Autoantibody epitopes to the smaller isoform of glutamate decarboxylase do not differ in Swedish and Japanese type 1 diabetes patients and may be associated with high-risk human leucocyte antigen class II alleles.

Type 1 diabetes (T1D) is an autoimmune disease with a strong human leucocyte antigen (HLA) class II association. Depending on geographic locations, the disease-associated HLA class II alleles vary. We evaluated the beta cell-specific autoimmunity reflected in autoantibodies directed to the smaller isoform of glutamate decarboxylase (GAD65) in Japanese and Swedish T1D patients. GAD65Ab epitope specificities were assessed using GAD65-specific recombinant Fab. GAD65Ab epitope specificities did not differ between Swedish and Japanese patients. Only recognition of the MICA-4-defined middle epitope was significantly stronger in the Japanese T1D patient group compared to the Swedish T1D patients (P = 0.001). Binding to the b96.11-defined middle epitope was substantial in both groups and showed significant associations with high-risk HLA class II haplotypes. In the Japanese T1D group the association was with haplotype DRB1*0802-DQB1*0302 (P = 0.0008), while in the Swedish T1D patients binding to the b96.11-defined epitope as associated with the presence of high-risk HLA genotypes DR3-DQB1*0201 and/or DR4-DQB1*0302 (P = 0.02). A significant association between reduction in binding in the presence of recombinant Fab (rFab) DPD and high-risk allele DQB1*0201 was found (P = 0.008) in the Swedish T1D patients only. We hypothesize that epitope-specific autoantibodies effect the peptide presentation on HLA class II molecules by modulating antigen uptake and processing. Molecular modelling of the high-risk HLA class II molecules will be necessary to test whether these different molecules present similar peptide-binding specificities.

Modulation of antigen presentation by autoreactive B cell clones specific for GAD65 from a type I diabetic patient.

We used a GAD65-specific human B-T cell line cognate system in vitro to investigate the modulation of GAD65 presentation by autoantibody, assessed in a proliferation assay. Generally, if the T cell determinant overlaps or resides within the antibody epitope, effects of presentation are blunted while if they are distant can lead to potent presentation. For three different autoreactive B-T cell line cognate pairs, the modulation of GAD65 presentation followed the mode of overlapping or distant epitopes with resultant potent or undetectable presentation. However, other cognate pairs elicited variability in this pattern of presentation. Notably, one B cell line, DPC, whose antibody epitope did not overlap with the T cell determinants, was consistently poor in presenting GAD65. Using the fluorescent dye Alexa Fluor 647 conjugated to GAD65 to study receptor-mediated antigen endocytosis showed that all the antigen-specific B cell clones were efficient in intracellular accumulation of the antigen. Additionally, multicolour immunofluorescence microscopy showed that the internalized GAD65/surface IgG complexes were rapidly targeted to a perinuclear compartment in all GAD-specific B cell clones. This analysis also demonstrated that HLA-DM expression was reduced strongly in DPC compared to the stimulatory B cell clones. Thus the capability of antigen-specific B cells to capture and present antigen to human T cell lines is dependent on the spatial relationship of B and T cell epitopes as well other factors which contribute to the efficiency of presentation.

Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of autoreactive T-cells in type 1 diabetes: report of phase II of the Second International Immunology of Diabetes Society Workshop for Standardization of T-cell assays in type 1 diabetes.

The identification, quantification, and characterization of T-cells reactive with the islet autoantigens GAD65, proinsulin (PI), and tyrosine phosphatase-like molecules IA-2 and phogrin are major research goals in type 1 diabetes. In the Immunology of Diabetes Society First Workshop on Autoreactive T-Cells, the quality of recombinant preparations of these autoantigens was identified as a significant weakness, a finding that may account for much of the inconsistency in published studies of peripheral blood T-cell reactivity to islet autoantigens. Poor antigen quality has also hampered the development of novel technologies for the detection of islet-reactive T-cells. For these reasons, in the present study, several preparations of GAD65, PI, and IA-2 were collected and evaluated for endotoxin content, ability to stimulate a panel of relevant T-cell clones, and inhibitory effects on proliferation to unrelated third-party antigens. Through this process, we have been able to identify preparations of GAD65 and IA-2, generated in insect cells using the baculovirus expression system, that stimulate relevant clones and display low inhibitory effects on third-party antigens. In addition, we characterized a PI preparation generated in bacteria as being free of effects on proliferation to third-party antigens and low in endotoxin content. These preparations are important to promote the development of robust and sensitive assays of islet-reactive T-cells in patients with type 1 diabetes or patients at high risk for developing the disease.

