Simple methods for preparing EAC1,4b,2a,3b and EAC4b,3b with human or guinea pig complement components using an anticomplementary agent, K-76 monocarboxylic acid.
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A protein with decay-accelerating activity for the classical C3 convertase, C4b2a, has been isolated on the basis of this function from guinea pig erythrocyte stroma. The isolation procedure for decay-accelerating factor of stroma (DAF-S) utilizes butanol extraction and chromatography on DEAE-Sephacel, hydroxylapatite, and phenyl Sepharose. Purified DAF-S has a m.w. of 60,000 and 65,000 on reduced and unreduced SDS gels, respectively, and exhibits m.w. of 30,000 and 175,000 on alkaline gradient gels, suggesting multiples of a 60,000-65,000 subunit. Purified DAF-S elicited a monospecific antiserum whose IgG fraction neutralized the decay-accelerating activity for C4b,2a affixed to 10(7) sheep erythrocytes (EAC1,4,2) in a dose-response fashion. The monospecific antiserum diluted up to 1:5120 agglutinated 1 x 10(6) guinea pig erythrocytes, but not sheep or human erythrocytes, suggesting that DAF-S, an integral membrane protein, has species-specific antigens that are expressed on the surface of the guinea pig erythrocyte.
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A polysaccharide (PS) purified from venom of the ant Pseudomyrmex sp. causes the activation of the classical complement (C) pathway in normal serum, but not in guinea pig serum. To investigate why C was not activated in guinea pig serum, we partially purified guinea pig C1 in the presence of the protease inhibitor p-nitrophenyl, p'-guanidinobenzoate (NPGB). This C1 preparation was activated (mu = 0.15, pH 7.5) by the PS in a dose-dependent reaction after NPGB was eliminated by dilution. The PS decreased the action of the C1 inhibitor for C1 in diluted guinea pig serum, and it also inhibited the activity of highly purified guinea pig C1 inhibitor for C1. There was a direct correlation between the concentration of the guinea pig C1 inhibitor and the loss of ability of the PS to activate C1 in mixtures of constant concentrations of purified guinea pig C1 and purified venom PS, and increasing concentrations of purified guinea pig C1 inhibitor. The activity of the human C1 inhibitor, either in diluted serum or highly purified, was not decreased by the PS. These results show that the PS does not activate guinea pig C1 in serum because its action is blocked by the C1 inhibitor.
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We report here a rapid assay for the complement enzymes CVFBb, C4b2a and Cls. This assay involves the use of a peptide substrate that releases a fluorescent coumarin derivative (AMC) upon cleavage by the convertase. The substrate, BocLeuGlyArgAMC, was chosen because its sequence is similar to the carboxyl terminus of C3a, and identical to that of C5a. The Km of this substrate are about 125 microM for the C3/5 convertase CVFBb, 169 microM for C4b2a, and 140 microM for C1s.
A mathematical expression has been derived that successfully correlates the kinetic data for the immune-mediated clearance of red blood cells. The expression resulted from the solution of differential equations arising from a clearance mechanism that was, essentially, consistent with that described by Schreiber and Frank. The mathematical expression correlated data for both IgG-and IgM-mediated reactions. Four different rate constants appear in the final kinetic equation; these constants, which measure the rates of the various steps in the clearance process, were evaluated by an iterative curve-matching process. The values of the rate constants were found to be dependent upon type of sensitizing immunoglobulin, number of C1-fixing sites, and several known immune system modifiers. Correlation of the derived rate expression with the experimental data provided a critical test for the Schreiber-Frank mechanism and the values of the rate constants provided additional insights into the immune clearance process.
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INTRODUCTION: Hereditary angio-oedema is a poorly understood disease. In more than 70 p. 100 of the cases, diagnosis is made 5 to 10 years after the first manifestations despite their typical nature. PATIENTS AND METHODS: We examined retrospectively 33 cases of hereditary angio-neurotic oedema treated at the Besançon University Hospital from 1973 to 1990. RESULTS: Laryngeal oedema was the most dangerous complication causing a critical episode in 22 cases. Ten patients were given danazol. DISCUSSION: This study emphasizes that preventive treatment is essential, based on danazol which should be prescribed at the lowest dose which gives a 50 p. 100 rise in the C2 complement fraction (usually 50 to 200 mg/day). Life-threatening attacks should be treated with purified C1 INH, although there are still problems with drug supply.