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A cobra venom factor (CVF)-induced C3 convertase activity in the hemolymph of Galleria mellonella.

A cobra venom factor (CVF)-induced C3 convertase has been generated from the hemolymph of Galleria mellonella. CVF was immobilized on Sepharose 4B and treated with cell-free hemolymph obtained from either unvaccinated G. mellonella larvae or larvae immunized with formalized Pseudomonas aeruginosa. The C3-cleaving activity was detected by the ability to cleave the alpha-chain of bovine C3 in a manner analogous to the CVF-induced mammalian C3 convertase, CVF,Bb. The insect-derived C3 convertase formed at 28 degrees C but not at 37 degrees C, then once formed was active at both 28 degrees C and 37 degrees C. EDTA did not inhibit the formation and action of the insect derived C3 cleaving activity.

Animals↗

Is primary biliary cirrhosis an immune complex disease?

Large immune complexes are present in the circulation of patients with primary biliary cirrhosis and result in the activation of complement by the classical pathway. Such large complexes are capable of producing tissue damage. The granulomatous lesions surrounding the small bile-ducts within the liver of patients with primary biliary cirrhosis and the vasculitis, rheumatoid arthritis, and associated lesions are all compatible with immune complex injury. It is postulated that such large complexes could be formed in the vicinity of the bile-ducts by an antigen absorbed from the bile or biliary epithelium. Complexes reaching the systemic circulation might be responsible for the associated extra-hepatic diseases.

Antibody Formation↗

Measles, histocompatibility leukocyte antigen polymorphism, and natural selection in humans.

Profound lymphocytopenia (greater than 2,000 lymphocytes/mm3) occurring within two days of rash in 69 South African black children with measles predicted either death or progression to chronic lung disease in 51 (77%) of 66 children who were followed for at least six weeks. Lymphocytopenia was significantly associated with the presence of histocompatibility leukocyte antigen (HLA) AW32 (P = 0.01), with a relative risk of 5.5. There was a trend toward an association between the presence of particular antigens in the HLA complex and the various indices of humoral and cellular immunity studied. These findings are discussed in terms of variation in the clinical spectrum of the disease and in relation to the evolution of HLA polymorphism.

Antibodies, Viral↗

Different activation patterns in the plasma kallikrein-kinin and complement systems during coronary bypass surgery.

Components of the plasma kallikrein-kinin and complement systems were determined in patients undergoing open heart surgery with cardiopulmonary bypass. Spontaneous kallikrein activity (KK), plasma prekallikrein (PKK), functional kallikrein inhibition capacity (KKI), C3 activation products (C3-act), and the terminal complement complex (TCC) were measured. A marked, transitory increase in KK and a decrease in PKK were found prior to cardiopulmonary bypass just after heparin injection. An additional decline in PKK and KKI during bypass with a return to near control levels in the postoperative period was observed. C3-act increased in all patients during bypass, reaching a peak value at wound closure. The TCC concentration also increased significantly during cardiopulmonary bypass, returned to control levels in the early postoperative period, and then increased again in the late postoperative period. It is concluded that activation of the kallikrein-kinin system started after injection of heparin, prior to cardiopulmonary bypass. Activation of both the initial and the terminal complement cascade, however, started only after onset of cardiopulmonary bypass.

Adult↗

Mycoplasma pneumoniae pneumonia associated with IgA nephropathy.

An 18-yr-old male developed Mycoplasma pneumoniae pneumonia associated with IgA nephropathy. In the biopsied renal tissue, immunofluorescence showed striking deposits of IgA, C3 and C3 activator in the mesangium, and electron microscopic examination showed electron-dense deposits in the mesangial region. When the patient's serum was preincubated with rabbit antiserum to human IgA, the passive hemagglutination titer for M. pneumoniae was much decreased. We suggest that in some cases, IgA nephropathy may follow infection by an organism which stimulates in IgA antibody response, leading to the formation of IgA antigen antibody immune complex deposits in the glomerular mesangium.

Adolescent↗

[Description of an unusual enzymic activity cleaving the third component of complement].

A large enzymatic complex cleaving C3 (C3'ase) in the absence of magnesium (EDTA) has been partially characterized in two patients. On gel filtration EDTA-C3'ase was found in an 800 000 fraction containing antigenic C4, IgA and IgG in addition to alpha-2-macroglobulin and IgM normally present. EDTA-C3'ase was specifically neutralized by antibodies to IgG, IgA and C4. The characteristics of this unusual enzymatic complex are compatible with that of a C4b2a complex stabilized by its specific binding to an auto-antibody of the IgG and/or IgA class.

Chromatography, Gel↗

Requirements for the production of high-titre C3 nephritic factor (NEF) antibody in vitro.

C3 nephritic factor (NEF) is an IgG autoantibody directed against neoantigenic determinants of the alternative C3 convertase (C3b.Bb). Structural and functional studies require important amounts of this antibody, which are difficult to obtain from patients' sera. We have developed a method for increasing NEF production in vitro. Epstein-Barr virus is a herpes virus which transforms B lymphocytes. Some authors were able to induce the production of NEF in vitro after infection with Epstein-Barr virus (EBV). These works were preformed without any previous cellular selection of B cells. We have performed a method of preselecting antigen-binding cells prior to EBV transformation. Non-preselected cells yielded 0.16 U/million cells in culture (U/M) of NEF antibody, whereas enriched cells for NEF antibody in eliminated 8 U/M (sheep erythrocytes coated with anti-IgG, A, M). Specific NEF synthesis can be increased, in peripheral blood mononuclear cells (PBMC) from patients by in vitro stimulation with the antigens recognized by NEF [C3b.Bb, 21,000 MW protein from patients' E membranes and 26,000 MW protein from sheep E membranes (ShE)]. The highest stimulation is induced by the C3b.Bb and by 26,000 MW protein, 21,000 MW protein had lowest stimulatory effect. In this work also we have shown that patients having NEF antibody in sera have an increase of the CD5-CD19 subset, when compared with the controls.

