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Human Mutation Jul 2010The complement C4 locus is in the class III region of the MHC, and exhibits copy number variation. Complement C4 null alleles have shown association with a number of...
The complement C4 locus is in the class III region of the MHC, and exhibits copy number variation. Complement C4 null alleles have shown association with a number of diseases including systemic lupus erythematosus (SLE). However, most studies to date have used protein immunophenotyping and not direct interrogation of the genome to determine C4 null allele status. Moreover, a lack of accurate C4 gene copy number (GCN) estimation and tight linkage disequilibrium across the disease-associated MHC haplotypes has confounded attempts to establish whether or not these associations are causal. We have therefore developed a high throughput paralog ratio test (PRT) in association with two restriction enzyme digest variant ratio tests (REDVRs) to determine total C4 GCN, C4A GCN, and C4B GCN. In the densely genotyped CEU cohort we show that this method is accurate and reproducible when compared to gold standard Southern blot copy number estimation with a discrepancy rate of 9%. We find a broad range of C4 GCNs in the CEU and the 1958 British Birth Cohort populations under study. In addition, SNP-C4 CNV analyses show only moderate levels of correlation and therefore do not support the use of SNP genotypes as proxies for complement C4 GCN.
Topics: Alleles; Complement C4; Gene Dosage; Gene Frequency; Genetic Association Studies; Genotype; Haplotypes; Humans; Linkage Disequilibrium; Lupus Erythematosus, Systemic; Major Histocompatibility Complex; Mutation; Polymorphism, Single Nucleotide; Reproducibility of Results; White People
PubMed: 20506482
DOI: 10.1002/humu.21259 -
Annals of Medicine Aug 2012Hantaviruses are important human pathogens that cause clinical diseases characterized by renal and cardiopulmonary manifestations. Their pathogenesis is currently poorly...
INTRODUCTION
Hantaviruses are important human pathogens that cause clinical diseases characterized by renal and cardiopulmonary manifestations. Their pathogenesis is currently poorly understood. We have studied the role of the complement system in the pathogenesis of Puumala (PUUV) hantavirus infection.
MATERIAL AND METHODS
We studied the activation of complement by measuring the terminal complement complex SC5b-9 and complement component C3 and C4 levels in patients with acute PUUV infection. Several laboratory parameters and clinical findings reflecting the severity of PUUV-HFRS were evaluated with regard to complement activation.
RESULTS
The levels of SC5b-9 were significantly increased and C3 decreased in the acute stage as compared to the levels at full recovery (P < 0.001). We found that SC5b-9 levels were higher in patients with chest X-ray abnormalities than in patients with a normal X-ray during the acute stage (P = 0.028). Furthermore, SC5b-9 and C3 levels showed significant correlation with several clinical and laboratory parameters that reflect the severity of the acute PUUV infection.
CONCLUSIONS
We showed that the complement system becomes activated via the alternative pathway in the acute stage of PUUV infection and the level of activation correlates with disease severity. The results further suggest that complement activation may contribute to the pathogenesis of acute PUUV infection.
Topics: Adult; Aged; Complement Activation; Complement C3; Complement C4; Complement Membrane Attack Complex; Female; Hemorrhagic Fever with Renal Syndrome; Humans; Male; Middle Aged; Prospective Studies; Puumala virus; Severity of Illness Index; Young Adult
PubMed: 21495786
DOI: 10.3109/07853890.2011.573500 -
American Journal of Human Genetics Oct 2002The complex genetics of human complement C4 with unusually frequent variations in the size and number of C4A and C4B, as well as their neighboring genes, in the major...
The complex genetics of human complement C4 with unusually frequent variations in the size and number of C4A and C4B, as well as their neighboring genes, in the major histocompatibility complex has been a hurdle for accurate epidemiological studies of diseases associated with C4. A comprehensive series of novel or improved techniques has been developed to determine the total gene number of C4 and the relative dosages of C4A and C4B in a diploid genome. These techniques include (1) definitive genomic restriction-fragment-length polymorphisms (RFLPs) based on the discrete duplication patterns of the RCCX (RP-C4-CYP21-TNX) modules and on the specific nucleotide changes for C4A and C4B isotypes; (2) module-specific PCR to give information on the total number of C4 genes by comparing the relative quantities of RP1- or TNXB-specific fragments with TNXA-RP2 fragments; (3) labeled-primer single-cycle DNA polymerization procedure of amplified C4d genomic DNA for diagnostic RFLP analysis of C4A and C4B; and (4) a highly reproducible long-range-mapping method that employs PmeI-digested genomic DNA for pulsed-field gel electrophoresis, to yield precise information on the number of long and short C4 genes in a haplotype. Applications of these vigorously tested techniques may clarify the roles that human C4A and C4B gene-dosage variations play in infectious and autoimmune diseases.
