Heterogeneous proteolytic specificity and activity of the house dust mite proteinase allergen Der p I

Background Exposure of the skin or respiratory tract to proteinases is frequently associated with allergic sensitization. This is of particular significance in the domestic indoor environment where the proteolytic activity of Der p I, the group I allergen of the house dust mite Dermatophagoides pteronyssinus, may influence the allergenicity of mites. Using class-specific proteinase inhibitors and active-site affinity chromatography, we have previously shown that Der p I exhibits a mixed cysteine-serine proteinase activity. Measurement of the amount of cleavage, however, did not determine whether the inhibitors used were targeting exactly the same proteolytic mechanism. Objective To resolve this issue, we have examined whether the cleavage specificity of the cysteine and serine proteinase activities of Der p I was the same. Methods HPLC and mass spectrometry were used to analyse and identify the products of a Der p I-digested peptide substrate and thus identify the peptide bonds cleaved. Results Der p I cleaves different peptide bonds, depending upon the class of proteolytic mechanism used. In the model peptide substrate insulin B chain, the cysteine and serine proteinase activities of Der p I showed preference for glutamic acid and arginine respectively in the P1 position. Conclusion These data suggest the existence of more than one mechanistic form of the allergen immunologically identified as Der p I. If proteolytic activity is indeed a function of allergenicity, this information may have important implications for the pathogenicity of Der p I and the ability of innate antiproteinase defences in the respiratory tract to prevent immune sensitization.     

颈前部皮神经构筑研究

目的　为研究皮肤疾病和损伤提供皮神经构筑资料。方法　用S 10 0蛋白免疫组化染色法研究正常成人颈前部胸锁乳突肌前、后缘皮肤各层内皮神经的数量、分布及构筑特点。结果　 ( 1)皮肤乳头层内皮神经的小分支多分布在皮突下和乳头内血管丛附近。 ( 2 )真皮网状层内神经多位于小血管附近和汗腺、皮脂腺以及毛囊周围 ,神经分支呈串珠状或波纹形。 ( 3 )皮下组织内神经干和神经网同时存在。结论　皮肤内感觉神经树的分布及构筑特点与血管树相似。     

Expression, purification and molecular modeling of recombinant fibrinogenase [IV], a metalloproteinase from Deinakistrodon acutus venom.

A novel metalloproteinase, recombinant fibrinogenase IV (rFIV(a)), was expressed and purified from Deinakistrodon acutus venom. It was a single chain protein with an apparent molecular weight 27 kDa and an isoeletric point of pH 7.1. RFIV(a) cleaved preferentially the Aalpha-chain and also cleaved Bbeta, gamma-chains of fibrinogen when the incubation time was prolonged. The proteolytic activity was inhibited by EDTA, l-cysteine, and DTT, indicating rFIV(a) was a metalloproteinase requiring disulfide bonds for its activity. It kept above 85% of the initial activity from pH 4.5-11, showed an equal maximum activity at the temperature range from 30 to 50 degrees C, and was inactivated by Zn2+, Cu2+ and Cd2+. Homology modeling of rFIV(a) showed that two highly conserved disulfide bonds (Cys159-Cys164 and Cys117-Cys197) was maintained from its structure, and it exhibited the characteristic conserved motif H142E143XXH146XXGXXH152, whose three histidine residues were involved in binding of the catalytically essential zinc ion. This work demonstrates the expression, purification and characterization of recombinant fibrinogenase IV, which belongs to class P-I metalloproteinase from D. acutus venom.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) but not granulocyte colony-stimulating factor (G-CSF) induces plasma membrane expression of proteinase 3 (PR3) on neutrophils in vitro

The theoretical risk of triggering vasculitis resulting from administration of G-CSF and GM-CSF to patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), such as Wegener's granulomatosis (WG), who develop agranulocytosis due to cytotoxic therapy, is unknown. Since there is strong evidence that activation of polymorphonuclear neutrophils (PMN) induced by binding of ANCA to PR3 or myeloperoxidase (MPO) expressed on their plasma membrane is involved in the pathogenesis of systemic vasculitides (SV), we studied the surface expression of PR3 and MPO on PMN from healthy donors in response to G-CSF and GM-CSF in vitro by flow cytometric analysis. Increasing doses of G-CSF did not alter PR3 expression on either untreated or tumour necrosis factor-alpha (TNF-alpha)-primed donor PMN significantly. In contrast, GM-CSF significantly increased PR3 membrane expression on both intact PMN and neutrophils primed with TNF-alpha. MPO expression was not significantly altered by either G-CSF or GM-CSF. In summary, these data demonstrate that GM-CSF, but not G-CSF, induces plasma membrane expression of PR3 on PMN in vitro. Since in AAV accessibility of the antigen (PR3 or MPO) to the antibody (ANCA) on the plasma membrane of PMN is thought to be essential for neutrophil activation by ANCA, the results of the present study suggest that administration of GM-CSF to patients with WG with neutropenia implies a definite theoretical risk of deterioration of vasculitis via this mechanism.     

