We investigated the presence of autoantibodies to baculovirusexpressed human recombinant 65-and 67-kD isoforms of glutamate decarboxylase (GAD65 and GAD67) in insulin-dependent diabetes mellitus (IDDM). In the immunoprecipitation test using I35Slmethionine-labeled GADs antibodies to GAD65 were detected in 13/15 (87%) islet cell antibody (ICA)-positive and in 1/35 (2.9%) ICA-negative first-degree relatives of patients with IDDM, in 6/11 (54.5%) ICA-positive nondiabetic schoolchildren, and in 35/50 (70%) patients with newly diagnosed IDDM. GAD67 antibodies were positive only in five (33%) of the ICA-positive relatives (P < 0.05) and in nine (18%) IDDM patients at onset (P < 0.00001). After onset of IDDM antibodies to GAD65 and GAD67 declined but were still positive in 25 and 9.4% of subjects with long-standing IDDM (> 10 yr). In all study groups antibodies to GAD67 were only detected in GAD65 antibody-positive sera. An immunotrapping enzyme activity assay for GAD65 antibodies was positive in 64/75 (85.3%) of sera that were GAD antibody positive in the immunoprecipitation test (r = 0.870, P < 0.0001). In two (2.7%) sera GAD65 antibodies that block GAD enzyme activity were found. Our data suggest that antibodies to GAD65 but not to GAD67 represent sensitive markers for preclinical and overt IDDM. The immunotrapping assay here described represents a valuable technique for specific and sensitive screening for GAD antibodies. (J.

Neuropeptides such as gonadotrophin releasing hormone (GnRH) are presumed to play an important role in the regulation of the function and growth of human placenta. Knowledge about the placental site of GnRH expression and the eventual co-localization of its peptide with the GnRH receptor (GnRH-R) is crucial for a better understanding of possible autocrine/paracrine mechanisms. We therefore investigated these questions by use of in-situ reverse transcription-polymerase chain reaction (RT-PCR) alone or in combination with immunocytochemistry in human first and third trimester placentae. Paraffin-embedded placental sections (7 microm thick), or single trophoblasts in monolayer cultures for up to 3 days, were treated with proteinase K. Following RT with GnRH or GnRH-R specific oligoprimers, PCR was performed employing primers with exon-exon overlaps to exclude non-specific DNA amplification. Detection of the amplicons was accomplished by nested PCR which was performed with digoxigenin-labelled dUTP and nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl-phosphate (NBT/BCIP) for substrate visualization. The GnRH peptide was detected using a sandwich-antibody assay. GnRH and GnRH-R gene expression was found in all first and third trimester placentae, with abundant signals for the GnRH and GnRH-R message both in the cyto- and syncytiotrophoblasts. Single trophoblasts of different gestational ages in culture also displayed GnRH expression in individual cytotrophoblasts and in syncytiotrophoblast-like fusionates. Additional immunostaining revealed GnRH peptide to be co-localized with GnRH-R message in trophoblast layers. Since messages for GnRH and GnRH-R were found in virtually all trophoblasts, we infer that GnRH and GnRH-R are co-expressed in identical cells. These data strongly suggest that the trophoblasts are the source of GnRH, and that there is autocrine/ paracrine regulation by GnRH in human placenta.

We have investigated the expression and localization of endothelium-derived nitric oxide synthase (eNOS) and the effect of eNOS on placental human chorionic gonadotrophin (HCG) release. eNOS mRNA was found to be expressed in all tissues, with its expression significantly (P < 0.05) increased across gestation. Compared to normal term gestation, placentae from term pregnancies with fetal retardation, or maternal diabetes, but not with maternal hypertension, displayed significantly more (P < 0.05) eNOS mRNA. By immunocytochemistry, we found staining for eNOS in both the cyto-and syncytiotrophoblasts of first trimester and a loss of cytotrophoblast eNOS staining in term placentae, while syncytiotrophoblasts at term were strongly eNOS positive. Additional staining was found in endothelium surrounding the vascular tree. HCG was found to colocalize with eNOS in trophoblasts, but not in endothelia. When placental explants were perifused, exposure to the NOS substrate, the NO donor, l-arginine and trinitroglycerol evoked a prompt, albeit transient, increase of HCG release. The NOS inhibitor delayed, but did not block arginine-induced HCG release. Thus, eNOS is expressed in the human placenta at increasing levels during gestation with further increases during some pathological conditions. A role for NO in the acute endocrine modulation of the placenta is suggested by the colocalization of eNOS with HCG in human trophoblasts and the prompt secretion of HCG in response to agents which increase NO concentrations.

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 1xyd6) 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.

Summary.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 1~5 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.