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Circulating immune complexes, complement and complement component levels in childhood Hodgkin's disease.

Serum levels of circulating immune complexes (CIC) assayed by the Raji cell radioimmunoassay, total haemolytic complement (TCH50), Clq and C3 were correlated with clinical stage, histological type, age, sex and treatment of eighty-six children with Hodgkin's disease over a period of 4 years. Most significant findings were the changes of levels of CIC, TCH50, Clq and C3 during disease activity and following treatment. Significant perturbations were also seen in association with relapse. Levels of C and CIC were significantly elevated (P less than 0.001) at the time of diagnosis prior to splenectomy and/or any treatment. In the group before treatment, 81 percent of CIC levels were above 16 micrograms/ml with a maximum value of 1120 micrograms/ml. During treatment 33 percent were still above normal with a maximum of 320 micrograms/ml. Within 1 year after cessation of treatment, 37 percent also remained above normal levels with a maximum of 240 micrograms/ml. At relapse prior to treatment, 63 percent were again elevated with a maximum of 1280 micrograms/ml. The most significant difference on TCH50 levels relates to treatment periods. Sera of patients with active disease who are previously untreated show elevation of TCH50 levels (P less than 0.001) (average 127 CH50 mu/ml. During and after treatment eht TCH50 levels drop to 96 and 102 CH50 mu/ml, as compared to normal control of 100 CH50 mu/ml. In sera of patients at the first, second or third relapse, the combined TCH50 levels are significantly different from controls and across treatment periods (P less than 0.005).

Adolescent↗

Complement mediated solubilization: role of the complement in the clearance of circulating immune-complexes.

In 1975, Miller and Nussenzweig described a new function of the Complement (C'): the Capacity of this substance to solubilize "in vitro" antigen/antibody complexes previously formed. Subsequent studies of these authors and other researches (particularly Takahashi and his group) demonstrated that this process is mediated by activation of the alternative way of C', although it is faster when the classic way is involved. Basically, thanks to the Takahashi and group studies, it has been possible to know in detail the intimate mechanism of this phenomenon, usually known demonstrated that the key of all this process lies in the generation, through the C' alternative way, of numerous molecules of C3b that deposit over the antigen/antibody complexes (Ag/Ac), causing the breakage of the necessary mesh for such complexes to precipitate, which leads to formation of small immunocomplexes (IC) very rich in C3b which turn soluble again. ICs solubilized in this way suffer important physicochemical and biological changes, and transform into substances without inflammatory power that are not able to activate C' nor to interact with C3b receptors present in a great number of blood cells. However, they are captured by Kupffer cells in the hepatic sinusoids and quickly catabolized. CMS is closely related with another similar phenomenon, the Immuno-Precipitation inhibition (IPI), described by Schiferli and his group in the early 80's.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Role of the P2 residue of complement 1 inhibitor (Ala443) in determination of target protease specificity: inhibition of complement and contact system proteases.

A dysfunctional C1 inhibitor (C1 INH) from a family in whom the propositus presented with systemic lupus erythematosus but without angioedema previously was shown to have diminished inhibitory activity toward isolated C1r and C1s, and intact C1. The mutation was identified as replacement of Ala443 (P2) with Val. This study further analyzed the reactivity of this mutant and characterized two mutants with Ser or Asp at this position. Ser at P2 does not interfere with binding of target proteases. However, the mutant with Asp at this position is unable to bind C1r and beta factor XIIa, and also has a decreased rate of reaction with C1s and kallikrein. Therefore, alteration of polarity alone had no effect on binding, while a bulky and/or charged side chain was not tolerated. Although defective in inhibition of C1r and C1s, the P2 A-->V mutant had acquired the ability to complex with trypsin. It also completely retained the ability to complex with kallikrein and factor XIIa. None of the 10 individuals expressing this mutant protein has ever had angioedema. This observation, combined with normal inhibition of contact system proteases and defective inhibition of complement proteases, suggests that angioedema is caused by bradykinin generated from contact system activation.

Alanine↗

Presence of a serine protease in the complement-activating component of the complement-dependent bactericidal factor, RaRF, in mouse serum.

