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PubMed1984

Cleavage of C2 in pathological serum and plasma studied by crossed immunoelectrophoresis.

A cleavage product of C2, C2a was demonstrated by crossed immunoelectrophoresis in serum and EDTA plasma from patients with active systemic lupus erythematosus (SLE), patients in the early phase of acute poststreptococcal glomerulonephritis (AGN), and from two males with congenital deficiency of C1(-) inactivator, one of whom had symptoms of hereditary angioedema. C2a was not found in normal serum and plasma. C2 was more stable in plasma than in serum with regard to the effects of storage in room temperature and of repeated freezing and thawing. C2a concentrations were higher in serum than in plasma samples from SLE and AGN patients. Expressed in per cent of the total C2 protein, C2a was inversely correlated (r = -0.91) with the C2 hemolytic activity in the samples, which explains discrepancies between the results of immunochemical and functional assays, when used to measure C2 concentrations in disease.

Adult↗
PubMed2005

Is immunity in diabetic patients influencing the susceptibility to infections? Immunoglobulins, complement and phagocytic function in children and adolescents with type 1 diabetes mellitus.

OBJECTIVE: Previous reports suggest an increased susceptibility of diabetes patients to infections, but little information is available on possible underlying immunologic dysfunctions. The aim of this study was to evaluate humoral factors in pediatric patients with type 1 diabetes mellitus. METHODS: There were 66 diabetic patients (39 males:27 females; 5-17 yr) classified into two groups according to levels of glycohemoglobin (limit 9%): Group C - controlled (n = 33) and Group UC - uncontrolled (n = 33). We evaluated five patients in C and six in UC who reported previous infections. Immunologic analysis included measurement of plasma concentrations of immunoglobulins (Ig), C3, and C4 levels (turbidimetry); functional hemolytic assays for complement evaluation (CPH for classical and APH for alternative pathways), quantification of C4 isotypes C4A and C4B (ELISA), phagocytosis assays, measurement of bactericidal activity against Staphylococcus aureus, as well as tests of fungicidal capacity for Candida albicans. RESULTS: The UC Group had higher mean age, received higher insulin doses, and had higher concentrations of glycohemoglobin than the C Group. No significant differences in duration of the disease or nutritional conditions were detected between the groups. Lower IgA values in C (10/33) and lower IgG levels in UC (23/33) were detected, and there were inverse relationship with HbA1c values. Analysis of CPH, APH, C3, and C4 showed normal levels in both groups and no statistical correlation with the HbA1c. However, 9/33 children of the UC Group had decreased C3 values. C4B levels were below the normal range in 8/20 and correlated with higher HbA1c. Both phagocytic assays for S. aureus and Candida albicans were within normal limits. CONCLUSIONS: Low IgG concentrations and to some degree reduction in C4B levels were related to impaired metabolic control. No strong link between the immunological alterations was found in diabetic patients and the occurrence of infections.

Adolescent↗
PubMed1980

Metabolism of C4 and factor B in rheumatoid arthritis. Relation to rheumatoid factor.

Metabolic turnover determined by radioiodide labeled C4 and Factor B was studied in 18 patients with rheumatoid arthritis (RA) and 19 normal control subjects as a means of estimating the relative ratio of consumption of components in the classical and alternative pathways of complement activation. Predominance of fractional catabolic rate (FCR) of C4 over Factor B was demonstrated with differentially labeled C4 and Factor B. The hypercatabolism occurred in the extravascular space. C4 FCR correlated significantly with rheumatoid factor (RF) determined in a hemolytic assay (rs = 0.72), measured as IgG RF (rs = 0.57), and as IgM RF (rs = 0.45). There were no significant correlations with several other antibodies measured. These results are consistent with the hypothesis that RA is a systemic, extravascular immune complex disease, in which RF immune complexes play a significant pathogenetic role principally via activation of the classical pathway of complement.

Adult↗
PubMed1991

Gene responsible for deficient activity of the beta subunit of C8, the eighth component of complement, is located on mouse chromosome 4.

