Oral probiotic administration induces interleukin-10 production and prevents spontaneous autoimmune diabetes in the non-obese diabetic mouse.

AIMS/HYPOTHESIS: Recent observations suggest the involvement of the gastrointestinal tract in the pathogenesis of islet autoimmunity. Thus, the modulation of gut-associated lymphoid tissue may represent a means to affect the natural history of the disease. Oral administration of probiotic bacteria can modulate local and systemic immune responses; consequently, we investigated the effects of oral administration of the probiotic compound VSL#3 on the occurrence of diabetes in non-obese diabetic (NOD) mice. METHODS: VSL#3 was administered to female NOD mice three times a week starting from 4 weeks of age. A control group received PBS. Whole blood glucose was measured twice a week. IFN-gamma and IL-10 production/expression was evaluated by ELISA in culture supernatants of mononuclear cells isolated from Peyer's patches and the spleen, and by real-time PCR in the pancreas. Insulitis was characterised by immunohistochemistry and histomorphometric studies. RESULTS: Early oral administration of VSL#3 prevented diabetes development in NOD mice. Protected mice showed reduced insulitis and a decreased rate of beta cell destruction. Prevention was associated with an increased production of IL-10 from Peyer's patches and the spleen and with increased IL-10 expression in the pancreas, where IL-10-positive islet-infiltrating mononuclear cells were detected. The protective effect of VSL#3 was transferable to irradiated mice receiving diabetogenic cells and splenocytes from VSL#3-treated mice. CONCLUSIONS/INTERPRETATION: Orally administered VSL#3 prevents autoimmune diabetes and induces immunomodulation by a reduction in insulitis severity. Our results provide a sound rationale for future clinical trials of the primary prevention of type 1 diabetes by oral VSL#3 administration.

Expression of endocrine autoantibodies in chronic hepatitis C, before and after interferon-alpha therapy.

Interferon-alpha (IFN-alpha) treatment for chronic hepatitis C (CHC) has been associated with thyroid autoimmunity and/or dysfunction. Only a few data concerning the prevalence of islet-cell or adrenal cortex autoantibodies in IFN-alpha-treated subjects are currently available. The aims of our study were to evaluate in CHC, 1) the prevalence and association of thyroid, islet-cell and adrenal autoantibodies, and 2) the appearance of endocrine dysfunction, before and after a 6 month IFN-alpha treatment. We analyzed serum samples from 203 adult patients at the time of clinical diagnosis of CHC and showed that the prevalence of thyroperoxidase (TPOAb), thyroglobulin (TGAb), TSH-receptor (TRAb), glutamic acid decarboxylase (GAD65Ab), IA-2/ICA512 (IA-2/ICA512Ab) and 21-hydroxylase (21OHAb) autoantibodies was similar to that observed among healthy control subjects of similar age and sex distribution. Among 99 patients with follow-up serum samples, 83 accepted and 16 refused IFN-alpha treatment. The IFN-alpha treatment was associated with increase of TPOAb levels in 3 subjects already positive at baseline, with progression to overt hypothyroidism in 2 of them. The de novo appearance of autoantibodies was observed in 5/80 (6%) cases for TPOAb, 1/81 (1.2%) for GAD65Ab and 2/81 (2.5%) for IA-2/ICA512Ab. Clinical or subclinical signs of either hyperthyroidism or hypothyroidism were demonstrated in 3/5 cases with de novo appearance of TPOAb. Four subjects, initially positive for either GAD65Ab or IA2/ICA512Ab, were all found negative after IFN-alpha-treatment. No subjects showed positivity for 21OHAb either at baseline or after the follow-up period. Our study suggests that, in CHC untreated patients, the prevalence of endocrine autoantibodies is similar to that observed in the general population. Furthermore, we demonstrate that IFN-alpha treatment is associated with the induction or enhancement of thyroid, but not of islet-cell or adrenal cortex autoimmunity.

Autoantibody recognition of COOH-terminal epitopes of GAD65 marks the risk for insulin requirement in adult-onset diabetes mellitus.

