67 (1.34-2.07, P = 0.001 for Amadori albumin). The SRE for urinary TGF-beta1 remained statistically significant when adjusted for HbA(1c), Amadori albumin, and blood pressure. Circulating TGF-beta1 was elevated in individuals with proliferative retinopathy compared with individuals without retinopathy (SRE 1.29 [1.07-1.550], P = 0.007). This result was attenuated to 1.16 (0.95-1.43, P = 0.2) in the multivariate model, largely because of HbA(1c).. Elevated levels of urinary TGF-beta1 in macroalbuminuria were associated with elevations in Amadori albumin and HbA(1c) and also in blood pressure. In contrast, only circulating TGF-beta1 was related to proliferative retinopathy, and HbA(1c) largely accounted for this. These findings may indicate novel pathways for understanding mechanisms and therapeutic interventions.

Topics: Adult; Albuminuria; Blood Pressure; Body Constitution; Case-Control Studies; Diabetes Mellitus, Type 1; Glycated Hemoglobin; Humans; Lipids; Reference Values; Serum Albumin; Transforming Growth Factor beta; Transforming Growth Factor beta1

To examine humoral and mucosal immune responses to food antigens and their relation to the pathophysiology of type 1 diabetes mellitus, IgA and IgG antibodies to cow's milk antigens (bovine serum albumin (BSA) and beta-lactoglobulin (BLG)) and another food antigen (ovalbumin, (OVA)) in human serum were assessed by enzyme-linked immunosorbent assay (ELISA). If anti-idiotype antibodies to the antibodies were present in serum, they might interfere with the ELISA assay, so suitable microtiter plates were employed to minimize such interference. The levels of IgA and IgG antibodies to the above antigens (P<0.001-P<0.01) and the prevalence of positive sera (P<0.001-P<0.05) in the patient group (n=52, aged 14.5+/-4.1 (S.D.) years) were significantly higher than those in the control group (n=41, aged 13.3+/-6.8 (S.D.) years). Interestingly, the levels of IgA antibodies to all the food antigens examined were elevated in 26 (50%) patients, while the elevation was seen in 3 (7%) healthy controls. The elevation of IgA antibodies in the patients was well correlated with increased concentrations of IgA and transforming growth factor (TGF)-beta, which induces IgA-producing B-cells, in serum. Although the cytokine TGF-beta is secreted from regulatory T-cells (Th3), and is related to oral tolerance, the interleukin-2 (IL-2, Th1)/IL-4 (Th2) ratio in the patient group was significantly elevated (P<0.001), which might indicate that the oral tolerance is impaired in patients. Thus, we demonstrated that both IgA and IgG antibodies to several food antigens are elevated in patients. We suggest that impairment of oral tolerance might be related to the pathogenesis of type 1 diabetes mellitus.

Topics: Adolescent; Animals; Asian People; Cattle; Diabetes Mellitus, Type 1; Female; Food; Humans; Immunoglobulin A; Immunoglobulin G; Japan; Male; Milk; Reference Values; Serum Albumin, Bovine; Transforming Growth Factor beta

Numerous immunostimulatory protocols inhibit the development of T cell-mediated autoimmune insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse model. Many of these protocols, including treatment with the nonspecific immunostimulatory agents CFA or bacillus Calmette-Guérin (BCG) vaccine, have been reported to mediate protection by skewing the pattern of cytokines produced by pancreatic beta-cell autoreactive T cells from a Th1 (IFN-gamma) to a Th2 (IL-4 and IL-10) profile. However, most of these studies have documented associations between such cytokine shifts and disease protection rather than a cause/effect relationship. To partially address this issue we produced NOD mice genetically deficient in IFN-gamma, IL-4, or IL-10. Elimination of any of these cytokines did not significantly alter the rate of spontaneous IDDM development. Additional experiments using these mice confirmed that CFA- or BCG-elicited diabetes protection is associated with a decreased IFN-gamma to IL-4 mRNA ratio within T cell-infiltrated pancreatic islets, but this is a secondary consequence rather than the cause of disease resistance. Unexpectedly, we also found that the ability of BCG and, to a lesser extent, CFA to inhibit IDDM development in standard NOD mice is actually dependent upon the presence of the Th1 cytokine, IFN-gamma. Collectively, our studies demonstrate that while Th1 and Th2 cytokine shifts may occur among beta-cell autoreactive T cells of NOD mice protected from overt IDDM by various immunomodulatory therapies, it cannot automatically be assumed that this is the cause of their disease resistance.

Topics: Adjuvants, Immunologic; Animals; BCG Vaccine; Cells, Cultured; Cytokines; Diabetes Mellitus, Type 1; Female; Freund's Adjuvant; Gene Deletion; Immunity, Innate; Injections, Subcutaneous; Interferon-gamma; Interleukin-10; Interleukin-4; Islets of Langerhans; Lymphocyte Activation; Male; Mice; Mice, Inbred NOD; Mice, Knockout; Muromonab-CD3; Receptors, Antigen, T-Cell; RNA, Messenger; Th1 Cells; Th2 Cells; Transforming Growth Factor beta

To study transforming growth factor (TGF)-beta1 secretion by peripheral blood mononuclear cells (PBMC) from Type 1 diabetic patients with and without nephropathy.. Thirty normoalbuminuric Type 1 diabetic patients (urinary albumin excretion rate (AER) < 20 microg/min), 12 microalbuminuric (AER 20-200 microg/min), 10 nephropathic (AER > 200 microg/min), and 13 non-diabetic individuals were recruited. TGF-beta1 secretion by PBMC was measured by enzyme immunoassay (EIA) after 48 h culture with and without phytohaemagglutinin (PHA) (5 microg/ml).. After 48 h culture, the highest TGF-beta1 levels secreted by unstimulated PBMC were found in patients with nephropathy (median 6.2 (range 0.9-20.0) ng/ml) when compared to patients with normal albumin excretion (4.1 (0.2-11.3) ng/ml), microalbuminuria (1.8 (0.2-6.4) ng/ml) and healthy controls (1.0 (0.2-7.0) ng/ml); P = 0.02 for the three diabetic groups and P = 0.006 for all groups. At 48 h, the PHA-stimulated TGF-beta1 levels were 12.4 (2.9-30.0) ng/ml in nephropathic, 7.3 (0.5-21.2) ng/ml in normoalbuminuric, and 5.5 (0.5-27.6) ng/ml in microalbuminuric patients (P = 0.05). A correlation was observed between TGF-beta1 and diastolic blood pressure in the subgroup of patients with incipient and overt nephropathy (r = 0.45, P = 0.04).. Type 1 diabetic patients with overt nephropathy show increased TGF-beta1 secretion by PBMC. Diastolic blood pressure levels correlated with TGF-beta1 secretion in diabetic patients with nephropathy. Increased TGF-beta1 secretion by PBMC may be associated with renal and vascular disease in Type 1 diabetes mellitus.

Topics: Adult; Albuminuria; Biomarkers; Blood Pressure; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Humans; Immunoenzyme Techniques; Lymphocyte Activation; Lymphocytes; Male; Reference Values; Transforming Growth Factor beta

Genetic background of the fetus contributes to the pathogenesis of congenital malformation after teratogen exposure. Such contribution is illustrated in left-right axis malformations observed in the F1 offspring of nonobese diabetic (NOD) mouse dams and sires from different strains. When sires of the NOD, ICR, or C57BL/6J were mated with NOD dams, incidence varied depending on the fetal genotype, with 65% in NOD x NOD, 24% in NOD x ICR, and 7% in NOD x C57BL/6J.. As a first step in elucidating the molecular basis of the interstrain differences in susceptibility to situs defects, we compared genomic sequences of six genes HNF3beta, Acvr2b, Nodal, ZIC3, Lefty1, and Smad2, which are involved in the normal development of left-right axis among NOD, ICR, and C57BL/6J strains.. The outbred strain ICR had 1) a 0.2-kb insertion in the putative promoter region of the isoform E of HNF3beta together with a G to A change that could create a potential splice acceptor in the exon 3 of HNF3beta (gene frequency P = 0.36), 2) five single base substitutions within the 5' controlling element and a proline to serine substitution (P2S) of Lefty1 (P = 0.77), and 3) a tyrosine to histidine substitution within the prodomain of Nodal (P = 0.48). The inbred strain NOD had the same G to A change as ICR and a three-base deletion in the putative promoter of isoform E of HNF3beta.. We suggest that sequence variations in HNF3beta, Lefty1, and Nodal might account, in part, for the interstrain differences in susceptibility to situs abnormalities among the offspring of diabetic dams.

