Human monoclonal islet specific autoantibodies share features of islet cell and 64 kDa antibodies

The first human monoclonal islet cell antibodies of the IgG class (MICA 1-6) obtained from an individual with Type 1 (insulin-dependent) diabetes mellitus were cytoplasmic islet cell antibodies selected by the indirect immunofluorescence test on pancreas sections. Surprisingly, they all recognized the 64 kDa autoantigen glutamate decarboxylase. In this study we investigated which typical features of cytoplasmic islet cell antibodies are represented by these monoclonals. We show by double immunofluorescence testing that MICA 1-6 stain pancreatic beta cells which is in agreement with the beta-cell specific expression of glutamate decarboxylase. In contrast an islet-reactive IgM monoclonal antibody obtained from a pre-diabetic individual stained all islet cells but lacked the tissue specificity of MICA 1-6 and must therefore be considered as a polyreactive IgM-antibody. We further demonstrate that MICA 1-6 revealed typical features of epitope sensitivity to biochemical treatment of the target tissue which has been demonstrated for islet cell antibodies, and which has been used to argue for a lipid rather than a protein nature of target antigens. Our results provide direct evidence that the epitopes recognized by the MICA are destroyed by methanol/chloroform treatment but reveal a high stability to Pronase digestion compared to proinsulin epitopes. Conformational protein epitopes in glutamate decarboxylase therefore show a sensitivity to biochemical treatment of sections such as ganglioside epitopes. MICA 1-6 share typical features of islet cell and 64 kDa antibodies and reveal that glutamate decarboxylase-reactive islet cell antibodies represent a subgroup of islet cell antibodies present in islet cell antibody-positive sera

Association between Antibodies to the MR 67,000 Isoform of Glutamate Decarboxylase (GAD) and Type 1 (Insulin-Dependent) Diabetes Mellitus with Coexisting Autoimmune Polyendocrine Syndrome Type II

By using an immunoprecipitation assay, we analysed reactivity of autoantibodies to human recombinant GAD65 and GAD67 in sera from patients with autoimmune polyendocrine syndrome Type II (APS II) with and without Type 1 (insulin-dependent) diabetes mellitus (IDDM) compared to patients with organ-specific autoimmunity. Overall antibodies to GAD65 were correlated with IDDM in all study groups, whereas GAD67 antibodies were associated with IDDM when APS II coexists. Antibodies to GAD65 and GAD67 were detected in 13 (44.8%) and 7 (24.1%) out of 29 APS II patients with IDDM, but in only 4 (13.8%) and 2 (6.9%) out of 29 APS II patients without IDDM, respectively (p < 0.05). In short-standing IDDM (< 1 year), antibodies to GAD67 were significantly more frequent in patients with APS II (5 of 9 [55.6%] subjects) compared to matched diabetic patients without coexisting polyendocrinopathy (1 of 18 [5.6%] subjects) (p < 0.02). The levels of GAD65 (142 ± 90 AU) and GAD67 antibodies (178 ± 95 AU) were significantly higher in patients with polyglandular disease than in patients with isolated IDDM (91 ± 85 AU and 93 ± 57 AU) (p < 0.02). Interestingly, all 11 GAD67 antibody positive subjects also had GAD65 antibodies (p < 0.0001), and in 10 of 11 anti-GAD67 positive sera the GAD67 antibodies could be blocked by either GAD67 or GAD65, suggesting the presence of cross-reactive autoantibodies. No correlation was observed between GAD antibodies and age, sex or any particular associated autoimmune disease, besides IDDM. GAD antibodies were present in only 1 of 6 (16.7%) patients with APS Type I, in 1 of 26 (3.9%) patients with autoimmune thyroid disease but in none of the patients with Addison's disease (n = 16), pernicious anaemia (n = 7) or normal controls (n = 50). Our data suggest distinct antibody specificities reactive to GAD isoforms in APS II and IDDM, which might reflect different mechanisms of autoimmune response in IDDM with coexisting autoimmune polyendocrine autoimmunity