Topics: Alleles; Complement C4a; Complement C4b; Gene Dosage; Genotype; Humans; Major Histocompatibility Complex; Molecular Sequence Data; Phenotype; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length
PubMed: 12224044
DOI: 10.1086/342778 -
The Journal of Experimental Medicine Apr 2010The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that...
The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutralization in solution. These studies define a novel immune evasion mechanism for restricting complement control of microbial infection.
Topics: Animals; Biocatalysis; CHO Cells; Complement C1; Complement C1 Inhibitor Protein; Complement C1s; Complement C3-C5 Convertases; Complement C3b; Complement C4; Complement C4b; Complement Factor I; Complement Hemolytic Activity Assay; Complement Pathway, Classical; Complement Pathway, Mannose-Binding Lectin; Cricetinae; Cricetulus; Dengue Virus; Enzyme Precursors; Guinea Pigs; Humans; Kinetics; Neutralization Tests; Protein Binding; Viral Nonstructural Proteins
PubMed: 20308361
DOI: 10.1084/jem.20092545 -
Kidney International Apr 1987Activation of alternative complement pathway is presumed to be important pathogenically in IgA nephropathy since renal biopsies usually exhibit glomerular deposition of...
Activation of alternative complement pathway is presumed to be important pathogenically in IgA nephropathy since renal biopsies usually exhibit glomerular deposition of C3 and P (properdin). Surprisingly, little is known about plasma complement activation in this disease, and the plasma C3 and C4 concentrations are usually normal or increased. We quantitated C3 activation in 202 plasmas from 81 patients with IgA nephropathy using a sensitive new assay that detects a neoantigen [iC3b-C3d neoantigen) which appears when C3b is inactivated to iC3b, C3dg, or C3d. This assay accurately quantitates small amounts of in vivo C3 activation. The concentration of iC3b-C3d neoantigen in plasma was significantly increased, indicating C3 activation in 37% of the pediatric and 57% of the adult plasmas assayed. When data from serial determinations in the patients were analyzed, 75% of the adult and 57% of the pediatric patients had C3 activation on at least one occasion. Classical pathway activation, quantitated by C4 activation was found in 20% of the adult and 5% of the pediatric plasmas. No association was found between elevated iC3b-C3d neoantigen concentration and history of macroscopic hematuria, chronic renal insufficiency or degree of proteinuria. These studies show that complement activation can frequently be detected in the plasma of IgA nephropathy patients. However, the pathophysiologic significance of this complement activation remains to be determined.
Topics: Adult; Child; Complement Activation; Complement C3; Complement C4; Complement Pathway, Alternative; Complement Pathway, Classical; Female; Glomerulonephritis, IGA; Humans; Male
PubMed: 3586493
DOI: 10.1038/ki.1987.101 -
Arthritis Research & Therapy 2007Systemic lupus erythematosus is a complement-mediated autoimmune disease. While genetic deficiencies of classical pathway components lead to an increased risk of... (Comparative Study)
Comparative Study
Systemic lupus erythematosus is a complement-mediated autoimmune disease. While genetic deficiencies of classical pathway components lead to an increased risk of developing systemic lupus erythematosus, end organ damage is associated with complement activation and immune complex deposition. The role of classical pathway regulators in systemic lupus erythematosus is unknown. C4 binding protein (C4bp) is a major negative regulator of the classical pathway. In order to study the role of C4bp deficiency in an established murine model of lupus nephritis, mice with a targeted deletion in the gene encoding C4bp were backcrossed into the MRL/lpr genetic background. Compared with control MRL/lpr mice, C4bp knockout MLR/lpr mice had similar mortality and similar degrees of lymphoproliferation. There were no differences in the extent of proteinuria or renal inflammation. Staining for complement proteins and immunoglobulins in the kidneys of diseased mice revealed no significant strain differences. Moreover, there was no difference in autoantibody production or in levels of circulating immune complexes. In comparison with C57BL/6 mice, MRL/lpr mice had depressed C4 levels as early as 3 weeks of age. The absence of C4bp did not impact serum C4 levels or alter classical pathway hemolytic activity. Given that immune complex renal injury in the MRL/lpr mouse is independent of Fc receptors as well as the major negative regulator of the classical pathway, new mechanisms for immune-complex-mediated renal injury need to be considered.