Topics: Animals; Diabetes Mellitus, Type 1; Diabetes, Gestational; DNA Mutational Analysis; DNA Primers; DNA-Binding Proteins; Female; Genetic Variation; Hepatocyte Nuclear Factor 3-beta; Homeodomain Proteins; Left-Right Determination Factors; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Inbred NOD; Multigene Family; Mutation; Nodal Protein; Nuclear Proteins; Pregnancy; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Situs Inversus; Smad2 Protein; Trans-Activators; Transcription Factors; Transforming Growth Factor beta

The aim of this study was to evaluate possible changes in the circulating levels of interferon (IFN)-gamma, interleukin (IL)-4 and transforming growth factor (TGF)-beta in association with the autoimmune process leading to type 1 diabetes. Expression levels of mRNAs specific for each cytokine were determined in peripheral blood mononuclear cells (PBMC) by a multiplex reverse transcription-polymerase chain reaction (RT-PCR) followed by hybridization reactions with lanthanide-labelled probes and detection by time-resolved fluorometry. Newly diagnosed diabetic children had lower levels of IFN-gamma, IL-4 and TGF-beta 1 signals compared to their age- and sex-matched controls (P < 0.02, P < 0.005 and P < 0.005, respectively) and also the autoantibody-positive subjects had significantly lower levels of IL-4 and TGF-beta 1 in comparison with their matched controls (P = 0.0013 and P = 0.012). No significant differences were observed when comparing matched pairs of diabetic children and autoantibody-positive subjects. Our results suggest a systemic bias towards reduced production of T-helper cell type 2 cytokines (IL-4 and TGF-beta 1) during the autoimmune process, but there was also a reduced level of IFN-gamma expression in the periphery at the onset of clinical diabetes.

Topics: Autoantibodies; Child; Child, Preschool; Cytokines; Diabetes Mellitus, Type 1; Female; Humans; Infant; Interferon-gamma; Interleukin-4; Leukocytes, Mononuclear; Male; RNA, Messenger; T-Lymphocytes; Transforming Growth Factor beta

B cells can serve dual roles in modulating T cell immunity through their potent capacity to present Ag and induce regulatory tolerance. Although B cells are necessary components for the initiation of spontaneous T cell autoimmunity to beta cell Ags in nonobese diabetic (NOD) mice, the role of activated B cells in the autoimmune process is poorly understood. In this study, we show that LPS-activated B cells, but not control B cells, express Fas ligand and secrete TGF-beta. Coincubation of diabetogenic T cells with activated B cells in vitro leads to the apoptosis of both T and B lymphocytes. Transfusion of activated B cells, but not control B cells, into prediabetic NOD mice inhibited spontaneous Th1 autoimmunity, but did not promote Th2 responses to beta cell autoantigens. Furthermore, this treatment induced mononuclear cell apoptosis predominantly in the spleen and temporarily impaired the activity of APCs. Cotransfer of activated B cells with diabetogenic splenic T cells prevented the adoptive transfer of type I diabetes mellitus (T1DM) to NOD/scid mice. Importantly, whereas 90% of NOD mice treated with control B cells developed T1DM within 27 wk, <20% of the NOD mice treated with activated B cells became hyperglycemic up to 1 year of age. Our data suggest that activated B cells can down-regulate pathogenic Th1 immunity through triggering the apoptosis of Th1 cells and/or inhibition of APC activity by the secretion of TGF-beta. These findings provide new insights into T-B cell interactions and may aid in the design of new therapies for human T1DM.

Topics: Adoptive Transfer; Animals; Antigen-Presenting Cells; Apoptosis; Autoantigens; B-Lymphocyte Subsets; Cell Communication; Cell Separation; Diabetes Mellitus, Type 1; Down-Regulation; Fas Ligand Protein; fas Receptor; Female; Islets of Langerhans; Leukocytes, Mononuclear; Ligands; Lipopolysaccharides; Lymphocyte Activation; Lymphocyte Transfusion; Membrane Glycoproteins; Mice; Mice, Inbred NOD; Mice, SCID; Prediabetic State; Spleen; T-Lymphocyte Subsets; Th1 Cells; Transforming Growth Factor beta

The alterations of TGF-beta1 production are believed to contribute to the development of insulin-dependent diabetes mellitus (IDDM) in animal models as well as in humans. There is also increasing evidence about the role of this cytokine in the pathogenesis of diabetic vascular complications. The aim of our study was to evaluate in vitro TGF-beta1 production by peripheral blood of newly diagnosed type 1 diabetes patients and subjects in the pre-clinical stage of the disease in comparison to healthy controls and relatives of IDDM patients with low genetic risk for diabetes development. The study was carried out in three groups of subjects: 22 patients with a recently diagnosed type 1 diabetes, their 24 first degree relatives with a different genetic risk of IDDM development and 18 healthy volunteers (control group). In all studied groups whole blood was taken for morphology parameters. HbA1C and for 72 h cultures with PHA stimulation for the estimation of TGF-beta1 in vitro production. TGF-beta1 concentration in supernatants were quantified by ELISA. In the first degree relatives HLA typing (for DR3, DR4 and DQB1*0602 alleles), measurements of anti-pancreatic antibodies (ICA, GADA, IA-2A, IAA) and intravenous glucose tolerance tests were performed. The levels of TGF-beta1 in the supernatants were significantly higher in diabetic patients (P < 0.0002) and in their first degree relatives (P < 0.05) in comparison to the control group. In the group of first degree relatives TGF-beta1 levels were highest in subjects with the presence of two or more pancreatic autoantibodies and/or with impaired insulin release in IVGTT, but lowest in relatives with protective DQB1*0602 alleles (P < 0.01). There was also a significant positive correlation between the TGF-beta1 levels and HbA1C in the IDDM subjects and first degree relatives (P < 0.03). Our study suggests that the alterations of TGF-beta1 levels could be associated with the activity of autoimmune process leading to pancreatic B cells destruction and may have a role in the pathogenesis of diabetic complications, but further studies in humans are needed.

Topics: Adolescent; Adult; Animals; Autoantibodies; Case-Control Studies; Diabetes Mellitus, Type 1; Female; Glucose Tolerance Test; HLA-D Antigens; Humans; In Vitro Techniques; Male; Pancreas; Prediabetic State; Risk Factors; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta1 is an important mediator in the pathogenesis of diabetic nephropathy. Urinary TGF-beta1 reflects TGF-beta1 production in the kidney, and alpha1-microglobulin tubular dysfunction. These 2 markers were studied in the early phases of type 1 diabetes.. There were 113 type 1 diabetic children and adolescents (mean +/- SD: age 14.1 +/- 2.9 years, and diabetes duration 7.4 +/- 2.9 years, HbA1c 9.3 +/- 1.5%) and 39 healthy subjects (age 13.8 +/- 2.8 years) who participated in the study. Of the diabetic patients, 105 were normoalbuminuric (2-3 consecutive overnight urinary albumin excretion rates [AERs] <20 microg/min) and 8 had microalbuminuria (at least 2 AERs 20-200 microg/min). Overnight urinary TGF-beta1 and alpha1-microglobulin levels were measured and the results expressed as the ratio to urinary creatinine concentration.. Data are medians (range). Diabetic patients had higher urinary TGF-beta1 levels than those of control subjects: 0.9 ng/mg (0.05-122.3) vs. 0.3 ng/mg (0.05-2.2) creatinine, respectively (P = 0.003). Urinary TGF-beta1 levels correlated with urinary glucose (r = 0.2, P = 0.03) and alpha1-microglobulin (r = 0.2, P = 0.02) levels, but not with HbA1c, AER, age, or duration of diabetes. In 43 patients with urinary TGF-beta1 above the control levels, urinary TGF-beta1 levels correlated with urinary glucose (r = 0.6, P < 0.001) and alpha1-microglobulin (r = 0.6, P < 0.001) levels. Diabetic patients had higher urinary alpha1-microglobulin levels than those of control subjects: 4.8 microg/mg (0.6-48.8) vs. 2.7 microg/mg (0.8-11.6) creatinine, respectively (P < 0.001). Alpha1-microglobulin levels correlated with AER (r = 0.2, P = 0.02), HbA1c (r = 0.3, P = 0.001), urinary glucose (r = 0.5, P < 0.001), and urinary TGF-beta1 levels.. An early rise in urinary TGF-beta1 levels was observed in young type 1 diabetic patients. Urinary TGF-beta1 is associated with 2 interrelated tubular markers, alpha1-microglobulin and urinary glucose.

