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Introduction: Methylmercury (MeHg) is an environmental contaminant that is of particular concern in Northern Arctic Canadian populations. Specifically, organic mercury compounds such as MeHg are potent toxicants that affect multiple bodily systems including the nervous system. Developmental exposure to MeHg is a major concern, as the developing fetus and neonate are thought to be especially vulnerable to the toxic effects of MeHg. The objective of this study was to examine developmental exposure to low doses of MeHg and effects upon the adult central nervous system (CNS). The doses of MeHg chosen were scaled to be proportional to the concentrations of MeHg that have been reported in human maternal blood samples in Northern Arctic Canadian populations., Method: Offspring were exposed to MeHg maternally where pregnant Sprague Dawley rats were fed cookies that contained MeHg or vehicle (vehicle corn oil; MeHg 0.02 mg/kg/body weight or 2.0 mg/kg/body weight) daily, throughout gestation (21 days) and lactation (21 days). Offspring were not exposed to MeHg after the lactation period and were euthanized on postnatal day 450. Brains were extracted, fixed, frozen, and sectioned for immunohistochemical analysis. A battery of markers of brain structure and function were selected including neuronal GABAergic enzymatic marker glutamic acid decarboxylase-67 (GAD67), apoptotic/necrotic marker cleaved caspase-3 (CC3), catecholamine marker tyrosine hydroxylase (TH), immune inflammatory marker microglia (Cd11b), endothelial cell marker rat endothelial cell antigen-1 (RECA-1), doublecortin (DCX), Bergmann glia (glial fibrillary acidic protein (GFAP)), and general nucleic acid and cellular stains Hoechst, and cresyl violet, respectively. Oxidative stress marker lipofuscin (autofluorescence) was also assessed. Both male and female offspring were included in analysis. Two-way analysis of variance (ANOVA) was utilized where sex and treatment were considered as between-subject factors (p* <0.05). ImageJ was used to assess immunohistochemical results., Results: In comparison with controls, adult rat offspring exposed to both doses of MeHg were observed to have (1) increased GAD67 in the cerebellum; (2) decreased lipofuscin in the locus coeruleus; and (3) decreased GAD67 in the anterior CA1 region. Furthermore, in the substantia nigra and periaqueductal gray, adult male offspring consistently had a larger endothelial cell and capillary perimeter in comparison to females. The maternal high dose of MeHg influenced RECA-1 immunoreactivity in both the substantia nigra and periaqueductal gray of adult rat offspring, where the latter neuronal region also showed statistically significant decreases in RECA-1 immunoreactivity at the maternal low dose exposure level. Lastly, males exposed to high doses of MeHg during development exhibited a statistically significant increase in the perimeter of endothelial cells and capillaries (RECA-1) in the cerebellum, in comparison to male controls., Conclusion: Findings suggest that in utero and early postnatal exposure to MeHg at environmentally relevant doses leads to long-lasting and selective changes in the CNS. Exposure to MeHg at low doses may affect GABAergic homeostasis and vascular integrity of the CNS. Such changes may contribute to neurological disturbances in learning, cognition, and memory that have been reported in epidemiological studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

Schizophrenia is a common mental disorder affecting patients' thoughts, behavior, and cognition. Recently, the NRG1/ErbB4 signaling pathway emerged as a candidate therapeutic target for schizophrenia. This study investigates the effects of aripiprazole and sertindole on the NRG1/ErbB4 and PI3K/AKT/mTOR signaling pathways in ketamine-induced schizophrenia in rats. Young male Wistar rats received ketamine (30 mg/kg, intraperitoneally) for 5 consecutive days and aripiprazole (3 mg/kg, orally) or sertindole (2.5 mg/kg, orally) for 14 days. The proposed pathway was investigated by injecting LY294002 (a selective PI3K inhibitor) (25 μg/kg, intrahippocampal injection) 30 min before the drugs. Twenty-four hours after the last injection, animals were subjected to behavioral tests: the open field test, sucrose preference test, novel object recognition task, and social interaction test. Both aripiprazole and sertindole significantly ameliorated ketamine-induced schizophrenic-like behavior, as expected, because of their previously demonstrated antipsychotic activity. Besides, both drugs alleviated ketamine-induced oxidative stress and neurotransmitter level changes in the hippocampus. They also increased the gamma-aminobutyric acid and glutamate levels and glutamate decarboxylase 67 and parvalbumin mRNA expression in the hippocampus. Moreover, aripiprazole and sertindole increased the NRG1 and ErbB4 mRNA expression levels and PI3K, p-Akt, and mTOR protein expression levels. Interestingly, pre-injecting LY294002 abolished all the effects of the drugs. This study reveals that the antipsychotic effects of aripiprazole and sertindole are partly due to oxidative stress reduction as well as NRG1/ErbB4 and PI3K/AKT/mTOR signaling pathways activation. The NRG1/ErbB4 and PI3K signaling pathways may offer a new therapeutic approach for treating schizophrenia in humans., (© 2022. The Author(s).)

Development of neuropsychiatric disorder is associated with stress-related increase in pro-inflammatory cytokines. Chrysophyllum albidum fruit is an edible tropical fruit containing vitamins and phenolic compounds, well known for their anti-inflammatory and antioxidant activities. This study was designed to investigate the neuroprotective effect of C. albidum fruit extract (CAFE) on stress and lipopolysaccharide (LPS)-induced behavioral and neurochemical impairments in mice. Male Swiss mice were divided into 6 groups (n = 6). Groups 1-3 were orally treated daily for 14 days with normal saline (0.1 mL/10 g), CAFE (100 mg/kg) and Ferulic acid (FA, 10 mg/kg), and left in home cage as controls. Groups 4-6 were treated similarly but subjected to repeated social defeat (RSD) stress using the resident-intruder model from days 1-14. The RSD-animals were injected with LPS (125 µg/kg, i.p) 60 min after each RSD session from days 8-14. Neurobehavioral functions: locomotor, cognitive and anxiety-like behaviors were assessed 24 h after the last treatment. Pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α), dopamine, acetylcholinesterase, glutamic acid decarboxylase (GAD), malondialdehyde, nitrites, and reduced glutathione (GSH) were determined in brain tissue. CAFE significantly attenuated RSD and LPS-induced hypolocomotion, cognitive impairment and anxiety-like behavior when compared to the control. Treatment with CAFE also significantly reversed the negative effects of RSD and LPS on pro-inflammatory cytokines, dopamine, acetylcholinesterase, GAD, and oxidative-nitrosative stress levels. The findings clearly indicated that Chrysophyllum albidum fruit demonstrated neuroprotective effects and can play a key role in mitigating against chronic stress and inflammation linked to neuropsychiatric disorders., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A prompt diagnosis and treatment of patients with autoimmune cerebellar ataxia (CA) with antibodies against glutamic acid decarboxylase (GAD-Abs) may lead to a better prognosis. Herein, we report prodromal transient neurological symptoms that should raise clinical suspicion of CA with GAD-Abs. We initially identified a 70-year-old man who presented a first acute episode of vertigo, diplopia, and ataxia lasting 2 weeks. Two months later, he experienced a similar episode along with new-onset gaze-evoked nystagmus. After 4 months, downbeat nystagmus, left limb dysmetria, and gait ataxia progressively appeared, and an autoimmune CA was diagnosed based on the positivity of GAD-Abs in serum and cerebrospinal fluid (CSF). We searched retrospectively for similar presentations in a cohort of 31 patients diagnosed with CA and GAD-Abs. We found 11 (35.4%) patients (all women, median age 62 years; 8/11 [72.7%] with autoimmune comorbidities) with transient neurological symptoms antedating CA onset by a median of 3 months, including vertigo in 9 (81.8%; described as paroxysmal in 8) and fluctuating diplopia in 3 (27.3%) patients. The identification of transient neurological symptoms of unknown etiology, such as paroxysmal vertigo and fluctuating diplopia, should lead to GAD-Abs testing in serum and CSF, especially in patients with autoimmune comorbidities.

