Functional consequences of HLA-DQ8 homozygosity versus heterozygosity for islet autoimmunity in type 1 diabetes.

Human leukocyte antigen (HLA) class II haplotypes are established risk factors in type 1 diabetes (T1D). The heterozygous DQ2/8 genotype confers the highest risk, whereas the DQ6/8 genotype is protective. We hypothesized that DQ2/8 trans-molecules composed of α and ß chains from DQ2 and DQ8 express unique ß-cell epitopes, whereas DQ6 may interfere with peptide binding to DQ8. Here we show that a single insulin epitope (InsB13-21) within the T1D prototype antigenic InsB6-22 peptide can bind to both cis- and trans-dimers, although these molecules display different peptide binding patterns. DQ6 binds a distinct insulin epitope (InsB6-14). The phenotype of DQ8-restricted T cells from a T1D patient changed from proinflammatory to anti-inflammatory in the presence of DQ6. Our data provide new insights into both susceptible and protective mechanism of DQ, where protecting HLA molecules bind autoantigens in a different (competing) binding register leading to 'epitope stealing', thereby inducing a regulatory, rather than a pathogenic immune response.

A predictive model of short cervix at 20-24 weeks using first-trimester cervical length measurement and maternal history.

OBJECTIVES: To develop a model for the prediction of short cervix ( ≤ 15 mm) at 20-24 weeks by combining maternal history and transvaginal ultrasonographic measurement of cervical length at 11-14 weeks. To explore the value of an additional ultrasound examination of the cervix at about 17 weeks. METHODS: Longitudinal prospective study in 800 unselected pregnant women presenting for first-trimester ultrasound assessment by nuchal translucency and serum biochemistry. Cervical length was evaluated transvaginally between 11 weeks and 13 weeks and 6 days (cx1), at 16-19 weeks (cx2) and 20-24 weeks (cx3). Backward multiple logistic regression analysis with cx3 ≤ 15 mm as the dependent variable was used to identify the predictors of a short cervix at 20-24 weeks. RESULTS: Cx1 and history of preterm delivery were significant independent contributors of a short cervix at 20-24 weeks [area under the curve (AUC 0.808, p < 0.001, Model) 1]. Furthermore, the cx1/cx2 ratio was a significant independent predictor of a short cervix at 20-24 weeks (odds ratio = 58.325 p = 0.012). The addition of the cx1/cx2 ratio improved the model (AUC = 0.878, p < 0.001, Model 2). CONCLUSIONS: A short cervix at 20-24 weeks can be predicted at the 11-14 weeks scan. The addition of a cervical measurement at about 17 weeks can improve the prediction model.

Attitudes of pregnant women regarding termination of pregnancy for fetal abnormality.

OBJECTIVE: To study the attitudes of pregnant women towards termination of pregnancy for fetal abnormality. MATERIALS AND METHODS: A questionnaire was completed by all pregnant women attending routine ultrasound scan. They were asked whether they would opt for termination of the pregnancy in case the fetus was diagnosed with one of the following abnormalities: lethal anomaly, anomaly causing developmental delay, anomaly causing physical handicap, anomaly causing disfigurement and severe anomaly diagnosed after 24 weeks of pregnancy. Logistic regression analysis was used to examine the effect of a variety of demographic and socio-economic characteristics in their choices. RESULTS: A total of 533 women completed the questionnaire out of which 447 (86%) would terminate the pregnancy in case of lethal fetal anomaly. The corresponding figures for anomaly causing developmental delay, anomaly causing physical handicap and anomaly causing disfigurement were 396 (77.8%), 332 (65.9%) and 228 (45.2%). A total of 313 (64.7%) would request late termination owing to severe anomaly. The only two statistically significant factors that influenced the maternal decision on pregnancy termination were religious beliefs and the frequency of practicing religious duties (p < 0.001). CONCLUSION: The majority of pregnant women would terminate pregnancy for lethal fetal anomaly and for an anomaly causing mental or physical handicap, even in late pregnancy.

Pulsed-field gradient nuclear magnetic resonance study of transport properties of fluid catalytic cracking catalysts.

