SummaryMolecular mimicry between viral antigens and host proteins was often suggested to be involved in induction of autoimmune diseases. In type 1 diabetes where pancreatic 13 cells are destroyed by autoimmune phenomena, a linear sequence homology between a major autoantigen, glutamate decarboxylase (GAD), and the 2C protein of coxsackie B4 was identified. In addition, a sequence homology between GAD and the mycobacterial heat shock protein 60 was described and the suggestions were made that molecular mimicry between GAD, coxsackievirus B4-2C protein, and/or heat shock protein 60 (hsp60) may be actively involved in an autoimmune reaction towards the pancreatic/g-cells. Our group was the first to isolate human monoclonal autoantibodies to GAD (MICA 1-6) from a patient with newly diagnosed type I diabetes. The MICA allowed a detailed characterization of the diabetes associated self-epitopes in GAD and represent a set of GAD autoantibodies present in sera from patients with type I diabetes. Using deletion mutants of GAD we demonstrated that the regions of GAD covering the homology sequences to coxsackievirus B4 and to the hsp60 were absolutely required for binding of the MICA to GAD. We now designed an antibody-based analysis to ask whether molecular mimicry between GAD and coxsackie B4-2C or hsp60 is relevant in type 1 diabetes. Since part of the MICA recognize conformational epitopes, they allow to test for conformational molecular mimicry in viruses that have been incriminated in the development of type I diabetes. Our data reveal no crossreactivity between the diabetes associated GAD epitopes defined by the MICA and hsp60, rubellavirus, cytomegalovirus, and coxsackie B1-B6 virus antigens. Neither coxsackie B4-specific antibodies in sera from normal individuals nor GAD-positive sera from patients with type 1 diabetes indicated a crossreactivity between coxsackie B4-2C and GAD. Although the regions in GAD homologous to coxsackie B4-2C and hsp60 represented parts of GAD indispensible for binding of diabetes associated autoantibodies they did not mediate a crossreactivity of autoantibodies between GAD and these two proteins. No evidence for molecular mimicry between GAD and a whole panel of foreign antigens was detected by autoantibodies in type 1 diabetes.

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.

The technique of DNA-based vaccination was used to generate a T-cell-dependent antibody response to glutamic acid decarboxylase (GAD) in BALB/c, C57BL/6, and non-obese diabetic (NOD) mice. Plasmids were constructed in which the expression of the rat GAD65 (rGAD65) or the rat GAD67 (rGAD67) gene was driven by the immediate early region promoter of the human cytomegalovirus (pCMV). This "naked" plasmid DNA was then injected into the regenerating muscles of the studied mice. In the vaccinated animals, antibody responses to GAD65 or to GAD67 were induced. Epitope recognition of GAD was studied by protein footprinting, a technique which makes use of a limited proteolysis of antibody-bound antigen. Different epitope recognition patterns were found, corresponding to strain-specific patterns. Mild trypsin treatment generated 50 kD, 46 kD, 40 kD, 30 kD, and 21 kD proteolytic fragments. In NOD mice, 50, 46 and 40 kD bands were the most prominent signals. In non-diabetes prone BALB/c mice, a faint 40 kD band appeared suggesting a rather weak protection of GAD from tryptic lysis. The pattern observed in C57BL/6 mice was more comparable to the NOD mice pattern with prominent 40 kD and 30 kD signals and a faint 21 kD fragment. Diabetes incidence was unchanged in NOD mice, and no diabetes was observed in C57BL/6 and BALB/c mice, respectively. The data demonstrate that genetic immunization is a suitable novel tool to stimulate and to manipulate an immune response against the diabetes-associated protein glutamic acid decarboxylase. Interestingly, our results indicate that, by genetic vaccination, distinct B-cell epitopes were generated in the various studied mouse strains.

Der Kehlkopf spielt eine wichtige Rolle bei der Regulierung des Gesamtatemwegswiderstandes (2, 13, 15), wobei auch Vagusreflexbahnen involviert zu sein scheinen (5). Normalerweise werden die Stimmlippen während der Inspiration weit gestellt und verengen sich geringgradig während der Exspiration. Diese Regulation ist bei einigen Formen von Stridor gestört, nämlich bei den funktionellen Laryngospasmen. Dabei lassèn sich in-und exspiratorische Formen unterscheiden, wobei manchmal die Abgrenzung zum Asthma bronchiale schwierig wird. Seit der Mitte des letzten Jahrhunderts wurden mehrere soicher Fãlle beschrieben (19), auch Sigmund Freud hat eine Kasuistik publiziert (6). Neues Interesse finden diese Stridorformen seit etwa 1974. Nahezu 30 Fälle wurden bisher beschrieben, wobei einige Autoren zu bedenken gaben, daI diese Erkrankungen weitaus häufiger sein dürften (Tab. 1). Wir konnten im Sommer 1983 eine besondere Form von funktionellem Laryngospasmus beobachten, die sich nur während körperlicher Belastung einstellte. Kasuistik Vom Hausarzt wurde uns eine l9jahrige Schiilerin zur Abklarung von Atemnotszuständen wahrend schwerer korperlicher Belastung (Schulsport) Erstmals im 11. Lebensjahr kam es während eines Wettschwimmens zu einer schweren Atemnot, was dazu führte, da1 sich das Kind nie wieder so stark korperlich belastete. Trotzdem waren

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