Sera from 73 strains of mice were tested for hemolytic activity through the classical and the alternative pathways (CP and AP) in a single radial hemolysis assay. Sera from 16 out of 45 laboratory inbred strains had no lytic activity, and Ouchterlony analysis with anti-C5 serum showed them to be C5-deficient. Sera from 2 out of 28 strains derived from wild mice also had no lytic activity, but the C5 molecule was detectable in both. The hemolytic activity of sera from these strains can be restored by the addition of partially purified human C8 or human serum deficient in C8 alpha-gamma, leading us to conclude that strains M.MOL-MSM (MSM) and Mae are deficient in the beta subunit of C8. Typing of (DBA/2J X MSM)F1 hybrids and of progeny of a backcross to MSM showed that this C8 deficiency is controlled by a single recessive gene, designated C8b; the allele with hemolytic activity is C8b1; and the allele with no activity C8b0. Because of synteny homologies in mouse and human, we looked for and found close linkage between C8b and Pgm-2. Typing of recombinant mice for Mup-1 mapped the C8b locus 2.3 centimorgans (cM) telomeric to Pgm-2 on mouse chromosome 4.

Animals↗
PubMed1983

A hemolytic assay for clinical investigation of human C2.

The heat lability of early acting components of human complement was studied in detail. Kinetic disappearance of individual components was monitored by hemolytic assay. C2 and factor B were the most heat labile components. We took advantage of the difference in heat stability between C2 and C1 to develop a hemolytic assay for human C2.

Animals↗
PubMed1981

Reactivity of human C-reactive protein with positively charged liposomes.

Interaction of CRP with liposomes was studied by agglutination, complement consumption, and the binding assay. Liposomes composed of phosphatidylcholine and stearylamine were agglutinated with CRP. The interaction of CRP and liposomes was further demonstrated by the consumption of hemolytic complement activity. Stearylamine could be substituted by cetyltrimethylammonium bromide, indicating that positively charged molecules were required for the interaction. Agglutination of liposomes caused by CRP was dependent on the fatty acid composition of phosphatidylcholine, cholesterol content and temperature. Differential scanning calorimetry measurement revealed that both the agglutination of liposomes and the consumption of complement caused by CRP required the fluid state of membranes. Particle electrophoresis measurement showed that stearylamine in fluid state membranes was more dispersed than in rigid state membranes indicating that lateral distribution of stearylamine affected the reactivity of liposomes. The mode of complement activation by the interaction of CRP with positively charged liposomes was similar to that observed in the system of protamine and heparin.

Agglutination Tests↗
PubMed2001

[Immunological features of renal lesion in chronic alcoholism].

One hundred and sixty males whose mean age was 42 years were examined. Of them there were 122 patients with stage II chronic alcoholism (CA), 92 with renal lesion following the type of chronic glomerulonephritis (CG) (Group 1); 30 patients with CA without renal lesion (Group 2), and 42 patients had CG alone (Group 3). Methods that characterize humoral immunity were used. These included detection of circulating immune complexes (CIC) by polyethylene glycol precipitation, measurement of the concentrations of IgA, IgM, and IgG by the Mancini radial immunodiffusion assay, detection DNA antibodies by the Farre test modified by V. V. Koshelev, that of serum anticomplement activity, measurement of the levels of complement by its hemolytic activity, determination of the activity of the lysosomal enzymes acid RNAase, acid DNAase, and cathepsin by the procedure of A. A. Pokrovsky et al. Complex estimation of the content of CIC, immunoglobulins, DNA antibodies and the activity of the lysosomal enzymes in patients with renal lesion makes it possible to evaluate the severity of a pathological process and to make its prognosis.

Adult↗
PubMed1994

Development of a C1q-ABO-ELISA to measure C1q binding by human anti-A alloantibodies.

Little is known about the correlation between the amount of anti-A antibodies and the respective complement activating capacity. We have therefore developed a new ELISA procedure for the study of complement activation by anti-A antibodies bound to immobilized blood group substance A through the classical pathway. C1q binding capacity of anti-A was then compared to both anti-A immunoglobulin (Ig) isotype serum concentrations and to the hemolysing capacity of O type sera. We have demonstrated that sera with low anti-A IgG and IgM levels produced low C1q binding activity and no hemolysis of human A1 red blood cells (RBC), whereas higher anti-A Ig levels resulted in either weak or strong C1q binding capacity as well as weak or strong RBC hemolysis. A correlation of rs = 0.755 was observed between C1q-ABO-ELISA and hemolysin assay results, although only 58% of the O type sera containing C1q binding anti-A lysed A1 type RBC under the used conditions. The C1q-ABO-ELISA is therefore a more sensitive assay for detecting 'dangerous' O type donors for blood transfusion to A type recipients than screening tests for hemolytic anti-A or determination of anti-A Ig levels alone.