Topics: Adolescent; Adult; Albuminuria; Child; Creatinine; Diabetes Mellitus, Type 1; Female; Glycoproteins; Glycosuria; Humans; Male; Membrane Glycoproteins; Reference Values; Serine Proteinase Inhibitors; Transforming Growth Factor beta; Trypsin Inhibitor, Kunitz Soybean

The recently discovered arachidonic acid derivatives, isoprostanes, are increased in pathological conditions associated with oxidative stress, such as diabetes. No role has yet been described for isoprostanes during the development of diabetic nephropathy. Cell culture in high ambient glucose has been used as a model in elucidating cellular mechanisms underlying diabetic nephropathy. Among the growth factors involved in the effect of high glucose, transforming growth factor-beta (TGF-beta) has been described as playing a key role in the development of nephropathy.. Streptozotocin-induced diabetic rats were supplemented in their diet with the antioxidant vitamin E (1000 U/kg diet). Blood and urine samples were taken to determine renal function and isoprostane concentration, as determined by gas chromatography/mass spectrometry. Glomerular mesangial and endothelial cells were cultured in high ambient glucose to determine the synthesis of isoprostanes and the role of isoprostanes in high glucose-induced synthesis of TGF-beta.. Streptozotocin-induced diabetic rats had marked increases in plasma levels and urinary excretion rates of F(2)-isoprostanes. Dietary supplementation with vitamin E normalized (plasma) and reduced (urine) isoprostane levels and, surprisingly, improved proteinuria and blood urea nitrogen (BUN) levels. High ambient glucose increased F(2)-isoprostane synthesis in glomerular endothelial and mesangial cells in culture. Incubation of glomerular cells with F(2)-isoprostanes stimulated the production of TGF-beta.. Increased F(2)-isoprostane synthesis during diabetes appears to be responsible in part for the increase in renal TGF-beta, a well-known mediator of diabetic nephropathy.

Topics: Animals; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Dinoprost; Endothelium; F2-Isoprostanes; Glomerular Mesangium; Glucose; Kidney Glomerulus; Male; Mice; Mice, Inbred Strains; Proteinuria; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

Testicular Sertoli cells protect pancreatic islet grafts from allo- and autoimmune destruction; however, the mechanism(s) of protection is unclear. The aim of this study was to determine whether Fas ligand (FasL) and/or transforming growth factor (TGF)-beta, immunoregulatory proteins produced by Sertoli cells, might mediate the protective effects of these cells against autoimmune destruction of islet beta-cells. Sertoli cells were purified from testes of NOD mice and implanted under the right renal capsule of diabetic NOD mice, whereas NOD islets were implanted under the left renal capsule. Of the mice that received islet and Sertoli cells grafts, 64% (9 of 14) remained normoglycemic at 60 days posttransplantation compared with 0% (0 of 6) of the mice that received islet grafts alone. Immunohistochemical examination of Sertoli cell grafts in normoglycemic mice revealed that TGF-beta1 expression by Sertoli cells remained high, whereas FasL expression by Sertoli cells decreased progressively posttransplantation. Also, plasma levels of TGF-beta1 were significantly elevated in mice that received Sertoli cells and islet grafts, and anti-TGF-beta1 antibody administration completely abrogated the protective effect of Sertoli cells on islet graft survival, whereas anti-FasL antibody did not. Islet graft destruction in anti-TGF-beta1-treated mice was associated with increases in interferon (IFN)-gamma-producing cells and decreases in interleukin (IL)-4-producing cells in the islet grafts. We conclude that 1) Sertoli cell production of TGF-beta1, not FasL, protects islet beta-cells from autoimmune destruction and 2) TGF-beta1 diverts islet-infiltrating cells from a beta-cell-destructive (IFN-gamma+) phenotype to a nondestructive (IL-4+) phenotype.

Topics: Animals; Antibodies, Monoclonal; Autoimmunity; Diabetes Mellitus, Type 1; Fas Ligand Protein; Graft Survival; Immunohistochemistry; Islets of Langerhans; Islets of Langerhans Transplantation; Kidney; Male; Membrane Glycoproteins; Mice; Mice, Inbred NOD; Sertoli Cells; Testis; Transforming Growth Factor beta

Early graft failure, graft rejection, and autoimmune recurrence remain unresolved issues in islet xenotransplantation in type 1 diabetes. The first aim of this study was to examine the existence of early graft failure in spontaneously diabetic autoimmune NOD mice after rat islet transplantation under technically controlled circumstances. The second aim was to examine the mediators of this early xenograft dysfunction. First, we demonstrated a higher percentage of early xenograft failure (48%) in spontaneously diabetic NOD mice as compared with chemically diabetic old NOD (13%, P < 0.05) and C57Bl/6 (7%, P < 0.01) mice. In addition, in spontaneously diabetic NOD mice, xenogeneic islets displayed early graft failure more frequently than allogeneic (23%, P < or = 0.05) or isogeneic islets (7%, P < 0.01). No early graft failure was observed in allotransplantation or isotransplantation in chemically diabetic mice. Reverse transcriptase-polymerase chain reaction analysis of cytokine mRNA in islet xenografts 8 h after transplantation showed higher levels of interleukin (IL)-1 mRNA in autoimmune diabetic mice compared with chemically diabetic old NOD mice (1.40 +/- 0.32 vs. 0.90 +/- 0.14 IL-1 copies/beta-actin copies, P < 0.05). In contrast, mRNA levels of transforming growth factor (TGF)-beta were lower in spontaneously diabetic NOD mice than in chemically diabetic old NOD mice (0.67 +/- 0.16 vs. 1.36 +/- 0.50 TGF-beta copies/beta-actin copies, P < 0.05). No differences in tumor necrosis factor-alpha, IL-6, and inducible nitric oxide synthase were seen between autoimmune and nonautoimmune diabetic mice. T-cell cytokines (IL-2, IL-4, IL-10, and gamma-interferon) were absent in all mice until 48 h after transplantation. These data suggest that early islet xenograft failure is more common in spontaneously diabetic NOD mice and could be due to a nonspecific inflammatory reaction locally in the grafts.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Female; Graft Rejection; Interleukin-1; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Rats; RNA, Messenger; Time Factors; Transforming Growth Factor beta; Transplantation, Heterologous; Transplantation, Homologous; Transplantation, Isogeneic

Topics: Animals; Apoptosis; Arthritis, Experimental; Arthritis, Rheumatoid; Autoimmune Diseases; Celiac Disease; Diabetes Mellitus, Type 1; Genetic Predisposition to Disease; Humans; Interleukin-10; Mice; Mice, Inbred MRL lpr; Mice, Knockout; Multiple Sclerosis; Risk Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Genetic susceptibility and resistance to most autoimmune disorders are associated with highly polymorphic genes of the MHC and with non-MHC-linked polygenic modifiers. It is known that non-MHC-linked polymorphisms can override or enhance the susceptibility to an autoimmune disease provided by pathogenic MHC genes, but the mechanisms remain elusive. In this study, we have followed the fate of two highly diabetogenic beta cell-specific T cell receptors (Kd and I-Ag7 restricted, respectively) in NOR/Lt mice, which are resistant to autoimmune diabetes despite expressing two copies of the diabetogenic MHC haplotype H-2g7. We show that at least two mechanisms of non-MHC-linked control of pathogenic T cells operate in these mice. One segregates as a recessive trait and is associated with a reduction in the peripheral frequency of diabetogenic CD8+ (but not CD4+) T cells. The other segregates as a dominant trait and is mediated by IL-4- and TGF-beta1-independent immune suppressive functions provided by lymphocytes that target diabetogenic CD4+ and CD8+ T cells, without causing their deletion, anergy, immune deviation, or ignorance. These results provide explanations as to how non-MHC-linked polymorphisms can override the susceptibility to an autoimmune disease provided by pathogenic MHC haplotypes, and demonstrate that protective non-MHC-linked genes may selectively target specific lymphoid cell types in cellularly complex autoimmune responses.

Topics: Amino Acid Sequence; Animals; CD8-Positive T-Lymphocytes; Cell Movement; Clonal Anergy; Clonal Deletion; Crosses, Genetic; Diabetes Mellitus, Type 1; Female; Genes, T-Cell Receptor beta; Immune Tolerance; Immunity, Innate; Interleukin-4; Islets of Langerhans; Lymphocyte Count; Major Histocompatibility Complex; Male; Mice; Mice, Inbred C57BL; Mice, Inbred MRL lpr; Mice, Inbred NOD; Mice, Transgenic; Molecular Sequence Data; Receptors, Antigen, T-Cell, alpha-beta; Transforming Growth Factor beta

The impact of exposure to lead on gut cytokine gene expression and oral tolerance was analyzed. Oral tolerization with ovalbumin (OVA) increased levels of IL-10 and TGF-beta in gut tissue while IFN-gamma mRNA levels remained unchanged in both autoimmune diabetes prone NOD and normal C57BL/6 mice. This shift towards Th2/Th3 type cytokine gene expression was completely abolished by concomitant treatment with PbCl2 (6 x 0.5 mg/kg) in NOD mice while the cytokine balance in C57BL/6 mice was unaffected. Suppression of Th2/Th3 type cytokine expression was associated with a dampened oral tolerance response to OVA as determined by T cell proliferation assays. We conclude that in autoimmunity prone NOD mice environmental toxicants may disturb immune homeostasis by targeting the gut immune system.

