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At least 19 recordsLinked to original sources
PubMed1992

Human keratinocytes and A-431 cells synthesize and secrete factor B, the major zymogen protease of the alternative complement pathway.

Biosynthetic radiolabeling studies demonstrate that human keratinocytes and A-431 cells, a human epidermoid carcinoma cell line, synthesize and secrete factor B as a monomeric 105-kD protein. Epithelial cell-derived factor B comigrates in SDS-PAGE with that produced by HepG2 cells, a human hepatoma cell line traditionally employed in studies of complement component biosynthesis. Comparative pulse-chase studies in A-431 and HepG2 cells show that this alternative pathway complement component is produced as co-migrating 100-kD intracellular proteins that are processed in both cell types to 105-kD extracellular factor B. Quantitatively, immunoprecipitable factor B accounts for 0.05% of radiolabeled proteins in A-431 cell culture media. Treatment of biosynthetically radiolabeled A-431 cell culture media with cobra venom factor and factor D for 60 min at 37 degrees C produces the specific factor B cleavage products Ba and Bb. These fragments are not identifiable in control culture media subjected to similar treatment in the absence of alternative pathway activators. Northern blot analysis of total cellular RNA from human keratinocytes, A-431 cells, and HepG2 cells reveals qualitative identity of a 2.8-kb factor B mRNA species in these three cell types. The relative level of factor B mRNA expression in these cells parallels their level of factor B protein synthesis (i.e., HepG2 cells greater than A-431 cells greater than human keratinocytes). Epithelial cell-derived factor B may play an important role in local inflammatory reactions and also directly interact with epithelial cell derived C3--a key classical and alternative pathway complement component recently shown to be produced by human keratinocytes.

Animals↗
PubMed1979

The quantitation of alternative pathway complement function by timed lysis assay.

A simple timed lysis assay is described for quantifying haemolytic complement activity in human serum. Classical pathway complement function was determined by measuring the time taken to lyse 50% of a standard suspension of antibody-coated sheep erythrocytes; the time required for 50% lysis of a standard rabbit erythrocyte suspension was similarly used to evaluate alternative pathway function. Because target erythrocytes prepared on different days gave slightly different 50% lysis times, it was necessary first to construct a series of calibration curves for converting 50% lysis times into CH50 U/ml. For this purpose, a range of dilutions of the standard human serum, of known haemolytic activity, was tested against erythrocytes prepared on 10 separate occasions. The standard serum was subsequently included with each batch of unknown sera and used to select the appropriate calibration curve for direct conversion of the 50% lysis time into CH50 U/ml. Eleven samples of normal human serum were tested by both the timed lysis assay and by the dilution methods of Mayer (1971) (classical) and Platts-Mills and Ishizaka (1974) (alternative pathway). Comparable results were obtained in all cases.

Animals↗
PubMed1992

Activation of the alternative complement pathway accompanies disease flares in systemic lupus erythematosus during pregnancy.

OBJECTIVE: To assess the activity of systemic lupus erythematosus (SLE) during pregnancy and to distinguish it from preeclampsia. METHODS: We prospectively measured the complement activation products Ba, Bb, SC5b-9, and C4d, as well as the conventional complement determinants C3, C4, and CH50, during pregnancy in 14 patients with SLE and 10 women with preeclampsia. RESULTS: Four of the 14 SLE patients were considered to have disease flares, 3 occurring in the second trimester and 1 postpartum. In these patients, significant abnormalities of Ba, Bb, SC5b-9, and CH50 were noted. In contrast, measures of C4d did not distinguish between pregnant patients who had flares and those whose SLE remained stable. Although decreased values of C3 were rarely seen in the patients with stable disease, normal values of C3 during lupus pregnancy were not reliably associated with stable disease. Three of 10 non-SLE patients with preeclampsia had elevated levels of Ba; however, in each case, the CH50 level was close to or within the normal range. This was in sharp contrast to the findings observed in the 4 patients with active SLE, in whom high levels of plasma Ba were always associated with low CH50 values. Moreover, the ratio of CH50 to Ba was significantly lower in the patients with lupus flares than in the non-SLE patients with preeclampsia. CONCLUSION: While a decline in the CH50 level alone could otherwise be attributed to decreased synthesis of complement components, these data demonstrate that ongoing activation of the alternative complement pathway can accompany disease flares in pregnant women with SLE.

Complement Activation↗
PubMed1976

Activation of the alternative complement pathway in systemic lupus erythematosus.