ABO Blood-Group System↗
PubMed1995

Automated homogeneous liposome-based assay system for total complement activity.

We developed an automated homogeneous immunoassay, based on immune lysis of dinitrophenyl (DNP)-labeled liposomes, for measuring total complement activity. Liposome lysis caused by complement activity was detected spectrophotometrically from entrapped glucose-6-phosphate dehydrogenase activity. Complement activity in human sera was quantified by comparison with a calibration curve. For ease of application to fully automated routine clinical analyzers, we adopted a two-reagent system, one reagent containing a homogeneous population of small DNP-labeled liposomes and one containing antibody/substrate. This system required calibration only once a week. Within-run and between-run CVs were 0.4-1.3% (n = 10) and 1.8-4.7% (n = 10), respectively. Serum results were linear upon dilution (with saline) over a twofold range. Bilirubin, hemoglobin, Intrafat, and serum proteins such as rheumatoid factor, M protein, IgG, and IgA did not affect the assay results. The results (y) correlated well with those from a hemolytic complement activity test (x): y = 1.05x - 1.14, r = 0.92, on 66 samples in the range < 10- > 50 kU/L. This method should therefore be of great use for the determination of complement activity.

Adolescent↗
PubMed2005

The effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate life-supporting kidney xenografts.

BACKGROUND: In pig-to-nonhuman primate solid organ xenotransplantation using organs from donors transgenic for human decay-accelerating factor (hDAF), the main type of rejection is antibody-mediated (acute humoral xenograft rejection, AHXR). This occurs despite the complement-regulatory function of the transgene, neutralization of natural antibodies to Galalpha1-3Gal (Gal) using soluble glycoconjugates, and chronic immunosuppression. As complement components play a major role in graft destruction after antibody binding, we evaluated the efficacy of chronic complement inhibition by soluble complement receptor type 1 (TP10). METHODS: Life-supporting hDAF-transgenic kidney transplantation was performed in cynomolgus monkeys, using cyclophosphamide induction, and maintenance immunosuppression with cyclosporin A, mycophenolate sodium, and tapering steroids. Rejection was treated with bolus steroid injections: if not successful animals were terminated. Three groups were studied: in group 1 (n=4) GAS914 (a soluble glycoconjugate comprising Gal on a poly-L-lysine backbone) was added before and after transplantation; group 2 (n=2) received GAS914 as in group 1 and in addition TP10 before and after transplantation; in group 3 (n=4) GAS914 was only given before transplantation and TP10 as in group 2. Monitoring included the regular assessment of anti-porcine antibodies, complement activity (soluble C5b-9), therapeutic drug monitoring, and graft histology. RESULTS: Survival in group 1 was 6, 12, 31 and 37 days, respectively, and in all four cases graft histology showed AHXR. The two animals in groups 2 survived 3 and 15 days, respectively, and similarly showed AHXR in graft histology. In group 3 two animals showed AHXR (10 and 37 days survival, respectively), and two others did not show AHXR (20 and 32 days survival, respectively). The diagnosis AHXR included the deposition of complement activation products in the graft, which were present at lower intensity in animals treated with TP10. In all animals GAS914 effectively neutralized circulating anti-Gal antibody. Antibodies were detectable in the circulation of all animals using porcine erythrocytes in a hemolytic assay, although at lower levels than before transplantation. Soluble C5b-9 was not detectable in the circulation of animals receiving TP10, and circulating TP10 concentrations in these animals were in a presumed pharmacologically active range. CONCLUSIONS: The inclusion of TP10 in the immunosuppressive protocol does not clearly lead to improved xenograft survival. Despite effective neutralization of anti-Gal antibodies and effective inhibition of systemic complement activity, AHXR was apparent in four of six animals under chronic TP10 treatment, including deposits of complement activation products in the graft. Apparently, effective systemic complement inhibition by TP10 in combination with local complement regulation by the hDAF transgene product does not necessarily result in effective inhibition of complement activation at locations in the xenograft upon binding of anti-porcine antibodies to the grafted endothelium.

Acute Disease↗
PubMed1990

Molecular and functional identification and purification of complement component factor D from urine of patients with chronic renal failure.