Pulsed-field gradient nuclear magnetic resonance (PFG NMR) has been applied to study molecular diffusion in industrial fluid catalytic cracking (FCC) catalysts and in USY zeolite for a broad range of molecular displacements and temperatures. The results of this study have been used to elucidate the relevance of molecular transport on various displacements for the rate of molecular exchange between catalyst particles and their surroundings. It turned out that this rate, which may determine the overall rate and selectivity of FCC process, is primarily related to the diffusion mode associated with displacements larger than the size of zeolite crystals located in the particles but smaller than the size of the particles. This conclusion has been confirmed by comparative studies of the catalytic performance of different FCC catalysts.

Structural analysis of two HLA-DR-presented autoantigenic epitopes: crucial role of peripheral but not central peptide residues for T-cell receptor recognition.

Specific and major histocompatibility complex (MHC)-restricted T-cell recognition of antigenic peptides is based on interactions of the T-cell receptor (TCR) with the MHC alpha helices and solvent exposed peptide residues termed TCR contacts. In the case of MHC class II-presented peptides, the latter are located in the positions p2/3, p5 and p7/8 between MHC anchor residues. For numerous epitopes, peptide substitution studies have identified the central residue p5 as primary TCR contact characterized by very low permissiveness for peptide substitution, while the more peripheral positions generally represent auxiliary TCR contacts. In structural studies of TCR/peptide/MHC complexes, this has been shown to be due to intimate contact between the TCR complementarity determining region (CDR) three loops and the central peptide residue. We asked whether this model also applied to two HLA-DR presented epitopes derived from an antigen targeted in type 1 diabetes. Large panels of epitope variants with mainly conservative single substitutions were tested for human leukocyte antigen (HLA) class II binding affinity and T cell stimulation. Both epitopes bind with high affinity to the presenting HLA-DR molecules. However, in striking contrast to the standard distribution of TCR contacts, recognition of the central p5 residue displayed high permissiveness even for non-conservative substitutions, while the more peripheral p2 and p8 TCR contacts showed very low permissiveness for substitution. This suggests that intimate TCR interaction with the central peptide residue is not always required for specific antigen recognition and can be compensated by interactions with positions normally acting as auxiliary contacts.

A sensitive and specific competition microELISA for the immunoactive polypeptide parathymosin and detection of this peptide in porcine tissues.

A sensitive and specific microELISA assay is described for the immunoactive polypeptide parathymosin. Antibodies against a synthetic peptide corresponding to the rat parathymosin sequence 5-30 were raised in rabbits immunised with this peptide conjugated to keyhole limpet hemocyanin (KLH). The useful range of the assay was 0.25-30 pmol (3-330 ng) of parathymosin and the assay was specific. The related immunoactive polypeptides prothymosin alpha or thymosin alpha 1 showed no cross-reactivity. In spiking experiments the recovery of the assay was found to be greater than 92% at all concentrations tested. The intra-assay variation was 17%, whereas the inter-assay variation was 26%. Using this assay the highest concentration of parathymosin was found in porcine liver, followed by kidney, lung, thymus and spleen. This assay compares favorably with one microELISA and two RIA methods already published, in that it is more sensitive by at least an order of magnitude, and it is simpler and quicker.

Novel approaches toward early diagnosis of islet allograft rejection.