Type 1 diabetes mellitus is an autoimmune metabolic disorder characterized by chronic hyperglycemia, the presence of autoreactive T and B cells and autoantibodies against self-antigens. A membrane-bound enzyme on the pancreatic beta-cells, glutamic acid decarboxylase 65 (GAD65), is one of the main autoantigens in type 1 diabetes. Autoantibodies against GAD65 are potentially involved in beta-cell destruction and decline of pancreatic functions. The human complement receptor type 1 (CD35) on B and T lymphocytes has a suppressive activity on these cells. We hypothesized that it may be possible to eliminate GAD65-specific B cells from type 1 diabetes patients by using chimeric molecules, containing an anti-CD35 antibody, coupled to peptides resembling GAD65 B/T epitopes. These molecules are expected to selectively bind the anti-GAD65 specific B cells by the co-cross-linking of the immunoglobulin receptor and CD35 and to deliver a suppressive signal. Two synthetic peptides derived from GAD65 protein (GAD65 epitopes) and anti-CD35 monoclonal antibody were used for the construction of two chimeras. The immunomodulatory activity of the engineered antibodies was tested in vitro using peripheral blood mononuclear cells (PBMCs) from type 1 diabetes patients. A reduction in the number of anti-GAD65 IgG antibody-secreting plasma cells and increased percentage of apoptotic B lymphocytes was observed after treatment of these PBMCs with the engineered antibodies. The constructed chimeric molecules are able to selectively modulate the activity of GAD65-specific B lymphocytes and the production of anti-GAD65 IgG autoantibodies by co-cross-linking of the inhibitory CD35 and the B cell antigen receptor (BCR). This treatment presents a possible way to alter the autoimmune nature of these cells., (© 2019 British Society for Immunology.)

GAD-alum given into lymph nodes to type 1 diabetes patients participating in an open-label pilot trial resulted in preservation of C-peptide similar to promising results from other trials. Here, we compared the immunomodulatory effect of giving GAD-alum directly into lymph nodes versus that induced by subcutaneous administration. Samples from T1D patients ( n = 6) who received 4  μ g GAD-alum into lymph nodes (LNs), followed by two booster injections one month apart, and from patients ( n = 6) who received two subcutaneous injections (SC) (20  μ g) given one month apart were compared. GADA, IA-2A, GADA subclasses, IgE, GAD 65 -induced cytokines, PBMC proliferation, and T cell markers were analyzed. Lower doses of GAD-alum into LN induced higher GADA levels than SC injections and reduced proliferation and IgG1 GADA subclass, while enhancing IgG2, IgG3, and IgG4. The cytokine profile was dominated by the Th2-associated cytokine IL-13, and GAD 65 stimulation induced activated CD4 T cells. Patients responding clinically best account for most of the immunological changes. In contrast, SC treatment resulted in predominant IgG1, predominant IFN- γ , higher proliferation, and activated CD4 and CD8 cells. Patients from the LN group with best metabolic outcome seemed to have common immune correlates related to the treatment. This trial is registered with DIAGNODE (NCT02352974, clinicaltrials.gov) and DIABGAD (NCT01785108, clinicaltrials.gov).

Background & Objectives: Type-1 diabetes mellitus (T1DM) and latent autoimmune diabetes in adults (LADA) share similar pathological features but differ in age of onset and progression. There is a scarcity of information on differences in CD4+ T-cell responses, particularly, cytokine secretion, between the two forms of autoimmune diabetes. Here proliferative potential and concentration of pro- and anti-inflammatory cytokines secreted by peripheral blood mononuclear cells (PBMCs) of T1DM and LADA patients were compared, after in vitro stimulation with β-cell autoantigens., Methods: A total of 19 patients with LADA, 37 with T1DM and 20 healthy controls were compared on the basis of lymphocyte proliferation and secretion of pro- and anti-inflammatory cytokines belonging to different T-helper types after in vitro stimulation of PBMCs with insulin and glutamic acid decarboxylase 65 (GAD65)., Results: Following insulin stimulation, LADA group secreted higher concentration of interleukin-17 (IL-17) (P=0.02) and had higher proportion of interferon gamma (IFN-γ) secretors (P<0.001) than T1DM group. Post-GAD65 stimulation, higher proportion of LADA patients secreted IL-23 than T1DM group (P=0.02). Proportion of responders , as well as levels of secreted IL-10, were significantly higher in LADA than T1DM group, following stimulation with both insulin (P=0.01) and GAD65 (P=0.03). A significant positive correlation was observed between body mass index and IL-17 levels (r=0.41, P=0.04) and fasting plasma C-peptide with IL-10 levels (r=0.37, P=0.04)., Interpretation & Conclusions: There are differences in the portfolio of cytokine secretion in diabetic subjects with varying rates of β-cell destruction as LADA subjects secrete higher levels of both pro- and anti-inflammatory cytokines on exposure to β-cell autoantigens, thus highlighting another distinguishing feature in the pathophysiology of the two forms of autoimmune diabetes.