A 100-kDa protein was isolated from the complement-activating component of mouse Ra-reactive factor. It generated a 29-kDa chain upon reduction. Partial amino acid sequences of the 29-kDa chain were determined after fragmentation with CNBr. A 386-bp-long probe was synthesized by the polymerase chain reaction with oligonucleotide primers designed by reference to the amino acid sequence. Using this probe, we cloned a 3.7-kb DNA from a mouse liver cDNA library. The amino acid sequence of the 29-kDa chain deduced from the nucleotide sequence of this cDNA was consistent with the amino acid sequences determined. The full sequence was found to have characteristics of a serine protease and exhibited 29% and 30% homologies with those of light chains of the C1r and C1s subcomponents of complement, respectively.

Amino Acid Sequence↗

A simple rosette assay for demonstration of complement receptor sites using complement-coated zymosan beads.

In the present article we describe a simple rosette assay for detection of C3 receptor-bearing B lymphocytes with complement (C)-coated zymosan (Zy) beads. Zy coated with murine or human C bound to a distinct population of human and mouse lymphocytes as well as to the majority of lymphoblastoid cells of several human established cell lines. Rosette formation was also observed with human red blood cells, with human monocytes and neutrophils. Experiments with anti-immunoglobulin sera, with other B and T cell markers and with mouse thymocytes proved that the capacity to bind C-coated Zy is primarily a feature of B lymphocytes. The following findings suggested that C-coated Zy is bound via receptor sites for the activated components of C3: (a) Zy coated with C3-deficient serum failed to bind, (b) comparable percentages of various lymphoid and nonlymphoid cells formed rosettes with C-coated Zy as well as with antibody and C-coated sheep red blood cells, and (c) antibodies against human or murine C3 inhibited the binding of C-coated Zy.

B-Lymphocytes↗

Studies on activation and levels of haemolytic complement of buffalo (Bubalus bubalis). 1. Classical complement pathway.

Optimum conditions for haemolytic complement (HC) assay in buffalo serum were standardized. In all, 11 indicator systems of red blood cells (RBC) and haemolysins were investigated. Maximum HC CH50 titre was obtained with rabbit RBC sensitized with goat haemolysin. The effect of pH, Ca2+ and Mg2+ concentration, ionic strength, time and temperature were studied. Of all the variables, ionic strength influenced the HC activity most significantly. The standard system for titrating the HC consisted of rabbit RBC sensitized with goat haemolysin, sucrose-veronal buffer with pH 7.5, ionic strength 0.023 M and Ca2+ and Mg2+ concentrations 6 x 10(-4) and 2 x 10(-3) M, respectively. Incubation at 37 degrees C for 2 h gave highest haemolytic activity. With this protocol 5-7-fold higher HC activity was recorded than with prestandardized conditions. Levels of HC were determined in the sera of 98 buffaloes aged from 1 month to 12 years. The lowest mean CH50 units of 401 +/- 0.35 per ml were recorded in buffalo calves below 3 months of age. The mean HC levels increased with age, reaching peak values of 2349 +/- 62.25 CH50 units/ml in 2-3-year-old buffalo. Animals in the age group 5-12 years had significantly decreased (P less than 0.05) mean HC levels of 1545 +/- 68.94.

Animals↗

Studies on the haemolytic complement of the dromedary camel (Camelus dromedarius). II. Alternate complement pathway haemolytic activity in serum.

Fresh camel serum caused lysis of unsensitised red blood cells (RBC) of chicken, rabbit and guinea pig. Homologous RBC were resistant to lysis. There was only minimal lysis of goat, sheep, rat and cattle RBC. Lysis of heterologous RBC was attributed to the presence of alternate complement activity (ACP) in the serum as adsorption with respective RBC and addition of 10 mM ethylene glycol-bistetraacetate (EGTA) in the SVBS diluent did not abrogate the haemolytic activity. Guinea pig RBC were the most sensitive to lysis, giving a mean ACP activity of 41.5 +/- 1.8 CH50 units ml-1. Clotting, followed by storing of blood between 0 and 37 degrees C for 1 h did not significantly affect ACP activity. However, considerable activity was lost when blood was clotted and stored at 44 degrees C for 1 h, or when serum was kept at 4 degrees C for 24 h. Treatment with zymosan, or incubation at 56 degrees C for 30 min inhibited ACP activity. Maximum ACP activity occurred in the presence of 8 mM Mg2+ in the SVBS-EGTA diluent, at pH 7.3 and incubation time of 2 h at 37 degrees C. Levels of ACP activity were determined in 79 healthy camels of different age groups, ranging from 3 months to 15 years. Calves between 3 months and 1 year of age had higher ACP activity than camels in the age group of 5 years and above. Highest mean ACP activity of 89 +/- 7.9 CH50 units ml-1 were recorded in 1-5 year old camels (P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