Urine proteins of normal subject and patients with impaired renal function were analyzed by two-dimensional polyacrylamide gel electrophoresis. As a result, a clear spot was detected specifically in urine from patients with obvious renal dysfunction. The isoelectrical point of this unique spot was pH 7.1-7.2 and the flow-rate (Rf) was 0.50-0.55 as that of albumin was 1.0. Partial amino acid sequence analysis revealed that the NH2-terminal to 22nd amino acid sequence was identical with that of complement factor D. We purified 22 mg of this protein (factor D) from 5000 ml of urine from a patient on hemodialysis by three chromatographic steps using DEAE-Sephadex A-50 and Sephacryl S-200. The purified urine factor D gave a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis at the position of 23 kD, and displayed normal factor D hemolytic activity. The concentrations of factor D estimated by hemolytic assay were 1.9 micrograms/ml of normal serum, less than 0.1 microgram/ml of normal urine, 15 micrograms/ml of patient serum and 50 micrograms/ml of patient urine.

Amino Acid Sequence↗
PubMed2004

In vitro immunomodulatory activity of plants used by the Tacana ethnic group in Bolivia.

One hundred and seventy-eight ethanolic plant extracts from the pharmacopoeia of the Tacana, an ethnic group from Bolivia, were screened for immunomodulatory activity using complement cascade inhibition and ADP-induced platelet aggregation inhibition assays. Six impaired both complement pathways (classical and alternative): stem bark from Astronium urundeuvea (Anacardiaceae), Cochlospermum vitifolium (Cochlospermaceae), Terminalia amazonica (Combretaceae), Triplaris americana (Polygonaceae), Uncaria tomentosa (Rubiaceae) and Euterpe precatoria (Arecaceae) roots. Inhibition of complement cascade was independent of essential ion complexation, and was not due to direct hemolytic activity on target red blood cells. For A. urundeuvea, C. vitifolium, and T. amazonica, anti-inflammatory activity relied on cyclo-oxygenase inhibition. Four of these species (A. urundeuva, T. americana, U. tomentosa and E. precatoria) are used traditionally to treat inflammatory processes.

Bolivia↗
PubMed1981

Meningococcal meningitis in familial deficiency of the fifth component of complement.

Absence of the fifth component of complement (C5) by immunochemical assay and marked deficiency by hemolytic assay (less than 0.1%) was found in a family in which the oldest male child had suffered severe and recurrent meningococcemia at age 15 years, two brothers developed meningococcal meningitis four years later (at ages 18 and 14 years), and a sister had the gonococcal arthritis-dermatitis syndrome. Although group-specific meningococcal antibody was present in the sera from all four siblings, serum bactericidal activity against Neisseria meningitidis could be demonstrated only in the presence of exogenous rabbit complement. Serum total hemolytic complement activity was undetectable in all four, but was restored to normal by the addition of purified C5. Subsequently, a second episode of group Y meningococcal meningitis was experienced by one brother and presumed gonococcal arthritis-dermatitis syndrome recurred in the sister. The family is the largest C5-deficient kindred to be reported and emphasizes the importance of C5 in host susceptibility to invasive Neisseria infections. In contrast to the peak incidence of N meningitidis disease in the general population in the first year of life, age of onset of meningococcal infection in these patients and in the 13 previously reported patients with terminal complement component deficiency has usually been in adolescence and early adulthood.

Adolescent↗
PubMed1980

Activation of the classic complement pathway in patients with the C3 nephritic factor.

5 patients, including 4 with partial lipodystrophy and 1 with dense deposit disease (type II mesangiocapillary glomerulonephritis; MCGN), had positive C3 nephritic factor assays by both a crossed-gel immunoelectrophoretic and a magnesium-dependent hemolytic assay. One or more early classic complement component was significantly decreased in the serum from each patient. The titer of the C3 nephritic factor did not correlate with the presence of dense intramembranous deposits or with the severity of the renal disease. One patient with partial lipodystrophy had ultrastructural evidence suggesting transformation from type I to type II MCGN. These findings do not support a strict separation of MCGN into subtypes according to current criteria.

Adolescent↗
PubMed1983

Variance in the activity of the fourth component of mouse complement.

