Pathogenesis Of Insulin-dependent Diabetes Research Articles

Evaluation of Colchicine Effect on Cytokine Production from Peripheral Blood Mononuclear Cells of Type I Diabetes = تقييم تأثير الكولشيسين على إنتاج السيتوكينات من الخلايا في الدم المحيطي وحيدات النوى من مرضى السكري النوع الأول

Problem: Type 1 diabetes mellitus (DM) results from autoimmune destruction of insulin-producing β cells and is characterized by the presence of insulitis and β-cell autoantibodies. Cytokines are known to play an important role in autoimmunity and have been suggested to be involved in the pathogenesis of insulin-dependent diabetes (IDDM). The aim of the present study was to evaluate the levels of major cytokines such as IL-2, IL-6, and IL-10 in Type I DM and in apparently healthy volunteers (control) and to study the effect of colchicine on the capacity of peripheral blood monocytes isolated from Type I diabetes patients and healthy volunteers with or without immune stimulation to produce IL-2, IL-6, and IL-10. Experimental approach: Fifty type I diabetes patients were randomly recruited from the Diabetes/Endocrine Clinics along with 15 healthy subjects. Levels of cytokines were measured from cultured blood monocytes using standard ELISA kits. Major findings: results revealed that basal levels of IL-2, IL-6, and IL-10 were significantly higher in diabetes group compared to healthy group (P < 0.05). LPS or PHA-induced production of these cytokines was significantly higher in diabetes group (P < 0.05). Moreover, pretreatment of stimulated monocytes of DM patients with colchicine inhibited cytokine levels to be comparable to that of cell cultured from healthy individuals. Conclusions: These results provide a new perspective on the protective role of colchicine against inflammatory long-term complications associated with type I DM.

Cytokines in sera from insulin-dependent diabetic patients at diagnosis

Cytokines are known to play an important role in autoimmunity and have been suggested to be involved in the pathogenesis of insulin-dependent diabetes (IDDM). In the present study we have measured IL-1, IL-2, IL-4, IL-6, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) (using both immunoassays and bioassays) in sera from 50 patients affected by IDDM at the time of clinical diagnosis and 51 age and sex matched controls. Detectable levels of IL-1, IL-2, IL-6 and IFN-gamma were found in the serum of a small percentage of subjects and were not significantly different between patients and controls. IL-4 was detectable in a higher number of both patients and controls and circulating TNF-alpha (greater than 1 U/ml) was found in a percentage of patients (24%) significantly higher than controls (P less than 0.01). Raised levels of TNF-alpha were detectable using an immunoenzymatic assay whereas TNF bioactivity in these samples was negligible. We conclude that the presence of immunoreactive TNF-alpha in the patient's sera may reflect an increased localized production of this cytokine at pancreatic level. However, the measurement in serum of other cytokines does not add information on the role that they may play in the pathogenesis of IDDM.

Unraveling the Pathogenesis of Type 1 Diabetes with Proteomics: Present And Future Directions

Type 1 diabetes (T1D) is the result of selective destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. T1D is due to a complex interplay between the beta-cell, the immune system, and the environment in genetically susceptible individuals. The initiating mechanism(s) behind the development of T1D are largely unknown, and no genes or proteins are specific for most T1D cases. Different pro-apoptotic cytokines, IL-1 beta in particular, are present in the islets during beta-cell destruction and are able to modulate beta-cell function and induce beta-cell death. In beta-cells exposed to IL-1 beta, a race between destructive and protective events are initiated and in susceptible individuals the deleterious events prevail. Proteins are involved in most cellular processes, and it is thus expected that their cumulative expression profile reflects the specific activity of cells. Proteomics may be useful in describing the protein expression profile and thus the diabetic phenotype. Relatively few studies using proteomics technologies to investigate the T1D pathogenesis have been published to date despite the defined target organ, the beta-cell. Proteomics has been applied in studies of differentiating beta-cells, cytokine exposed islets, dietary manipulated islets, and in transplanted islets. Although that the studies have revealed a complex and detailed picture of the protein expression profiles many functional implications remain to be answered. In conclusion, a rather detailed picture of protein expression in beta-cell lines, islets, and transplanted islets both in vitro and in vivo have been described. The data indicate that the beta-cell is an active participant in its own destruction during diabetes development. No single protein alone seems to be responsible for the development of diabetes. Rather the cumulative pattern of changes seems to be what favors a transition from dynamic stability in the unperturbed beta-cell to dynamic instability and eventually to beta-cell destruction.