HLA-DR53 molecules restrict glutamic acid decarboxylase peptide presentation to T cells of a Type I diabetes patient: specification of the trimolecular HLA-peptide/T-cell receptor complex.

AIMS/HYPOTHESIS: Our aim was to define the molecular specificity of glutamic acid decarboxylase-specific T-cells isolated from a patient (patient 40) with recent onset Type I (insulin-depent) diabetes mellitus. METHODS: The peptide epitope was defined using synthetic peptides to identify the minimal sequence required for T-cell activation and to determine the amino acids that contribute either to MHC binding or T-cell receptor signaling. The MHC class II-restricted peptide presentation was determined using a panel of allogeneic antigen-presenting cells and murine fibroblast-cell lines transfected to express individual human class II alleles and by blocking studies with monoclonal antibodies. The T-cell receptor was also molecularly characterized. RESULTS: Despite that patient 40 carries high-risk alleles of the DRB1 and DQB1 loci, his T-cells recognize a glutamic acid decarboxylase-derived peptide in association with class II, DR53, molecules. Although anchor residues for DR53 molecules have not yet been determined, it was possible to model epitope binding based on sequence comparisons with other class II molecules associated with susceptibility or protection for Type I diabetes. CONCLUSION/INTERPRETATION: The complete molecular specification of the MHC-peptide ligand and the T-cell receptor complex of glutamic acid decarboxylase-specific T-cells will enable analysis of strategies designed to alter T-cell function. For example, the role of altered peptide ligands or T-cell receptor-specific peptides can be studied using a model whose components reflect the natural affinities of MHC-peptide and T-cell receptor-ligand interactions selected in response to this important autoantigen.

Intradermal administration of GAD & evaluation of diabetes incidence in mice: possible relevance for skin tests in humans.

In vitro cell mediated reactivity to Glutamic Acid Decarboxylase (GAD) has been reported in man and in the non-obese diabetic (NOD) mouse. The demonstration of such reactivity in vivo using GAD in a simple intradermal skin test would be useful for mass screening of subjects at risk of Type 1 diabetes. Such a skin test could be simply applied to the forearm, then signs of local reaction would indicate patients at risk. However, in order to safely apply a skin test of this type it must be certain that administration of the antigen does not itself provoke or start the process leading to diabetes in susceptible individuals. In the present study the NOD mouse model was used. GAD and two peptides of GAD, which may have relevance to the disease process, were applied intradermally to these mice to determine whether a local reaction could be seen and to see if the diabetes rate was altered. Moreover, Balb/c mice, which can be considered to be at zero risk of developing the disease, were also injected with the same GAD and GAD peptides. No significant differences were seen in the diabetes incidence of the treatment groups compared to the control groups in either the NOD or Balb/c mice although a local swelling was seen in female NOD mice susceptible to diabetes after GAD administration in the footpad. We conclude that the administration of GAD and/or GAD peptides does not provoke or accelerate diabetes incidence in the NOD mouse and that an intradermal skin-test with GAD may be suitable for preliminary trials aimed at large scale screening of humans for their potential to develop type 1 diabetes.

Human monoclonal antibodies isolated from type I diabetes patients define multiple epitopes in the protein tyrosine phosphatase-like IA-2 antigen.