There are two methods for hemolytic titration of C4. One is a method using EAClgp, oxyC2hu and EDTA-Cgp (E-C method) and the other using EAClgp, oxyC2hu and C4Dgp (C4D method). The C4 titers obtained by these two methods differed depending on whether the C4 exists in plasma or in serum. In the case of Ssh strain, serum C4 activity assayed by the E-C method was higher than that by the C4D method, while plasma C4 activity assayed by the E-C method was lower than that by the C4D method. It was hypothesized that these results would occur from the difference in the state of C4 molecule present in serum or plasma. From chromatographic analysis, it was apparent that the C4 molecule in serum existed in at least four different molecular states; free C4 molecule with low Ss antigenicity, hemolytically inactive Ss protein, active C4 with high molecular weight and low Ss antigenicity, and inactive Ss protein with high molecular weight. Their expression of Ss antigen and the hemolytic C4 activity were different, and each type of C4 molecule behaved differently in the assay of the two methods. Furthermore an important role of mouse C5 in the hemolytic assay of mouse C4 is discussed.

Animals↗
PubMed1988

Serum complement activation in central nervous system disease in Sjögren's syndrome.

PURPOSE: Central nervous system disease and vasculitis are extraglandular manifestations of Sjögren's syndrome. In our experience, central nervous system disease develops in approximately 70 percent of patients with Sjögren's syndrome and biopsy documented peripheral vasculitis. In order to further investigate the pathogenesis of central nervous system disease and its relationship to peripheral vasculitis in Sjögren's syndrome, we examined sera of patients with Sjögren's syndrome with and without focal central nervous system involvement for evidence of terminal complement pathway activation. PATIENTS AND METHODS: Patients were classified as having active focal central nervous system involvement only when they had focal neurologic deficits on physical examination, plus at least one abnormal neurodiagnostic test result. Two thirds of these patients also had cognitive or psychiatric dysfunction. Patients were classified as having peripheral vasculitis if they had clinical and histopathologic documentation of vascular inflammation. Serum SC5b-9 was measured by a sensitive enzyme-linked immunoabsorbent assay. Total hemolytic complement assay, measurement of serum C3 and C4 by radial immunodiffusion, and determination of immune complexes were performed. RESULTS: Fluid-phase terminal complement complexes (SC5b-9) were detected in the sera of 25 of 30 (83 percent) patients with focal central nervous system involvement, but in only seven of 21 (33 percent) patients with Sjögren's syndrome without focal central nervous system disease (p = 0.00084 by Yates' chi-square analysis). Four of these seven patients without focal central nervous system disease, but who had serum SC5b-9, had psychiatric or cognitive dysfunction. SC5b-9 was also detected in sera from 14 of 15 (93 percent) patients with active biopsy-documented peripheral vasculitis in contrast to 18 of 36 (50 percent) patients without clinical evidence of peripheral vasculitis (p = 0.0094). Serum SC5b-9 was a more sensitive indicator of complement activation than circulating immune complex or complement assays. CONCLUSION: These findings suggest that terminal complement activation may participate in the pathophysiology of both central nervous system and peripheral vasculitis in Sjögren's syndrome. Serum SC5b-9 appears to be a useful diagnostic indicator of vascular inflammation in Sjögren's syndrome and appears to identify those patients at risk for central nervous system complications.

Antigen-Antibody Complex↗
PubMed2004

Novel monoclonal antibodies against mouse C3 interfering with complement activation: description of fine specificity and applications to various immunoassays.

The role of complement proteins in various pathophysiological settings has been studied primarily using mouse models of disease. However, the specific contribution of C3-derived fragments to these biologic processes has not been addressed in a rigorous manner because of a lack of antibodies that can selectively recognize mouse C3 or any of its degradation fragments. Here we report the generation and characterization of a panel of rat monoclonal antibodies reacting with mouse C3 and its degradation products. We describe their performance in various immunological assays such as ELISA, Western blotting, flow cytometry and immunohistochemistry. Of all the antibodies generated, one selectively recognized the C3a anaphylatoxin, and all other reacted with C3c. Furthermore, two monoclonal antibodies preferentially reacted with the cleaved C3 fragments C3b/iC3b/C3c but not native C3. Except for the one recognizing C3a, all antibodies were suitable for detecting C3 deposited on cells and tissues, two effectively inhibited the hemolytic activity of mouse complement and one enhanced C3-deposition to the cell membrane. These novel monoclonal antibodies may serve as useful reagents for elucidating functions mediated by C3-derived fragments in various pathophysiological conditions.

Animals↗