Infants diets and insulin-dependent diabetes: evaluating the “cows' milk hypothesis” and a role for anti-bovine serum albumin immunity.

Insulin-Dependent Diabetes (IDD) results from an autoimmune destruction of the insulin secreting pancreatic beta cells. The immunological mechanisms underlying the development of IDD as well as the role of environmental agents (e.g., diet, viruses, stress) in the pathogenesis of the disease are the subject of considerable research efforts. Significant attention has recently been directed to a hypothesis that consumption of cows' milk in infancy may trigger the autoimmune process underlying IDD. Early evidence supporting this “cows' milk hypothesis” included case-control studies surveying infant nutrition practices (i.e., breast feeding versus consumption of infant formula) and the subsequent development of IDD. However, intense media interest surrounding a report indicating anti-bovine serum albumin (BSA) immunity as the cause of IDD has lead to heightened public awareness of the issue, and, together with the epidemiological data, prompted The American Academy of Pediatrics to modify its guidelines for infant feeding practices. However, less public and scientific attention has been given toward the observations that many of these case-control studies were retrospective in design and subject to recall bias, narrow in scope in terms of collecting dietary information, and that similar results have not been duplicated in other more recent (and better designed) investigations. Furthermore, the immunological report implicating anti-BSA immunity with the disease has become controversial due to difficulties in conforming the findings, and experiments in animal models closely resembling human IDD have not uniformly supported a role for anti-BSA immunity in the pathogenesis of IDD. Given the significant morbidity and mortality associated with IDD, an improved understanding of the cause of this disorder as well as identifying possible methods for its prevention are essential. However, without additional supporting information, modification of the cows' milk/BSA composition of diets in order to avoid the disease may be premature. Further studies are needed to clearly establish a role for diet in the pathogenesis of IDD.

DNA-damage and NAD(+)-depletion are initial events in oxygen radical induced islet cell death.

Previous studies in vitro and in animal models indicate that reactive oxygen intermediates (ROI) contribute to the destruction of pancreatic islet cells in the pathogenesis of insulin dependent diabetes (Mendola et al., 1989; Rabinovitch et al., 1992). During islet inflammation endothelial cells and infiltrating macrophages are thought to release cyto-toxic amounts ROI (Zweier et al., 1988; Brenner et al., 1993). In addition it was shown, that islet cells are highly susceptible to ROI since they possess only low levels of oxygen radical defense systems (Grankvist et al., 1981; Malaisse et al., 1982). We recently have described an in vitro model of oxygen-radical induced islet cell death where ROI are generated by xanthine oxidase in the presence of hypoxanthine (Burkart et al., 1992). In the present study we investigated the initial events of ROI-induced pancreatic islet cell destruction. We observed that DNA strand breaks and the activation of poly(ADP-ribose) polymerase in islet cell nuclei are prominent early consequences of ROI action. The subsequent islet cell death occured several hours after ROI treatment, which appeared to result from depletion of cellular NAD+ pools. Inhibition of poly(ADP-ribose) polymerase by nicotinamide resulted in the preservation of the NAD+ pool and in islet cell survival.

Cellular immunity to a determinant common to glutamate decarboxylase and coxsackie virus in insulin-dependent diabetes.

