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Results for “Complement Pathway, Alternative”

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PubMedDate not supplied

[In vitro effect of rainbow trout (Oncorhynchus mykiss) serum in Tylodelphys sp. (trematoda, Diplosromatidae) metacercariae].

Here, it has been described that normal fresh sera from rainbow trout (Oncorhynchus mykiss) were able to lyse Tylodelphys sp. metacercariae isolated from Galaxias maculatus (Jenyns, 1842) (Teleostei, Galaxiidae) brain. This effect was time dependent, and progressively increased since 1 h up to 18 h incubation. There were oscillations in the ability of different sera samples to lyse these parasites. Similar values were achieved with other salmonid sera, and also with normal human sera pool. The lytic activity was abolished by heat-inactivated sera, by zymosan treatment and by incubation in the presence of EDTA. However, the mean values obtained in the presence of EGTA-Mg++ did not significantly differ from those found with sera as a source of complement. The results of this study suggested the role of complement alternative pathway on Tylodephys sp. in vitro lysis, under our experimental conditions.

Animals↗

PubMed1978

Prolonged complement activation in mice.

Because antibody responses to the alternative complement pathway activator, cobra venom factor, are T-dependent and B mice therefore do not develop resistance to its action, it was possible to examine whether renal injury occurs under circumstances of protracted third-phase alternative pathway activation. After periods of up to three months, no evidence from measurements of blood urea or proteinuria or from examinations with light microscopy immunofluorescence or electron microscopy was obtained to indicate a directly nephrotoxic effect of this type of complement activation.

Animals↗

PubMed1985

Complement-mediated solubilization of immune complexes and their interaction with complement C3 receptors.

Some of the molecular events in the complement (C)-mediated solubilization of immune complexes (IC) have been clarified in recent years. The solubilization is primarily mediated by alternative C pathway proteins whereas factors in the classical pathway accelerate the process. Components of the membrane attack complex do not participate in the reaction. Besides affecting the size and solubility of circulating IC the interaction with C factors influences the reactivities of the complexes towards fluid phase reactants and mediates the reversible binding of IC to cellular C3 receptors. Our knowledge of the cellular localization, expression and structure of the C3 receptors, especially the C3b (CR1) receptor, has been considerably extended in the last few years, whereas our understanding of the physiological role of these receptors is still fragmentary. However, it is becoming increasingly evident that impaired solubilization of IC in patients with compromised C function may permit the complexes to deviate from their normal pattern of interaction with C3 receptors probably influencing both the organ distribution and clearance of IC and thereby also their phlogistic potentials.

Antigen-Antibody Complex↗

PubMed1997

Hyperacute lung rejection in a pig-to-human transplant model: the role of anti-pig antibody and complement.

BACKGROUND: The physiology of hyperacute rejection of pig lung by human blood and the role of antispecies antibody and complement in this phenomenon have not previously been characterized. METHODS: Human blood was perfused through an ex vivo pig heart-lung preparation. In the treatment groups, blood was either unmodified or modified to deplete alternative pathway complement (heat treatment), anti-pig antibody, or both. Control experiments were performed with unmodified and heat-treated pig blood. Physiologic parameters, organ survival, and immunohistology were the primary outcome measures assessed. RESULTS: Pig lung was consistently damaged by human blood within 45 min (median 20 min), as evidenced by elevated pulmonary vascular resistance and parenchymal injury. Immunohistologic studies of perfused lungs showed prominent deposition of IgM and classical pathway component, C4, and weaker deposition of alternative pathway component, properdin. Heat treatment did not impede the rise in pulmonary vascular resistance or significantly prolong survival. Depletion of anti-pig antibody prolonged survival (median 90 min) and attenuated the rise in pulmonary vascular resistance. Antibody absorption, combined with heat treatment of plasma, prevented the elevation in pulmonary vascular resistance and yielded median graft survival (210 min) similar to pig blood perfusion (approximately 240 min). CONCLUSIONS: These results show that elevated pulmonary vascular resistance and pulmonary parenchymal injury are mediated at least in part by antispecies antibody and heat-sensitive pathways. They are consistent with the hypothesis that complement activation contributes significantly to acute lung damage in the pig-to-human species combination.

Animals↗

PubMed2000

Different regulation of factor H and FHL-1/reconectin by inflammatory mediators and expression of the two proteins in rheumatoid arthritis (RA).