Protein tyrosine phosphatase-like IA-2 autoantigen is one of the major targets of humoral autoimmunity in patients with insulin-dependant diabetes mellitus (IDDM). In an effort to define the epitopes recognized by autoantibodies against IA-2, we generated five human mAbs (hAbs) from peripheral B lymphocytes isolated from patients most of whom had been recently diagnosed for IDDM. Determination and fine mapping of the critical regions for autoantibody binding was performed by RIA using mutant and chimeric constructs of IA-2- and IA-2beta-regions. Four of the five IgG autoantibodies recognized distinct epitopes within the protein tyrosine phosphatase (PTP)-like domain of IA-2. The minimal region required for binding by three of the PTP-like domain-specific hAbs could be located to aa 777-979. Two of these hAbs cross-reacted with the related IA-2beta PTP-like domain (IA-2beta aa 741-1033). A further PTP-like domain specific hAb required the entire PTP-like domain (aa 687-979) for binding, but critical amino acids clustered in the N-terminal region 687-777. An additional epitope could be localized within the juxtamembrane domain (aa 603-779). In competition experiments, the epitope recognized by one of the hAbs was shown to be targeted by 10 of 14 anti-IA-2-positive sera. Nucleotide sequence analysis of this hAb revealed that it used a V(H) germline gene (DP-71) preferably expressed in autoantibodies associated with IDDM. The presence of somatic mutations in both heavy and light chain genes and the high affinity or this Ab suggest that the immune response to IA-2 is Ag driven.

Humoral and cellular immune parameters before and during immunosuppressive therapy of a patient with stiff-man syndrome and insulin dependent diabetes mellitus.

OBJECTIVES: Humoral and cellular immune reactivity are reported for two neuroendocrine autoantigens-glutamic acid decarboxylase (GAD) and the protein tyrosine phosphatase IA-2-in a patient with the autoimmune type of stiff-man syndrome and insulin dependent diabetes (IDDM). METHODS: Antibodies and T cell proliferation against GAD and IA-2 and cytokine release of antigen stimulated T cells (IFN-gamma) were determined before and several times during immunosuppressive therapy with prednisolone. RESULTS: Raised GAD antibodies against full length GAD65 or chimeric constructs were detected before therapy and they remained at a high concentration despite a marked clinical improvement during cortisone treatment. Antibodies to IA-2 were undetectable, but weak T cell responses to both GAD and IA-2 were seen before therapy and once on reduction of high cortisone dosages when the patient showed signs of clinical deterioration. Cytokine profiles showed increased IFN-gamma production after stimulation with GAD or IA-2 suggesting increased activation of TH1 cells. CONCLUSION: Immunosuppressive therapy --even with extremely high doses of 500 mg a day--does not lead to the reduction of antibody concentrations in the periphery nor to a switch in epitope recognition of such antibodies despite clinical improvement. The amount of T cell reactivity to various antigens, however, may be a useful marker to monitor the effectiveness of immunotherapy.

Comparison of a new anti-glutamic acid decarboxylase (GAD) enzyme-linked immunosorbent assay (ELISA) with radioimmunoassay methods: a multicenter study.

Several methods are available for the measurement of antibodies to glutamic acid decarboxylase (anti GAD). These antibodies are valuable tools for the immunodiagnosis of insulin-dependent (type 1) diabetes mellitus (IDDM) and for the assessment of risk for the future development of IDDM. We here describe a new enzyme-linked immunosorbent assay (ELISA) for the detection of anti-GAD which was tested in a multicenter study. The results of the new anti-GAD ELISA correlate well with those obtained by radioimmunoassays (RIA) and they have a higher sensitivity (69%) and specificity (98%) compared to other anti-GAD enzyme immunoassays as determined in the IDW Proficiency Test Program for the detection of GAD antibodies. The new ELISA is simple and easy to perform, with convenient handling of the reagents. Quantitative and reproducible test results are available within approximately four hours. The new anti-GAD ELISA can be used for large scale population screening to indicate a prediabetic state as well as to diagnose autoimmune diabetes in adults (LADA) and the risk for IDDM in pregnant women with gestational diabetes.

Identification of naturally processed T cell epitopes from glutamic acid decarboxylase presented in the context of HLA-DR alleles by T lymphocytes of recent onset IDDM patients.