Topics: Administration, Oral; Animals; Antigens; Cytokines; Diabetes Mellitus, Type 1; Female; Gene Expression; Immune Tolerance; Intestine, Small; Lead; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Ovalbumin; RNA, Messenger; Spleen; T-Lymphocyte Subsets; Transforming Growth Factor beta

Insulin-dependent or Type 1 diabetes mellitus (IDDM) has been associated with an increased severity of periodontal disease. Because periodontal ligament (PDL) cells play a significant role in maintenance and regeneration of mineralized tissue, the success of procedures, such as guided tissue regeneration, is directly related to the ability of these cells to augment mineralized tissue. The objective of this study was to examine the ability of PDL cells from long-standing IDDM patients to form mineralized tissue and to determine whether these cells would exhibit altered responses to exogenously added growth factors.. PDL cells were isolated from 4 patients with IDDM treated with insulin for at least 5 years and from systemically healthy donors. The cell isolates were tested for their ability to form mineralized nodules in vitro and to express alterations in alkaline phosphatase activity in response to exogenously added growth factors (transforming growth factor-beta (TGF-beta), platelet-derived growth factor-BB (PDGF-BB), and insulin-like growth factor-1 (IGF-1). Alkaline phosphatase activity was determined spectrophotometrically.. Although all PDL cell isolates formed mineralized nodules in vitro, PDL cells from diabetics formed mineralized nodules more slowly than did the controls. Alkaline phosphatase activity was not altered by exposure of diabetic PDL cells to TGF-beta for 9 days. In contrast, non-diabetic isolates exhibited increased levels of activity with increasing concentrations, from 0.5 to 1.0 ng/ml. Alkaline phosphatase activity was significantly higher in non-diabetic, but not in diabetic, cell isolates exposed to TGF-beta at 1.0 ng/ml, when compared to non-treated controls. Diabetic cell isolates exhibited significantly lower alkaline phosphatase activity than the non-diabetic isolates when exposed to either TGF-beta, PDGF-BB, IGF-1 or a combination of PDGF-BB and IGF-1.. These results suggest that the populations of PDL cells in insulin-dependent diabetics may be altered in their ability to form mineralized tissue and to respond to growth factors, functions affecting the maintenance and regeneration of the periodontium.

Topics: Adult; Alkaline Phosphatase; Becaplermin; Calcification, Physiologic; Cells, Cultured; Diabetes Mellitus, Type 1; Female; Growth Substances; Humans; Insulin-Like Growth Factor I; Male; Middle Aged; Periodontal Ligament; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Recombinant Proteins; Spectrophotometry; Time Factors; Transforming Growth Factor beta

In the present study, we examined the effect of glucose concentration on the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta (TGF-beta) mRNA using reverse transcriptase-polymerase chain reaction (RT-betaCR) in normal healthy leukocytes in vitro and in leukocytes from patients with type 1 diabetes mellitus. In vitro, the level of TGF-beta mRNA was altered in response to the glucose concentration (maximum at 10 mmol/L), while bFGF mRNA remained relatively constant and VEGF mRNA varied with no clear correlation with the glucose concentration. Leukocytes from type 1 patients showed no difference in bFGF or TGF-beta mRNA levels compared with age-matched healthy controls. However, VEGF mRNA was significantly lower in type 1 patients compared with controls (P < .05). When the patients were subtyped according to the severity of retinopathy, the level of TGF-beta mRNA was elevated selectively in patients with evidence of active new retinal vessels (P < .01) and VEGF121 mRNA was reduced in patients with mild to moderate retinopathy. Thus, leukocyte growth factor mRNAs respond to acute changes in the glucose concentration in vitro, and are differentially expressed in type 1 diabetic patients during the course of the disease.

Topics: Adult; Diabetes Mellitus, Type 1; Diabetic Retinopathy; Electrophoresis, Agar Gel; Endothelial Growth Factors; Female; Fibroblast Growth Factor 2; Gene Expression Regulation; Glucose; Glycated Hemoglobin; Humans; Leukocytes; Lymphokines; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Adult; Case-Control Studies; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Female; Humans; Male; Smoking; Transforming Growth Factor beta

Oral administration of antigens has been proposed in the prevention and treatment of autoimmune diseases. We reported that oral administration of 0.8 mg of recombinant human insulin to 6-week-old NOD mice every other day for a month generated regulatory T-cells that were able to reduce the severity of insulitis and the percentage of clinical diabetes in naive irradiated recipients when co-injected with diabetogenic T-cells. In the present study, immunohistochemical analysis of the pancreatic glands revealed that injection of T-cells from insulin-fed mice upregulated the number of interleukin (IL)-4-secreting cells within the islets. Using two strains of NOD mice congenic at the Tbeta, or Thy1, locus, we observed a higher proportion of T-cells from insulin-fed mice in both the spleen (7.73 +/- 0.3 vs. 5.57 +/- 0.2%; P < 0.001) and the pancreatic lymph nodes (10.1 +/- 0.8 vs. 7.2 +/- 0.7%; P < 0.05) of cotransferred mice. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, mice reconstituted with T-cells from insulin-fed animals had detectable amounts of IL-4 mRNA, specifically in the pancreatic lymph nodes (8 of 9 experimental mice vs. 1 of 9 control mice) and the pancreas (3 of 3 experimental mice vs. 0 of 3 control mice). Gamma-interferon mRNA was detectable in all cotransferred animals, but IL-10 mRNA and transforming growth factor beta mRNA were undetectable. These results suggested a shift from a T-helper 1 (Th1) to a Th2 pattern of cytokine expression and underlined the role of pancreatic lymph nodes in the protection. Repeated injections of 500 microg s.c. of anti-IL-4 monoclonal antibody led to an accentuation of the severity of islet infiltration and to the development of clinical diabetes. We concluded that oral administration of insulin can induce the presence of regulatory T-cells in the pancreas and the corresponding draining lymph nodes, initiate the secretion of IL-4 in this microenvironment sufficiently to suppress the activity of Th1 autoreactive T-cell clones, and ultimately provide protection against autoimmune diabetes.

Topics: Administration, Oral; Animals; Cytokines; Diabetes Mellitus, Type 1; Female; Humans; Immunohistochemistry; Insulin; Interferon-gamma; Interleukin-4; Lymph Nodes; Mice; Mice, Inbred NOD; Pancreas; Polymerase Chain Reaction; Recombinant Proteins; RNA, Messenger; T-Lymphocytes; Transforming Growth Factor beta

Previous studies have shown that cultured skin fibroblasts (SFs) from insulin-dependent diabetic mellitus (IDDM) patients with diabetic nephropathy (DN) exhibit both increased proliferation and Na+/H+ antiporter activity. The present study correlated the growth rate and mRNA expression of integrin subunits, extracellular matrix molecules, and transforming growth factor-beta in cultured SFs, with the biopsy determined rate of development of DN lesions ranging from slow to rapid in nine IDDM patients. These varying rates of development of DN lesions were expressed by a mesangial expansion score as estimated by the rate of change in mesangial fraction volume per year. Cultured SF proliferation by direct cell counts positively correlated with mesangial expansion score (r = 0.65; P < 0.05). Expression of cultured SF alpha3 integrin subunit mRNA levels, as well as type I collagen mRNA (P < 0.05 for both), but not transforming growth factor-beta mRNA levels (Northern blot analysis), were also positively correlated with mesangial expansion score. We postulate that these observations of correlations between activities of cultured SFs and the rate of progression of DN lesions may be predictive of the risk to develop clinical DN in IDDM, may be in part genetically regulated, and may be of pathogenetic importance.

Topics: Adult; Biopsy; Blotting, Northern; Cell Division; Cells, Cultured; Collagen; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Fibroblasts; Humans; Integrins; Kidney; Male; Skin; Transforming Growth Factor beta

TGF-beta1, expressed in the pancreatic islets, protects the nonobese diabetic (NOD) mouse from insulin-dependent diabetes mellitus (IDDM). The islet antigen-specific T cell response of ins-TGF-beta1 mice relied on different antigen-presenting cells (APC) from those used by NOD T cells. T cells from NOD mice utilized B cells to present islet antigen, whereas T cells from ins-TGF-beta1 mice utilized macrophages. In addition, the islet antigen-specific T cell repertoire of ins-TGF-beta1 mice was distinct and deviated toward an IL-4-producing Th2 phenotype. When ins-TGF-beta1 mice were treated with anti-iL-4 antibody, islet antigen-specific IFNGamma-producing Th1 cells were unleashed, and the incidence of diabetes increased to the level of NOD mice. This suggests active suppression of a diabetogenic T cell response. This study describes a novel mechanism in which expression of TGF-beta1 in the context of self-antigen shifts APC preference, deviating T cell responses to a Th2 phenotype, preventing IDDM.