Antigens, CD↗

Autoantibody to the alternative pathway C3/C5 convertase and its anti-idiotypic response. A study in affinity.

In an effort to understand the development and control of autoantibody production, we studied the affinity of autoantibody to the alternative pathway C3/C5 convertase (C3 nephritic factor (C3NeF)) and its autoanti-idiotypic antibodies, Ab2 alpha and Ab2 beta. These were isolated and purified from newborns, normal adults, and patients with membranoproliferative glomerulonephritis. In all cases, both IgG and IgM C3NeF were available for study. The affinity of IgG and IgM C3NeF for their natural Ag (10(8) liters/mol) as well as for the internal image of that Ag displayed on Ab2 beta was high (10(10) liters/mol). Furthermore, the affinity of IgG C3NeF was nearly 100-fold higher in patients than in newborns, whereas there were no significant changes with IgM C3NeF. By contrast, there were not differences in the affinity of IgG Ab2 alpha (which does not display any likeness to the native Ag) from normal adults and patients to any C3NeF isolate. There was, however, a progressive increase in affinity between both Ab2 alpha preparations and IgG C3NeF from newborns, adult normal subjects, and patients, implying an alteration in C3NeF to account for the changes in affinity. These data suggest that Ag-driven affinity maturation occurs with autoantibody but may not occur within the idiotypic network. These data also indicate that as autoantibody affinity matures, it appears to modify its idiotype, perhaps in an effort towards autoregulation.

Adult↗

[Effect of Expafusion (HES 40/0.5) on the corpuscular elements of blood and inhibitors of blood coagulation].

The present study reports on an investigation of 32 normal subjects who were given intravenously 500 ml of hydroxyethyl starch (HES) over 30 min. In 16 normal subjects, 500 ml of blood had been previously withdrawn over 20 min. Tolerance proved to be good. Side effects such as hyperergic reactions, influence on circulatory or cardiac function were not observed. Changes in corpuscular elements of blood did not exceed the dilution effect and mostly returned to the initial value 60 min after termination of HES infusion. The change in thrombocyte function, immunoglobulins and the inhibitors alpha 1-antitrypsin, alpha 2-macroglobulin, as well as C1-inactivator and C3-activator only slightly, but not more pronounced than it could have been expected by the dilution effect. A favorable effect could be recorded in erythrocyte filtration according to Schmidt-Schoenbein. Here, we found an improved flow property which exceeded the hemodilution effect and lasted over the 4-hour test period. In addition, thrombocyte aggregation during ADP administration in the Born chamber did not show any change which suggests that a clinically relevant effect on thrombocyte function and thus the danger of hemorrhage during HES administration can be excluded.

Adolescent↗

Characterization of three monoclonal antibodies against C3 with selective specificities.

We raised 15 mouse monoclonal antibodies against human C3 from two separate fusions. Mouse plasmacytoma cells were fused with spleen cells from mice immunized either with a mixture of C3, C3b and C3c, or with a mixture of C3dg and C3d. Three of the 15 monoclonals were characterized in detail. N-7A reacted with native C3 as well as C3b and C3c. Two other monoclonals, C-5G and G-3E, recognized neoantigenic determinants on C3c and C3dg, respectively. In ELISA, C-5G reacted with C3b and C3c but not with native C3 nor with C3dg; and on the other hand G-3E reacted only with C3dg. The selective specificities of these monoclonals were further confirmed in a binding assay to C3 fragments formed on cellular intermediates. C-5G bound exclusively to EC3b, and G-3E bound to EiC3b, EC3dg and EC3d. N-7A bound only very poorly to EC3b. C-5G inhibited both the haemolytic activity of C5 convertase and also the CR1-mediated rosette formation of B-enriched peripheral mononuclear cells with EC3b. G-3E inhibited the CR2-mediated EC3dg-rosette formation of Raji cells. These monoclonals, with selective specificities, can distinguish the state of activation and degradation of the C3 molecule.

Antibodies, Monoclonal↗

Purification and characterization of decay-accelerating factor (DAF) from Raji cells.

Decay-accelerating factor (DAF), a membrane protein that regulates the complement system, was purified to homogeneity from lymphoblastoid (Raji) cells (DAF-R). It exhibited almost the same molecular weight as DAF from stroma of erythrocytes (DAF-S). Purified DAF-R, which could be reincorporated into cell membranes, accelerated the decay of the C3 convertases, in both the classical (C4b2a) and the alternative (C3bBb) pathways. This activity was completely inhibited by a monoclonal anti-DAF antibody, 1C6. From these results, DAF-R and DAF-S can not be distinguished; however, the decay-accelerating activity of DAF-R was much higher than that of DAF-S. 1C6 enhanced the binding of C3 to Raji cells by incubating with six purified components of the alternative pathway, whereas it did not induce the killing of Raji cells after a short incubation period. When antibodies against Raji cells were added to the above system, the blocking of DAF activity by 1C6 resulted in efficient killing of Raji cells by autologous complement. From these results, it is clear that DAF on nucleated cells plays an important role in protecting these cells from the damage caused by autologous complement.

Burkitt Lymphoma↗