Tolerogenic dendritic cells (tDC) constitute a promising therapy for autoimmune diseases, since they can anergize T lymphocytes recognizing self-antigens. Patients with type 1 diabetes mellitus (T1D) have autoreactive T cells against pancreatic islet antigens (insulin, glutamic acid decarboxylase 65 -GAD65-). We aimed to determine the ability of tDC derived from T1D patients to inactivate their insulin- and GAD65-reactive T cells. CD14+ monocytes and CD4+CD45RA- effector/memory lymphocytes were isolated from 25 patients. Monocyte-derived DC were generated in the absence (control, cDC) or presence of IL-10 and TGF-β1 (tDC), and loaded with insulin or GAD65. DC were cultured with T lymphocytes (primary culture), and cell proliferation and cytokine secretion were determined. These lymphocytes were rechallenged with insulin-, GAD65- or candidin-pulsed cDC (secondary culture) to assess whether tDC rendered T cells hyporesponsive to further stimulation. In the primary cultures, tDC induced significant lower lymphocyte proliferation and IL-2 and IFN-γ secretion than cDC; in contrast, tDC induced higher IL-10 production. Lymphocytes from 60% of patients proliferated specifically against insulin or GAD65 (group 1), whereas 40% did not (group 2). Most patients from group 1 had controlled glycemia. The secondary cultures showed tolerance induction to insulin or GAD65 in 14 and 10 patients, respectively. A high percentage of these patients (70-80%) belonged to group 1. Importantly, tDC induced antigen-specific T-cell hyporesponsiveness, since the responses against unrelated antigens were unaffected. These results suggest that tDC therapy against multiple antigens might be useful in a subset of T1D patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Growing insight into the pathogenesis of type 1 diabetes (T1D) and numerous studies in preclinical models highlight the potential of antigen-specific approaches to restore tolerance efficiently and safely. Oral administration of protein antigens is a preferred method for tolerance induction, but degradation during gastrointestinal passage can impede such protein-based therapies, reducing their efficacy and making them cost-ineffective. To overcome these limitations, we generated a tolerogenic bacterial delivery technology based on live Lactococcus lactis (LL) bacteria for controlled secretion of the T1D autoantigen GAD65370-575 and the anti-inflammatory cytokine interleukin-10 in the gut. In combination with short-course low-dose anti-CD3, this treatment stabilized insulitis, preserved functional β-cell mass, and restored normoglycemia in recent-onset NOD mice, even when hyperglycemia was severe at diagnosis. Combination therapy did not eliminate pathogenic effector T cells, but increased the presence of functional CD4(+)Foxp3(+)CD25(+) regulatory T cells. These preclinical data indicate a great therapeutic potential of orally administered autoantigen-secreting LL for tolerance induction in T1D., (© 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Aim: The balance between T helper cell subsets is an important regulator of the immune system and is often examined after immune therapies. We aimed to study the immunomodulatory effect of glutamic acid decarboxylase (GAD) 65 formulated with aluminium hydroxide (GAD-alum) in children with Type 1 diabetes, focusing on chemokines and their receptors., Methods: Blood samples were collected from 70 children with Type 1 diabetes included in a phase II clinical trial with GAD-alum. Expression of CC chemokine receptor 5 (CCR5) and CCR4 was analysed on CD4+ and CD8+ lymphocytes after in vitro stimulation with GAD(65) using flow cytometry, and secretion of the chemokines CCL2, CCL3 and CCL4 was detected in peripheral blood mononuclear cell supernatants with Luminex., Results: Expression of Th1-associated CCR5 was down-regulated following antigen challenge, together with an increased CCR4/CCR5 ratio and CCL2 secretion in GAD-alum-treated patients, but not in the placebo group., Conclusion: Our results suggest that GAD-alum treatment has induced a favourable immune modulation associated with decreased Th1/Tc1 phenotypes upon antigen re-challenge, which may be of importance for regulating GAD(65) immunity., (© 2012 The Authors. Diabetic Medicine © 2012 Diabetes UK.)

Objective: Immunotherapy using peptides from the β-cell antigen GAD65 can preserve glucose homeostasis in diabetes-prone NOD mice; however, the precise mechanisms that arrest islet-reactive T cells remain unresolved. Our previous work revealed that a dominant GAD65 epitope contained two overlapping I-A(g7)-restricted determinants, 524-538 and 530-543, with the former associated with amelioration of hyperglycemia. Here, we sought to discover whether p524-538-specific T cells could directly regulate islet-reactive T cells., Research Design and Methods: Prediabetic NOD mice were used to determine the relationship between peptide p524-538-induced interleukin (IL)-13 and regulation of islet autoimmunity. Pancreatic lymph node (PLN) cells from mice at distinct stages of islet inflammation, peri-insulitis versus invasive insulitis, were harvested to establish the expression pattern of IL-13 receptor α1 (IL-13Rα1) on islet-associated T cells., Results: Peptide p524-538 preferentially induced IL-13-producing T cells that antagonized the release of γ-interferon by spontaneously arising GAD65 autoimmunity, and recombinant human IL-13 inhibited proliferation of islet-reactive clonotypic T cells. A subset of CD4(+) T cells in NOD and NOD.BDC2.5 T cell receptor transgenic mice expressed functional IL-13Rα1, which induced phosphorylation of signal transducer and activator of transcription 6 in the presence of cognate cytokine. Notably, the number of IL-13Rα1(+) T cells was heightened in the PLN of young NOD mice when compared with older female counterparts with advanced insulitis. Immunization with p524-538 preserved IL-13Rα1 expression on PLN T cells., Conclusions: IL-13 may be important for regulating autoimmunity in the early stages of insulitis, and the loss of IL-13Rα1 on islet-reactive T cells may be a biomarker for fading regional immune regulation and progression to overt diabetes.

A phase II clinical trial with glutamic acid decarboxylase (GAD) 65 formulated with aluminium hydroxide (GAD-alum) has shown efficacy in preserving residual insulin secretion in children and adolescents with recent-onset type 1 diabetes (T1D). We have performed a 4-year follow-up study of 59 of the original 70 patients to investigate long-term cellular and humoral immune responses after GAD-alum-treatment. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with GAD(65). Frequencies of naïve, central and effector memory CD4+ and CD8+ T cells were measured, together with cytokine secretion, proliferation, gene expression and serum GAD(65) autoantibody (GADA) levels. We here show that GAD-alum-treated patients display increased memory T-cell frequencies and prompt T-cell activation upon in vitro stimulation with GAD(65), but not with control antigens, compared with placebo subjects. GAD(65)-induced T-cell activation was accompanied by secretion of T helper (Th) 1, Th2 and T regulatory cytokines and by induction of T-cell inhibitory pathways. Moreover, post-treatment serum GADA titres remained persistently increased in the GAD-alum arm, but did not inhibit GAD(65) enzymatic activity. In conclusion, memory T- and B-cell responses persist 4 years after GAD-alum-treatment. In parallel to a GAD(65)-induced T-cell activation, our results show induction of T-cell inhibitory pathways important for regulating the GAD(65) immunity.

Context: Mesenchymal stem cells (MSCs) exert an immunosuppressive effect on the immune system. However, studies on the immunomodulatory potential of MSCs in type 1 diabetes are lacking., Objective: We aimed to evaluate whether human MSCs may inhibit in vitro pancreatic islet antigen-specific T cell activation in type 1 diabetes., Design: Human MSCs were isolated and characterized. Peripheral blood mononuclear cells (PBMCs) were obtained from nine type 1 diabetic patients at disease onset and 13 healthy control subjects. IFN-gamma, IL-10, and IL-4 enzyme-linked immunospot responses of lymphocytes incubated with glutamic acid decarboxylase 65 (GAD65) were investigated in PBMC cultures and PBMC/MSC cocultures. Levels of prostaglandin E2 (PGE2), IFN-gamma, IL-4, and IL-10 in supernatants were measured by ELISA. PGE2 inhibition experiments with NS-398 and indomethacin were also performed., Results: Five diabetic patients were identified with a positive PBMC IFN-gamma response to GAD65 and negative IL-10 and IL-4 response. PBMC/MSC cocultures resulted in a significant decrease in the number of spots and in detection of IL-4-secreting cells. PGE2 inhibitors abrogated the immune-suppressive effect, indicating an involvement of PGE2 production, and the constitutive production of PGE2 by MSCs was enhanced in PBMC/MSC coculture. Moreover, in GAD-responder patients, GAD-stimulated PBMC/MSC cocultures significantly decreased secretion of IFN-gamma and IL-10 and increased secretion of IL-4., Conclusions: These results provide evidence that human MSCs abrogate in vitro a proinflammatory T helper type 1 response to an islet antigenic stimulus in type 1 diabetes. MSCs induce IL-4-producing cells, suggesting a possible switch to an antiinflammatory T helper type 2 signaling of T cells.