Glutamic acid decarboxylase (GAD) has been defined as a major target antigen in insulin-dependent diabetes mellitus (IDDM). To identify the molecular ligands triggering a T cell response to GAD, a panel of human GAD65-specific T lymphocyte lines was generated from peripheral blood of three recent onset IDDM patients. All lines derived from a patient expressing the high-risk-conferring HLA-DR*0301/ *0401 haplotypes recognized a single epitope localized between amino acid positions 270 and 283 of GAD65, a stretch that is located in close proximity to the homology region shared with Coxsackie virus P2-C protein. All lines with this specificity were restricted to the DRA, B1*0401 product of the DR4 haplotype. Analysis of the GAD-specific T cell response in a second patient homozygous for DR4 haplotypes demonstrated that the same DRA, B1*0401 allele selected T cells specific for a different determinant. The T cell response profile in a third patient showed that DR*1501/ *1601-encoding haplotypes could present at least three different epitopes to GAD65-specific T lymphocytes. One of these epitopes was presented by a DR allele associated with the resistance-conferring DRB1*1501 haplotype. GAD-specific T cell lines could not be isolated from HLA class II-matched normal individuals. Our data reveal that (a) the T cell response to GAD65 is quite heterogenous in recent onset IDDM patients; (b) HLA-DR, not DQ, seems to be the principal restriction element used by T cells present at the onset of the disease; and (c) T cells responding to epitopes containing identical sequences to Coxsackie virus P2-C protein were not detected.

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.

Human monoclonal islet cell antibodies from a patient with insulin-dependent diabetes mellitus reveal glutamate decarboxylase as the target antigen.

The autoimmune phenomena associated with destruction of the beta cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

Quantitative analysis of CD6, CD4 and CD8 cell surface molecules compared to the absolute numbers of CD6+, CD4+ and CD8+ T-cells in peripheral blood in patients with HIV-infection.

T lymphocyte subsets were determined on blood samples from 16 HIV-seropositive patients with manifest AIDS (CDC IV), 24 HIV-seropositive patients with lymphadenopathy syndrome (LAS, CDC III), 16 HIV-seropositive clinical healthy persons (CDC II) and 11 HIV-seronegative homosexuals as control group. Absolute numbers of T-cells (CD6+), T-helper/inducer-cells (CD4+) and T-suppressor/cytotoxic-cells (CD8+), obtained by immunofluorescence staining were compared with the absolute amount of subset specific surface molecules, obtained by a T-cell-ELISA. With both, indirect immunofluorescence technique and ELISA technique a highly significant decrease of the absolute numbers of CD4+ cells and the absolute amount of CD4 surface molecules, respectively, was found in asymptomatic HIV-infection, LAS and in manifest AIDS. In all HIV-seropositive groups the relative decrease of CD4 surface molecules was significantly greater than the decline of CD4+ cells. This phenomenon however was not seen in HIV-seronegative homosexuals. The absolute number of CD6+ cells and the amount of CD6 surface molecules were found significantly lowered in AIDS compared to HIV-seronegative homosexuals. No significant changes were found for CD8+ cell numbers and CD8 surface molecule in the progression of the HIV-infection.

A new ELISA-based assay for quantitation of human T-lymphocyte subpopulations.

In order to circumvent the problems associated with the available methods, we have developed a simple, reliable ELISA for quantitation of T-cells and their subpopulations, T-helper/inducer and T-suppressor/cytotoxic cells. Standard curves with three concentrations of sheep anti-mouse IgG-coupled beads for each T-cell population were used for the determination of the T-cell content in blood. Enzyme-labelled monoclonal antibodies against T-Pan, T-S and T-H cell surface markers were readily able to bind to such beads and the test system was calibrated with T-lymphocytes by comparing cytofluorographic and enzyme immunometric results. Purified preparations of monocytes and granulocytes were negative in the test. Lymphocytes from 50 healthy blood donors gave results which correlated closely with cytofluorograph determinations.

Determination of cell wall teichoic acid structure of staphylococci by rapid chemical and serological screening methods.

Investigations of cell wall teichoic acid structures of various staphylococci were carried out by a rapid method based on the gas-liquid chromatographic separation of products obtained after treatment of phenol-extracted cells with 70% hydrofluoric acid. In most of the strains teichoic acids of the poly(glycerolphosphate) and/or poly(ribitol-phosphate) type were found. Teichoic acids of the poly(glycerolphosphate-N-acetylglucosaminephosphate) type and polymers consisting of N-acetylglucosaminephosphate were present in few strains. The results obtained by the rapid chemical screening method were compared with data obtained by serological analysis of teichoic acid structures using specific antisera and the lectin wheat germ agglutinin. Teichoic acid components occurring in low concentrations could only be detected with the chemical and not with the serological method. A number of strains of species of the genus Staphylococcus have been studied using these rapid methods. With a few exceptions, the teichoic acid structure proved to be a constant marker within a given species.

