Conventionally, complement activation by biomedical polymers has been evaluated by determining the C3a concentration in the fluid phase only. According to this criterion, biomaterials such as hemodialysis membranes made from cellulosic or various synthetic polymers were classified as activators or nonactivators of complement. Since certain membranes bind large quantities of C3a from the fluid phase, classification based on fluid-phase C3a concentration has in some instances been inaccurate. As follows from the comparison of complement activation by cuprophane and polyacrylonitrile membranes, the capacity of a biomedical polymer to activate complement is not determined by the number of potential covalent binding sites on its surface. Biomaterial itself may lack hydroxyl and/or amino groups, and yet it may activate C3 in human serum very efficiently. Some of the biomaterials may also bind unactivated/unfragmented C3 whether in the absence or presence of other serum proteins. In addition, binding of factor B (a promotor of C3 activation) and binding of factor H (an inhibitor of C3 activation) to certain biomaterials have been found to be independent of complement activation and unaffected by the presence or absence of C3. Thus, it is becoming apparent that the requirements for the formation and stability of the C3 convertase on artificial surfaces differ from those on biological membranes, and that the relative magnitude of binding of factor B and factor H to the surface per se cannot be used as a reliable indicator of the capacity of the biomaterial to activate complement. Further studies are necessary to elucidate the molecular mechanisms of C3 and C5 activation on the surfaces of biomedical polymers.(ABSTRACT TRUNCATED AT 250 WORDS)
A population of 98 healthy Black Americans was studied in order to determine normal ranges for total C4, C4A and C4B. Mean total C4 in Blacks measured by an enzymelinked immunoassay (EIA) was 44 +/- 12.8 mg/dl which was significantly different (p less than 0.001) from Caucasian levels of 31.7 +/- 11.5 mg/dl. The difference in total C4 was due to increased levels of C4B in Blacks (means = 24.4 mg/dl) as compared to Whites (means = 15.7 mg/dl; p less than 0.001). These results remained significantly different even when 8 samples having the C4A 91 phenotype were excluded. Since EIAs using monoclonal antibodies with Ch 1 specificity may yield false results, C4 allotyping is recommended when quantitating C4A and C4B levels in Blacks.
Frequencies for HLA class I and II histoglobulins and C4A, C4B, BF complement proteins were performed for 59 sarcoidosis patients. The DR5 allele was present in 55.9% of patients as compared to 31.5% of controls. We noticed that its increase was more relevant in males and in those with a poor prognosis. BF F allele was significantly over-represented in patients (29.09% vs. 19.15% of control), especially in women. Special emphasis was given to BF F subtyping, to define an association between a particular BF F subtype and patient's sex or disease outcome.
Using apparatus available in any laboratory we developed a semiautomated kinetic technique for complement activity assay. Hemolysis of sensitized red blood cells is performed in the thermostated microflow cell of a spectrophotometer connected to a computer. The computer controls, displays on the screen, and analyzes all the different phases of the assay. After definition of optimal operating conditions, we compared the results obtained by this technique and by Kabat and Mayer's. On 221 patients' sera the regression coefficient was 0.94. The values for samples deficient in one fraction or after in vitro activation were very similar. The coefficient of variation was close to 1% for within series studies and better than 3% between series. This technique is very easy to perform even in a routine nonspecialized laboratory and up to 30-40 sample/h can be tested.
The cleavage of purified, functionally active rabbit C3 by cobra venom factor and trypsin was analysed by reducing and non-reducing sodium dodecyl sulphate electrophoresis and autoradiography. The specific aim of the study was to compare these reactions to those that occur with human C3. Analysis showed that the pattern of breakdown was very similar to that for the human protein: while the beta-chain remained intact, there was step-wise degradation of the alpha-chain to form C3a, C3b, iC3b and C3c, all of which could be identified by gel analysis. The metabolic behaviour of three of these cleavage products, C3a, C3b and iC3b, was then examined in vivo using dual isotope techniques. Rabbits were studied simultaneously with I-131-C3 and I-125-labelled C3 breakdown products. Analysis of plasma and urine radioactivity for the subsequent 72 h showed that all three breakdown proteins had rapid rates of catabolism in vivo compared to the native molecule. Specifically, 93 and 98% of C3b and iC3b, respectively, were eliminated from the plasma compartment within 10 h of injection. C3a was completely eliminated within 10 h. By comparison, native C3 showed a half-life of 29 +/- 3 h (mean +/- SD) and a fractional catabolic rate of 4.30 +/- 0.75%/h. The data support the use of this species in studies of complement behaviour in models of human immune disease and further clarify the basis for changes in plasma C3 concentration that accompany active immune complex- and antibody-mediated activity, in vivo.