Topics: Animals; Antigen-Presenting Cells; Autoantigens; Diabetes Mellitus, Type 1; Glutamate Decarboxylase; Immune Tolerance; Interleukin-4; Islets of Langerhans; Mice; Mice, Inbred NOD; Mice, Transgenic; Phenotype; Spleen; Th2 Cells; Transforming Growth Factor beta

Paracrine effect of transforming growth factor-beta1 (TGF-beta1) on autoimmune insulitis and diabetes was studied by transgenic production of the active form of porcine TGF-beta1 (pTGF-beta1) in pancreatic islet (islet) alpha cells in nonobese diabetic (NOD) mice under the control of rat glucagon promoter (RGP) (NOD-RGP-TGF-beta1). None of 27 NOD-RGP-TGF- beta1 mice developed diabetes by 45 wk of age, in contrast to 40 and 71% in male and female nontransgenic mice, respectively. None of the NOD-RGP-TGF-beta1 mice developed diabetes after cyclophosphamide (CY) administration. Adoptive transfer of splenocytes of NOD-RGP-TGF-beta1 mice to neonatal NOD mice did not transfer diabetes after CY administration. Adoptive transfer of three types of diabetogenic lymphocytes to NOD-RGP-TGF-beta1 and nontransgenic mice after CY administration led to the lower incidence of diabetes in NOD-RGP-TGF-beta1 mice versus that in nontransgenic mice: 29 vs. 77% for diabetogenic splenocytes, 25 vs. 75% for islet beta cell-specific Th1 clone cells, and 0 vs. 50% for islet beta cell-specific CD8(+) clone cells, respectively. Based on these, it is concluded that autoimmune diabetes in NOD mice is not a systemic disease and it can be completely prevented by the paracrine TGF-beta1 in the islet compartment through protection against CD4(+) and CD8(+) effector lymphocytes.

Topics: Adoptive Transfer; Animals; Autoimmune Diseases; Clone Cells; Cyclophosphamide; Diabetes Mellitus, Type 1; Enzyme-Linked Immunosorbent Assay; Female; Genes, Synthetic; Glucagon; Islets of Langerhans; Male; Mice; Mice, Inbred NOD; Mice, Transgenic; Mutagenesis, Site-Directed; Organ Specificity; Polymerase Chain Reaction; Promoter Regions, Genetic; Rats; Recombinant Fusion Proteins; RNA, Messenger; Spleen; Swine; T-Lymphocyte Subsets; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Th1 Cells; Transforming Growth Factor beta

Nonobese diabetic (NOD) mice develop insulitis and diabetes through an autoimmune process. Since TGF-beta1 down-regulates many immune responses, we hypothesized that TGF-beta1 could prevent disease in NOD mice and that there would be several advantages to cytokine delivery by a somatic gene therapy approach. We opted for i.m. injection of a naked plasmid DNA expression vector encoding murine TGF-beta1 (pCMV-TGF-beta1). Treatment with pCMV-TGF-beta1 resulted in the retention and expression of the vector in muscle cells, associated with a considerable elevation in the plasma levels of TGF-beta1, that was not observed in control vector-treated mice. The levels of TGF-beta1 produced were sufficient to exert immunosuppressive effects. Delayed-type hypersensitivity responses were suppressed, and autoimmunity-prone NOD mice were protected from insulitis and diabetes in models of cyclophosphamide-accelerated and natural course disease. In pCMV-TGF-beta1-treated mice, pancreatic IL-12 and IFN-gamma mRNA expression was depressed, and the ratio of IFN-gamma to IL-4 mRNA was decreased, as determined by semiquantitative reverse-transcription PCR. In contrast, NOD mice injected with a vector encoding the proinflammatory cytokine IFN-gamma developed diabetes earlier. Intramuscular administration of cytokine-encoding plasmid vectors proved to be an effective method of cytokine delivery in these mice, and altered autoimmune disease expression.

Topics: Animals; Autoimmunity; Diabetes Mellitus, Type 1; Gene Transfer Techniques; Genetic Therapy; Mice; Mice, Inbred NOD; Transforming Growth Factor beta

Type I diabetes mellitus is a chronic disorder that results from autoimmune destruction of the insulin-producing pancreatic beta cell. The non-obese diabetic mouse is a model of the human autoimmune disease Type I diabetes [1-3]. We have previously shown that ingested type 1 interferon inhibits chronic relapsing experimental autoimmune encephalomyelitis and the adoptive transfer of experimental autoimmune encephalomyelites by T cells, and decreases both antigen-specific and mitogen-induced pro-inflammatory cytokine secretion in this disorder. We therefore tried to determine whether ingested murine interferon alpha inhibits insulinitis and suppresses Type I diabetes mellitus in non-obese diabetic mice. Murine interferon alpha, given daily, decreased islet inflammation and suppressed diabetes. It increased the concanavalin A and ionomycin plus myristic acid palmitic ester-induced production of interleukin 4 and 10 and interferon gamma-secretion in spleen cells from treated mice. Adoptive transfer of unstimulated splenocytes secreting interleukin 4 and interleukin 10 from fed interferon alpha donors suppressed spontaneous diabetes mellitus in recipients. The protective effect of adoptively transferred unstimulated splenocytes shows the presence of ingested interferon alpha-activated regulatory splenic cell populations that may work via increased interleukin 4 or interleukin 10 production. Ingested interferon alpha administered during vulnerable periods in at-risk populations may potentially provide a continuous, convenient, non-toxic and effective treatment for Type I diabetes.

Topics: Adoptive Transfer; Animals; Concanavalin A; Diabetes Mellitus, Type 1; Female; Interferon-alpha; Interferon-gamma; Interleukin-10; Interleukin-4; Interleukins; Ionomycin; Mice; Mice, Inbred NOD; Myristic Acid; Palmitic Acid; Spleen; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

PCR assays were established for easy and fast analysis of two transforming growth factor-beta1 (TGF-beta1) gene mutations, a C to T transition at position 76 in exon 5 resulting in a change from threonine to isoleucine in position 263 (Thr263Ile) of the propeptide and a deletion of a C in the intron sequence eight bases prior to exon 5 (713-8delC). These mutations were evaluated in insulin-dependent diabetes mellitus (IDDM) patients (n = 137) and control subjects (n = 105) and in IDDM patients with (n = 170) and without (n = 99) nephropathy. After evaluating intra- and interindividual variation in TGF-beta1 expression levels, the TGF-beta1 mRNA level in phorbol 12-myristate-13-acetate-stimulated (1 ng/ml) lymphocytes from individuals with different TGF-beta1 genotypes was also studied. No association of the two TGF-beta1 sequence variations with IDDM in general was found. However, a weak but significant association of the Thr263Ile mutation with diabetic nephropathy was found (P = 0.03). No correlation between TGF-beta1 transcription level and genotype of any of the two studied polymorphisms was found. However, significant interindividual differences in TGF-beta1 mRNA levels were observed between the tested individuals (P < 0.0001) compatible with a genetic control mechanism of TGF-beta1 synthesis at the mRNA level.

Topics: Amino Acid Substitution; Denmark; Diabetes Mellitus, Type 1; Diabetic Nephropathies; DNA Mutational Analysis; DNA, Complementary; Gene Expression Regulation; Genotype; Introns; Point Mutation; Polymerase Chain Reaction; Polymorphism, Genetic; Sequence Deletion; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

Certain diets can have major effects on the development of IDDM in DP-BB rats, but data are scant on the timing, dose, and mechanisms involved. We therefore determined the dose response, timing, and duration of exposure required to induce diabetes, and characterized the effects of nutritionally adequate diets with widely different diabetogenicity on the pancreatic islet area and cytokines. DP-BB rats were fed a diabetogenic, cereal-based, NIH-07 (NIH) diet or a protective, casein or hydrolyzed casein (HC)-based, semipurified diet. Rats were fed from weaning to 50 or 100 days with the HC diet and then switched to the NIH diet, or fed the NIH diet from weaning to 50 days and switched to the HC diet. Pancreas histology and diabetes outcome were determined. Semiquantitative morphometric analyses of hematoxylin and eosin-stained sections of pancreas from 41-day-old rats were also carried out. Diet-induced effects on pancreatic cytokine levels were measured at 70 days using reverse transcriptase-polymerase chain reaction analysis of gamma-interferon (IFN-gamma), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-beta). Long-term daily exposure, particularly around the beginning of puberty to late adolescence (50-100 days), was important for development of diabetes. DP-BB rats could be rescued from diabetes development by feeding them a low-diabetogen HC diet as late as 50 days. Diabetes frequency was highest in rats fed 70% and 100% NIH diets. By age 41 days, before classic insulitis, the islet area in HC-fed DP-BB rats was 65% greater than in NIH-fed rats. By 70 days, when mononuclear cells were visible in the islets of most NIH-fed, but not HC-fed rats, the more pronounced inflammatory process in NIH-fed rats was associated with a Th1 cytokine pattern (high IFN-gamma and low IL-10 and TGF-beta), whereas the pancreases of HC-fed rats showed fewer infiltrating cells, low levels of IFN-gamma, and high levels of TGF-beta, typical of a Th2 cytokine pattern. Thus dietary modification can occur as late as puberty. Further, long-term exposure to sufficient amounts of food diabetogens between 50 and 100 days was required for maximum diabetes induction. The islet area was modified by diet before signs of classic insulitis. Pancreatic inflammation in NIH-fed animals is a Th1-dependent phenomenon. The HC diet inhibited insulitis and was associated with a Th2 cytokine pattern in the pancreas, protecting diabetes-prone rats from developing diabetes.