BACKGROUND: The inability to diagnose early rejection of an islet allograft has previously proved to be a major impediment to progress in clinical islet transplantation. The need to detect early rejection will become even more relevant as new tolerance-inducing protocols are evaluated in the clinic. We explored three novel approaches toward development of early diagnostic markers of islet rejection after islet allotransplantation. METHODS: (a) Canine islet allograft transplant recipients were immunosuppressed for 1 month, then therapy was withdrawn. Serum glutamic acid decarboxylase antigen (GAD65), an endogenous islet protein, was monitored daily with a CO2 release assay. (b) Rodent islets were genetically engineered to express a unique foreign protein (beta-galactosidase) by using adenoviral vectors, and after allograft transplantation, the viral-specific protein was measured in serum using optical luminescence. (c) Rodents receiving islet allografts were immunosuppressed temporarily, and daily glucose tolerance tests were followed until graft failure occurred. RESULTS: (a) Although serum monitoring of GAD65 antigen demonstrated elevated levels preceding loss of graft function in preliminary studies, the effect was not reproducible in all animals. (b) Genetically engineered rodent islets demonstrated normal insulin kinetics in vitro (insulin stimulation index 2.57+/-0.2 vs. 2.95+/-0.3 for control islets, P=ns), and purified viral protein products had a stable half-life of 8 hr in vivo. After islet allotransplantation, there were two peak elevations in serum viral proteins, confirming that an intra-islet "sentinel signal" could be detected serologically during acute rejection. There was no lead-time ahead of hyperglycemia, however. (c) Daily sequential intravenous glucose tolerance (IVGT) tests demonstrated evidence of allograft dysfunction (decline in KG) with a 2-day lead time to hyperglycemia (2.58+/-0.3 vs. 1.63+/-0.2%/min, respectively, P<0.001), with an accuracy of 89%, sensitivity of 78%, and specificity of 95%. CONCLUSIONS: Of the three diagnostic tests, metabolic assessment with an abbreviated IVGT was the most effective method of demonstrating early islet dysfunction due to rejection.

No specific reactivity to E. coli glutamic acid decarboxylase from sera of newly-diagnosed insulin dependent diabetic patients.

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.

RGD sequences in several receptor proteins: novel cell adhesion function of receptors?

In the process of homology modelling of the 3-dimensional structure of alleles of the human histocompatibility protein HLA-DQ, we discovered that its RGD tripeptide (beta 167-169) forms part of a loop. A search through protein sequence data bases, revealed this cell adhesion motif in 67 integral plasma membrane proteins (in 48 extracellularly, and in the remaining 19 intracellularly), which are bona fide receptors, and none of them has thus far been considered as a cell adhesion protein. The 3-dimensional structure of one of these, the rat neonatal Fc receptor, is known and its extracellular RGD sequence is in an adhesion-like loop, a fact that went unnoticed in the original papers. In a few other cases, e.g. rat and mouse growth hormone receptor, and mouse CD40 ligand, homology modelling by ourselves and others reveals that the said sequences are part of a loop, in similarity to all RGD sequences found in proteins with established adhesion function and known 3-dimensional structure. Likewise, inspection of all known protein 3-dimensional structures containing an RGD sequence, and not having a documented cell adhesion function (total of 65 separate entries) shows that such sequence is mostly (52/65 or 80% of cases) part of a loop. We therefore call attention to these surprising findings, discuss the possible cell adhesion role of these receptor proteins, and draw an analogy from the two well characterised examples, that of soluble IGF binding protein 1 and the transcriptional activator protein Tat of HIV, where their RGD sequences have been shown by site-directed mutagenesis to participate in cell-adhesion interactions, without prior knowledge of the location of the tripeptide, or the 3-dimensional structure of the respective protein.

Possible viral implication in the pathogenesis of Sjögren's syndrome.

How then could the observed pathological changes in the exocrine glands of patients with pSS be related to the various in vitro and in vivo laboratory findings? To begin with, the fact of inappropriate HLA-D/DR expression in epithelial cells in the absence of any infiltrating T cells or residual IFN-gamma must argue for an exogenous agent such as a virus that modulates the gene expression of epithelial cells. Such inappropriate expression is probably accompanied by the expression of other molecules that promote adhesion of lymphocytes in the proximal endothelium and infiltration into the exocrine gland. The autoimmune response takes place in these very exocrine glands and probably is initiated by the presentation of antigen(s) to the CD4+ T cells by the HLA-D/DR+ epithelial cells. The HLA-D/DR association shown in patients with pSS probably has to do with the antigenic fragments of the autoantigens that these molecules can preferentially bind to. The activated CD4+ T cells can then deliver help to specific B lymphocytes leading them to autoantibody-secreting plasma cells. In addition the extent of polyclonal activation of B lymphocytes and their potential for cytokine secretion and proliferation in vitro, is consistent with the view of virally-induced activation of these cells. The cross-reactivity to retroviral antigens observed in the sera of pSS patients, and the retroviral sequences detected in the salivary glands argue for some type of a retroviral infection of pSS patients in the course of the disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymorphic structural features of modelled HLA-DQ molecules segregate according to susceptibility or resistance to IDDM.