Some type 2 diabetic subjects develop secondary failure to sulphonylurea treatment and require insulin therapy. To test the diagnostic sensitivity and specificity of epitopes of GAD65 autoantibodies (GAD65Ab) for insulin requirement, in patients with latent autoimmune diabetes of the adult, we studied 569 adult subjects with a clinical diagnosis of type 2 diabetes mellitus. All the patients had been initially treated with hypoglycemic agents and/or diet for at least 1 yr. The presence of GAD65Ab (61/569, 10.7%) depended on insulin therapy (P<0.0001), low BMI (P<0.0001), and low basal C-peptide (P = 0.01). The majority of GAD65Ab-positive subjects (47/61, 77%) had antibodies directed to both middle (GAD65-MAb) and COOH-terminal (GAD65-CAb) epitopes. However, GAD65-CAb were more frequent in insulin-treated subjects (92% of GAD65Ab+ individuals) than in subjects treated with hypoglycemic agents and/or diet (18.2% of GAD65Ab+ individuals), while the exclusive presence of GAD65-MAb was more frequent in subjects treated with hypoglycemic agents and/or diet (81.8% vs. 8%) (P<0.0001). The presence of GAD65-CAb had a diagnostic specificity for insulin requirement as high as 99.4% (compared with 96.9% of GAD65Ab as measured in the traditional radiobinding assay) and identified a subgroup of patients with low BMI, low basal C-peptide values, and a need for insulin therapy. Subjects carrying only GAD65-MAb were phenotypically indistinguishable from GAD65Ab-negative patients. Patients positive for GAD65-M+CAb, but not those positive for GAD65-MAb only, showed an increased risk for thyroid autoimmunity, as revealed by the presence of thyroid peroxidase autoantibodies. Our study demonstrates that the use of epitope-specific antibody assays improves the diagnostic specificity of GAD65Ab, and that the presence of GAD65Ab binding to COOH-terminal epitopes is strongly associated with a need for insulin requirement.

Levels of adrenocortical autoantibodies correlate with the degree of adrenal dysfunction in subjects with preclinical Addison's disease.

To test the hypothesis that levels of adrenal autoantibodies correlate with the degree of adrenal dysfunction, we followed up adrenal cortex autoantibody (ACA) titers and 21-hydroxylase (21OH) autoantibody (21OHAb) levels in 19 ACA-positive subjects with preclinical Addison's disease. On enrollment, all the 19 ACA-positive subjects were positive for 21OHAb. At follow-up, the concordance rate for simultaneous presence/absence of both ACA and 21OHAb was as high as 91% and a strong, positive correlation between 21OHAb levels and ACA titers was observed (P < 0.0001). The levels of adrenal autoantibodies were positively associated with the severity of adrenal dysfunction (ANOVA, P < 0.0001 for both 21OHAb and ACA): the 21OH index was significantly lower at stage 0 or 1 than at stage 2+3 (corrected P < 0.001 andP < 0.05) or stage 4 (corrected P < 0.001 and <0.01). Similarly, ACA titer at stage 4 was significantly higher than stage 0 (P < 0.001), stage 1 (P < 0.001), and stage 2+3 (P < 0.05); and ACA titer at stage 2+3 was higher than stage 0 (P < 0.001) and stage 1 (P < 0.05). In subjects with progression of adrenal dysfunction (n = 14), levels of 21OHAb and ACA increased significantly (P = 0.041 and P = 0.002) during the follow-up period. In 5 subjects, the remission of biochemical signs of adrenal dysfunction was associated with the disappearance of both ACA and 21OHAb. Our study shows that the levels of adrenal autoantibodies correlate with the degree of adrenal dysfunction, and this suggests that production of high-level 21OHAb strongly signals the destructive phase of the autoimmune disease process.

Etiological diagnosis of primary adrenal insufficiency using an original flowchart of immune and biochemical markers.