Local neutrophil influx following lateral fluid-percussion brain injury in rats is associated with accumulation of complement activation fragments of the third component (C3) of the complement system.

Traumatic brain injury can lead to locally destructive secondary events mediated by several inflammatory components. Following lateral fluid-percussion (FP) brain injury in rats, we examined cortical and hippocampal sections for neutrophil infiltration and accumulation of complement component C3. Neutrophil influx into the brain after injury was detected by an improved myeloperoxidase (MPO) microassay and manual cell counting, while C3 accumulation was detected using immunocytochemistry. MPO levels were elevated in the injured cortical tissue, whereas C3 immunoreactivity was increased in both injured cortical and ipsilateral hippocampal sections. These results show that the FP model of head injury leads to an intense local inflammatory reaction and subsequent tissue destruction.

Animals↗

Inhibition of the formation of the complement membrane-attack complex by a monoclonal antibody to the complement component C8 alpha subunit.

The effect of nine monoclonal antibodies to complement component C8 on the interaction of C9 with preformed cell-surface C5b-8 complexes and on the functional insertion of C8 into the membrane-attack complex (MAC) was investigated. None of the antibodies prevented C9 insertion into a preformed C5b-8 complex. One antibody (F1) directed to the C8 alpha subunit clearly inhibited formation of a functional MAC. It is proposed that this antibody prevents the C8 alpha subunit unfolding and distorting the bilayer to allow C9 insertion.

Animals↗

Identity of the segment of human complement C8 recognized by complement regulatory protein CD59.

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The inhibitory function of CD59 derives from its capacity to interact with both the C8 and C9 components of MAC, preventing assembly of membrane-inserted C9 polymer. MAC-inhibitory activity of CD59 is species-selective and is most effective when both C8 and C9 derive from human or other primate plasma. Rabbit C8 and C9, which can substitute for human C8 and C9 in MAC, mediate virtually unrestricted lysis of human cells expressing CD59. In order to identify the segment of human C8 that is recognized by CD59, recombinant peptides containing human or rabbit C8 sequence were expressed in Escherichia coli and purified. CD59 was found to specifically bind to a peptide corresponding to residues 334-385 of the human C8 alpha-subunit, and to require a disulfide bond between Cys345 and Cys369. No specific binding was observed to the corresponding sequence from rabbit C8 alpha (residues 334-386). To obtain functional evidence that this segment of human C8 alpha is selectively recognized by CD59, recombinant C8 proteins were prepared by co-transfecting COS-7 cells with human/rabbit chimeras of the C8 alpha cDNA, and cDNAs encoding the C8 beta and C8 gamma chains. Hemolytic activity of MAC formed with chimeric C8 was analyzed using target cells reconstituted with CD59. These experiments confirmed that CD59 recognizes a conformationally sensitive epitope that is within a segment of human C8 alpha internal to residues 320-415. Our data also suggest that optimal interaction of CD59 with this segment of human C8 alpha is influenced by N-terminal flanking sequence in C8 alpha and by human C8 beta, but is unaffected by C8 gamma.

Amino Acid Sequence↗

The embryotrophic activity of oviductal cell-derived complement C3b and iC3b, a novel function of complement protein in reproduction.