Insulin-dependent diabetes (IDD) results from the autoimmune destruction of the insulin-producing pancreatic beta cells. Autoreactive T-lymphocytes are thought to play a pivotal role in the pathogenesis of IDD; however, the target antigens of these cells, as well as the inductive events in the disease, are unclear. PBMC in persons with or at increased risk for IDD show elevated reactivity to the beta cell enzyme glutamate decarboxylase (GAD). To identify the T-lymphocyte-reactive determinants of GAD, an overlapping set of synthetic peptides was used to stimulate the PBMC from these individuals, PBMC responsiveness to GAD peptides was not restricted to those with IDD, and a number of peptides elicited responses in PBMC. However, the major determinant of GAD recognized by persons at increased risk for IDD was amino acids 247-279, a region which has significant sequence similarity to the P2-C protein of Coxsackie B virus (47% of 15 increased risk [islet cell autoantibody-positive relatives]; 25% of 16 newly diagnosed IDD patients; and 0% of 13 healthy control subjects). Responses to tetanus and insulin antigens were not different between the study groups. In addition, PBMC from individuals responding to GAD peptides within 247-279 also responded to a Coxsackie viral peptide (i.e., P2-C amino acids 32-47), an observation supporting potential molecular mimicry in this immune response. Although the role of environmental agents in the pathogenesis of the disease remains unclear, these cellular immunological findings support the epidemiological evidence suggesting an inductive role for enteroviruses like Coxsackie B in the autoimmunity underlying IDD.

Histological study of pancreatic beta-cell loss in relation to the insulitis process in the non-obese diabetic mouse

Non-obese diabetic (NOD) mice spontaneously develop an autoimmune diabetes with higher incidence in females than in males. In order to elucidate possible factors involved in the different incidence of diabetes between male and female mice, we studied the progression of pancreatic beta-cell loss in relation to mononuclear cell infiltration of the pancreas (insulitis). We examined the pancreas of 76 NOD mice (39 males and 37 females) of different ages. The beta-cell content was assessed by immunoperoxidase staining of sections with an anti-insulin serum and the severity of insulitis was determined by haematoxylin staining of the same sections. A semi-quantitative criterion was used to grade both parameters. The results showed that females have a faster loss of beta-cell mass, which progressively decreases with the increase of severity of insulitis. In males, a medium to severe degree of insulitis is required before initial loss of beta cells occurs. Under the age of 10 weeks there was a significantly lower content of beta cells in females than males (2.84 +/- 0.03 vs 2.67 +/- 0.07; P = 0.02). Since we never observed a significant difference in the degree of mononuclear cell infiltration in age-matched males and females, these data support the hypothesis of weaker beta-cell resistance to immunological attack in female mice. Thus beta-cell sensitivity, in addition to immunological activity, is an important factor in the pathogenesis of insulin dependent diabetes.

A membrane form of brain L-glutamate decarboxylase: identification, isolation, and its relation to insulin-dependent mellitus.

A membrane form of L-glutamate decarboxylase (GAD) was identified and purified to apparent homogeneity from hog brain. The purified GAD was established as an integral membrane protein by phase-partitioning assay, charge-shift electrophoresis, and chromatography on a hydrophobic interaction column. This membrane GAD has a native molecular mass of 96 +/- 5 kDa and is a homodimer of 48 +/- 3-kDa subunits. Immunoprecipitation and immunoblotting tests revealed the presence of antibodies against this membrane GAD in sera from patients with insulin-dependent diabetes mellitus. Since this form of GAD appears to be an integral membrane protein and is presumed to have extracellular domains exposed, it seems reasonable to suggest that membrane GAD is more likely than soluble GAD to be involved in the pathogenesis of insulin-dependent diabetes and related autoimmune disorders such as stiff-man syndrome.

ANTI-RETROVIRUS ANTIBODIES IN INSULIN-DEPENDENT DIABETIC CHILDREN AT DIAGNOSIS AND DURING 1–2.6 YRS FOLLOW-UP

Viruses have long been implicated in the pathogenesis of insulin-dependent diabetes (IDDM). Recently it has been observed that β-cell specific expression of endogenous retrovirus is associated with the development of insulins and diabetes in non-obese diabetic (NOD) mice. The aim of our study was to evaluate the presence of retroviral markers in 27 newly diagnosed IDDM children, aged 9.6±3.8 yrs (range 3-15.9 yrs). In all patients anti-islet cell (ICA), anti-insulin (IAA), anti-thyroid (TgA and MsA) antibodies were evaluated and HLA typing was performed. Virological studies included the search for anti-retrovirus (HTLVs) antibodies using the western immunoblotting (WIB) and the HTLV-I DNA amplification by polymerase chain reaction (PCR) for tax/rex pol, and gag (PIS) regions. At onset of IDDM ICA were present in 19 patients (70%) and IAA in 2 of them, while all patients were negative for TgA and MsA. HTLVs antibodies were detected in 10 out of the 27 patients (37%) while no HTLV-I DNA sequencies were detected using our PCR assay. Interestingly, we have observed a frequent association between the presence of HTLVs antibodies and the presence of HLA-DR3 and DQ2 antigens. Until now, during 1-2.6 yrs follow-up we could revalue 13 children, four with HTLVs antibodies and 9 without antibodies. In 2/4 patients the HTLVs antibodies disappeared and in another boy, with five antibodies at onset of IDDM (directed against p 19, rgp21, p24, p26, p36) only one antibody (directed against rgp 21) was constantly present.