A genetic influence of the major histocompatibility complex (MHC) on the susceptibility and the development of the different clinical forms of paracoccidioidomycosis (PCM) has been postulated. In the present investigation allotypes of MHC-coded class III gene products (complement components C2, BF, C4A, and B) were determined in 69 Brazilian PCM patients and 225 healthy control individuals matched for ethnic and geographic origin. The frequency of the non-expressed C4B allele (C4B*Q0) was significantly elevated in comparison to the controls (p less than 0.01; Fisher's exact test). Three out of 69 patients had a complete C4B deficiency as against 2 among 223 control individuals. The C4A*Q0 allele was also more frequent in the patients. Other C4 alleles were not seen to differ between the two groups. The analysis of BF allotypes showed a non-significant predominance of the rarer allele BF*S07 in the patients, whereas no difference in the distribution of C2 alleles was seen. The data on MHC class III association may support the hypothesis of immune response modulation in PCM and suggest a functional genetic role of complement action against the fungus and in the outcome of PCM infection. We conclude that MHC class III products, especially C4B*Q0, are associated with chronic uni- or multifocal PCM and may influence the course of the infection.
64 members of a large kindred with inherited deficiency of the seventh component of complement, C7, were studied for plasma levels of antigenetic and functional components of complement as well as for clinical manifestations of infections and autoimmune diseases. Thirty-six individuals showed a low level of C2, C7, C8, and/or C9, including null alleles for C4A and C4B. Two subjects had a complete C7 deficiency. One of them concomitantly presented a low C2 level and a C4BQ0 allele. HLA allotyping strongly suggested C2 depression associated with a C4BQ0 allele. The 2 individuals with total absence of C7 suffered from fulminant disseminated meningococcal infections. The partial depression of one or more complement components associated with apparent good health. These results may indicate that simultaneous partial depressions of up to four complement components do not lead to clinical manifestation of infectious and autoimmune disease.
Conventionally, complement activation by biomedical polymers has been evaluated by determining the C3a concentration in the fluid phase only. According to this criterion, biomaterials such as hemodialysis membranes made from cellulosic or various synthetic polymers were classified as activators or nonactivators of complement. Since certain membranes bind large quantities of C3a from the fluid phase, classification based on fluid-phase C3a concentration has in some instances been inaccurate. As follows from the comparison of complement activation by cuprophane and polyacrylonitrile membranes, the capacity of a biomedical polymer to activate complement is not determined by the number of potential covalent binding sites on its surface. Biomaterial itself may lack hydroxyl and/or amino groups, and yet it may activate C3 in human serum very efficiently. Some of the biomaterials may also bind unactivated/unfragmented C3 whether in the absence or presence of other serum proteins. In addition, binding of factor B (a promotor of C3 activation) and binding of factor H (an inhibitor of C3 activation) to certain biomaterials have been found to be independent of complement activation and unaffected by the presence or absence of C3. Thus, it is becoming apparent that the requirements for the formation and stability of the C3 convertase on artificial surfaces differ from those on biological membranes, and that the relative magnitude of binding of factor B and factor H to the surface per se cannot be used as a reliable indicator of the capacity of the biomaterial to activate complement. Further studies are necessary to elucidate the molecular mechanisms of C3 and C5 activation on the surfaces of biomedical polymers. Its understanding should lead to the development of new or modification of existing artificial membranes which would cause less adverse effects for patients during extracorporeal procedures.
Thirty narcoleptic patients (29 out of 30 of Mediterranean origin) were studied for HLA polymorphisms (only 24 were investigated for HLA class III). We found that these patients are characterized by DR2, DQW2 phenotype (p less than 0.0001) as those of Anglo-Saxon origin so far studied and by the complotype: BfS, C4A3, C4B1. Just 1 of 7 BfF patients has the Fb subtype detected by isoelectric focusing technique (p = 0.007). The only patient of Black origin (his mother is Eritrean) is completely different from other patients (DR3,5; DQW2,W3; C4A4,4; C4B1,2), supporting the hypothesis that it is not the DR2, DQW1 phenotype per se involved in this syndrome. Subdividing the subjects according to different clinical features, we cannot demonstrate genetic heterogeneity.