Factor H and the FHL-1/reconectin protein are two human plasma proteins that act as important regulators of the alternative complement pathway. Each protein is encoded by a unique transcript, but both mRNAs are derived from the factor H gene by means of alternative processing. In order to address potential functional differences between the two proteins we analysed their expression in hepatic and non-hepatic cells and studied their regulation by inflammatory mediators. We demonstrate that factor H and FHL-1/reconectin transcripts which are regulated by the same gene promoter and are initiated at the same transcription start site are differently expressed. Expression of the molecules is induced and regulated by the inflammatory mediators interferon-gamma (IFN-gamma) and the anti-inflammatory glucocorticoid dexamethasone. Both factor H and FHL-1/reconectin are expressed and secreted by synovial fibroblasts and are present in synovial fluid derived from patients suffering from rheumatoid or reactive arthritis. The local synthesis in synovial fibroblasts and their induction by IFN-gamma and dexamethasone, but not by tumour necrosis factor-alpha, suggests for each of the two complement regulators a protective role in RA.

Alternative Splicing↗

PubMed1977

Effect of heparin on complement activation and lysis of paroxysmal nocturnal hemoglobinuria (PNH) red cells.

The effect of heparin upon the binding of the third component of complement (C3) to PNH red cells in vitro and their subsequent hemolysis is described. Heparin, in increasing concentrations, progressively inhibits membrane C3 fixation and hemolysis when the classic complement pathway is activated by anti-red cell antibodies. Heparin has a biphasic effect upon membrane C3 fixation and hemolysis when complement is activated in serum at decreased ionic strength (sucrose lysis) or in serum at decreased pH (Ham test). Heparin in concentrations above 2 U/ml inhibits C3 binding and hemolysis while lower concentrations of heparin enhance the consequences of complement activation by these two procedures. This enhanced complement activation may explain the increased hemolysis sometimes reported in PNH patients treated with heparin, and suggests that heparin may aggravate the consequences of pathologic alternative pathway complement activation in other diseases.

Binding Sites, Antibody↗

PubMed1981

Acute-phase C56-forming ability and concentrations of complement components in normotensive and hypertensive pregnancies.

A prospective study of 61 women who were normotensive at booking revealed an increased incidence of C56-forming ability in those who later developed hypertension. Similar changes were found before delivery in a separate group of women admitted with latent hypertension and in each of 11 women with severe pre-eclampsia and a surviving fetus. The C56-forming ability, an acute phase feature, often preceded the hypertension and the findings suggest an indirect relation between complement changes and the mechanism causing hypertension in pregnancy. Concentrations of complement components in hypertensive and normotensive pregnancies were also compared with each other and with those of normal healthy non-pregnant women. The hypertensive patients had increased total alternative pathway function and higher factor B concentrations. The C56-negative sera from hypertensive patients tended to have C7 concentrations higher than normal whereas C56-positive patients had higher C5 concentrations. The aetiological and pathological implications are discussed.

Complement Activation↗

PubMed2002

The genetics and pathogenesis of haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura.

PURPOSE OF REVIEW: In recent years there has been a substantial increase in the understanding of the genetics and pathogenesis of haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura. RECENT FINDINGS: In diarrhoeal associated haemolytic uraemic syndrome it has been established that the virulence of Escherichia coli O157 is related to intimin adhesion and the transport of verocytotoxin on polymorphonuclear cells. It has been shown that early changes in the coagulation pathway predate the onset of diarrhoeal haemolytic uraemic syndrome. Mutations in factor H, a fluid-phase regulator of the alternative complement pathway, have been identified in 10-20% of patients with both familial and sporadic (non-diarrhoeal-associated) haemolytic uraemic syndrome. The mutations mainly cluster in the C terminal part of factor H, a region that is important for both binding to C3b and also polyanionic structures on cell surfaces. The identification of antibodies against a plasma metalloproteinase responsible for cleaving ultralarge von Willebrand factor multimers in thrombotic thrombocytopenic purpura has been followed by the elucidation of the identity of the proteinase. It has been shown to be a member of the ADAMTS family, and mutations have been identified in the gene in families with inherited thrombotic thrombocytopenic purpura. SUMMARY: The molecular pathogenesis of haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura is an exciting and rapidly evolving field. These recent advances will lead to logical, targetted changes in the management of these conditions.

Diarrhea↗

PubMed1988

Biochemical characterization of a factor produced by trypomastigotes of Trypanosoma cruzi that accelerates the decay of complement C3 convertases.