Progression to Diabetes in Nonobese Diabetic (NOD) Mice with Transgenic T Cell Receptors

The T cell receptor (TCR) requirements in the pathogenesis of insulin-dependent diabetes were examined with transgenic NOD mice bearing nondisease-related TCR alpha and beta chains. In both TCR beta and TCR alpha beta transgenic NOD mice the beta chain transgene was expressed by > 98% of peripheral T cells. The alpha chain transgene was also highly expressed. Insulitis developed in both sets of transgenic animals with most of the lymphocytes in the lesion expressing the transgenic beta chain and with depletion of the endogenous TCR V beta genes. Nonetheless, NOD animals transgenic for TCR beta and TCR alpha beta developed diabetes similar to controls. Thus, skewing the TCR repertoire did not diminish autoimmune susceptibility in NOD mice.

Insulin-secreting β-cells possess specific receptors for interleukin-1β

The effect of the cytokine interleukin-1β on the insulin secretory responsiveness of single β-cells (HIT-T15) was investigated. In the short-term, IL-1β induced a dosage-dependent stimulation of insulin release. In contrast, in the long-term, IL-1β, inhibited both basal and secretagogue-stimulated insulin secretion. We also demonstrate the simultaneous presence of specific high and low affinity binding sites for IL-1β on β-cells. IL-1β, which has been implicated in the pathogenesis of insulin-dependent diabetes, may therefore mediate its opposing effects on β-cells through a specific plasma membrane receptor.

Induction and therapy of autoimmune diabetes in the non-obese diabetic (NOD/Lt) mouse by a 65-kDa heat shock protein.

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta cells of the pancreas. The results described here indicate that a beta-cell target antigen in non-obese diabetic (NOD/Lt) mice is a molecule cross-reactive with the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis. The onset of beta-cell destruction is associated with the spontaneous development of anti-hsp65 T lymphocytes. Subsequently hsp65 cross-reactive antigen becomes detectable in the sera of the prediabetic mice and some weeks later anti-hsp65 antibodies, anti-insulin antibodies, and anti-idiotypic antibodies to insulin antibodies become detectable. The hsp65-cross-reactive antigen, the autoantibodies, and the T-cell reactivity then decline with the development of overt insulin-dependent diabetes. The importance of hsp65 in the pathogenesis of insulin-dependent diabetes was confirmed by the ability of clones of anti-hsp65 T cells to cause insulitis and hyperglycemia in young NOD/Lt mice. Moreover, hsp65 antigen could be used either to induce diabetes or to vaccinate against diabetes, depending on the form of its administration to prediabetic NOD/Lt mice. Other antigens such as the 70-kDa heat shock protein (hsp70) had no effect on the development of diabetes.

Initial pathogenic events in IDDM.

A workshop sponsored by the National Institute of Child Health and Human Development was held in June 1988 to discuss the initial events in the pathogenesis of insulin-dependent diabetes and to make recommendations for future studies. Better definition of immunological markers to reliably predict the disease will enable the detection and study of the earliest pathogenic events involved. The precise autoimmune mechanisms and the role of the environment, both in the initiation of the disease process and precipitation of clinically overt disease, need to be accurately determined to define strategies that might eventually lead to its prevention.

Dietary protein restriction reduces the frequency and delays the onset of insulin dependent diabetes in BB rats.