Interindividual variation and consistency of migratory behavior in the Eurasian woodcock

Diverse spatio-temporal aspects of avian migration rely on relatively rigid endogenous programs. However, flexibility in migratory behavior may allow effective coping with unpredictable variation in ecological conditions that can occur during migration. We aimed at characterizing inter- and intraindividual variation of migratory behavior in a forest-dwelling wader species, the Eurasian woodcock Scolopax rusticola, focusing on spatio-temporal consistency across repeated migration episodes. By satellite-tracking birds from their wintering sites along the Italian peninsula to their breeding areas, we disclosed a remarkable variability in migration distances, with some birds flying more than 6,000 km to Central Asian breeding grounds (up to 101\ub0E). Prebreeding migration was faster and of shorter duration than postbreeding migration. Birds moving over longer distances migrated faster during prebreeding migration, and those breeding at northernmost latitudes left their wintering areas earlier. Moreover, birds making longer migrations departed earlier from their breeding sites. Breeding site fidelity was very high, whereas fidelity to wintering areas increased with age. Migration routes were significantly consistent, both among repeated migration episodes and between pre- and postbreeding migration. Prebreeding migration departure date was not significantly repeatable, whereas arrival date to the breeding areas was highly repeatable. Hence, interindividual variation in migratory behavior of woodcocks was mostly explained by the location of the breeding areas, and spatial consistency was relatively large through the entire annual cycle. Flexibility in prebreeding migration departure date may suggest that environmental effects have a larger influence on temporal than on spatial aspects of migratory behavior

Utilização de Azatioprina no tratamento de Trombocitopenia imunomediada em cão

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Simultaneous Detection of Circulating Autoreactive CD8+ T-Cells Specific for Different Islet Cell–Associated Epitopes Using Combinatorial MHC Multimers

textabstractOBJECTIVE - Type 1 diabetes results from selective T-cell-mediated destruction of the insulin-producing β-cells in the pancreas. In this process, islet epitope-specific CD8+T-cells play a pivotal role. Thus, monitoring of multiple islet-specific CD8+T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive. RESEARCH DESIGN AND METHODS - Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B10-18, prepro-insulin (PPI)15-24, islet antigen (IA)-2797-805, GAD65114-123, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)265-273, and pre-pro islet amyloid polypeptide (ppIAPP)5-13-specific CD8+T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients. RESULTS - Using this kit, islet autoreactive CD8+T-cells recognizing insulin B10-18, IA-2797-805, and IGRP265-273were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI15-24proved to be the most sensitive epitope. Applying the "Diab-Q-kit" to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell-derived epitopes that were associated with disease activity and correlated with clinical outcome. CONCLUSIONS - A kit was developed that allows simultaneous detection of CD8+T-cells reactive to multiple HLA-A2-restricted β-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples

The synaptic vesicle-associated protein amphiphysin is the 128-kD autoantigen of stiff-man syndrome with breast cancer

Stiff-Man syndrome (SMS) is a rare disease of the central nervous system (CNS) characterized by progressive rigidity of the body musculature with superimposed painful spasms. An autoimmune origin of the disease has been proposed. In a caseload of more than 100 SMS patients, 60% were found positive for autoantibodies directed against the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). Few patients, all women affected by breast cancer, were negative for GAD autoantibodies but positive for autoantibodies directed against a 128-kD synaptic protein. We report here that this antigen is amphiphysin. GAD and amphiphysin are nonintrinsic membrane proteins that are concentrated in nerve terminals, where a pool of both proteins is associated with the cytoplasmic surface of synaptic vesicles. GAD and amphiphysin are the only two known targets of CNS autoimmunity with this distribution. This finding suggests a possible link between autoimmunity directed against cytoplasmic proteins associated with synaptic vesicles and SMS