Topics: Age Factors; Animals; Cytokines; Diabetes Mellitus, Type 1; Diet; Disease Models, Animal; Dose-Response Relationship, Drug; Interferon-gamma; Interleukin-10; Pancreas; Rats; Rats, Inbred BB; T-Lymphocytes, Helper-Inducer; Th1 Cells; Th2 Cells; Time Factors; Transforming Growth Factor beta

A new type of CD4+ T cell clone (NY4.2) isolated from pancreatic islet-infiltrated lymphocytes of acutely diabetic non-obese diabetic (NOD) mice prevents the development of insulin-dependent diabetes mellitus (IDDM) in NOD mice, as well as the recurrence of autoimmune diabetes in syngeneic islet-transplanted NOD mice. It has been demonstrated that the cytokine TGF-beta, secreted from the cells of this clone, is the substance which prevents autoimmune IDDM. This investigation was initiated to determine the molecular role TGF-beta plays in the prevention of autoimmune IDDM by determining its effect on IL-2-induced signal transduction in Con A-activated NOD mouse splenocytes and HT-2 cells. First, we determined whether TGF-beta, secreted from NY4.2 T cells, inhibits IL-2-dependent T cell proliferation in HT-2 cells (IL-2-dependent T cell line) and NOD splenocytes. We found that TGF-beta suppresses IL-2-dependent T cell proliferation. Second, we determined whether TGF-beta inhibits the activation of Janus kinases (JAKs), as well as signal transducers and activators of transcription (STAT) proteins, involved in an IL-2-induced signalling pathway that normally leads to the proliferation of T cells. We found that TGF-beta inhibited tyrosine phosphorylation of JAK1, JAK3, STAT3 and STAT5 in Con A blasts from NOD splenocytes and HT-2 cells. Third, we examined whether TGF-beta inhibits the cooperation between STAT proteins and mitogen-activated protein kinase (MAPK), especially extracellular signal-regulated kinase 2 (ERK2). We found that TGF-beta inhibited the association of STAT3 and STAT5 with ERK2 in Con A blasts from NOD splenocytes and HT-2 cells. On the basis of these observations, we conclude that TGF-beta may interfere with signal transduction via inhibition of the IL-2-induced JAK/STAT pathway and inhibition of the association of STAT proteins with ERK2 in T cells from NOD splenocytes, resulting in the inhibition of IL-2-dependent T cell proliferation. TGF-beta-mediated suppression of T cell activation may be responsible for the prevention of effector T cell-mediated autoimmune IDDM in NOD mice by TGF-beta-producing CD4+ suppressor T cells.

Topics: Acute-Phase Proteins; Animals; Calcium-Calmodulin-Dependent Protein Kinases; CD4-Positive T-Lymphocytes; Cell Line; Diabetes Mellitus, Type 1; DNA-Binding Proteins; Down-Regulation; Enzyme Activation; Interleukin-2; Janus Kinase 1; Janus Kinase 3; Lymphocyte Activation; Mice; Mice, Inbred NOD; Milk Proteins; Phosphorylation; Protein-Tyrosine Kinases; Receptors, Interleukin-2; STAT3 Transcription Factor; STAT5 Transcription Factor; T-Lymphocytes, Regulatory; Trans-Activators; Transforming Growth Factor beta

Transgenic mice whose pancreata express transforming growth factor-beta (TGF-beta) directed by an insulin promoter (Ins-TGF-beta mice) were used to assess the effect of local TGF-beta1 on allograft rejection and on autoimmune diabetes occurring as a cross-reaction to viral antigens. Pancreatic TGF-beta1 did not delay allograft rejection, nor did it inhibit autoimmune diabetes after lymphocytic choriomeningitis infection of double transgenic mice (LCMV/TGF-beta1 mice). These results suggest that local TGF-beta1 does not serve as an immunosuppressive agent for allograft rejection or virus-mediated autoimmune diabetes.

Topics: Animals; Antigen Presentation; Diabetes Mellitus, Type 1; Graft Rejection; Lymphocytic Choriomeningitis; Male; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Mice, Transgenic; Pancreas Transplantation; Transforming Growth Factor beta; Transplantation, Homologous

Topics: Animals; Diabetes Mellitus, Type 1; Mice; Mice, Transgenic; Pancreas; Pancreatitis; Transforming Growth Factor beta

In diabetes prone BB rat pancreas the Th1/ Th2 cytokine balance and the expression of inducible nitric oxide synthase (iNOS) was determined by mRNA analysis before and after the onset of insulitis. Specific mRNA was amplified by reverse transcriptase polymerase chain reaction, quantitated with radiolabelled probes by phosphoimaging and calibrated with the amount of co-amplified beta-actin mRNA. At 50 days of age, prior to recognizable insulitis, there was already significantly enhanced expression of both, Th1 and Th2 cytokines, and of iNOS mRNA, when compared to Wistar rat pancreas (p < 0.001). This supports the concept of an inconspicuous early phase of islet infiltration by single immunocytes, called single cell insulitis. At 70 days of age mononuclear infiltration of islets had begun and was associated with upregulation of interferon gamma (IFN gamma) and iNOS, but downregulation of interleukin-10 and transforming growth factor beta mRNA (p < 0.001). These findings correlate the onset of insulitis with a shift of the Th1/Th2 cytokine balance towards Th1 cell reactivity. Indeed there was a close correlation of the Th1/Th2 cytokine ratio but not of absolute IFN gamma mRNA levels with the insulitis score. Vaccination at day 50 with tetanus toxoid did not affect cytokine gene expression while diphtheria toxoid and even more strongly BCG administration induced a shift towards Th2 reactivity (p < 0.001) while iNOS mRNA was decreased (p < 0.01). Oral dosing with immunostimulatory components of Escherichia coli also changed the quality of inflammation. Oral lipopolysaccharide (LPS) from E. coli and OM-89, an endotoxin free extract containing immunostimulatory glycolipopeptides and heat shock protein (hsp) 65, both downregulated IFN gamma mRNA while only OM-89 in addition suppressed iNOS mRNA and enhanced Th2 cytokine gene expression (p < 0.001). We conclude that the onset of insulitis is associated with a shift towards Th1 cytokine and iNOS gene expression. Diphtheria toxoid and BCG vaccination stimulates Th2 reactivity but does not downregulate Th1. The latter can be achieved through oral administration of LPS or a glycopeptide fraction (OM-89) from E. coli.

Topics: Administration, Oral; Animals; Bacterial Vaccines; Cytokines; Diabetes Mellitus, Type 1; Diphtheria Toxoid; Disease Models, Animal; Escherichia coli; Gene Expression Regulation; Interferon-gamma; Interleukin-10; Lipopolysaccharides; Mycobacterium bovis; Nitric Oxide Synthase; Pancreas; Random Allocation; Rats; Rats, Inbred BB; Rats, Wistar; Regression Analysis; RNA, Messenger; Specific Pathogen-Free Organisms; Tetanus Toxoid; Transforming Growth Factor beta

Diabetes mellitus is a systemic disease with profound effects on oral health and periodontal wound healing. Uncontrolled diabetes adversely affects surgical wound healing and is often associated with abnormal proliferation of fibroblasts, excessive angiogenesis and poor bone regeneration. Human gingival fibroblasts and periodontal ligament cells from both diabetics and non-diabetics were evaluated for growth responses following culture in 20 mM glucose, a concentration compatible with blood glucose levels in uncontrolled diabetics. Gingival fibroblasts derived from 9 non-diabetic patients and 3 insulin-dependent diabetics either proliferated or showed little change of growth in elevated glucose. Enhanced proliferation was observed following 1 wk of culture in glucose. Growth of periodontal ligament cells from 5 non-diabetic patients was inhibited by 20 mM glucose. Fibroblasts that were markedly growth stimulated were probed for expression of basic fibroblast growth factor (bFGF) using a reverse-transcribed polymerase chain reaction (RT-PCR). Results indicate that fibroblasts exhibiting the greatest increase in growth in response to high glucose also exhibited increased expression of bFGF. No changes were observed in mRNA expression for platelet-derived growth factor-AA, platelet-derived growth factor-BB, insulin-like growth factor and transforming growth factor-beta 1. Mitogenic effects induced by the cytosol of fibroblasts exhibiting increases of growth in 20 mM glucose were abrogated by neutralizing antibodies to bFGF. In addition, some periodontal ligament cells that were growth inhibited by high glucose had reduced expression of bFGF. These data suggest that bFGF may play a role in the abnormal wound healing associated with periodontal surgery of uncontrolled diabetics.

