Bibliothek

Notizen: Studies have shown that the dust mites Dermatophagoides pteronyssinus and D. farinae contain several serine proteases, two of which have been shown to be allergenic, and to include trypasin and chymotrypsin, corresponding to the groups III and VI mite allergens. However, mites also contain other serine proteases, and the data reported in this study show that an elastase-like enzyme is present in both species. This enzyme was differentiated from the other serine proteases, particularly chymotrypsin, on the basis of charge, substrate specificity, and inhibition by copper and mercury cations. Its apparent mol. mass, as judged by gel filtration, was similar to those previously described for trypsain and chymotrypsin, i.e., 30 kDa. Several isoforms were detected by isoelectric focusing, but the isoelectric points of the major forms in both D. pteronyssinus and D. farinae were 10.5 and 9.8, respectively, contrasting with the acidic mite chymotrypsins. All three serine proteases were detected in whole mite and faecally enriched extracts, but the activities of trypsin and the elastase-like enzyme were greater in the latter type of extract. These data were similar to those obtained by quantitative immunochemical analysis of the D. farinae group III allergen in appropriate extracts, suggesting that culture conditions may modulate protease production. A monoclonal antibody affinity matrix specific for the group III allergen from D. farinae was shown to bind mite trypsin. However, a small amount of mite chymotrypsin also bound, suggesting limited immunologic cross-reactivity, a finding consistent with known sequence data.

Notizen: Recent studies indicate that the majority of clinically important aeroallergens are biochemically active, A diverse range of properties have been demonstrated but most possess either enzymatic activity (principally hydrolytic), enzyme inhibitory activity, low molecular weight ligand transporting or regulatory properties. In addition, some allergens are glycosylated and/or are structurally similar to proteins which have evolved to function in the respiratory system per se. Little attention has been given to the possibility that the biochemical activity of an allergen or any post-translational modifications contribute to sensitization. In this review, mechanisms with the potential to influence immunogenicity are discussed including interaction with respiratory secretions, epithelial disruption, interactions with immunocompetent cells and receptor mediated endocytosis. Given that many aeroallergens are structurally and functionally similar to a variety of endogenous (e. g. lysosomal enzymes) and exogenous proteins (e. g. microbial enzymes and glycoproteins), particular attention has been directed to the latter. This process represents an important non-adaptive defence mechanism which has evolved to recognize and process such proteins and it is feasible that it plays a similar role in the processing of some allergens entering the respiratory system.

Notizen: The acarofauna and Der p I allergen concentrations in dust samples from mattresses and lounge room carpets obtained from 20 homes from two coastal cities, Perth and Bunbury, were determined. All samples were shown to contain mites and the geometric mean numbers of total mites/g of mattress and carpet dust for Perth and Bunbury were 480 and 263, and 585 and 992, respectively. Carpets from both centres had a significantly (P 〈 0.02) greater mean number of mite species (Perth 9.1, Bunbury 9.0) than mattresses (Perth 5.2, Bunbury 5.7). The predominant mite species were D. pteronyssinus, E. maynei and Tarsonemus spp. D.farinae was found to be absent from all dust samples examined. E. maynei was present in the 10 Bunbury homes and in 50% of the Perth homes, ranging from 0 to 81% of mites identified. The arithmetic mean Der p I concentrations in the mattresses and carpets in Perth and Bunbury were 4.2 and 4.1, and 3.8 and 9.2 μg/per gram of fine dust, respectively, and Der p I concentration correlated with mite counts (r = 0.75; P 〈 0.001). The concentration of Der p I equivalent per 100 mites was 1.5 μg. The data are consistent with the view that asthmatic patients in Western Australia have significant exposure to a variety of house dust mites and that E. maynei may be clinically significant.

Notizen: The presence of a high molecular weight glycoprotein component bearing the hapten phosphorylcholine (Pc) has been demonstrated in some but not all extracts of house dust mite using a phosphorylcholine specific IgA myeloma protein (S107). The Pc bearing component was isolated from house dust mite extracts by precipitation at equivalence using whole myeloma serum and the isolated fraction was found to be allergenic as judged by a direct RAST assay using a highly house dust mite allergic human serum pool. RAST inhibition studies using free hapten and direct RAST studies using pneumococcal polysaccharide C-substance indicated that the Pe moiety did not contribute significantly to the immunodominant portion of the isolated allergen. Further studies indicated that the S107 isolated material was immunochemically different from the tridacnin reactive material and the con A reactive material previously isolated from extracts of house dust mite.