Unlabelled: Perfect maternal diabetes compensation is crucial for the outcome of the baby. However, little is known how hyperglycaemia influences the specific immune response. Furthermore, babies of type 1 diabetes (T1D) mothers have less risk of development T1D than babies with a T1D father. This study aimed to analyze the effect of maternal hyperglycaemia on newborns with focus on the response to diabetes-associated autoantigens., Populations: (1) Newborns of T1D mothers split into groups according to maternal diabetes compensation during the 3rd trimester: perfect (n = 15) or acceptable (n = 25) compensation. (2) newborns with T1D father (n = 12) (3) newborns with a mother treated for either gestational or type 2 diabetes (n = 10) (4) control newborns (n = 25). Spontaneous as well as diabetes-associated autoantigen-stimulated production of 23 cytokines and chemokines were tested using protein microarray. In addition, the influence of glucose on cytokine and chemokine responsiveness was analyzed in vitro. The study groups differed in their spontaneous as well as stimulated cytokine and chemokine spectra. A prominent Th1 response (high IFN-gamma) from autoantigen stimulation was observed especially in babies of T1D fathers (P = 0.001) and also in mothers with perfect diabetes compensation during the 3rd trimester (P = 0.016) in comparison with control newborns. By contrast, cord blood mononuclear cells cultivated in vitro in high glucose concentration decreased the diabetogenic stimulated Th1 cytokine response. Maternal 'sweet' as well as 'autoimmune environment' may both lead to lower occurrence of T1D within their offspring. Further studies will reveal the exact immunological mechanism of this observation.

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.

Although the majority of agents with antiexcitotoxic action act as glutamate receptor antagonists, enzymatic degradation of glutamate can also be neuroprotective. The very low specific activity of the mammalian form of glutamate decarboxylase (GAD), the enzyme that catalyzes the formation of gamma-aminobutyric acid (GABA) from glutamate in neurons, is likely to limit its utility as an antiglutamate neuroprotectant. In contrast, the bacterial form of GAD can be isolated with relatively high specific activity and is most active in acidic environments. We have expressed and purified GAD from Escherichia coli (bGAD) and tested the ability of the enzyme to protect against glutamate excitotoxicity. Incubation of rat hipppocampal slices with the potassium channel antagonist tetraethyl ammonium (TEA) resulted in widespread excitotoxic death of pyramidal and granule cell neurons. bGAD alone showed no significant neurotoxicity and significantly reduced excitotoxicity induced by TEA. We hypothesize that bGAD may be internalized into the synaptic vesicle compartment by nonspecific endocytosis, where both the appropriate pH and high glutamate concentrations are present. Targeting of this enzyme to the interior of synaptic vesicles may enhance its potency as a neuroprotectant against excitotoxicity.

Study Design: A prospective in vivo animal study., Objectives: To examine the effect of the proenkephalin and glutamic acid decarboxylase (GAD)-expressing herpes simplex virus (HSV)-based vectors in a rodent model of lumbar radiculopathy., Summary of Background Data: We have previously shown that nonreplicating HSV-based vectors coding for proenkephalin or GAD can be used to transduce dorsal root ganglion (DRG) neurons in vivo to produce enkephalin or gamma-aminobutyric acid. HSV-mediated gene transfer of proenkephalin or GAD to DRG reduces pain-related behavior in rodent models of peripheral neuropathic pain., Methods: We created a model of lumbar radiculopathy by ligation of the dorsal and ventral lumbar roots proximal to the DRG. Three days later, we inoculated nonreplicating HSV-based vectors coding for proenkephalin or GAD subcutaneously in the foot., Results: Subcutaneous inoculation of either vector 3 days after ligation of the dorsal and ventral L5 lumbar roots resulted in a substantial and significant reduction in pain-related behavior (mechanical allodynia). Vector-mediated reduction in pain-related behavior was higher in magnitude and longer in duration after inoculation of the GAD-expressing vector than that achieved by the inoculation of the proenkephalin-expressing vector., Conclusions: HSV-mediated gene transfer provides a novel method for treating chronic neuropathic pain related to lumbar root injury in rodents.

Type 1 diabetes (T1D) is an autoimmune disease suggested to be of a T helper (Th)1-like origin. This study aimed to investigate the Th1-like and Th2-like profile in high-risk individuals during the prediabetic phase and the immunologic effect of treatment with nicotinamide. High-risk first-degree relatives of T1D patients participating in the European Nicotinamide Diabetes Intervention Trial (ENDIT) were treated with either nicotinamide or placebo. Peripheral blood mononuclear cells (PBMC) were obtained during the prediabetic phase and close to the onset of manifest T1D and from nondiabetic high-risk individuals. Using the sensitive enzyme-linked immunospot (ELISPOT) technique to distinguish Th1-like from Th2-like lymphocytes, secretion of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) was analyzed from PBMCs spontaneously and after in vitro stimulation with the diabetes-associated autoantigens, glutamic acid decarboxylase 65 (GAD65, protein and peptide, aa 247-279), recombinant tyrosine phosphatase (IA-2), and heat shock protein (HSP, aa 437-460). High-risk individuals showed high spontaneous as well as autoantigen-induced IFN-gamma secretion. Secretion of IFN-gamma and the IFN-gamma/IL-4 ratio, induced by autoantigens, decreased in individuals developing T1D (p < 0.05), whereas nondiabetic individuals showed an increased IL-4 response (p < 0.05). Thus, a Th1-dominated cytokine profile observed in high-risk individuals inclined toward a diagnosis of diabetes. Nicotinamide caused decreased spontaneous (p = 0.05) and in vitro autoantigen-induced IFN-gamma secretion (p < 0.05) and may play a role in immune regulation, even though it has not been shown to prevent T1D.

Clinical intervention trials evaluating the efficacy of antibody immunotherapy in type 1 diabetes are in progress. We tested effects on prediabetic islet antigen-specific autoreactive T cells of antithymocyte globulin (ATG) and humanized monoclonal antibodies against CD3 (ChAglyCD3) or CD25 (daclizumab) with regard to downmodulation of the target protein, proliferation, cytokine production, complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC), and survival. ATG leads to depletion of autoreactive CD4+ T cells by ADCC, CDC, and apoptosis, whereas anti-CD3 and anti-CD25 inhibited T-cell autoreactivity in a nondepleting fashion. ATG treatment led to a cytokine burst of Th1- and Th2-associated cytokines. Modulation of cytokine release through humanized monoclonal antibodies was moderate and selective: anti-CD25 led to increased release of IL-2 and reduced production of TNFalpha, whereas anti-CD3 decreased release of interferon-y and IL-5 and increased secretion of IL-10. ATG and the humanized monoclonal antibodies displayed contrasting mechanisms of action.