The structural features of HLA-DQ alleles which are susceptible and resistant to insulin-dependent diabetes mellitus (IDDM) have been examined using a model of their three-dimensional structure obtained by energy minimisation, based on the published structure of HLA-DR1. The model shows DQ molecules to have an overall shape nearly identical to that of DR molecules, but with significant differences in the fine structure: 1) the antigen-binding groove of DQ molecules has a polymorphic first pocket; this pocket can be either amphiphilic or hydrophilic, 2) The beta 49-56 dimerisation domain of DQ is polymorphic: hydrophobic, or amphiphilic, or hydrophilic and positively charged, leading to spontaneous or T-cell receptor-induced homodimer formation, or T-cell receptor-induced homodimer formation, or difficulty of the formation of such dimers, respectively; 3) a prominent Arg-Gly-Asp loop is formed by some DQ alleles (beta 167-169) and probably functions in cell adhesion. There are also small differences in the residues and sequences implicated in CD4 binding (mostly in DQ beta 134-148) but the significance of these differences cannot be evaluated at present. All seven DQ alleles which confer susceptibility to IDDM possess a hydrophilic first pocket in the antigen-binding groove, a hydrophobic or amphiphilic beta 49-56 dimerisation patch that allows for spontaneous or T-cell receptor-induced dimerisation, and the Arg-Gly-Asp loop. By contrast, in the protective alleles at least one of these three features is absent. This segregation of phenotypes according to susceptibility or resistance can well explain the model of tighter autoantigen binding by the protective alleles compared to the susceptible alleles, previously proposed for the pathogenesis of IDDM.

IL-6-mediated MHC class II induction on RIN-5AH insulinoma cells by IFN-gamma occurs via the G-protein pathway.

Major histocompatibility complex (MHC) class II antigen expression has been implicated in the pathogenesis of autoimmune type 1 diabetes. In this study we examined the role of various cytoldnes that may induce MHC class II surface antigen expression, using the rat insulinoma line RIN-5AH as a pertinent model system. As in another study, the ability of IFN-gamma to amplify MHC class II antigen expression 4-fold is demonstrated. At the same time we noted a 5-fold increase of these histocompatibility antigens by IL-6. Signal transduction analysis reveals that IL-6-induced MHC class II expression is specifically mediated by the G-protein system (activation of p21(ras) by IL-6) since mevalonic acid lactone (a Gprotein inhibitor) abolishes the action of IL-6. In contrast, IFN-gamma, which does not activate p21(ras), is not inhibited by protein kinase C (PKC) inhibitors but by those of the G-protein pathway. This finding raises the possibility that IFN-gamma induces RIN cells to secrete IL-6 (as shown previously, as well as in this paper) which, in turn, increases class II antigen expression via the G-protein pathway. This action may be unique to IL-6 or in synergy with IFN-gamma. Other cytokines such as IL-1alpha and beta, and TNF-alpha induce a smaller increase in MHC class II antigens on RIN cells, and appear to activate both the G-protein and the PKC signal transduction pathways to varying degrees. Therefore, injury of pancreatic beta-cells and possible induction of autoimmune type 1 diabetes via various cytokines may be caused by IL-6 or IFN-gamma, or by their ability to induce MHC class II antigen upregulation.

Immunological versatility and carbon regulation of Cellulomonas fimi endo-1,4-beta-glucanases.