Infective- and vertebrate-stage trypomastigotes of Trypanosoma cruzi resist serum killing by the alternative complement pathway, whereas noninfective vector-stage epimastigotes, from which trypomastigotes derive, are serum-sensitive. This form of developmental preadaption is commonly observed in protozoan parasites, but its mechanisms are poorly understood. We have demonstrated previously that trypomastigotes spontaneously shed molecules which interfere with formation and accelerate the intrinsic decay of complement C3 convertases, a finding which may explain the evasion of complement lysis by trypomastigotes. We now describe the partial purification and characterization of the T. cruzi C3 convertase inhibitor from the supernatant of culture metacyclic and tissue culture trypomastigotes. Decay-accelerating activity for both classical and alternative pathway C3 convertases copurifies on anion-exchange fast protein liquid chromatography and chromatofocusing with 35S-labeled molecules of 87-93 kDa, pI 5.6-5.8. The labeled components are destroyed by papain and retained on concanavalin A-Sepharose, procedures which remove functional decay-accelerating activity from the supernatant. The 87-93-kDa components are immunoprecipitated by sera from patients chronically infected with T. cruzi, but not by antisera to any known regulatory proteins of the human complement cascade. Lytic activity for tissue culture trypomastigotes in chagasic sera is associated with antibody reactivity against the 87-93-kDa 35S-labeled components and with inhibition of decay-accelerating activity. The T. cruzi factor is the first developmentally regulated microbial complement inhibitor to be biochemically characterized.

Animals↗

PubMed1989

Evasion of Trypanosoma cruzi from complement lysis.

This review outlines: a) the main biochemical and biological properties of the complement system (C) components; b) the manner through which they interact in the two distinct routes of C activation, the classical and the alternative pathways, to generate the enzymes C3 and C5 convertases responsible for release of the peptides C4a, C3a and C5a endowed with the properties of mediating the early events of the inflammatory process or the potentially cytolytic complex C5b-C9; c) the main features of control of these activation processes; d) the identification of cell surface components present in the trypomastigote forms of Trypanosoma cruzi possibly involved in the mechanisms developed by this parasite to evade C lysis; e) the inactivation or removal of these cell surface components by enzymatic (trypsin or papain), chemical (periodate) or physical (heating at 45 degrees C) treatments; f) isolation of these components by chromatographic methods; and, g) demonstration that some of these cell surface components interfere with C3 convertase formation or action in a manner similar to the decay accelerating factor (DAF).

Animals↗

PubMed1986

Effect of Trypanosoma cruzi membrane components on the formation of the classical pathway C3 convertase.

When T. cruzi trypomastigote forms are held at 45 degrees C for 10 min in saline they become susceptible to lysis by the alternative complement pathway. As the result of heating trypomastigotes, but not epimastigotes, substances are released into the fluid phase which inhibit EAC1,4,2,3 by inducing decay. The inhibition was most pronounced at the level of the decay of the C14b2a complex. C3 convertase of the classical pathway was also inhibited by live trypomastigote forms. These data suggest that the trypomastigote but not epimastigote forms of T. cruzi have cell surface modulators of C3 convertase that permit them to evade the lytic action of complement.

Animals↗

PubMed2004

Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non-immunosuppressed baboons.

Human membrane cofactor protein (CD46) controls complement activation and when expressed sufficiently as a transgene protects xenografts against complement-mediated rejection, as shown here using non-immunosuppressed baboons and heterotopic CD46 transgenic pig kidney xenografts. This report is of a carefully engineered transgene that enables high-level CD46 expression. A novel CD46 minigene was validated by transfection and production of a transgenic pig line. Pig lymphocytes were tested for resistance to antibody and complement-mediated lysis, transgenic tissues were characterized for CD46 expression, and kidneys were transplanted to baboons without immunosuppression. Absorption of anti-Galalpha(1,3)Gal epitope (anti-GAL) serum antibodies was measured. Transgenic pigs expressed high levels of CD46 in all tissues, especially vascular endothelium, with stable expression through three generations that was readily monitored by flow cytometry of transgenic peripheral blood mononuclear cells (PBMC). Transgenic PBMC pre-sensitized with antibody were highly resistant to human complement-mediated lysis which readily lysed normal pig PBMC. Normal pig kidneys transplanted without cold ischemia into non-immunosuppressed adult baboons survived a median of 3.5 h (n = 7) whereas transgenic grafts (n = 9), harvested at approximately 24-h intervals, were either macroscopically normal (at 29, 48 and 68 h) or showed limited macroscopic damage (median > 50 h). Microscopic assessment of transplanted transgenic kidneys showed only focal tubular infarcts with viable renal tissue elsewhere, no endothelial swelling or polymorph adherence and infiltration by lymphocytes beginning at 3 days. Coagulopathy was not a feature of the histology in four kidneys not rejected and assessed at 48 h or later after transplantation. Baboon anti-GAL serum antibody titers were high before transplantation and, in one extensively analyzed recipient, reduced approximately 8-fold within 5.5 h. The data demonstrate that a single CD46 transgene controls hyperacute kidney graft rejection in untreated baboons despite the presence of antibody and complement deposition. The expression levels, tissue distribution and in vitro functional tests indicate highly efficient CD46 function, controlling both classical and alternative pathway complement activation, which suggests it might be the complement regulator of choice to protect xenografts.