The "gold standard" for evaluation of immunoassays is blinded testing, using coded samples and antigens. Although assays for autoreactive T cells are no exception to this rule, it is nonetheless rarely applied in this context. To facilitate such investigations, we coded a panel of 10 peptides representing T cell epitopes reported to be of relevance to islet autoimmunity. These were deployed in a novel cytokine ELISPOT assay, in which the use of immunoglobulin-free medium reduces background reactivity and thus potentially enhances the specificity and sensitivity of detection of autoreactive T cells. Significant IFN-gamma production against GAD65 (554-575), insulin (B9-23), and IA-2 (709-736) peptides were observed in type 1 diabetic patients, whereas no significant changes from background were detected in healthy controls. These results confirm the utility of the blinded performance of T cell assays as the most robust platform for assessing technologies to T cell autoreactivity.

HLA-DQ is strongly associated with genetic predisposition to type 1 diabetes. It is assumed that HLA-DQ molecules exert their effects on the disease via the presentation of peptides from islet autoantigens to CD4(+) T-cells, but little information regarding HLA-DQ-restricted, islet antigen-specific, autoreactive T-cells is available. To investigate the role of HLA-DQ in the immune response to islet autoantigens, we measured T-cell proliferation to insulin and GAD65 in the presence and absence of monoclonal antibodies that block HLA-DQ-mediated antigen presentation in recent-onset type 1 diabetic patients and their siblings. Positive proliferative T-cell responses to GAD65 were observed in 60% of type 1 diabetic patients and 52% of siblings. This proliferation was significantly reduced in the presence of anti-DQ antibody, demonstrating the presence of primed, effector HLA-DQ-restricted T-cell responses to GAD65. Positive proliferative responses to insulin were observed in 25% of type 1 diabetic patients and 10% of siblings. However, blocking HLA-DQ-restricted T-cell responses led to a significant increase in proliferation to insulin, implying the presence of primed suppressive HLA-DQ-restricted T-cell responses to insulin. These results indicate that HLA-DQ acts as a restriction element for both proliferative and suppressor cells, with the relative balance of these cells dependent on the nature of the autoantigen.

The neural network of the inferior colliculus (IC), implicated in the generation of defensive behavior to aversive acoustic stimuli, is under tonic GABAergic control. Dopamine also seems to have a modulatory role in these neural circuits. It is still unclear how such changes in transmission of acoustic information influence the motor expression of the defensive behavior. Startle reaction to a sudden noise has been used as an effective way to measure the motor reactivity of rats to fearful acoustic stimuli. In this work we examined the processing of sensorial information--assessed by the recording of auditory evoked potentials (AEP)--and the behavioral effects--evaluated by the freezing and startle responses--during the reduction of GABA levels caused by microinjections of semicarbazide (SMC, 6 microg/0.2 microl), a glutamic acid decarboxylase inhibitor, into the IC. These data were compared to the effects of the overall arousal elicited by apomorphine (APO, 0.5 mg/kg, i.p.). The results obtained show that IC microinjections of SMC induced freezing behavior, enhanced the AEP and impaired the startle reaction to a loud sound. On the other hand, APO changed neither the AEP nor the startle in the same experimental conditions. These results suggest that the release of GABAergic control of the neural substrates of aversion in the IC results in an increased processing of auditory information along with an inhibitory influence on the motor pathways responsible for the startle response.

Gamma-aminobutyrate (GABA) is a ubiquitous four-carbon, non-protein amino acid synthesized by glutamate decarboxylase. Previous research suggests that the endogenous synthesis of GABA, a naturally occurring inhibitory neurotransmitter at neuromuscular junctions, serves as a plant resistance mechanism against invertebrate pests. In this study, two homozygous transgenic tobacco lines constitutively overexpressing a single copy of a full-length chimeric glutamate decarboxylase cDNA and possessing enhanced capacity for GABA accumulation (GAD plants), a homozygous transgenic line lacking the gene insert, and wild-type tobacco were employed. Tobacco budworm larvae were presented with plant attached wild type and transgenic leaves for 4 hr in a feeding preference study. Larvae consumed six to twelve times more leaf tissue from wild-type plants than from GAD plants. These results suggest that leaf GABA accumulation, which is known to occur in response to insect larval walking and feeding, represents a rapidly deployed local resistance mechanism.

The 65 kD isoform of Glutamic Acid Decarboxylase (GAD), is one of the major autoantigens in human type 1 diabetes mellitus. This enzyme shares amino acid identity, in select regions already determined as antigenic with its counterpart from E. coli. We tested the reactivity of diabetic and normal sera and an E. coli GAD-specific monoclonal antibody (2D9) to E. coli GAD by solid phase and competition ELISA, as well as immunoblotting to check for cross-reactivity of autoantibodies to the two antigens. Specific antibodies for E. coli GAD are present in diabetics and normal subjects without any differences in frequency and titer. The reactivity of such antibodies in ELISA could be blocked in a dose-dependent manner by the addition of excess antigen in the liquid phase. Furthermore, the monoclonal antibody against E. coli GAD does not recognise human recombinant GAD65 in an ELISA. We conclude that there is no basis for cross-reactivity between the two antigens, and antibody reactivity to GAD65 in man cannot arise from cross-reactivity to the E. coli enzyme.

Anti-glutamate decarboxylase autoantibodies (GAD-A) are associated with a group of patients with progressive cerebellar ataxia. We reported previously that cerebellar GABA(A)-mediated synaptic transmission was presynaptically depressed by GAD-A in the cerebrospinal fluid (CSF). Using whole-cell recording of rat cerebellar slices, we found in the present study that CSF immunoglobulins from ataxic patients reduced gamma-aminobutyric acid (GABA) release from cerebellar interneurons, thereby attenuating presynaptic inhibition on neighboring excitatory synapses through GABA(B) receptors (GABA(B)Rs). Our results suggest that in in vitro slices, GAD-A elicited the pathophysiological action of reduction in GABA release, which subsequently resulted in dual synaptic impairment in the cerebellar circuit, by depression of GABA(A) receptor (GABA(A)R)-mediated inhibitory synaptic transmissions, and attenuation of GABA(B) receptor-mediated inhibition of excitatory transmissions.

Nonobese diabetic (NOD) mice spontaneously develop diabetes as a consequence of an autoimmune process that can be inhibited by immunotherapy with the 60-kDa heat shock protein (hsp60), with its mycobacterial counterpart 65-kDa (hsp65), or with other Ags such as insulin and glutamic acid decarboxylase (GAD). Microbial infection and innate signaling via LPS or CpG motifs can also inhibit the spontaneous diabetogenic process. In addition to the spontaneous disease, however, NOD mice can develop a more robust cyclophosphamide-accelerated diabetes (CAD). In this work, we studied the effect on CAD of DNA vaccination with constructs encoding the Ags human hsp60 (phsp60) or mycobacterial hsp65 (phsp65). Vaccination with phsp60 protected NOD mice from CAD. In contrast, vaccination with phsp65, with an empty vector, or with a CpG-positive oligonucleotide was not effective, suggesting that the efficacy of the phsp60 construct might be based on regulatory hsp60 epitopes not shared with its mycobacterial counterpart, hsp65. Vaccination with phsp60 modulated the T cell responses to hsp60 and also to the GAD and insulin autoantigens; T cell proliferative responses were significantly reduced, and the pattern of cytokine secretion to hsp60, GAD, and insulin showed an increase in IL-10 and IL-5 secretion and a decrease in IFN-gamma secretion, compatible with a shift from a Th1-like toward a Th2-like autoimmune response. Our results extend the role of specific hsp60 immunomodulation in the control of beta cell autoimmunity and demonstrate that immunoregulatory networks activated by specific phsp60 vaccination can spread to other Ags targeted during the progression of diabetes, like insulin and GAD.