Approximately 70-80% of cases of primary adrenal insufficiency are classified as idiopathic. An effective protocol for the etiological diagnosis of primary adrenal insufficiency is needed to ensure correct patient management. With the aim of developing an algorithm for the etiological diagnosis of primary adrenal insufficiency, we studied 56 Italian patients with nonsurgical primary adrenal insufficiency and 24 French patients with X-linked adrenoleukodystrophy (ALD) for serum levels of adrenal cortex, steroid-21-hydroxylase (21OHAb), islet cell (ICA), glutamate decarboxylase (GAD65Ab), IA2/ICA512 (ICA512Ab), thyroid peroxidase (TPOAb) autoantibodies, and plasmatic concentrations of very long chain fatty acids (VLCFA). High levels of 21OH and adrenal cortex antibodies were found in 35/42 (83%) and 17/42 (40%) Italian patients with idiopathic adrenal insufficiency, respectively. Levels of adrenal autoantibodies correlated inversely with disease duration (P < 0.0001). Elevated VLCFA were found in 4/42 (10%) idiopathic patients. A total of 34/35 (97%) idiopathic patients with a disease duration of less than 20 yr was positive for either 21OHSAb or elevated levels of VLCFA. None of 14 patients with posttuberculosis adrenal insufficiency had elevated levels of either adrenal antibodies or VLCFA. ICA, GAD65Ab, ICA512Ab, and TPOAb were found in 6/56 (11%), 8/56 (14%), 4/56 (7%), and 23/56 (41%) patients, respectively. None of 24 French ALD patients with adrenal insufficiency was positive for organ-specific autoantibodies. The measuring of 21OH antibodies and plasma VLCFA levels enabled a correct diagnosis of autoimmune (89%) and ALD (8%) in 97% of patients with idiopathic primary adrenal insufficiency of less than 20 yr of duration. The results of our study have important therapeutic and prognostic implications.

Protection from autoimmune diabetes by adjuvant therapy in the non-obese diabetic mouse: the role of interleukin-4 and interleukin-10.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease that is characterized by the destruction of insulin-producing beta-cells in the pancreatic islets. A single administration of CFA prevents clinical hyperglycaemia in non-obese diabetic (NOD) mice. We have previously shown that CFA administration does not eliminate insulitis in the pancreas of the treated animals, but diverts the disease process from a destructive to a non-destructive pathway. We provide evidence that this phenomenon may be under cytokine control. Neutralizing monoclonal antibodies against IL-4 and IL-10 were injected, singularly or in combination, into CFA-treated NOD mice. Antibody treatment did not lead to the development of overt diabetes; however, a marked impairment of glucose tolerance was shown in about one half of the mice treated with a combination of the two antibodies at the end of the study. This functional abnormality correlated with the histological loss of pancreatic islet tissue. These studies suggest a role for IL-4 and IL-10 in CFA-induced protection from diabetes in the NOD mouse. They also suggest that, in this animal model, the nature of the autoimmune response to islet tissue (either destructive or non-destructive) may reflect the relative proportion of Th1- and Th2-type cytokines produced in the lesions.

Effect of adjuvant therapy on development of diabetes in mouse and man.

Freund's complete adjuvant (CFA) and BCG vaccine modulate the development of type 1 diabetes in animal models. In non-obese diabetic mice, CFA and BCG significantly reduced the proportion developing diabetes compared with controls. Histological examination showed that autoimmune disease still developed but had been diverted to become nondestructive. In a preliminary trial in 17 newly diagnosed, type 1 diabetic patients, intracutaneous administration of 0.1 mL of BCG 1 mg/mL led to clinical remission in 11 (65%)--by week 4 in 6. Remission has been sustained in 3 for 6-10 months. No side-effects were reported. A double-blind trial of BCG is warranted.

Preventive effects of azathioprine (AZA) on the onset of diabetes mellitus in NOD mice.