The oviduct-derived embryotrophic factor, ETF-3, enhances the development of trophectoderm and the hatching process of treated embryos. Monoclonal anti-ETF-3 antibody that abolishes the embryotrophic activity of ETF-3 recognized a 115-kDa protein from the conditioned medium of immortalized human oviductal cells. Mass spectrometry analysis showed that the protein was complement C3. Western blot analysis using an antibody against C3 confirmed the cross-reactivities between anti-C3 antibody with ETF-3 and anti-ETF-3 antibody with C3 and its derivatives, C3b and iC3b. Both derivatives, but not C3, were embryotrophic. iC3b was most efficient in enhancing the development of blastocysts with larger size and higher hatching rate, consistent with the previous reported embryotrophic activity of ETF-3. Embryos treated with iC3b contained iC3b immunoreactivity. The oviductal epithelium produced C3 as evidenced by the presence of C3 immunoreactivity and mRNA in the human oviduct and cultured oviductal cells. Cyclical changes in the expression of C3 immunoreactivity and mRNA were also found in the mouse oviduct with the highest expression at the estrus stage. Molecules involving in the conversion of C3b to iC3b and binding of iC3b were present in the human oviduct (factor I) and mouse preimplantation embryo (Crry and CR3), respectively. In conclusion, the present data showed that the oviduct produced C3/C3b, which was converted to iC3b to stimulate embryo development.

Animals↗

Effect of continuous complement inhibition using soluble complement receptor type 1 on survival of pig-to-primate cardiac xenografts.

A single bolus of soluble complement (C) receptor type 1 (sCR1, TP-10) has been shown to delay hyperacute rejection (HAR) of porcine cardiac xenografts (Xgs) by primate recipients. In these recipients, C activity slowly returned and C deposition was noted in the Xgs at rejection. To evaluate the effect of sustained C inhibition using sCR1 on HAR, two additional cynomolgus monkeys received porcine cardiac Xgs and a continuous infusion of sCR1. In the first recipient, Xgs survival was 5 days (120+ hr), whereas in the second, Xg survival was 7 days (168+ hr). Serial biopsies of the Xgs were remarkable for an increasing cellular infiltrate composed predominantly of neutrophils and macrophages, and the development of edema, hemorrhage, and myocyte necrosis. These findings suggest that once C-mediated HAR has been inhibited, infiltration of the Xg by these cells may lead to accelerated acute rejection, which is an additional barrier to successful longer term Xg survival.

Animals↗

Antigenic changes of complement components: relevance for the analysis of complement activation.

The present paper briefly reviews some general principles in the field of activation-dependent epitopes in the complement cascade. The advantage of monoclonal antibodies in the study of these epitopes is pointed out and available antibodies are listed. Application of such antibodies in reliable and valid assays to detect activation at all levels of the cascade is addressed. The importance of these assay in the study of various pathophysiological and clinical conditions is briefly discussed.

Antibodies, Monoclonal↗

Purification and partial characterization of the third component of the complement system from porcine serum (C3) and of a crystallizable degradation product of the fourth component of the complement system from human serum (C4). Study of the tryptic digestion products of human and porcine C3 and of human C4.

The third component of the porcine complement system (C3) was isolated. The protein with 190000 molecular weight is composed of two chains alpha and beta of Mr 116000 and 74000. The amino-acid composition is very similar to that of human C3. A degradation product of human C4 was also isolated and characterized. This protein was crystallized. The degradation product is characterized by a molecular weight of 128000; the major part of the alpha chain is lost. Native human and porcine C3 and human C4 were submitted to tryptic digestion. The digestion products were isolated and characterized in terms of molecular weight, subunit organization and amino-acid composition. The present study confirms the high homology among these proteins.

Amino Acids↗

Possible mechanism for in vitro complement activation in blood and plasma samples: futhan/EDTA controls in vitro complement activation.