Notizen: There have been only a few studies of how allergens cross the airway epithelium to cause allergic sensitization. House dust mite fecal pellets (HDMFP) contain several proteolytic enzymes. Group 1 allergens are cysteine peptidases, whilst those of groups 3, 6 and 9 have catalytic sites indicative of enzymes that mechanistically behave as serine peptidases. We have previously shown that the group 1 allergen Der p 1 leads to cleavage of tight junctions (TJs), allowing allergen delivery to antigen presenting cells.In this study we determined whether HDMFP serine peptidases similarly compromise the airway epithelium by attacking TJs, desmosomes and adherens junctions.Experiments were performed in monolayers of MDCK, Calu-3 or 16HBE14o-epithelial cells. Cell junction morphology was examined by 2-photon molecular excitation microscopy and digital image analysis. Barrier function was measured as mannitol permeability. Cleavage of cell adhesion proteins was studied by immunoblotting and mass spectrometry.HDMFP serine peptidases led to a progressive cleavage of TJs and increased epithelial permeability. Desmosomal puncta became more concentrated. Cleavage of TJs involved proteolysis of the TJ proteins, occludin and ZO-1. This was associated with activation of intracellular proteolysis of ZO-1. In contrast to occludin, E-cadherin of adherens junctions was cleaved less extensively. Although Calu-3 and 16HBE14o-cells expressed tethered ligand receptors for serine peptidases, these were not responsible for transducing the changes in TJs.HDMFP serine peptidases cause cleavage of TJs. This study identifies a second general class of HDM peptidase capable of increasing epithelial permeability and thereby creating conditions that would favour transepithelial delivery of allergens.

Notizen: Background Asthmatic inflammation results in increased oxygen free radical generation and assessment of the activity of the selenitim (Se) dependent anti-oxidant enzyme, glutathione peroxidase (GSH-Px) in asthma may therefore be important. Objective To test the hypothesis that reduced GSH-Px activity and Se intake contribute to asthmatic infiammation, platelet and whole blood GSH-Px activities and serum and whole blood Se concentrations were measured and compared in atopic and non-atopic asthmatic patients and non-asthmatic control subjects.Methods GSH-Px activities of whole blood and isolated platelets were assessed in 41 asthmatic patients (33 atopic) and 41 age- and sex-matched non-asthmatic sttbjects (15 atopic) by spectrophotometric assay based oti the oxidation of NADPH. Se concentrations were determined by semi-automated fluorimetric assay. Results Mean (± sd) platelet GSH-Px activity was lower in asthmatic (89.5 ± 45.7 μmol NADPH oxidized min−1 g−1 of protein) than in non-asthmatic subjects (109,9 ± 41.9; P= 0.038) and in atopic (89.7 ± 45.1, n = 48) compared with non-atopie subiects (113.7 ± 40.9, n= 34: P= 0.016). Mean whole blood GSH-Px activity was also lower in atopic (12.2 ± 5.2 μmol NADPH oxidized min−1 g−1 of Hb) than in non-atopic subjects (14.5 ± 4.2; P= 0.038). In non-asthmatic subjects, the mean whole blood GSH-Px activity was lower in men (9.9 ± 3.5) than in women (14.5 ± 3.7; P = 0.0004) and was positively correlated with age (r= 0.51; P = 0.0006). Mean serum Se was lower in asthmatic (1.07 ± 0.12 μmol/L) than in non-asthmatic subjects (1.16 ± 0.31; P = 0.036), Using multiple linear regression, asthma was an independent predictor of decreased platelet GSH-Px after gender, age and serum Se were taken into account (P = 0.048) while atopy was a significant predictor of low whole blood GSH-Px independent of asthma, gender, age and whole blood Se (P = 0.033).Conclusions In addition to Se status, atopy, gender and uge all appear to influence GSH-Px activity, although the relative importance of these factors may difler in asthmatic and non-asthmatic populations. It seems likely that the reduced activity of this enzyme in platelets und hiood may reflect mechanisms associated with the pathogenesis and severity of asthma.