We have studied the effects of long-term treatment with azathioprine (AZA) vs cyclosporin A (CSA) vs placebo (PL), in three groups of 10 week old, prediabetic NOD mice. One of 8 AZA, none of 8 CSA and 7 of 11 PL treated mice developed overt diabetes (IDDM). Quantitative morphometric analysis conducted on mouse pancreatic histologic sections documented that extent and degree of islet beta-cell damage were incomparably less severe in the mice that received AZA or CSA compared to those treated with PL. Since early and prolonged treatment with AZA seems to prevent the onset of DM in NOD mice as nearly effectively as CSA, AZA, which is significantly safer than CSA, could replace the latter as a potential approach for the immunotherapy of IDDM.

Detection of cell-mediated immunity in type I diabetes mellitus.

Type I insulin dependent diabetes mellitus (IDDM) is thought to result from chronic, cell-mediated, 'autoimmune' islet damage. Antibody testing is extensively used to define and follow the pre-diabetic population. However, the assay for cell mediated immunity (CMI) should be more predictive of impending disease. This report shows that it is possible to detect human islet reactive CMI in the IDDM patient. Groups of athymic CD-1 nu/nu mice were injected intraperitoneally with either mononuclear blood cells (MBCs) or plasma from 10 newly diagnosed Type I diabetic patients and 10 normal control subjects. Both glycemic control and histopathology were used to assay islet specific CMI in diabetic individuals. None of the injected mice showed any impairment of glycemic control. However, MBCs from six of 10 diabetic patients, but from none of the 10 normal subjects, induced significant mononuclear cell infiltrate in the pancreas of the recipient mice (P = 0.005). The infiltrate was focused on the islet tissue and no damage was seen in control tissues. No histological abnormalities were observed when plasma was transferred. We conclude that cellular reactivity seen in this model is tissue specific and disease associated. Our findings provide evidence that CMI to human islet tissue can be detected in IDDM patients.

Facilitation of specific tolerance induction in adult mice by RS-61443.

RS-61443 is an immunosuppressive agent that facilitates pancreatic islet allograft acceptance in two mouse strain combinations (BALB/c----CBA and C57Bl/6J----BALB/c). A remarkable feature of this agent is its ability to facilitate long-term graft acceptance after a short (30 days) period of treatment; following withdrawal of the agent 40-70% of islet allografts are maintained for an indefinite period. This long-term graft acceptance has been shown to result from specific tolerance induction in the recipient animal. The state of specific tolerance is an active rather than a passive form of tolerance, such as clonal deletion or anergy. Active tolerance induction is cyclosporine-sensitive, although cyclosporine enhances graft acceptance when used in combination therapy with RS-61443, this agent inhibits tolerance development under the influence of RS-61443.

Immunoprotection of pancreatic islet grafts within artificial microcapsules.

To circumvent pancreatic islet graft-directed immune destruction we enveloped porcine islets within highly biocompatible and selectively permeable algin/polyaminoacid microcapsules. These special microspheres were deposited between the inner (permeable) and the outer (impermeable) layers of an artificial, coaxial vascular prosthesis, directly anastomized to blood vessels. Five dogs with spontaneous, insulin-dependent diabetes received microencapsulated porcine islets in arterio-vein iliac prosthesis by-passes. One showed complete and the remainder partial, sustained reversal of hyperglycemia. Microencapsulation may be a potential solution to immunological problems related to islet transplantation.

[Islands of Langerhans transplant in the treatment of diabetes mellitus: current status, indications, future perspectives]. / Il trapianto delle insule di Langerhans nella terapia del diabete mellito: stato attuale, indicazioni, prospettive future.

Islet cell transplantation is a potential and attractive alternative to exogenous insulin administration for the therapy of IDDM. Large scale clinical applicability of this approach has been hampered, so far, by technical problems such as separation of massive islet concentrations and immune rejection. Microencapsulation within algin/polyaminoacids has provided islets with selective permeable biomembranes thus allowing prevention of the host's immune response and circumvention of general immunosuppression of the recipient. This progress could offer new opportunities for islet cell transplantation in patients with IDDM.

A method for the massive separation of highly purified, adult porcine islets of Langerhans.