Chemical composition and structure of cell wall teichoic acids of staphylococci.

The cell wall teichoic acid structures of 22 staphylococci including 13 type strains were determined. Most of the strains contain a poly(polyolphosphate) teichoic acid with glycerol and/or ribitol as polyol component. The polyolphosphate backbone is partially substituted with various combinations of sugars and/or amino sugars. Most of the substituents occur in a monomeric form but some strains also contain dimers of N-acetylglucosamine as substituents. Staphylococcus hyicus subsp. hyicus NCTC 10350 and S. sciuri DSM 20352 revealed rather complex cell wall teichoic acids. They consist of repeating sequences of phosphate-glycerol-phosphate-N-acetylglucosamine. The amino sugar component is present in this case as a monomer or an oligomer (n less than or equal to 3). Moreover, the glycerol residues are partially substituted with N-acetylglucosamine. The cell wall teichoic acid of S. auricularis is a poly(N-acetylglucosaminyl-phosphate) polymer similar to that found in S. caseolyticus ATCC29750. The cell wall teichoic acid structures for type strains of S. auricularis, S. capitis, S. cohnii, S. haemolyticus, S. hominis, S. hyicus subsp. hyicus, S. sciuri, S. xylosus and S. warneri were determined for the first time in detail. The structures of some of the previously described teichoic acids had to be revised (S. epidermidis, S. simulans, S. aureus phage type 187).

[Intravaginal treatment of colpitis maculosa with an oestriol-containing vaginal cream (author's transl)]. / Intravaginale Behandlung der Colpitis maculosa mit einer östriolhaltigen Vaginalcreme.

43 patients with colpitis maculosa (average age 60.2 years) were selected for an open control therapeutic study with Ortho-Gynest vaginal cream (Ortho-Cilag). The cream contains 0.5 mg of oestriol per single applicator filling. The treatment lasted from 3 to 4 weeks, success being evaluated by clinical documentation and cytological evaluation of vaginal smears before and after treatment. 10 patients (23.3%) were treated successfully, 29 (67.4%) showed a distinct improvement both clinically and cytologically, whilst the remaining 4 (9.3%) showed only moderate improvement. Hence, 39 patients (90.7%) were classified as having been successfully or partly successfully treated. Severe symptoms disappeared completely or were greatly alleviated in 91.4% cases. Moderate symptoms vanished in 59.7%. 58.1% showed a complete normalisation of the former atrophic vaginal skin. Blood spotting and reddening of the vaginal wall vanished completely. A change from dry to moist vagina occurred in 77.3% patients. Discharge vanished completely in 80.6% cases. No untoward side effects were recorded.

[Results of a questionnaire issued to expectant and delivered mothers (author's transl)]. / Ergebnisse einer Schwangeren- und Wöchnerinnenbefragung.

At the Department of Obstetrics and Gynaecology, Wilhelminenspital der Stadt Wien, Vienna, 98% of all deliveries are continuously monitored. 30% (1979) received epidural anaesthesia. The presence of the husband in the delivery room and partial rooming-in is available to all mothers. To find out if the service to expectant and delivered mothers is according to the requirement of our patients, questionnaires were distributed indiscriminately over a two-month period to 350 pregnant women and to 240 women in the puerperium. Fetal monitoring was valued positively in the majority of cases, the presence of the husband during delivery is requested in a minority of cases only, but would be welcomed by a higher proportion of puerperal women for the next delivery. Expectant mothers wished epidural anaesthesia in 50% of cases. Not only pregnant, but also delivered women demanded rooming-in, the latter group agreeing in the main with our partial form of rooming-in. Nearly 90% of mothers with rooming-in felt well prepared for baby care on leaving the hospital. We believe in the possibility of a synthesis of continuous monitoring to achieve optimum safety of delivery and family-orientated obstetrics in the hospital management of labour and the puerperium.