Serum factors activating the alternative pathway of complement in vitro, independent of classical pathway activation was demonstrated in six of eleven patients with systemic lupus erythematosus (SLE). These serum factors were detected by lysis of gluthathione-sensitized human erythrocytes and by C3 and factor B conversion in the presence of EGTA (10 mM) and MgCl2 (0-3 mM), conditions which blocked activation of the classical pathway but permitted activation of the alternative pathway. In order to determine if in vivo activation of the alternative pathway of complement was present in SLE, highly-purified factor B was labelled with radioactive iodine (125I), and its metabolism studied in the eleven patients with SLE and in twelve control subjects. All six patients with serum factors capable of activating the alternative pathway in vitro, had in vivo evidence of alternative pathway activation as measured by increased fractional catabolic rate (FCR) of factor B. Two patients without demonstrable alternative pathway activating factors in their sera had an elevated FCR of factor B. Six of the patients with increased FCR of factor B had disease limited to skin or joint and one had lupus nephritis which was inactive at the time of study. One of the four patients who were in clinical remission had elevated FCR. This study demonstrates that a significant number of patients with SLE of relatively mild disease activity had evidence of alternative complement pathway activation. This activation did not appear to be limited to patients with lupus nephritis and raises the possibility that it could also be related to some of the extra-renal manifestations of SLE.

Adolescent↗
PubMed1979

Deficient alternative complement pathway activity in newborn sera.

Neonatal susceptibility to overwhelming bacterial infection is commonly attributed to a relative deficiency in serum opsonic activity. However, few studies have compared the functional capacity of the classical complement pathway with that of the alternative complement pathway in the neonate. The opsonic activity of nine maternal infant serum pairs were studied by determining percent uptake of radiolabeled Escherichia coli. Seven mother-infant paired sera were studied using E. coli strains known to be opsonized via the alternative complement pathway: the mean percent uptake of E. coli opsonized in neonatal sera was 16.8%; of those opsonized in maternal sera, 54%; and of those opsonized in control sera, 45% (P less than 0.005). Two E. coli strains requiring the classical complement pathway for opsonization were phagocytized equally well in maternal and infant sera of seven mother-infant pairs. Determination of anti-O hemagglutination inhibition (HI) antibody titers in six maternal sera for one classical complement pathway activating and one alternative complement pathway strain showed no correlation between percent phagocytosis and HI antibody titer. These data would suggest that serum levels of classical pathway components are probably adequate for opsonization of E. coli via the classical pathway, but that low alternative complement pathway activity in neonatal sera may contribute to the newborn's increased susceptibility to bacterial sepsis.

Complement Activation↗
PubMed1979

Activation of the alternative pathway of complement by antiserum to factor B.

Monospecific rabbit and goat antisera to human complement proteins and human immunoglobulins were tested for their ability to activate the alternative complement pathway. This activation was detected by two methods where classical pathway activation was blocked with EGTA and alternative pathway activation was promoted with added magnesium ions. These two methods consisted of lysis of GSHE and conversion of factor B into split products. C1q-depleted serum was used in a third assay system. Only antiserum to human factor B was able to activate the alternative pathway in the various systems used. None of the other anticomplement sera showed such activity. When antiserum to factor B was fractionated by ammonium sulfate and column chromatography, activation of the alternative pathway was found in the IgG fraction, and this activity was completely removed by absorption with purified factor B but not with other purified complement components.

Animals↗
PubMed1978

[Activity of the alternative complement pathway by various axenic strains of Entamoeba histolytica].

The activation of the alternative pathway of complement by axenic cultures of E. histolytica is reported. The activation is carried out in different proportions by several axenic strains, i.e., HK9:NIH, HM1:IMSS, HM2:IMSS and HM3:IMSS; the end result consists of a destruction of the parasite as well as consumption of the serum complement. The participation of the alternative pathway was studied in the presence of EGTA and Mg++, zymosan, heat-inactivation at 50 degree C for 20 min and immunoelectrophoretic studied with anti-factor B.

Animals↗
PubMed1979

Classical and alternate complement pathway activities in paired dairy cow--newborn calf sera.

Hemolytic assays were used to compare alternate and classical C pathway activities in sera obtained from clinically normal newborn dairy calves and their mothers at the time of delivery. Mean alternate and classical CH50 concentrations in sera from newborn calves were both significantly lower than in their dams (P less than 0.001). The titer of alternate C pathway activity, expressed as CH50 units/ml, in sera from 17 calves was 12.9 +/- 5.5, whereas for the cows it was 25.8 +/- 6.2. The ratio of cow: calf serum alternate CH50 titers averaged 2.25 +/- 0.80 and ranged from 0.88 to 4.14. Classical CH50 titers were 78.0 +/- 42.7 units/ml in calf sera and 246.0 +/- 44.5 in cow sera. The ratio of cow: calf serum classical CH50 titers averaged 3.71 +/- 1.49 and ranged from 1.19 to 6.87. The wide range of values, noted for both the alternate and classical C pathways, within maternal and neonatal groups was assumed to reflect the biologic variability of complement levels in bovine serum. The possible relationships between deficient levels of alternate and classical CH50 activity in newborn calves and their susceptibility to infections is discussed.