BACKGROUND: Ongoing in vitro complement (C) activation in citrate or EDTA plasma has prevented an accurate analysis of C-activation products generated in vivo. The aim of this study was to characterize handling and storage conditions required to prevent in vitro C activation in blood and plasma samples collected with Futhan/EDTA. METHODS: Biotrak(TM) RIAs were used to quantitatively measure C3a and C4a in blood and/or plasma samples from healthy individuals (controls) and from liver transplant patients. Blood samples were routinely drawn into either EDTA (1 g/L) tubes or into tubes containing both EDTA (1 g/L) and Futhan (0.1 g/L) and immediately centrifuged at 2000g for 15 min at 4 degrees C. RESULTS: In controls, C4a, but not C3a, in fresh samples (time 0) was higher in EDTA plasma than in Futhan/EDTA plasma (n = 20; P = 0.002). Futhan/EDTA prevented C3a and C4a generation in blood and plasma samples held at room temperature (22-23 degrees C) for 1 h and in plasma held for 24 h at 4 degrees C or -70 degrees C. The mean C3a concentration (1.76 mg/L; n = 19) at time 0 in EDTA plasma samples from liver transplant patients was significantly higher than for controls (0.34 mg/L; n = 11). In these patients, the mean C3a in EDTA samples increased to 13.8 mg/L after 60 min at room temperature, but there was no change in the C3a concentration of an EDTA plasma from a control. In the patients, C3a concentrations were lower in Futhan/EDTA plasma than in EDTA at time 0 and after 60 min at room temperature (1.40 and 2.02 mg/L, respectively). The mean patient C4a was 4.02 mg/L in EDTA plasma at time 0 vs 0.24 mg/L for controls; it increased to 16.9 mg/L after 60 min at room temperature compared with 0.76 mg/L for controls. The mean patient C4a was 0.83 mg/L in Futhan/EDTA plasma at time 0 vs 0.1 mg/L for controls. Neither patient nor control C4a concentrations increased vs time in Futhan/EDTA. CONCLUSION: The combination of Futhan (0.1 g/L) and EDTA (1 g/L) eliminates in vitro C activation.

Adult↗

Reiter protein complement fixation test; a preliminary comparison of the TPI test with the complement fixation test employing a soluble protein antigen derived from the Reiter strain.

A comparative study of the specificity of the Reiter protein complement fixation (RPCF) test and the Treponema pallidum immobilization (TPI) test on 180 sera showed that the results in 178 instances or 98.9 per cent were in agreement. The sensitivity of the RPCF test, when compared to the TPI test, on 189 sera from patients known to have syphilis was in agreement in 182 or 96.3 per cent, assuming a correlation exists between positive TPI and both the "reactive" or "weakly reactive" RPCF test results. As to reproducibility of results, the RPCF test results agreed in 84 of the 87 sera tested (95.4 per cent). The sera were tested at least three times on different days. Anticomplementary reactions were observed in three of 91 normal sera.

Complement Fixation Tests↗

Mutagenesis of the Arg-Gly-Asp triplet in human complement component C3 does not abolish binding of iC3b to the leukocyte integrin complement receptor type III (CR3, CD11b/CD18).

The leukocyte integrin complement receptor type III (CR3, CD11b/CD18) binds the C3 cleavage product iC3b. Many other integrins bind their ligands via an Arg-Gly-Asp (RGD) triplet. Both the RGD-containing C3 peptide 1390TRYRGDQDATMS1401 (pro-C3 numbering) and the RGD-like fibrinogen peptide GGAKQAGDV, which binds to the platelet integrin glycoprotein IIb-IIIa, were shown to inhibit the iC3b-CR3 interaction, suggesting that this binding is also RGD-mediated (Wright, S.D., Weitz, J.I., Huang, A. J., Levin, S.M., Silverstein, S.C., and Loike, J.D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7734-7738). However, unlike other integrin-ligand interactions, that of CR3 and iC3b is unaffected by the hexapeptide GRGDSP, and substitutions in the RGD triplet of C3 from other species appear to be tolerated. It was, therefore, proposed (Grossberger, D., Marcuz, A., du Pasquier, L., and Lambris, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1323-1327) that the highly conserved DATMS portion of the inhibitory C3 peptide may have been responsible for its binding. To address these inconsistencies and directly assess the role of the 1390-1401 segment within the complete iC3b molecule in mediating binding to CR3, a human C3 cDNA was altered by site-directed mutagenesis and the expressed recombinant proteins were examined in a CR3-specific assay. Replacement of RGD by AAA did not abolish rosetting of the corresponding iC3b-coated erythrocytes to human CR3-bearing leukocytes. In addition, mutant iC3b molecules in which the positively charged R1391 (corresponding to K in the fibrinogen peptide) and the highly conserved 1397DATMS sequence were replaced by Q and NAAMA respectively, were still bound by CR3. We conclude that the iC3b-CR3 interaction is not mediated by the RGD triplet or its neighboring residues.

Amino Acid Sequence↗