Environmental agents have been implicated in the pathogenesis of insulin dependent diabetes (IDD). These studies were designed to learn if dietary protein influences the development of IDD in the BB rat. Specifically, analysis involved the effects of substituting a modified, semi-synthetic diet (AIN-76) containing soy protein as the sole protein source for the standard chow containing a mixture of animal and non-animal protein. IDD was less frequent (73% vs. 38%, P less than or equal to 0.01), and the onset of diabetes was retarded (110 +/- 11.0 vs. 92 +/- 15.5 days, P less than or equal to 0.01) in rats fed the study diet versus standard chow, respectively. The frequency of thyroid collodal autoantibodies was also significantly decreased in rats fed the study diet (56% vs. 23%, P less than or equal to 0.04), whereas frequencies of smooth muscle and gastric parietal cell autoantibodies were less frequent, but not significantly so. Lymphocyte counts and subsets were unaffected. In non-diabetic rats at greater than 180 days of age, insulitis was less severe in the experimental group. These findings suggested that dietary protein may influence the development of IDD in the BB rat.

AUTOIMMUNITY TO ANTERIOR PITUITARY CELLS AND THE PATHOGENESIS OF INSULIN-DEPENDENT DIABETES MELLITUS

An immunofluorescence study using unfixed cryostat sections of human pituitary glands was carried out on sera from patients with type-Ia (juvenile-onset) diabetes (61 recent onset, 48 longstanding). 63 of their selected high-risk first-degree relatives and 117 patients with type-Ib ("polyendocrine") diabetes were tested for comparison. Healthy controls included 48 sera from laboratory staff and students. Pituitary-cell antibodies were found in none of the controls, in 2% of patients with longstanding diabetes, in 16·6% of patients with diabetes of recent onset, and in 36·6% of genetically predisposed relatives with islet-cell antibodies in their sera (of whom 7 became diabetic during a 3-year follow-up period, 4 of them reacting with pituitary cells for 1-3 years before the onset of diabetes). Thus pituitary antibodies tended to disappear after onset of symptoms. Many of the sera reacted with multiple anterior-pituitary cell types. These findings suggest a wider involvement of the endocrine-organ system in the pathogenesis of insulin-dependent diabetes and are in accordance with clinical observations showing excess growth in prepubertal boys at onset of diabetic symptoms and with the results of experiments on virus-induced diabetes in mice. The connection of these pituitary antibodies with autoimmune lymphocytic hypophysitis is at present unknown.

Humoral autoimmunity to gut-related endocrine cells

An immunofluorescence study using unfixed cryostat sections of human pituitary glands was carried out on sera from patients with type-Ia (juvenile-onset) diabetes (61 recent onset, 48 longstanding). 63 of their selected high-risk first-degree relatives and 117 patients with type-Ib ("polyendocrine") diabetes were tested for comparison. Healthy controls included 48 sera from laboratory staff and students. Pituitary-cell antibodies were found in none of the controls, in 2% of patients with longstanding diabetes, in 16·6% of patients with diabetes of recent onset, and in 36·6% of genetically predisposed relatives with islet-cell antibodies in their sera (of whom 7 became diabetic during a 3-year follow-up period, 4 of them reacting with pituitary cells for 1-3 years before the onset of diabetes). Thus pituitary antibodies tended to disappear after onset of symptoms. Many of the sera reacted with multiple anterior-pituitary cell types. These findings suggest a wider involvement of the endocrine-organ system in the pathogenesis of insulin-dependent diabetes and are in accordance with clinical observations showing excess growth in prepubertal boys at onset of diabetic symptoms and with the results of experiments on virus-induced diabetes in mice. The connection of these pituitary antibodies with autoimmune lymphocytic hypophysitis is at present unknown.

Antibodies to viruses and to pancreatic islets in nondiabetic and insulin-dependent diabetic patients.

Autoimmunity is frequently involved in the pathogenesis of insulin-dependent diabetes, and viral infections have been implicated in some cases. We have investigated the possibility that islet cells and viruses share antigenic determinants with the result that antiviral antibodies would cross-react with islet cells. Antibody titers to Coxsackie B2, B3, B4, and B5, Influenza A and B, and mumps viruses were compared with islet cell antibody (ICA) titers in newly diagnosed insulin-dependent diabetic patients and in some diabetic patients followed prospectively for 1 yr postdiagnosis. Nondiabetic patients, with culture-proven Coxsackie B4 infections and large rises in Coxsackie B4 antibody titers, were evaluated for islet cell antibodies. No relationship between ICA and viral antibody titers was found either in diabetic or nondiabetic patients. We conclude that it is unlikely that islet cells and the viruses tested share antigenic determinants and other mechanisms relating viral infection and autoimmunity in insulin-dependent diabetes must be sought.