Animals↗
PubMed1979

Activation of the alternative complement pathway by Torulopsis glabrata.

Torulopsis glabrata is a yeast-like fungus with increasing importance as an opportunistic human pathogen. The role of serum complement in resistance to this infection was studied by chemotactic and opsonic assays. The results indicate that T. glabrata activates complement via the alternative pathway. This may be an important mechanism in host immunity to this fungus.

Candida↗
PubMed1978

[Determination of the alternative complement pathway in patients with amebic liver abscess].

The activation of the alternative pathway of complement in the sera of patients with amebic liver abscess was studied. Of 11 sera examined, only six showed an activation of the alternative pathway. However, all sera showed diminished levels of complement (CH50) and C3, in spite of normal levels of C1q. The results suggest that the sera of patients with invasive amebiasis have a substance capable of activating the properdin pathway. The significance of these observations is not completely clear at the present time.

Complement Activation↗
PubMed1979

Temperature dependent activation of the alternate complement pathway by an IgG cryoglobulin.

A patient with chronic membranoproliferative glomerulonephritis is presented whose serum contains a monoclonal IgG3 cryoglobulin. The presence of persistent hypocomplementemia suggested the possibility that the cryoglobulin, upon cold-induced precipitation, was capable of activating the complement system. Because visible cryoprecipitation commenced in vitro at 30 degrees C, the patient's serum and normal serum had been added the isolated cryoglobulin were repeatedly cooled to 30 degrees C and rewarmed to 37 degrees C. This reproduction of the in vivo counterpart of blood circulating through an extremity exposed to the cold resulted in activation of C3-proactivator (properdin factor B), C3 cleavage, and a 78% reduction in total hemolytic complement. This study demonstrates that IgG is capable of activating complement via the alternate pathway and reveals a mechanism through which this can occur in vivo; namely, by means of temperature dependent polymerization. In addition, we postulate that episodic complement activation initiated by the cryoglobulin contributed to the development of glomerulonephritis in this patient.

Aged↗
PubMed1975

Pneumococcal type-associated variability in alternate complement pathway activation.

Opsonization of Streptococcus pneumoniae may be mediated by the alternate complement pathway. To study the importance of this interaction to human disease, complement consumption by pneumococci of various serotypes was measured in humwn serum chelated with ethyleneglycoltraacetic acid, a substance that blocks the classic but not the alternate complement pathwway. Serotype I, in contrast to all other types studied, lacked ability to consume complement in this system. The ability for serotypes III, IV, and VIII to activate the alternate pathway could be eliminated by prior serum absorption at O C with they type in question, a condition that would remove antibody but not complement. Types VII, XII, XIV, and XXV readily activated the alternate pathway in unabsorbed and absorbed sera. Differences could not be related to properties of the capsules. It was concluded that types I, III, IV, and VIII lack intrinsic ability to activate and thus be opsonized by the alternate complement pathway, although types III, IV, and VIII can do so in concert with specific antibody. The fact that these same types are especially prominent in human disease suggests that the ability to evade opsonization by the alternate complement pathway in pre-antibody phases of infection may be a virulence factor in pneumococci.

Antigen-Antibody Complex↗
PubMed1979

A sensitive microassay for the murine alternative complement pathway.

A microhemolytic assay for measurement of murine alternative complement pathway activity is described. The assay uses 51Cr release from neuraminidase-treated rabbit erythrocytes incubated with Mg2+ EGTA-chelated murine serum. Neuraminidase pretreatment of rabbit erythrocytes increases the sensitivity of the assay 8--10-fold, enable the use of small volumes of individual mouse sera. The assay affords a simple, sensitive and reproducible method for measuring murine alternative complement pathway activity. Significant differences were found between strains alternative complement pathway activity of serum from various inbred murine lines was measured.

Animals↗
PubMed1979

Consumption of C3 via the classical and alternative complement pathways by sera and synovial fluids from patients with rheumatoid arthritis.

Five sera and four synovial fluids from patients with rheumatoid arthritis were found to contain substances which consumed hemolytic C3 in normal human serum (NHS) and in normal guinea pig serum (NGPS). These fluids were then tested for ability to activate the alternative pathway by incubating them with NHS containing MgEGTA and C4 deficient GPS. All sera and two synovial fluids depleted C3 in these reagents, indicating direct activation by the alternative pathway. Density gradient ultracentrifugation demonstrated C3 fixing activity in some specimens in the greater than 19s regions. These substances may also activate complement similarly in vivo and participate in the development of inflammatory processes associated with this disease.