More than 10 protein molecules with endo-1,4-beta-glucanase activity were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram in Cellulomonas fimi culture supernatants, grown in CMC as carbon source. These molecules are shown to belong to at least four immunologically different groups, against three of which polyclonal antibodies were raised. The protein species used as antigens showed significant differences in cross reactivity, carbon regulation, and affinity to crystalline cellulose. Three intracellular precursors of the first group were detected, two of which were under carbon catabolite control with the third apparently being synthesized constitutively. In the extracellular environment this group showed the largest versatility in protein molecules. The second group appeared to originate from two intracellular precursors both synthesized constitutively and subject to minor extracellular modifications as compared to the first group. The main extracellular protein of this group showed high affinity toward crystalline cellulose. One intracellular precursor was identified for the third group, which was subject to carbon catabolite control. Only one extracellular molecule without binding ability to crystalline cellulose corresponded to this precursor, indicating that the latter was resistant to proteolytic modifications after excretion. It appears that the C. fimi cellulases are more complex than expected and reconstitution of the whole system will be difficult.

IL-1 beta transduces different signals than IL-1 alpha leading to class II antigen expression on beta-insulinoma RIN-5AH cells through specific receptors.

Like most cytokines, IL-1 transduces its signals for growth, differentiation and diverse cellular functions after binding to specific receptors on the cell surface. Up to now two IL-1 receptors have been reported, type I which induces signal transduction and type II which binds IL-1 but does not transduce signalling. By using the rat insulinoma RIN-5AH cell line that expresses both types of receptor mRNA, and computer-assisted binding analysis, we show that interleukin-1 beta (IL-1 beta) binds to a single class of high affinity receptors with a Kd of 155 pmol/l. The average number of receptors on adherent cell layer is calculated to be 7300 per cell. 125I-IL-1 beta binding can be competed out by unlabelled IL-1 beta. 125I-IL-1 alpha binding can be also obtained and is subject to competition by cold IL-1 alpha. Its saturation curve, however, varies within experiments due to differential receptor up-regulation. These results have also been confirmed by FACS analysis using specific antibodies to type I and II IL-1 receptors, where type I receptor antibody binds strongly to RIN-5AH cells, and type II receptor antibody shows weak staining, also due to inadequate receptor up-regulation. In order to determine whether functional signal transduction occurs via the receptors detected, it is shown that IL-1 beta is able to induce MHC class II antigen expression on the surface of the RIN cells, whereas IL-1 alpha is unable to do so, indicating different signal reception by the cells. IL-1 beta-induced class II upregulation shows moderate signs of p21ras or/and PKC dependency, whereas IL-1 alpha strongly activates both pathways that probably regulate different functions. Finally, both IL-1 alpha and beta induce nitric oxide (NO) production in a time-dependent fashion which appears to be unrelated to the signals and pathways described, but may be involved in the onset of autoimmune type 1 diabetes.

Modelling of the MHC II allele I-A(g7) of NOD mouse: pH-dependent changes in specificity at pockets 9 and 6 explain several of the unique properties of this molecule.

AIMS/HYPOTHESIS: We modelled the three-dimensional structure of I-A(g7), the chief genetic component of diabetes in non-obese diabetic mice, to understand the unusual properties of this molecule. METHODS: Modelling was done, in complex with established antigenic peptides, based on the structure of I-A(k). RESULTS: The selectivity of the I-A(g7) molecule changes greatly at pockets 9 and 6 but hardly at all at pockets 1, 4 and 7, between endosomal pH (5.0) and extracellular pH (7.0), in agreement with previous results. This selectivity is attributed to the unique combination of beta9His, beta56His and beta57Ser. The positive charges in and around pocket 9 at pH 5, favour binding by negatively charged residues. At pH 7 however, the uncharged alpha68, beta9 and beta56 histidines favour the accommodation of the bulky residues lysine, arginine, phenylalanine and tyrosine at pocket 9. The combination of beta9His and alpha66Glu is responsible for the pH-dependent selectivity at pocket 6. Furthermore, the lack of repulsion between beta56His and alpha76Arg at pH 7 leads to a more stable ternary complex. CONCLUSION/INTERPRETATION: These results reconcile previous conflicts over the peptide binding ability of I-A(g7) and its motif. They furthermore provide possible explanations for the short lifetime of cell-surface I-A(g7) complexes in vivo, the higher threshold of thymic negative selection and inherent self-reactivity shown by immunocytes in these mice and the protection from diabetes afforded to them by several transgenically expressed mouse class II alleles. This contributes to an understanding of the pathogenesis of Type I (insulin-dependent) diabetes mellitus in this animal.