A method for the massive and reproducible isolation of highly purified, adult porcine islets of Langerhans is described. The successful combination of donor animal-strain selection with original procedures for pancreas retrieval and enzymatic digestion permitted us to separate uniquely massive concentrations of pure porcine islets with no need for mechanical disruption of the pancreatic tissue. Following our procedure, porcine islets, which fully retain viability and function, can be harvested easily and rapidly. Xenotransplantation of such islets, immunoprotected within algin/polyaminoacidic microcapsules, was associated with complete reversal of hyperglycemia in rodents with either spontaneous or streptozotocin-induced diabetes mellitus.

Insulin resistance in porphyria cutanea tarda.

It has been reported that patients with porphyria cutanea tarda (PCT) develop carbohydrate (CHO) intolerance and manifest diabetes melitus (DM) more frequently than the normal population. In order to verify whether this is due to insulin resistance we studied 5 patients with PCT and 5 normal subjects matched for age, sex and weight. In all the patients an evaluation consisted of the glycemic curve and insulin response to an iv glucose tolerance test (IVGTT: 0.33 g/kg) as well as of an evaluation of the circulating monocyte insulin receptors. Blood samples were drawn in the basal state to measure plasma levels of NEFA, glycerol, and intermediate metabolites. The patients with PCT showed normal glucose tolerance which was obtained, however, at the expense of the elevated insulin levels: therefore a condition of insulin resistance was demonstrated in these subjects. An involvement of the lipid metabolism, observed by the raised levels of plasma NEFA and glycerol, was also evident. The insulin binding to circulating monocytes was reduced but not enough to justify the degree of insulin resistance observed. Therefore, it could be hypothesized, in agreement with similar studies, that a postreceptor defect is responsible for the insulin-resistance observed in patients with PCT and that the reduction of insulin receptors is determined by the down regulation in response to elevated insulinemic levels. An alteration of the porphyrin metabolism might be responsible for this disorder.

The effect of asymptomatic nocturnal hypoglycemia on glycemic control in diabetes mellitus.

To assess the effect of asymptomatic nocturnal hypoglycemia on glycemic control in insulin-dependent diabetes mellitus, we studied, on three nights, 10 patients receiving their usual regimens of continuous subcutaneous insulin infusion. During a control night, the patients' mean (+/- SE) plasma glucose level reached a nadir of 4.5 +/- 0.2 mmol per liter at 3 a.m.; the fasting glucose level was 5.9 +/- 0.3 mmol per liter at 7:30 a.m., and a peak glucose level of 8.6 +/- 0.3 mmol per liter was reached at 10 a.m., after breakfast. During nights two and three, supplemental insulin was infused intravenously from 10 p.m. to 2 a.m. to simulate a clinical overdose of insulin. On these nights, either hypoglycemia (2.4 +/- 0.2 mmol per liter) was permitted to occur or a nearly normal glucose level (5.5 mmol per liter) was maintained by infusion of glucose. The subjects were asymptomatic on all three nights. Despite comparable plasma free insulin levels from 4 to 11 a.m., both fasting (7.3 +/- 0.2 mmol per liter) and postbreakfast (12.5 +/- 0.4 mmol per liter) plasma glucose levels were significantly higher after hypoglycemia than when hypoglycemia was prevented (6.2 +/- 0.2 mmol per liter and 8.7 +/- 0.4 mmol per liter, respectively; P less than 0.001 in both cases). Fasting levels of plasma glucose correlated directly with overnight plasma levels of epinephrine (r = 0.78, P less than 0.001), growth hormone (r = 0.57, P less than 0.009), and cortisol (r = 0.52, P less than 0.02) but correlated inversely with the overnight nadir of plasma glucose (r = -0.62, P less than 0.005). We conclude that asymptomatic nocturnal hypoglycemia can cause clinically important deterioration in glycemic control (the Somogyi phenomenon) in patients receiving intensive insulin therapy, and should therefore be considered in the differential diagnosis of unexplained morning hyperglycemia.