Acute Disease↗

PubMed1991

Evasion of Yersinia enterocolitica serotype 03 from complement-mediated killing.

Using isogenic strains of Y. enterocolitica serotype 03 (isolate 75) differing in LPS side chains (S and R), plasmid content (p+ and p-) or selective failure of YOP1 expression (YOP1-) we observed comparable C9 consumption via the alternative pathway by all strains. Differences became apparent in the bactericidal assays in which the 75S p+ strain was resistant whereas the plasmid-negative S and R strains were killed. Increased but submaximal resistance was observed with the 75R p+ and 75S YOP1- strains indicating that LPS side chains and plasmid-encoded factors other than YOP1 also contribute to serum resistance. Deposition of C9 and terminal complement complex (TCC) formation were greatly reduced on the resistant 75S p+ strain compared to the sensitive 75S p- strain. The 75S YOP1- variant behaved like the 75S p- strain with respect to C9 deposition and TCC formation suggesting that YOP1 prevents terminal C activation. A comparison of TCC deposited on resistant and sensitive Y. enterocolitica revealed no differences with respect to their salt- and protease-resistance and their size. We conclude from our experiments that serum resistance of 75S p+ depends on several surface components YOP1 being the most important. The YOP1 protein clearly interferes with C9 deposition and TCC formation and thereby contributes to serum resistance of plasmid-positive, YOP1 expressing Y. enterocolitica.

Centrifugation, Density Gradient↗

PubMed2002

Isolation and characterization of a novel rat factor H-related protein that is up-regulated in glomeruli under complement attack.

The factor H family in humans is composed of seven distinct proteins, including factor H-related proteins (FHR) 1-5. All members contain tandemly arranged short consensus repeats (SCR) typical of the regulators of complement activation gene family. FHR-5 is unusual for this group of proteins, as it was initially identified as a component of immune deposits in glomerular diseases. During our cloning of the cDNA for rat factor H from glomerular epithelial cells (GEC), we identified an alternative 2729-bp cDNA transcript. The translated sequence encoded a protein containing 11 SCRs, most similar to SCRs 7-15 and 19-20 in native rat factor H, which is the same basic structure of human FHR-5. As such, this rat protein was termed FHR. Recombinant rat FHR produced in a eukaryotic expression system had a molecular mass of 78 kDa. In functional studies, recombinant FHR bound C3b and inhibited the complement alternative pathway in a dose-dependent fashion. Given the prominent expression of FHR-5 in human membranous nephropathy, a disease in which complement activation occurs in the vicinity of GEC, the expression of FHR in a rat model of this disease was evaluated. In both in vitro and in vivo models of complement activation on the GEC, FHR mRNA was up-regulated by a factor of 3-6-fold compared with controls in which complement could not be activated. Thus, we have identified a novel factor H family member in rats. This FHR protein is analogous to human FHR-5, both in structure and in potential involvement in glomerular immune complex diseases.

Amino Acid Sequence↗

PubMed1985

Familial occurrence of complement dysfunction in Crohn's disease: correlation with intestinal symptoms and hypercatabolism of complement.