Topics: Antibodies; Becaplermin; Bone Regeneration; Cell Division; Cells, Cultured; Culture Media; Diabetes Mellitus, Type 1; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression Regulation; Gingiva; Glucose; Humans; Insulin-Like Growth Factor I; Mitogens; Neovascularization, Pathologic; Periodontal Ligament; Platelet-Derived Growth Factor; Polymerase Chain Reaction; Proto-Oncogene Proteins c-sis; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Wound Healing

Oral administration of autoantigens suppresses development of autoimmunity in several animal models, and is being tested in clinical trials in patients with autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. Non-obese diabetic (NOD) mice spontaneously develop insulin-dependent diabetes mellitus at 15 to 20 weeks of age, after mononuclear cell (MNC) infiltration of the pancreatic islets of Langerhans and destruction of insulin-producing beta cells. We have previously shown that oral administration of insulin suppresses insulitis and development of diabetes in the NOD mouse. Oral insulin has no metabolic effect on blood glucose. Oral insulin mediates its effect through a T cell-dependent mechanism as shown by adoptive transfer and T cell depletion experiments, but the mechanisms responsible have not been fully explored. We now report a serial analysis of the cells and cytokines associated with development of diabetes in NOD mice, and contrast this with the findings in animals fed equine insulin or a control protein (ovalbumin). Animals were fed 1 mg twice a week for 5 weeks, beginning at 5 weeks of age. Marked insulitis in naive or ovalbumin-fed NOD mice occurred at 10 weeks, at which time a dense peri-islet and intra-islet MNC infiltration was observed. Immunohistological studies using monoclonal antibodies showed that infiltrating MNC consisted mainly of CD4+ T cells ( > 75% of leukocytes) plus smaller numbers of macrophages and CD8+ T cells. These cells displayed evidence of immune activation with expression of receptors for interleukin-2 (IL-2R) plus Th1 cytokines; dense labeling for IFN-gamma and tumor necrosis factor-alpha, plus lesser amounts of IL-2, was observed. MNC lacked labeling for IL-4, IL-10, prostaglandin-E, or transforming growth factor-beta. By contrast, at 10 weeks, pancreatic tissues from NOD mice fed insulin showed considerably less insulitis, and the residual MNC, although still largely CD4+ T cells plus macrophages, showed dense labeling for IL-4, IL-10, prostaglandin-E, and transforming growth factor-beta and an absence of IL-2, IFN-gamma or tumor necrosis factor-alpha Taken together with our previous findings, these data indicate that oral administration of insulin affects the development of diabetes in NOD mice through the generation of cells that elaborate immunoregulatory cytokines within the target organ and shift the balance from a Th1 to a Th2 pattern of cytokine expression.

Topics: Administration, Oral; Animals; Diabetes Mellitus, Type 1; Female; Insulin; Interleukin-10; Interleukin-4; Islets of Langerhans; Mice; Mice, Inbred NOD; Prostaglandins E; Transforming Growth Factor beta

Nonobese diabetic (NOD) mice spontaneously develop immune-mediated insulin-dependent diabetes mellitus and nephropathy, providing an opportunity to study the early molecular events in a model of diabetic glomerulosclerosis. The expression of several genes coding for growth factors and extracellular matrix was examined in microdissected glomeruli, by the use of reverse transcription-competitive polymerase chain reaction, in diabetic NOD mice (mean duration of diabetes, 28.5 +/- 7 days) and age-matched nondiabetic NOD mice with normal glucose tolerance. The levels of mRNA coding for transforming growth factor-beta 1, tenascin, and laminin B1 increased 1.9-, 2.0-, and 1.7-fold, respectively, whereas platelet-derived growth factor (PDGF)-B, alpha 1(IV) collagen, 72-kd collagenase, alpha-smooth muscle actin, and beta-actin mRNA remained stable in the diabetic mice. The kidney advanced glycosylation end-products levels increased 2.1-fold in the diabetic mice, and the diabetic glomeruli showed an accumulation of tenascin and laminin but not of type IV collagen by immunofluorescence microscopy. There was no increase in cell number per glomerulus after the onset of diabetes, a finding consistent with stable PDGF-B and alpha-smooth muscle actin mRNA levels. These findings provide evidence that increased glomerular transforming growth factor-beta 1, but not PDGF-B, mRNA is associated with the up-regulation of tenascin and laminin expression after advanced glycosylation endproduct accumulation, early after the onset of diabetes.

Topics: Animals; Base Sequence; Cell Adhesion Molecules, Neuronal; Diabetes Mellitus, Type 1; Extracellular Matrix Proteins; Female; Gene Expression; Kidney Glomerulus; Laminin; Mice; Mice, Inbred NOD; Microscopy, Fluorescence; Molecular Probes; Molecular Sequence Data; RNA, Messenger; Tenascin; Transforming Growth Factor beta

We have generated transgenic mice overexpressing TGF-beta 1 in pancreatic beta cells. This resulted in massive fibrosis of the pancreas; in adult mice, most of the acini were replaced by fibrotic and adipose tissues. A conspicuous disorganization of the islets of Langerhans was also observed; however, the number of beta cells was not decreased and the mice were normoglycemic. Backcrossing to transgenic mice overexpressing TNF-alpha in their islet beta cells (which also remain normoglycemic, (1)) yielded double transgenics, most of which became diabetic by the age of 4 months; histological analysis revealed a dramatic decrease in insulin-containing beta cells.

Topics: Amylases; Animals; Base Sequence; Chronic Disease; Diabetes Mellitus, Type 1; DNA Primers; Insulin; Islets of Langerhans; Mice; Mice, Transgenic; Molecular Sequence Data; Pancreas; Pancreatitis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Studies of ocular immunity showed that incubation of peritoneal exudate cells with antigen in the presence of aqueous humor containing TGF-beta, conferred upon them the ability to systemically inhibit antigen-specific cellular immunity when injected into naive recipients. Since cell mediated immunity has been implicated in the destruction of the islets of Langerhans in diabetes, it was theorized that injection of naive diabetes prone BB/W or rats with peritoneal exudate cells pre-cultured in the presence of islet antigen and TGF-beta might similarly inhibit their anti-islet immune reactions and prevent their development of diabetes. 34.2% (13 of 38) of experimental recipient diabetes prone rats developed diabetes while 78.4% (29 of 37; p < 0.0005 compared to experimentals), 72.2% (13 of 18; p < 0.03 compared to experimentals), 68.8% (11 of 16; p < 0.09 compared to experimentals), and 77.7% (7 of 9; p < 0.08 compared to experimentals) of controls receiving peritoneal exudate cells pre-cultured alone, with TGF-beta, with TGF-beta and pheochromocytoma (PC12) cells, or with islets + TGF-beta + anti-TGF-beta antibody, respectively, became diabetic. Experimental treatment did not markedly alter recipient spleen cell subsets, and spleen cells from protected rats did not confer disease protection when transferred into naive recipients. These data demonstrate that the above approach is efficacious and represents a unique strategy for preventing the development of autoimmune type I diabetes in an animal model.

Topics: Animals; Aqueous Humor; Cell Communication; Cells, Cultured; Diabetes Mellitus, Type 1; Exudates and Transudates; Flow Cytometry; Islets of Langerhans; Lymphocyte Transfusion; Lymphocytes; Rats; Rats, Inbred BB; Spleen; Thyroid Gland; Transforming Growth Factor beta

Renal hypertrophy is an early feature of diabetes, and it may predispose the kidney to the eventual development of parenchymal dysfunction. Since we have previously demonstrated that short-term culture in high glucose concentration stimulates production of transforming growth factor-beta 1 (TGF-beta 1) in proximal tubular and glomerular mesangial cells, we postulated that this cytokine, which has potent regulatory effects on cellular growth and extracellular matrix production, is important in mediating diabetic renal disease. In this study we evaluated the gene and protein expression of TGF-beta 1 in the kidney of two rodent models of spontaneous insulin-dependent diabetes mellitus [the biobreeding (BB) rat and the nonobese diabetic (NOD) mouse]. In association with the appearance in both models of significant renal hypertrophy, TGF-beta 1 mRNA levels were increased threefold in the kidney of the diabetic BB rat after 3 days of diabetes and also threefold after 7-9 days in the NOD mouse. There was no increase in TGF-beta 1 transcripts in the livers of the diabetic animals, suggesting that this response is tissue specific. Immunohistochemical studies revealed that TGF-beta 1 protein is concordantly elevated in the cortical tubular cells of the diabetic kidney in both models. These results suggest that the stimulated expression of renal TGF-beta is an early manifestation of the involvement of the kidney by diabetes. Whether increased TGF-beta production in the diabetic kidney is a key promoter of diabetic renal manifestations (e.g., hypertrophy) deserves additional studies.