Type 1 diabetes is a T cell-mediated disease in which B cells serve critical Ag-presenting functions. In >95% of type 1 diabetic patients the B cell response to the glutamic acid decarboxylase 65 (GAD65) autoantigen is exclusively directed at conformational epitopes residing on the surface of the native molecule. We have examined how the epitope specificity of Ag-presenting autoimmune B cell lines, derived from a type 1 diabetic patient, affects the repertoire of peptides presented to DRB1*0401-restricted T cell hybridomas. The general effect of GAD65-specific B cells was to enhance Ag capture and therefore Ag presentation. The enhancing effect was, however, restricted to T cell determinants located outside the B cell epitope region, because processing/presentation of T cell epitopes located within the autoimmune B cell epitope were suppressed in a dominant fashion. A similar effect was observed when soluble Abs formed immune complexes with GAD65 before uptake and processing by splenocytes. Thus, GAD65-specific B cells and the Abs they secrete appear to modulate the autoimmune T cell repertoire by down-regulating T cell epitopes in an immunodominant area while boosting epitopes in distant or cryptic regions.

Soluble HLA-DR401 or -DR404 tetramers containing a peptide corresponding to an immunodominant epitope from human GAD65 were used to analyze peripheral blood T-cells of newly diagnosed type 1 diabetic patients and at-risk subjects. Peripheral blood mononuclear cells were expanded on antigen-presenting cells presenting GAD65 peptide and subsequently activated with specific plate-bound class II-peptide monomers. T-cell activation defined in flow cytometry by CD4(high) and/or CD25 markers were observed in all type 1 diabetic patients and some at-risk subjects, but not in normal control subjects. The activated T-cells stained positive with tetramers containing the GAD65 epitope 555-567. Tetramer-positive cells were CD4(high) T-cells with high avidity for an immunodominant GAD65 T-cell epitope. Phenotyping of T-cells utilizing HLA class II tetramers provides a new tool to characterize the autoimmune response in type 1 diabetes.

Insulin-dependent type 1 diabetes is an autoimmune disease mediated by T lymphocytes recognizing pancreatic islet cell antigens. Glutamic acid decarboxylase 65 (GAD65) appears to be an important autoantigen in the disease. However, T cells from both patients with type 1 diabetes and healthy subjects vigorously proliferate in response to GAD65 stimulation ex vivo, leading us to postulate that the critical event in the onset of human diabetes is the activation of autoreactive T cells. Thus, we investigated whether GAD65-reactive T cells in patients with diabetes functioned as previously activated memory T cells, no longer requiring a second, costimulatory signal for clonal expansion. We found that in patients with new-onset type 1 diabetes, GAD65-reactive T cells were strikingly less dependent on CD28 and B7-1 costimulation to enter into cell cycle and proliferate than were equivalent cells derived from healthy controls. We hypothesize that these autoreactive T cells have been activated in vivo and have differentiated into memory cells, suggesting a pathogenic role in type 1 diabetes. In addition, we observed different effects with selective blockade of either B7-1 or B7-2 molecules; B7-1 appears to deliver a negative signal by engaging CTLA-4, while B7-2 engagement of CD28 upregulates T cell proliferation and cytokine secretion.

Objective: To investigate the effects of antisense glutamic acid decarboxylase (GAD(67)) oligodeoxynucleo-tide (ODN) on behavior, seizure threshold and EEG of hippocampus in the epileptic rats induced by pentylenetetrazol (PTZ)., Methods: A model of chronic epilepsy in rats was established by PTZ. The inhibition of GAD(67) mRNA expression in hippocampus was selectively induced by antisense oligodeoxynucleotide of GAD(67). The effect of antisense GAD(67) ODN on behavior, seizure threshold and EEG recording of kindled rats was examined., Results: Antisense GAD(67) ODN could inhibit the expression of GAD(67) mRNA and the concentration of GABA. It also could significantly shorten the latencies of seizure and increase the level of seizure and the frequency of epileptiform discharges., Conclusion: The gene of GAD(67) may be an anti-seizure gene, which might inhibit epileptiform discharge. The treatment of epilepsy by GAD(67) gene will have a bright future.

A workshop on autoreactive T-cell responses in NOD mice was held to optimize autoreactive T-cell detection methodologies. Using different proliferation assay protocols, 1 of the 11 participating laboratories detected spontaneous T-cell responses to GAD(524-543) and insulin(9-23) in their NOD mice. Two other laboratories were able to detect autoreactive responses when using enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) analysis of cytokines in culture supernatants, suggesting that these assays provided greater sensitivity. To address the divergent findings, a follow-up mini-workshop tested NOD mice from four different colonies side-by-side for T-cell proliferative responses to an expanded panel of autoantigens, using the protocol that had enabled detection of responses in the 1st International NOD Mouse T-Cell Workshop. Under these assay conditions, 16 of 16 NOD mice displayed proliferative responses to whole GAD65, 13 of 16 to GAD(524-543), 9 of 16 to GAD(217-236), 7 of 16 to insulin(9-23), and 5 of 16 to HSP277. Thus, spontaneous proliferative T-cell responses can be consistently detected to some beta-cell autoantigens and peptides thereof. Overall, the results suggest that more sensitive assays (e.g., ELISPOT, ELISA analysis of cytokines in supernatants, or tetramer staining) may be preferred for the detection of autoreactive T-cells.

We have reported previously that nonobese diabetic (NOD) fetal pancreas organ cultures lose the ability to produce insulin when maintained in contact with NOD fetal thymus organ cultures (FTOC). Initial studies indicated that exposure to glutamic acid decarboxylase (GAD65) peptides in utero resulted in delay or transient protection from insulin-dependent diabetes mellitus (IDDM) in NOD mice. We also found that exposure of young adult NOD mice to the same peptides could result in acceleration of the disease. To more closely examine the effects of early and late exposure to diabetogenic Ags on T cells, we applied peptides derived from GAD65 (GAD AA 246-266, 509-528, and 524-543), to our "in vitro IDDM" (ivIDDM) model. T cells derived from NOD FTOC primed during the latter stages of organ culture, when mature T cell phenotypes are present, had the ability to proliferate to GAD peptides. ivIDDM was exacerbated under these conditions, suggesting that GAD responsiveness correlates with the ivIDDM phenotype, and parallels the acceleration of IDDM we had seen in young adult NOD mice. When GAD peptides were present during the initiation of FTOC, GAD proliferative responses were inhibited, and ivIDDM was reduced. This result suggests that tolerance to GAD peptides may reduce the production of diabetogenic T cells or their capacity to respond, as suggested by the in utero therapies studied in NOD mice.