Alpha 1-antitrypsin deficiency and anti-proteinase 3 antibodies in anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis.

alpha 1-antitrypsin (alpha 1-AT) is a naturally occurring inhibitor of proteinase 3 (PR3) and elastase, two of the target antigens of anti-neutrophil cytoplasmic antibodies (ANCA). An increased incidence of alpha 1-AT phenotypes associated with dysfunctional alpha 1-AT or low serum levels has been reported in patients with anti-PR3 antibodies. We have studied the relationship between ANCA, and phenotypes and serum levels of alpha 1-AT. Phenotypes usually associated with a moderate or severe reduction in alpha 1-AT serum levels or in dysfunctional activity were found more often in individuals with anti-PR3 antibodies than in the general population: four of the 31 patients (13%) with anti-PR3 antibodies had phenotypes MZ (n = 2), S (n = 1) or Z (n = 1) (P < 0.05). However, the corresponding alpha 1-AT serum levels were normal (n = 3) or elevated (n = 1). None of the 31 sera with anti-PR3 antibodies had low levels of alpha 1-AT. No abnormal alpha 1-AT phenotype was demonstrated in seven patients with anti-elastase antibodies, despite a low level of alpha 1-AT in one serum. Anti-myeloperoxidase antibodies are common in patients with ANCA, but no abnormal phenotype or low serum alpha 1-AT level was demonstrated in any of 29 sera containing these antibodies. Finally anti-glomerular basement membrane (GBM) antibodies occur occasionally in patients with ANCA-associated diseases, but again none of 10 sera had an abnormal alpha 1-AT phenotype or low serum level. ANCA were not demonstrated by indirect immunofluorescence in any serum from 73 patients with abnormal alpha 1-AT phenotypes. These results confirm that patients with anti-PR3 antibodies often have alpha 1-AT phenotypes that are usually associated with low serum levels of alpha 1-AT or with dysfunctional protein. Nevertheless, the incidence of anti-PR3 antibodies in patients with abnormal alpha 1-AT phenotypes is very low. This probably reflects the rarity of Wegener's granulomatosis, the major disease associated with anti-PR3 antibodies, and the relative frequency of abnormal alpha 1-AT phenotypes. The mechanism for the development of anti-PR3 antibodies in patients with abnormal alpha 1-AT phenotypes is not clear, but may relate to the increased propensity of unbound and uninhibited PR3 to stimulate autoantibody production.     

Inhibition of proliferative responses of mouse spleen lymphocytes by bovine milk κ‐casein digests

The inhibitory effects of bovine milk κ‐casein and its enzymatic digests on the proliferative responses of mouse spleen lymphocytes induced or not induced by mitogens were studied with a colorimetric assay using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT). κ‐Casein and its glyco‐macropeptide (residues 106–169) inhibited the lipopolysaccharide (LPS)‐induced proliferative response, but the inhibitory effect was lost significantly after neuraminidase and chymotrypsin digestions. In contrast, trypsin and pronase digestions of K‐casein increased inhibitory effects. The pronase digest inhibited the proliferative responses not only induced by LPS but also in the absence of mitogen or when induced by concanavalin A and phytohemagglutinin. The pronase digest seemed to possess weak cytotoxity for lymphocytes. The inhibitory peptide was a glycopeptide(s) having specific size, and the inhibitory effects were reduced significantly by neuraminidase and chymotrypsin digestions. Moreover, similar inhibitory effects on proliferation of lymphocytes were observed in pronase‐digested bovine milk αs₁‐casein and β‐casein. These findings suggest that some peptides produced from milk caseins by the action of gastrointestinal proteinases might contribute to down‐regulate the immune response of neonates.     

SKALP/elafin gene polymorphisms are not associated with pustular forms of psoriasis

Psoriasis is a multifactorial skin disease characterised by epidermal abnormalities and infiltration by lymphocytes and polymorphonuclear leukocytes (PMN). Skin-derived antileukoproteinase (SKALP), also known as elafin, is a potent inhibitor of human leukocyte elastase and proteinase 3, two PMN-derived proteinases implicated in tissue destruction and leukocyte migration. We have shown that, at least at the protein level, SKALP is significantly decreased in lesional skin of patients with pustular psoriasis compared with plaque-type psoriasis. This finding raised the possibility that SKALP could be one of the candidate genes for pustular forms of psoriasis. We therefore performed single strand conformation polymorphism (SSCP) analysis on the SKALP gene to screen for mutations/polymorphisms in the exons of 30 patients with plaque-type psoriasis, 15 patients with pustular psoriasis and 48 healthy controls. In exon 1 a polymorphism was detected at position + 43 relative to the translation start site, resulting in a substitution of threonine for alanine in the signal peptide. In the promoter region a dinucleotide repeat polymorphism was identified. Both polymorphisms were not associated with pustular psoriasis, or psoriasis in general. Our data indicate that the decrease in SKALP activity in pustular psoriasis is not caused by mutations in the coding region of the gene, and that there is no allelic association between pustular psoriasis and SKALP gene polymorphisms.     

Induction of biologically active antineutrophil cytoplasmic antibodies by immunization with human apoptotic polymorphonuclear leukocytes

Translocation of intracellular components to the cell surface during the priming or apoptosis of polymorphonuclear leukocytes (PMN) is an important mechanism for interaction of antineutrophil cytoplasmic antibodies (ANCA) with these antigens. To test the capacity of apoptotic PMN to trigger production of ANCA, six groups of mice were immunized with either live or apoptotic lymphocytes, or with live, apoptotic, formalin-fixed, or lysed PMN. Mice immunized with both live and apoptotic neutrophils developed high titers of antibodies which gave a granular cytoplasmic immunofluorescent pattern. These antibodies were specific for lactoferrin and myeloperoxidase. Following a second intravenous infusion of apoptotic PMNs, mice developed anti-PR3 antibodies. Vasculitis lesions were not found in mice which developed ANCA. The ANCA-containing IgG fraction induced superoxide production by human PMNs. These results support the hypothesis that neutrophil-specific antigens presented on the cell membranes of apoptotic PMN may induce ANCA in the proper conditions.