Acta pathologica, microbiologica, et immunologica Scandinavica. Supplement最新文献

C1q-precipitins (C1q-p) are comprised of 7S IgG with C1q-binding activity found in sera of patients with hypocomplementemic vasculitis urticaria syndrome ( HVUS ) and systemic lupus erythematosis (SLE). We have utilized C1q-coated polystyrene beads to selectively isolate C1q-p and to establish a sensitive and quantitative assay of C1q-p and immune complex activity. Purified C1q-p was comprised of polyclonal IgG which retained 7S sedimentation and solid phase C1q-binding activity at physiological ionic strength both in the presence and absence of normal human sera. No precipitation interaction was observed between C1q-p and fluid-phase C1q or C1 under the conditions tested. Purified C1q-p had no activity in the Raji cell immune complex activity. C1q-p activity also was observed in and purified from SLE serum; this activity was distinguishable from 7S immune complex activity detected by Raji cells which was also present in SLE serum. These studies indicate that C1q-p is a 7S IgG molecule found in HVUS as well as some SLE sera and has activity in C1q-binding but not in Raji cell-binding immune complex assays. These data also suggest that C1q-p is a monomeric, polyclonal IgG with preferential affinity for bound C1q. In addition to its potential role in immune complex disease, C1q-p may also provide an important tool for studying the interaction of immunoglobulin and C1q, and should contribute important information to understanding the pathobiology of immune complex disease.

A method is described for isolating both functionally and highly pure C2 from normal human serum or plasma. Functionally pure C2 was obtained by a two-step method of ammonium sulfate (AS) fractionation of plasma or serum and chromatography on AH-Sepharose containing a bound lectin of Euonymus europeus. The functionally pure C2 can be isolated in 2 days, and is used routinely as a reagent for functional hemolytic titrations of C3 and C4 in the Clinical Immunology Laboratory. Highly pure C2 was isolated by the two-step fractionation with AS followed by chromatography on CM-cellulose, aged CNBr-activated Sepharose 4B, and AH-Sepharose-lectin. The major difficulty for isolating highly pure C2 was its separation from Factor B of the alternative complement pathway. The yield of C2 varied from 30 to 40 per cent. Following electrophoresis and staining on polyacrylamide gels, the single band was hemolytically active C2.

Proteins antigenically related to CoF, the anticomplementary protein of the venom of the Indian hooded cobra (Naja naja), were found in a variety of elapid and viper venoms but not in the venom of Brazilian crotalids . In keeping with this finding was the weak ability of Brazilian snake venom to convert C3 in human serum. All snake serums tested, including Brazilian crotalids , contained a beta-globulin antigenically related to CoF. This serum protein in Brazilian snake serum had a number of characteristics of mammalian C3, including conversion on storage or on incubation of this serum with endotoxin, zymosan or mammalian antigen-antibody precipitates. The serum protein did not, however, convert on incubation with hydrazine. Brazilian crotalid serum did not, as did cobra serum, have the ability to inactivate CoF's ability to activate complement in normal human serum. The crotalid serum had hemolytic activity for rabbit antibody-sensitized and unsensitized sheep red blood cells that was active in the presence of Ca++ and Mg++ or Mg++ alone but greater with Ca++ present, suggesting the presence of both classical and alternative pathways of complement activation. This activity was maximal at 37 degrees C, but was destroyed or inactive after heating at 50 degrees C for 1 hr, incubation with hydrazine or by addition of EDTA. A marked reduction of hemolytic activity of Bothrops serum occurred after removal of the CoF-like protein. These findings suggest that Brazilian snake venom has little CoF-like material, but its serum contains a CoF-like protein with many characteristics of C3.(ABSTRACT TRUNCATED AT 250 WORDS)

Lysis of autologous sheep red cells (labeled with methotrexate as the target hapten) by neat autologous serum was studied. The results indicated that the efficiency of lysis under physiological concentrations of cells and serum was about the same as lysis of the same target cells sensitized with autologous antibody and guinea pig serum as the source of complement. The data also indicate that the one-hit mechanism of immune lysis operates under near physiological conditions.

The effect of S-protein on the polymerization of C9 during assembly of the C5b-9 complex was examined. Utilizing SDS polyacrylamide gradient slab gel electrophoresis, tubular poly C9 was quantitated as SDS resistant protein of 1.1 to 1.3 X 10(6) molecular weight. Poly C9 formation occurred upon incubation of purified C5b-6, C7, C8 and C9 at molar ratios 1:1:1:12. Addition of purified S-protein to the protein mixture or to preassembled C5b-7 or C5b-8 blocked formation of poly C9 in a dose dependent fashion and gave rise to SC5b-9. SC5b-9 assembled from purified proteins or in zymosan-activated serum was visualized in the electron microscope as a wedge-shaped structure of 350 to 400 A length and 30 to 250 A width which lacked tubular poly C9 seen in images of the membrane attack complex (MAC). Using biotinyl-S-protein and colloidal gold particles coated with avidin, S-protein was located at the wide end of the wedge-like SC5b-9 complex. It is concluded that S-protein has a dual function in SC5b-9 assembly. It blocks the membrane site of C5b-7 and it inhibits C9 polymerization by SC5b-8. Accordingly, the main structural difference between SC5b-9 and the MAC is the lack of tubular poly C9.

The activation of the C-attack phase does not necessarily involve the components of the C5 convertases. C--56 hemolytic activity was generated from the same source of C7 depleted serum by the alternative pathway convertase or by freezing and thawing resp. In contrast to activation by the convertase, biological activities of C5a (chemotaxis, serotonin release) were not detected following activation by freezing. The yields of C--56 hemolytic activities were similar and the properties of the activated products were identical. No difference was found in the molecular weight, in the hydrophobicity or with respect to charge. The two activities were in the absence of C7 stable at 37 degrees C and decayed rapidly in the presence of C7. It is proposed that a conformational change in the tertiary structure of the molecule(s) is the critical event in the formation of an active C--56 complex. In this light the cleavage of C5a from the native molecule by the convertase appears as a side reaction, not by itself essential for activation.

A sufficient explanation for the observations that HAE is a dominantly transmitted disease and that the hemizygotes have levels of the normal protein of only in the region of 15%-20% of normal can be given by proposing that a substantial proportion of the catabolism of C1(-)-esterase inhibitor involves the prior formation of a complex with one of the enzymes with which the inhibitor reacts. This part of the catabolism will be largely independent of inhibitor concentration, i.e. of zero order, and for this reason occurs similarly in normals and in hemizygotes. Estimates of the extent of this zero order metabolism can be obtained from turnover data with normal and dysfunctional C1(-)-inhibitor and the results are consistent with the observed levels. In the form of the disease associated with the dysfunction protein the dysfunctional protein makes up more than 85% of the total protein found for the same reason. The extent of the enzyme inhibitor complex dependent catabolism (RO) can be determined in vivo by simultaneous turnovers of dysfunctional and normal inhibitor and gives a measure of the extent of activation of this group of enzymes. The value of this technique in clinical practice is described elsewhere.