Glutamic acid decarboxylase autoimmunity was investigated by immunizing female BALB/c, C57B1/6, National Marine Research Institute (NMRI) and non-obese diabetic (NOD) mice once or twice with glumatic acid decarboxylase, GAD65, bovine serum albumin, or phosphate-buffered saline in incomplete Freunds adjuvant, or not treating. Mice immunized with GAD65, showed splinic T-cell reactivity to GAD 65 in vitro assessed by cytokine secretion. However untreated NOD mice did not. NOD mice showed a vigorous IFN-gamma response after one immunization, whereas NMRI mice showed a lower response. IL-4 and IL-10 were only detected after two immunizations with higher levels in BALB/c, NMRI and NOD mice, compared to C57B1/6 mice. High levels of GAD65 antibodies were detected in all mice immunized with GAD65, though lower levels were found in C57B1/6 mice. Histological analysis of pancreata revealed that no control mice, regardless of treatment, had mononuclear cell infiltration in the islets. In NOD mice, peri-insulitis was detected in all groups, but less so in GAD65 and bovine serum albumin (BSA) immunized animals. These data demonstrate that NOD mice respond more vigorously to immunization with GAD65 than non-diabetic mice strains. Furthermore, immunization with GAD65 is not sufficient to provoke onset of diabetes in NOD mice or induce islet cell pathology in non-diabetes prone mice., (Copyright 1998 Academic Press)

IDDM is a T-cell-mediated autoimmune disease in which the insulin-producing beta-cells are destroyed. The disease process is complex, involving the recognition of several beta-cell autoantigens. One of these, GAD65, appears to have a critical and not fully defined role in IDDM in humans and in the NOD mouse. We provide evidence that an ongoing diabetogenic response in NOD mice can be suppressed after intravenous administration of GAD65, but not by other beta-cell autoantigens. Furthermore, suppression of the diabetogenic response is mediated by the induction of GAD65-specific CD4+ regulatory T-cells. Finally, cytokine analysis indicates that these CD4+ regulatory T-cells have a T-helper 2 phenotype.

The lymphopenia gene (lyp) on rat chromosome 4 is closely linked to autoimmune diabetes in the BioBreeding (BB) rat. Lyp controls the number of peripheral lymphocytes by reducing T cells of the RT6+ phenotype by almost 90%. Following nine cycle of marker-assisted cross-intercross breeding we have developed congenic lyp/lyp, lyp/+ and +/+ (wildtype) rats on the background of DR rats. Prediabetic and insulitis free lyp/lyp, lyp/+ and +/+ rats were used to determine the effect of lyp on cytokine expression in the thymus. In situ hybridization of thymus cryosections showed that the interferon gamma (IFN gamma) mRNA expression was highest in lyp/lyp rats and the hybridization signal was restricted to the medullary compartment. The frequency of IFN gamma and interleukin (IL)-10 mRNA expressing cells in isolated thymocytes determined by quantitative image analysis, demonstrated an increased IFN gamma: IL-10 ratio in thymocytes from lyp/lyp homozygotes compared to lyp/+ and +/+ rats. This confirmed a lyp gene dose-dependent segregation of the IFN gamma high phenotype. Recombinant human glutamic acid decarboxylase (GAD65) increased the number of IFN gamma and IL-10 mRNA expressing thymocytes after in vitro culture. We conclude that the quantitative ratio of cytokine producing thymocytes is associated with the lyp genotype. These potentially autoreactive thymocytes may explain the establishment of beta-cell directed autoimmunity in the BB rat despite peripheral lymphopenia.

The M(r) 65,000 isoform of glutamic acid decarboxylase (GAD65) has been implicated as the initiating islet cell antigen in the pathogenesis of diabetes, primarily based on studies in non-obese diabetic (NOD) mice. To test the role of this islet cell autoantigen in the pathogenesis of spontaneously occurring diabetes in another animal model, purified recombinant human islet GAD65 was injected i.v. at 200 micrograms/animal into 18-day-old diabetes-prone BB rats. For controls, bovine serum albumin (BSA), which has also been implicated in the pathogenesis of diabetes, or buffer alone was injected into age matched BB rats. At 210 days of age there were no differences in diabetes incidence in the 3 groups, i.e. 73% (11 of 15) in the GAD65-treated, 81% (13 of 16) in the BSA-treated and 65% (11 of 17) in the buffer-treated animals, or in the median age at onset of disease, i.e. 79 days (range 65-111), 87 days (range 60-107) and 86 days (range 74-109), respectively. The lack of protection against diabetes following GAD65 treatment could hypothetically be explained by no or by an aberrant expression of GAD in BB-rat islet cells. However, immunohistochemistry of pancreata and immunoblotting analysis of isolated islets showed that the expression of GAD65 and GAD67 was similar in BB and Lewis rats. In conclusion, these data indicate that neither GAD65 nor BSA autoimmunity is important for the development of diabetes in BB rats, in contrast to the situation in NOD mice, and further emphasizes that extrapolation from only one animal model to autoimmune diabetes in general may not be appropriate.

Background: Insulin (1) and glutamic acid decarboxylase (GAD) (2) are both autoantigens in insulin-dependent diabetes mellitus (IDDM), but no molecular mechanism has been proposed for their association. We have identified a 13 amino acid peptide of proinsulin (amino acids 24-36) that bears marked similarity to a peptide of GAD65 (amino acids 506-518) (G. Rudy, unpublished). In order to test the hypothesis that this region of similarity is implicated in the pathogenesis of IDDM, we assayed T cell reactivity to these two peptides in subjects at risk for IDDM., Materials and Methods: Subjects at risk for IDDM were islet cell antibody (ICA)-positive, first degree relatives of people with insulin-dependent diabetes. Peripheral blood mononuclear cells from 10 pairs of at-risk and HLA-DR matched control subjects were tested in an in vitro proliferation assay., Results: Reactivity to both proinsulin and GAD peptides was significantly greater among at-risk subjects than controls (proinsulin; p < 0.008; GAD; p < 0.018). In contrast to reactivity to the GAD peptide, reactivity to the proinsulin peptide was almost entirely confined to the at-risk subjects., Conclusions: This is the first demonstration of T cell reactivity to a proinsulin-specific peptide. In addition, it is the first example of reactivity to a minimal peptide region shared between two human autoimmune disease-associated self antigens. Mimicry between these similar peptides may provide a molecular basis for the conjoint autoantigenicity of proinsulin and GAD in IDDM.