Adult↗
PubMed1979

[Alternative complement pathway (author's transl)].

The dual role of the alternative complement pathway in recognition of foreign substances by a non-immune host and in the intrinsic regulation of the complement sequence is now well recognized. Activation of this pathway occurs through escape from its regulatory mechanisms induced by the activating principle; its functional expression depends on the respective levels of the component proteins C3, factor B, factor D and properdin, and on the control proteins beta 1H and C3bINA. This article presents recently acquired knowlege on the molecular mechanisms of activation, regulation and behaviour under pathological conditions of the alternative complement pathway.

Complement Activation↗
PubMed1979

Alternate complement pathway activation by group A streptococci: role of M-protein.

Avirulent strains of group A streptococci readily activate the complement system in normal human serum via the alternate complement pathway (ACP). Virulent M-positive group A streptococci are much less potent as activators of the ACP. The ability of M-positive streptococci to activate the ACP is enhanced by trypsinization or mild peptic digestion. The latter treatment removes the serologically active and antiphagocytic type-specific moieties of M protein, but retains the surface fuzzy layer. The phagocytosis of avirulent streptococci is markedly enhanced by preopsonization in serum chelated with Mg-ethylene glycol tetraacetic acid (classic complement pathway blocked) but not in serum devoid of heat-labile factors. These studies suggest that the function of M protein as a virulence factor may be mediated, at least in part, by its ability to retard interaction of ACP components with structures present on the streptococcal cell surface.

Bacterial Proteins↗
PubMed1990

Complement alternative pathway activation and control on membranes of human lymphoid B cell lines.

Membrane regulatory molecules normally prevent complement activation by autologous cells, therefore we compared the membrane control system of human lymphoid cell lines which activate or not human complement through the alternative pathway (AP). Membrane expression of decay-accelerating factor (DAF), membrane cofactor protein (MCP), complement receptors (CR)1, CR2 and H was measured either by radioimmunoassay or enzyme-linked immunosorbent assay on cell lysates. Soluble extracts of isolated membranes were tested functionally for their ability to accelerate the decay of C3bBb C3-convertase and allow the cleavage of C3b by factor I. Both regulatory functions were detected in solubilized membranes of Ramos cells, which do not activate the AP, as well as on the potent AP activator, Raji. Raji cells were found to express CR2, DAF and MCP molecules, while MCP was the only known regulatory protein detected on Ramos cells which expressed neither CR1, nor CR2, H or DAF. The I-cofactor activity of both Raji and Ramos cells was immunoprecipitated by anti-MCP, but the decay-accelerating activity was not adsorbed by anti-DAF nor by any of the available antibodies. Two EBV genome-negative cell lines (BJAB, BL41) were tested before and after in vitro conversion by EBV. As previously shown, EBV-converted cell lines activate the AP more efficiently than EBV- cell lines. At the same time, EBV superinfection induces an increase of both AP regulatory functions of cell membranes and enhances the expression of DAF, MCP and CR2. The results of this study show that complement activation by lymphoid cell lines is not related to an impaired autologous control of these cells, but that the expression of regulatory molecules increases together with the appearance of activating structures on the cell surface. Our results also suggest the occurrence of a new factor involved in the decay-accelerating activity on BL lines.

Antigens, CD↗
PubMed1978

Antibody-dependent cytotoxicity against Trypanosoma rhodesiense mediated through an alternative complement pathway.

A quantitative in vitro method was used to examine the role of classical and alternative pathways of complement activation in cytotoxicity against African trypanosomes by immune serum. This assay is based on the estimation of the extent of antibody-mediated cytotoxicity by measurement of inhibition of incorporation of [(3)H]leucine as an indicator of trypanosome metabolic integrity. To determine which pathway(s) is activated during cytotoxic events, complements sufficient in all components (C4S) and deficient in C4 (C4D) were used. Immune inhibition of [(3)H]leucine uptake by trypanosomes was observed in the presence of both complement sources. Treatment of C4S or C4D serum with cobra venom factor or disodium ethylenediaminetetraacetic acid abolished antibody-mediated cytotoxicity as well as immune hemolysis, thus suggesting the requirement for late-acting complement components. Ethyleneglycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid had no effect on inhibition of leucine incorporation, whereas immune hemolysis was inhibited, suggesting that cytotoxicity did not require C1 activation, a Ca(2+)-dependent event. The dependence of the cytotoxic process on Mg(2+) and not Ca(2+) ions and the fact that C4D guinea pig serum is fully active in trypanosome cytotoxicity indicate that an alternative pathway of complement activation is sufficient for activity.

Animals↗