Complement was studied in Crohn's disease probands with early onset and in their first degree relatives. Controls included 24 healthy volunteers and 24 patients with ulcerative colitis or peptic ulcers. Subnormal generation of chemotactic activity by the alternative pathway was shown in eight of 21 probands and in six of 33 relatives, a frequency in both groups significantly different from controls (p less than 0.005), with a strong connection between findings in patients and relatives. As previously shown in patients with Crohn's disease, the subnormal generation was related to decreased utilisation of complement C3 in relatives. Raised levels of circulating complement C3c split products suggested complement involvement in Crohn's disease probands. In contrast, plasma C3c was normal in all relatives, and none of the six cases with complement dysfunction had gastrointestinal symptoms or a history of inflammatory bowel disease. Our data suggest, that complement abnormality seen in Crohn's disease patients does not simply reflect mucosal inflammation or hypercatabolism of complement.

Adolescent↗

PubMed1999

Sex-limited protein: in vitro and in vivo functions.

Mouse complement component C4 exists in two isoforms, C4 and a protein with expression restricted to male animals called sex-limited protein (Slp). Although Slp is about 95% homologous to C4, it is generally believed to be non-functional, at least in conventional haemolytic complement assays. In a previous study, however, we showed that Slp is haemolytically active in a C1-inhibitor (C1INH)-regulated, EDTA-resistant mouse complement activation pathway. To study other possible implications of this finding, we generated constitutively expressing Slp-transgenic mice. The transgene was crossed into otherwise Slp-deficient C57Bl/6J and NZB mice. Members of the third backcross generation of C57Bl/6J mice were tested for functional Slp and classical and alternative complement pathway activities (CH50 and AP50 levels, respectively). Slp-transgenic C57Bl/6J mice showed enhanced CH50, but normal AP50 levels when compared with non-transgenic littermates. To discover a possible protective role for Slp in spontaneous systemic lupus erythematosus (SLE) in NZBxNZW (NZBxW) mice, the third backcross generation of Slp-transgenic NZB mice was mated with NZW mice and the development of SLE in the female offspring was followed. In these introductory experiments, Slp-transgenic NZBxW animals presented with a significantly extended life span. Our results imply that Slp is a mouse complement component with functions which partially resemble some of those of human C4A.

Animals↗

PubMed1984

Experimental bullous pemphigoid in guinea pigs: the role of pemphigoid antibodies, complement, and migrating cells.

Dermal-epidermal separation (DES) could be produced in vivo, 6 h after the injection of sera taken from patients with bullous pemphigoid into the dorsal skin of Hartley guinea pigs. DES did not occur when antihuman IgG rabbit serum was injected prior to the injection of the sera taken from patients with bullous pemphigoid. When IgG fraction from the patient's sera was injected, DES was also observed. Histologic findings in the skin specimens in vivo with concentrated sera or IgG fraction have shown DES, some cell infiltrations of neutrophils, eosinophils, and some lymphocytes similar to the skin lesions of bullous pemphigoid patients. Various reagents, such as colchicine, cytochalasin B, EDTA, and steroid were injected prior to the IgG fraction injection. DES and the migration of polymorphonuclear (PMN) leukocytes were inhibited by the preinjection of these reagents. These observations suggest that the migration of PMN leukocytes was necessary for the formation of DES. When IgG fraction was injected into C3-inactivated guinea pigs with cobra venom factor, DES was inhibited, whereas DES was not completely inhibited when IgG fraction was injected into C4-deficient guinea pigs. These results suggested that an alternative pathway may be necessary for DES.

Animals↗

PubMed1992

Immunofluorescent localization of pig complement component 3, regardless of the presence or absence of detectable immunoglobulins, in hyperacutely rejected heart xenografts.

Rabbit heart xenografts transplanted into the neck of newborn pigs were all hyperacutely rejected within two hours regardless of the presence or absence of detectable endogenous immunoglobulins (Ig). Cryostat tissue sections were prepared from the rejected rabbit hearts and incubated with sheep polyclonal antibodies against pig complement component 3 (C3), pig IgG and pig IgM. Specific immunoreaction was visualized by fluorescein-conjugated antibodies to sheep IgG. C3 was localized mainly on the surfaces of vascular endothelial as well as myocardial cells, and the localization was not dependent upon the presence of pig immunoglobulins within the same tissue. Both pig IgG and IgM were detected only in the heart xenografts transplanted into suckled pigs, whereas no trace of immunoglobulin was found in those transplanted into circulating antibody-free presuckled pigs. Treatment with cobra venom factor (which inhibits complement activity) prior to transplantation prolonged xenograft survival and completely abolished C3 immunostaining. The results provide new evidence at the histochemical level that the alternative pathway of complement is involved in hyperacute xenograft rejection of the species combination (rabbit to pig) used in this study.

Animals↗