We have postulated that a defect in specific antigenic induction of suppressor T lymphocytes may account for the immunoregulatory disorder in autoimmune thyroid disease. In this context, we have measured the proliferative responses of peripheral blood mononuclear cells (PBMC) to the synthetic peptides corresponding to the extracellular domain of the TSH receptor (TSHR) and recombinant glutamate decarboxylase (GAD65) by means of 3H thymidine incorporation. We have also studied the antigenic activation of CD4+ and CD8+ T lymphocytes by measuring human leukocyte antigen-DR (HLA-DR) expression on the cell surface by flow cytometric analysis. PBMC obtained from 47 patients with Graves' disease (GD) [including 19 hyperthyroid GD (hyper GD)], 18 with Hashimoto's thyroiditis (HT), 7 with nontoxic nodular goiter (NG), 18 with insulin-dependent diabetes (IDDM), and 20 normal controls (N), were cultured for 7 days in the presence or absence of the pool peptides representing 3 different segments of TSHR or GAD65 at final concentration of 30 micrograms/mL or 10 micrograms/mL. The proportion of subjects whose PBMC gave a positive proliferative response with a stimulation index (SI) of over 2.3 (i.e. above the mean +2 SD for N) to TSHR peptides was significantly higher in the hyper GD group than among euthyroid GD (eu GD), HT, IDDM, and N group. The corresponding differences in mean SI provided analogous results, showing significant responses above normal in only hyper GD. The CD4+ T lymphocytes from hyper GD group were significantly more activated by TSHR peptides compared to eu GD, HT, IDDM, and N, and this induction correlated to their thyroid hormone levels. Quite differently, the activation of CD8+ T lymphocytes from both hyper GD and eu GD group in response to TSHR peptides was impaired compared to HT, IDDM, and the N group; in contrast to the findings with CD4+ T lymphocytes, this was independent of thyroid hormone levels. On the other hand, while the CD8+ T lymphocytes from GD and N groups were activated equally by GAD65, the activation of CD8+ T lymphocytes from the IDDM group by GAD65 was impaired compared to the GD and N groups. In conclusion, the activation of CD8+ T lymphocytes from GD and IDDM by relevant antigens (i.e. TSHR peptides for GD and GAD65 for IDDM) was impaired, but not by irrelevant antigens (i.e. GAD65 for GD and TSHR peptides for IDDM). There was also a modest stimulation of CD8+ T cells from all groups by tetanus toxoid and cardiac myosin light chain peptide, both irrelevant antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Type 1 diabetes is the result of an ongoing autoimmune response to specific proteins expressed by the insulin producing beta cells. Recently, a number of beta cell autoantigens have been identified. However, their role in mediating the diabetogenic response is not known. Here we assess the relative importance of a panel of beta cell autoantigens in the disease process. The approach was to inhibit T cell proliferation to a given autoantigen by either i.t. or i.v. injections, and then determine the effect this had on the diabetogenic response. We show that administering murine glutamic acid decarboxylase (GAD) to 3-week-old NOD females can reduce the frequency of insulitis and prevent the onset of diabetes. In contrast, carboxypeptidase H or peripherin do not induce a similar protective effect, suggesting that GAD has a critical role in the diabetogenic response. These results also suggest that GAD may provide a useful target for antigen-specific immunotherapy.

Type 1 diabetes in man and the NOD (H-2g7) mouse is frequently associated with an autoimmune response to two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67. GAD-specific autoantibodies produced by B cells and GAD-specific T cells have been observed in both species. In the current study, the response to a GAD65-derived peptide, GAD65 524-543, previously reported to be an epitope recognized by spleen cells obtained from 3-week-old NOD mice, was assessed in NOD MHC and non-MHC congenic strains. Although spontaneous reactivity to GAD65 524-543 was not observed in NOD mice, the peptide was immunogenic in NOD mice as well as in two NOD congenic strains which are both H-2g7, B10.H-2g7 and NOD.B6Il2-Tshb. This was surprising since the B10.H-2g7 strain does not develop diabetes or insulitis and fewer than 3% of NOD.B6Il2-Tshb mice develop diabetes. The response to GAD65 524-543 was shown to be controlled by the MHC since neither the B10 nor the NOD.H-2b strain, both of which are H-2b, responded to the peptide. This study demonstrates that T cell responsiveness to GAD-derived peptides can be elicited in strains of mice that are resistant to the development of spontaneous diabetes, suggesting that peripheral tolerance to GAD is not associated with protection from diabetes.

Cytoplasmic islet cell antibodies are well-established predictive markers of IDDM. Although target molecules of ICA have been suggested to be gangliosides, human monoclonal ICA of the immunoglobulin G class (MICA 1-6) produced from a patient with newly diagnosed IDDM recognized glutamate decarboxylase as a target antigen. Here we analyzed the possible heterogeneity of target antigens of ICA by subtracting the GAD-specific ICA staining from total ICA staining of sera. This was achieved 1) by preabsorption of ICA+ sera with recombinant GAD65 and/or GAD67 expressed in a baculovirus system and 2) by ICA analysis of sera on mouse pancreas, as GAD antibodies do not stain mouse islets in the immunofluorescence test. We show that 24 of 25 sera from newly diagnosed patients with IDDM recognize islet antigens besides GAD. In contrast, GAD was the only islet antigen recognized by ICA from 7 sera from patients with stiff man syndrome. Two of these sera, however, recognized antigens besides GAD in Purkinje cells. In patients with IDDM, non-GAD ICA were diverse. One group, found in 64% of the sera, stained human and mouse islets, whereas the other group of non-GAD ICA was human specific. Therefore, mouse islets distinguish two groups of non-GAD ICA and lack additional target epitopes of ICA besides GAD. Longitudinal analysis of 6 sera from nondiabetic ICA+ individuals revealed that mouse-reactive ICA may appear closer to clinical onset of IDDM in some individuals. Mouse-reactive ICAs, however, remained absent in 36% of the patients at diagnosis of IDDM.

Insulin-dependent diabetes is characterised by autoantibodies to several pancreatic-islet-cell antigens, including glutamate decarboxylase. We measured the proliferative responses to this antigen of peripheral-blood mononuclear cells from patients with newly diagnosed insulin-dependent diabetes, relatives of diabetic patients, and healthy controls. The likelihood of a positive response was substantially greater among the diabetic patients and relatives positive for islet-cell autoantibodies (ICA) than among subjects at low risk of diabetes (controls and ICA-negative relatives). Glutamate decarboxylase may have a pathogenetic role in insulin-dependent diabetes.

Glutamate decarboxylase from Escherichia coli was immobilized on inorganic macroporous carriers by the glutaraldehyde, carbodiimide and bromacetyl methods, on silicagel coated with a layer of a glutaraldehyde and m-phenylene diamine copolymer, and by polyacrylamide gel incorporation. The efficiency of the above methods of immobilization was evaluated. The bromacetyl method was found to be the most efficient. The dependence of activity of soluble and immobilized Glu-decarboxylase upon pH, temperature, substrate concentration, and stability was established. The differences in the properties of soluble and immobilized Glu-decarboxylase were due to the substrate diffusion in pores of the carrier. The immobilized Glu-decarboxylase obtained showed high activity and stability.

The concentration of gamma-aminobutyric acid (GABA), the activities of related enzymes, i.e. glutamate decarboxylase and GABA transaminase, as well as the level of specific GABA binding sites were determined in ovaries and fallopian tubes obtained surgically from 31 women. None of the biochemical parameters examined showed a correlation with the age and hormonal background (serum estradiol and progesterone levels) of the patients. The respective ovarian and tubal values did not differ significantly in groups operated on because of uterine myoma and carcinoma. In organs from pregnant women, however, most GABAergic markers altered significantly. These findings indicate some gestation-related role for the ovarian and tubal GABA systems in humans.