Soluble interleukin 2 receptor molecules in the serum of patients with autoimmune diseases.

Lymphocytes upon activation release a soluble form of interleukin 2 receptor (IL-2R). Systemic autoimmune disorders are characterized by immune system disregulation associated with cellular activation; therefore we sought to determine the levels of soluble IL-2R molecules in the serum of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and primary Sjogren's syndrome (1 degree SS). Utilizing an enzyme immunoassay method we found increased serum levels of soluble IL-2R in 65.4% (34/52) of RA, in 34.9% (15/43) of SLE, and in 25.0% (13/52) of 1 degree SS patients, compared to 4.2% (1/24) of healthy individuals. High serum levels of soluble IL-2R correlated with several indices of disease activity in RA and SLE patients, as well as with disease progression to extraglandular involvement and to pseudolymphoma or lymphoma in patients with 1 degree SS. By gel filtration analysis, the soluble IL-2R circulating in the serum of a RA patient corresponded to a high molecular weight molecule (greater than 90 kDa) compared to the 65-kDa soluble IL-2R molecule released by phytohemagglutinin-stimulated normal peripheral lymphocytes.

Novel structural features of the human histocompatibility molecules HLA-DQ as revealed by modeling based on the published structure of the related molecule HLA-DR.

Structural modeling of the HLA-DQ molecules, a group of human histocompatibility antigens linked to autoimmune diseases and immunosuppression-based on the structure of the homologous molecule DR1, has revealed an overall shape typical of the class II histocompatibility molecules, yet with several novel features. These are unique to HLA-DQ and include: (1) an antigen-binding groove with a polymorphic first pocket and anchoring in the second and/or fifth pocket, (2) a polymorphic beta 49-56 dimerization patch, and (3) in many alleles a prominent Arg-Gly-Asp loop (beta 167-169), probably involved in cell adhesion, as it exhibits an architecture similar to identical sequences involved in such function. The alpha 2 beta 2 dimerisation domain and the CD4-binding region are nearly identical to their counterparts in the structure of HLA-DR1. The significance of the few substitutions in the CD-4 binding region remains to be evaluated. The polymorphic first antigen-binding pocket and the anchoring in the second and/or fifth pocket point to differences in antigenic fragment selection compared to HLA-DR antigens, while the polymorphism in the beta 49-56 homodimerization patch implies either ease of spontaneous or T lymphocyte receptor-induced homodimerization or difficulty in the latter. As homodimerization appears to be an obligatatory intermediate in the activation of cognate DQ-restricted T lymphocytes and DQ-bearing antigen-presenting cells, the dimerization properties of DQ allels signify the respective ease or difficulty of activation of these two cell types. The RGD loop confers cell adhesion possibilities to those DQ allels that possess it, yet its putative ligand cannot be defined at present. These features are suggestive of the probable mechanisms through which some of the unique immunological properties of the HLA-DQ molecules are effected.

Comparative studies on the fine structure of purple membrane from Halobacterium Cutirubrum and Halobacterium Halobium.

Direct comparison of the absorption and circular dichroic spectra of dark- and light-adapted purple membrane from Halobacterium cutirubrum and Halobacterium halobium indicated no apparent species differences. In addition, sequential bleaching and regeneration of the purple membrane with concomitant monitoring of the absorption and circular dichroic spectra showed no species differences as well. Furthermore, perturbation of the structure of the purple membrane from either species with a detergent, Triton X-100, yielded similar spectral changes. It was concluded: (i) no apparent differences exist in the molecular organization and protein fine structure of the two purple membranes, (ii) if exciton interaction among the retinal chromophores is a reasonable possibility in the case of the purple membrane from Halobacterium halobium, it must be similarly so for the membrane from Halobacterium cutirubrum, (iii) the effects of light adaptation on the membrane structure of both species are essentially the same, and (iv) the underlying molecular mechanisms for the bleaching and regenerative processes must be similar, if not identical, for the purple membranes of the two species.