Circulating immune complexes in diabetes.

Over the last few years more sensitive, reproducible and in some instances more specific techniques for the detection of circulating immune complexes (AgAb) have been developed [1-4]. Using these newer techniques immunological aspects of various diseases have been reappraised, and the presence of AgAb in diabetes has also been investigated. The interest in immune phenomena in diabetes has been increasing progressively. In the 1960s several studies, originating from the immunological implications of heterologous insulin administration, suggested that immune mechanisms could be of importance in the aetiology of diabetic microangiopathy. Morphological similarities with other immunological disorders were described [5-7], immune components were found in microangiopathic vessels [8-11], and diabeticlike lesions were experimentally induced by immune mechanisms especially in the renal glomeruli [12-14]. In the 1970s works by different authors provided a completely new outlook on the pathogenesis of insulin dependent diabetes. Clinical studies had shown a suggestive relationship between insulindependent diabetes and other organ specific autoimmune diseases [15, 16]. It was then reported that in insulin-dependent diabetes [17-18] there was a modification in the cell-mediated immune response [19-21], an association with particular HLA antigens [22-24], and finally the presence of autoantibodies against islet cells was demonstrated [25-27]. In 1977 Irvine et al. using a single sensitive method [28] reported an increase in circulating immune complexes (AgAb) in some insulin-dependent diabetics at the time of diagnosis as well as in treated diabetics. Since then other investigators have confirmed an increase in AgAb in many diabetics, and have shown a correlation between the presence of AgAb and various diabetic conditions [29-39]. Before proceeding further it is necessary to delineate precisely what can be measured by the AgAb methods. The solid phase CIq binding test, the Raji cell radioimmune assay and the conglutinin binding assay are some of the most sensitive techniques presently available [4, 40]. These methods are antigen non specific and may be influenced by immunoglobulin aggregates but not by DNA, bacterial endotoxins and heat induced aggregates. The CIq method detects mainly complexes in antigen excess through the Fc portion of immunoglobulin aggregates, whilst the other two methods reveal complexes near the equivalence point, mostly through the third component of the complement. The complexes detected by all these methods are potentially harmful since they may activate the complement system or react with those cells bearing receptors for complexed immunoglobulins or the complex bound complement (namely lymphocytes and macrophages). The antigens involved in the complex formation may be either part of a cell membrane and secondarily shed into the circulation as a complex or non-cellbound antigens. In the latter situation complexes may be formed either in the circulation, with subsequent localisation in vessel walls or perivascular tissues, or locally in certain organs or tissues. As the methods presently available do not distinguish between complexes related to specific disease processes and those found in other more common conditions, e.g. viral infections, it is not surprising that circulating AgAb are also found in some apparently healthy subjects. Thus in evaluating the presence of AgAb in a particular disease it is important to compare the results with those found in a large normal population and to select an appropriate limit of positivity [41]. Differences in these basic points may explain some of the discrepancies between the various reported data. In 1978 the increase in circulating AgAb in newly diagnosed insulin-dependent diabetics was confirmed in a large series of patients compared with agematched healthy controls using two different and sensitive methods [35]. In most of the patients AgAb

Parallel Dysfunctions of Pancreatic A, B and PP Cells in Insulin Dependent Diabetes

To test the possibility that insulitis might play an etiological role in the pathogenesis of insulin dependent diabetes, functions of 3 kinds of islet constituting cells (A, B and PP cells) were estimated by quantifying secretory responses of glucagon-, C-peptide-and pancreatic polypeptide-producing cells to hyperglycemia and hypoglycemia. In insulin dependent diabetes, all 3 hormonal responses were severely impaired to the same extent. On the other hand, 3 islet cell functions were uniformly but less severely impaired in insulin independent diabetics without a diabetic family history. These results suggest that A, B, and PP cells of islet of Langerhans are evenly destroyed in parallel fashion at least in insulin dependent diabetes and in some insulin independent diabetes, suggesting insulitis as a possible cause of these types of diabetes.