Topics: Animals; Diabetes Mellitus, Type 1; Female; Gene Expression Regulation; Glyceraldehyde-3-Phosphate Dehydrogenases; Hypertrophy; Immunohistochemistry; Kidney; Kidney Cortex; Kidney Tubules; Male; Mice; Mice, Inbred NOD; Rats; Rats, Inbred BB; RNA, Messenger; RNA, Ribosomal, 18S; Transforming Growth Factor beta

Diabetic nephropathy is characterized by excessive glomerular matrix accumulation, basement membrane thickening and sclerosis. Although it is clear that systemic metabolic disturbances precipitate such renal changes, the signals and pathways involved in this process are not fully elucidated. Recent evidence suggests that growth factors/cytokines are intimately involved in the pathogenesis of diabetic nephropathy. Because of its prosclerotic properties, transforming growth factor-beta (TGF-beta) is a prime candidate mediator of diabetic nephrosclerosis. We examined perfused kidney tissues isolated from spontaneously diabetic, non-obese diabetic mice (NOD) for TGF-beta content. By using murine isotype specific TGF-beta probes, we demonstrate that within 5 to 10 days of hyperglycuria renal TGF-beta 2 mRNA and protein content increases. By immunohistochemical analysis, de novo TGF-beta immunoreactivity was detected within both glomeruli and the interstitium. In order to determine the signals involved in promoting kidney TGF-beta content in vivo, TGF-beta regulation was examined in renal mesangial cells in vitro. Murine mesangial cells stimulated with glycosylated protein secrete bioactive TGF-beta and demonstrate a disproportionate increase in the steady state levels of TGF-beta 2 mRNA. These data suggest that a major early renal response in NOD mice to hyperglycemia or to glycosylated proteins is characterized by increases in TGF-beta.

Topics: Animals; Blotting, Northern; Blotting, Western; Cells, Cultured; Diabetes Mellitus, Type 1; Glomerular Mesangium; Glycation End Products, Advanced; Immunoenzyme Techniques; Kidney; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; RNA, Messenger; Transforming Growth Factor beta

Type 1, insulin-dependent diabetes mellitus (IDDM) appears to result from a T cell-dependent destruction of insulin-producing pancreatic beta cells. In non-obese diabetic (NOD) mice and in other rodent models of human IDDM, final expression of disease may be controlled by protective, as well as, destructive T cell influences. Previously, a CD8+ T cell clone, I.S. 2.15, was isolated directly from islets of disease-resistant male NOD mice. Upon transfer to young NOD recipients, the non-cytolytic I.S. 21.5 T cell clone, confers in vivo protection from two forms of accelerated IDDM. The present study demonstrates that I.S. 2.15 T cells induce in vitro immunosuppression. The suppressive effects of I.S. 2.15 T cells are mediated through soluble factor(s) and are independent of T cell activation, cell contact, antigen specificity or the major histocompatibility complex (MHC). By polymerase chain reaction (PCR), I.S. 2.15 T cells contain mRNA species encoding for the potentially immunosuppressive cytokines, interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta). The T cell suppressive effects engendered by I.S. 2.15 T cells closely mimic those observed with TGF-beta. Moreover, I.S. 2.15-induced immunosuppression correlates with intracellular levels of TGF-beta mRNA. These results establish that immunoregulatory T cells are present within islets in IDDM-resistant NOD mice and may impact on final disease expression through the production of soluble mediator(s).

Topics: Animals; Base Sequence; CD8 Antigens; Clone Cells; Cytokines; Diabetes Mellitus, Type 1; Interleukin-2; Islets of Langerhans; Lymphocyte Culture Test, Mixed; Mice; Mice, Inbred NOD; Molecular Sequence Data; Receptors, Interleukin-2; RNA, Messenger; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The objective of this study was to determine whether transforming growth factor beta (TGF-beta) can inhibit the destruction of islet cells by either spleen cells from acutely diabetic BB/Wor rats, or by the cytokines tumor necrosis factor (TNF) and interferon gamma (IFN-gamma). It has previously been reported that effector spleen cells from diabetic BB/Wor rats and humans can specifically lyse islet cell targets in a 51Cr release assay; further evidence in BB rats suggests that these cells are natural killer (NK) cells. Similarly, TNF and IFN-gamma have been shown to act synergistically to destroy islet cell monolayers. TGF-beta inhibits NK cell generation and activity, and was shown to inhibit the production and/or activity of various cytokines. A 51Cr release assay was used to evaluate the effects of TGF-beta on both the lymphocyte- and cytokine-mediated destruction of islet cells. Results showed that TGF-beta specifically inhibits lymphocyte-mediated islet cell lysis at concentrations of 10 and 1 ng/ml. Preincubation of islets with TGF-beta for 24 h prior to the addition of 51Cr-labeled target cells markedly increased the percent inhibition of islet cell lysis. TGF-beta (10 ng/ml) was also shown to significantly inhibit the TNF and IFN-gamma-induced lysis of islet cell monolayers. These findings demonstrate that TGF-beta can significantly reduce both the diabetic spleen cell, and cytokine-mediated lysis of islet cells and suggest that it may play a role in preventing islet damage in vivo.

Topics: Animals; Cells, Cultured; Cytokines; Cytotoxicity, Immunologic; Diabetes Mellitus, Type 1; Dose-Response Relationship, Drug; Islets of Langerhans; Rats; Rats, Inbred BB; Spleen; Transforming Growth Factor beta

The objective of this study was to determine whether pre-incubation of islet cells with transforming growth factor beta (TGF-beta) can inhibit their destruction by spleen cells from acutely diabetic BB/Wor rats. It was previously reported that the lysis of islet cells by spleen cells from diabetic BB/Wor rats can be significantly inhibited by TGF-B. The inhibition of spleen cell mediated lysis of islet cells was found to be greater when the effector lymphocytes were pre-treated with TGF-beta. The current study was designed to determine whether the lysis of islet cells by spleen cells from diabetic BB/Wor rats can be reduced if the islets are pre-treated with TGF-beta. The results revealed that pre-incubation of islets with TGF-beta significantly reduced their susceptibility to lysis by diabetic spleen cells. This protection was still demonstrable when, after incubation of the islets in TGF-beta for almost 2 weeks, the TGF-beta was removed 3 days prior to the assay. These data establish that TGF-beta can reduce the susceptibility of islet cells to autoimmune destruction and, in conjunction with its previously demonstrated direct inhibitory effects on diabetic spleen cells, suggest that it may be useful in preventing the disease in vivo.

Topics: Animals; Cells, Cultured; Cytotoxicity, Immunologic; Diabetes Mellitus, Type 1; Islets of Langerhans; Lymphocytes; Rats; Rats, Inbred BB; Spleen; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that can either stimulate or inhibit cellular proliferation depending on cell type and culture conditions. The immunohistochemical localization of TGF-beta was investigated in human retinas and choroids using streptavidin peroxidase immunohistochemistry and a polyclonal rabbit antibody directed against the N-terminal 30 amino acids of TGF-beta 1. This antibody recognizes the beta 1 form of TGF-beta but not beta 2. TGF-beta localization was observed exclusively in photoreceptors in all adult non-diabetic and non-insulin dependent diabetic eyes, and 4 of 6 insulin dependent eyes. It was determined that TGF-beta was associated with both rods and cones using localization of peanut agglutinin (PNA), a lectin which binds to cone sheaths, on serial sections. Chondroitinase ABC digestion of sections prior to immunohistochemistry did not reduce TGF-beta immunoreactivity, suggesting that binding was not to glycosaminoglycans in the interphotoreceptor matrix. TGF-beta immunoreactivity was not observed in 2 premature human eyes in which photoreceptor outer segments had not yet developed. Localization in photoreceptors was also not observed in photocoagulation scars, in atrophic regions in a diabetic retina, nor in detached areas of retina from a young victim of head trauma. Based on PNA binding, succinate dehydrogenase enzyme histochemistry and phase contrast microscopy on adjacent sections, the TGF-beta negative areas of these retinas did not appear to have viable photoreceptors. This work demonstrates that TGF-beta is found exclusively in viable adult human retinal photoreceptors. It's function in these cells is currently not known.

Topics: Adult; Aged; Aged, 80 and over; Biotin; Cicatrix; Diabetes Mellitus, Type 1; Humans; Immunohistochemistry; Lectins; Light Coagulation; Middle Aged; Peanut Agglutinin; Photoreceptor Cells; Postmortem Changes; Postoperative Complications; Tissue Distribution; Transforming Growth Factor beta