While preformed BSA-anti-BSA immune complexes (PIC) bind efficiently to human RBC after their interaction with human complement, nascent BSA-anti-BSA immune complexes (NIC) formed in the presence of complement do not bind to autologous RBC. The same results were obtained with tetanus toxoid-anti-tetanus toxoid PIC and NIC. In order to elucidate the causes of this marked difference between the RBC-binding properties of PIC and NIC, the profile of complement activation induced by them was compared using haemolytic assays and sensitive ELISA tests. BSA-anti-BSA NIC activated C1 more efficiently than PIC. This was reflected in a higher C4 content of the isolated NIC and higher C1 INH-C1s and lower C1q-fibronectin complex level in the NIC-treated serum as compared to the PIC-treated ones. Although isolated NIC contained more C3 than isolated PIC did, there was no significant difference in the AP activation. These findings suggest that the failure of NIC to bind to RBC is not due to a lack of C4-binding, or C3-binding and/or activation, but rather to the special structure of this type of complex.
We studied the activation and C1 inactivator-dependent dissociation of the first component of complement, the C1q(C1r-C1s)2 complex, in relation to recruitment of the classical activation pathway in the circulation of 24 patients with systemic lupus erythematosus (SLE). The patients were divided into three groups on a clinical basis, and were investigated during flares of disease activity. Group I had mild symptoms, group II major extrarenal manifestations, and group III manifest renal disease. High serum concentrations of trimer complexes containing C1 inactivator, activated C1r and zymogen C1s(C1 IA-C1r-C1s) were found in the majority of the patients. Some patients with high C1 IA-C1r-C1s concentrations showed no evidence of classical pathway activation, indicating that C1 activation was controlled by the action of C1 IA at the C1r level. By contrast, formation in serum of tetramer complexes in which C1 IA was firmly bound to both C1r and C1s (C1 IA-C1r-C1s-C1 IA) was associated with C2 and C3 cleavage in EDTA plasma, and with manifest hypocomplementemia. Low C1 IA-C1r-C1s-C1 IA values were observed in conjunction with substantial C2 cleavage in a few patients. Thus, C1 IA-C1r-C1s-C1 IA may not always be a sensitive indicator of classical pathway activation. Efficient recruitment of the classical pathway was related to disease severity, with some overlap between the clinical groups. In conclusion, C1 dissociation with formation of C1 IA-containing complexes was consistently found in patients with active SLE. The results suggested that C1 IA-dependent control of C1 activation was of biological significance in the disease.
Comparison of amino acid sequences of the alpha-chain fragment of human C4, C4d, has shown C4A- and C4B-specific sequences at residues 1101-1106 in which the aspartic acid-histidine substitution at position 1106 may be related to the amide and ester bond forming properties of these molecules. Peptides containing twelve amino acid residues of the C4A- or C4B-specific sequences were synthesized and injected into female Balb/c mice. Serum from 2 mice, one immunized with the C4A-specific peptide and the other with the C4B-specific peptide, gave strong isotype-specific responses in an enzyme-linked immunosorbent assay against affinity-purified C4A3 and C4B2B1. Spleen cells from these mice were fused with the mouse myeloma SP2/0-Ag 14, and two cloned cell lines, AII-1 and BII-1, were established from hybrids. Enzyme-linked immunosorbent assay and western blotting of monoclonal antibodies AII-1 and BII-1 show that the former reacts with the C4A but not with the C4B alpha-chain and the latter with C4B but not with the C4A alpha-chain. Furthermore, immunoblotting of C4 allelic variants showed that AII-1 reacted with all C4A allotypes tested, including A6, A4, A3 and A2, whereas BII-1 reacted with all C4B allotypes tested, including B5, B3, B2, and B1.
Human blood platelets are not aggregated by C5a-desArg. They are brought to aggregation, however, when human polymorphonuclear leukocytes (PMN) are present in the platelet suspension. Then, mixed aggregates form upon activation with C5a-desArg. The platelets do not adhere only to PMN but also stick to each other, indicating that they are activated. This is also evident from their morphology which shows pseudopod formation. The formation of mixed aggregates requires the presence of Ca2+ and Mg2+, it does not occur at temperatures below 20 degrees C. The results suggest that the platelets are activated indirectly, by a mediator released from the PMN upon stimulation with C5a-desArg. When mixed with platelets, PMN partially aggregate already upon addition of Ca2+. This effect is not seen in pure PMN suspensions. C5a-desArg causes additional aggregation which includes the platelets. The indirectly stimulated platelets in turn enhance the aggregation of the PMN in the mixtures incubated with C5a-desArg. This may cause a positive feedback.
