Biomedical subjects
Publications and source records attributed to B Utecht.
Elafin and antileukoprotease are potent peptide-like inhibitors of human leukocyte elastase and have been isolated from human skin and bronchial mucus. Elafin proved to be a potent inhibitor of proteinase 3, whereas other inhibitors of human leukocyte elastase such as antileukoprotease and eglin C proved to be much less effective.
Elafin, a human skin derived inhibitor of human leukocyte elastase, was tested for inhibitory activity against proteinase 3, an elastin degrading proteinase of neutrophils. The inhibitory activity of elafin was compared with antileukoprotease and eglin C. Elafin proved to be a potent inhibitor of elastin-FITC degradation showing an IC 50 of 9.5 x 10(-9) M. Potency was found to be more than 100-fold higher as compared with antileukoprotease and eglin C.
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The target antigen of anti-neutrophil cytoplasm antibodies (ACPA; also known as ANCA) was isolated by affinity chromatography from supernatants of human neutrophils, stimulated with phorbol ester to induce degranulation. Sequence analysis of the antigen revealed 17 NH2-terminal amino acids (IVGGHEAQPHIRPIYMA), which have considerable sequence homology with known serine proteinases. Investigation of the enzymatic activity showed that the antigen is a neutral serine proteinase that is able to cleave elastin. Since the molecular weight of the antigen, its substrate specificity, and its inhibitor profile reported in this study are identical with those reported recently for proteinase 3, we conclude that ACPA are most probably directed against proteinase 3.
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Autoantibodies directed against cytoplasmic components of neutrophil granulocytes and monocytes (ACPA) have previously been described as a disease-specific marker for Wegener's granulomatosis (WG). We have developed an ELISA for determining and quantifying ACPA using an affinity-purified antigen preparation. The antigen was purified from supernatants of human neutrophils stimulated with phorbol ester to induce degranulation, by means of affinity chromatography with naturally occurring human autoantibodies. The established ELISA was sufficiently specific and sensitive, and the ACPA concentrations obtained with it correlated significantly with the ACPA titres determined by an indirect immunofluorescence technique (Spearman's rank correlation coefficient = 0.85, P less than 0.001, n = 105 WG patients). This ELISA provides precise ACPA quantitation and should prove valuable for monitoring disease activity in WG.
The in vitro analysis of B-cell function has become more important recently. For this purpose, polyclonal B-cell activators (PBAs) and adequate test systems are now available. We compared the protein A plaque assay which is applied to determine the number of immunoglobulin(Ig)-secreting cells and the ELISA (enzyme linked immunosorbent assay) which is employed for the measurement of the amounts of secreted Ig. The number of Ig-secreting cells and the amounts of Ig in the culture supernatants after stimulation of mononuclear cells with PBAs correlated significantly, i.e. a B-cell activation can be detected by both test systems. The variation coefficients of the protein A plaque assay were higher than those of the ELISA. Furthermore, the protein A plaque assay is a very time-consuming method; therefore, an ELISA should be applied for the routine diagnostic of immunodeficiencies. Nevertheless, the protein A plaque assay remains important for the research of immunopathological diseases and for the determination of in vivo activated B-cells (spontaneous plaques) which can increasingly be observed in immunological disorders like AIDS (acquired immunodeficiency syndrome) or systemic lupus erythematosus.