Topics: Animals; Complement C4; Histocompatibility Antigens; Kidney Diseases; Lupus Erythematosus, Systemic; Mice; Mice, Inbred C57BL; Mice, Inbred MRL lpr; Mice, Knockout; Species Specificity
PubMed: 17971229
DOI: 10.1186/ar2320 -
Clinical and Experimental Immunology Oct 1999Human intestinal epithelial cells have been established as local sites for complement biosynthesis. In this study, we investigated the effects of IFN-gamma and sodium...
Sodium butyrate blocks interferon-gamma (IFN-gamma)-induced biosynthesis of MHC class III gene products (complement C4 and factor B) in human fetal intestinal epithelial cells.
Human intestinal epithelial cells have been established as local sites for complement biosynthesis. In this study, we investigated the effects of IFN-gamma and sodium butyrate on biosynthesis of MHC class III gene products (complement C4 and factor B) in the human fetal intestinal epithelial cell line INT-407. IFN-gamma induced a dose- and time-dependent increase in C4 and factor B secretion. However, sodium butyrate dose-dependently inhibited IFN-gamma-induced C4 and factor B secretion. These effects were also observed at the mRNA level. Immunoblotting indicated that IFN-gamma induced a rapid activation of Stat1alpha, and fluorescence immunohistochemistry detected a translocation of Stat1alpha into the nucleus within 1 h. However, the translocation of Stat1alpha was not affected by the addition of sodium butyrate. Nuclear run-on assay indicated that IFN-gamma induced a weak increase in the transcription rate of factor B gene, and sodium butyrate did not affect this response. IFN-gamma and sodium butyrate induced a counter-regulatory effect on C4 and factor B secretion: IFN-gamma acted as a potent inducer, but sodium butyrate potently abrogated these responses. These are mainly regulated through the post-transcriptional mechanism.
Topics: Blotting, Northern; Butyrates; Cell Line; Complement C4; Complement Factor B; Culture Media, Conditioned; DNA-Binding Proteins; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Humans; Immunoblotting; Interferon-gamma; Intestinal Mucosa; Phosphorylation; RNA, Messenger; STAT1 Transcription Factor; Time Factors; Trans-Activators; Transcription, Genetic
PubMed: 10540154
DOI: 10.1046/j.1365-2249.1999.01004.x -
Arthritis and Rheumatism Apr 2008To determine whether key features of systemic lupus erythematosus (SLE), namely, production of non-nuclear antibodies (anti-C1q and anticardiolipin antibodies [aCL]) and...
OBJECTIVE
To determine whether key features of systemic lupus erythematosus (SLE), namely, production of non-nuclear antibodies (anti-C1q and anticardiolipin antibodies [aCL]) and depletion of complement components C3 and C4, aggregate in families. In addition, we examined relationships between anti-C1q and C3 and C4 levels.
METHODS
The study cohort comprised 1,037 predominantly white (82%) nuclear families in which at least 1 member had SLE. Associations of antibody measurements between probands and their unaffected siblings were examined using parametric and nonparametric analyses, along with associations between unaffected siblings and their parents. The heritability of anti-C1q, C3, and C4 was estimated, and interdependencies between these factors were examined in a regression model accounting for the family structure of the data set.
RESULTS
We demonstrated associations between siblings for anti-C1q (odds ratio [OR] 3.74, 95% confidence interval [95% CI] 2.65, 5.28) and IgG and IgM aCL (OR 4.08, 95% CI 1.83, 5.13 and OR 2.06, 95% CI 1.46, 2.91, respectively) and, for anti-C1q, association between unaffected parents and their unaffected offspring (OR 4.34, 95% CI 2.16, 8.72). We also demonstrated significant heritability of anti-C1q, C3, and C4 (approximately 45%). Anti-C1q was negatively associated with C3 and C4 in SLE probands but not in their healthy relatives.
CONCLUSION
Non-nuclear antibodies and C3 and C4 cluster within the families of SLE probands, suggesting that specific autoantibody formation is partly genetically determined, even if the total genetic effect in unaffected relatives is insufficient to cause disease. Anti-C1q antibodies accelerate C3 and C4 depletion in patients with SLE but have no effect in the absence of disease.
Topics: Adult; Aged; Aged, 80 and over; Autoantibodies; Cluster Analysis; Cohort Studies; Complement C1q; Complement C3; Complement C4; Female; Genetic Predisposition to Disease; Humans; Lupus Erythematosus, Systemic; Male; Middle Aged; Odds Ratio; Pedigree; Phenotype; United Kingdom
PubMed: 18383369
DOI: 10.1002/art.23400 -
Journal of Clinical Laboratory Analysis 2004The lack of credible reference materials and satisfactory methods for quantifying serum levels has limited the bedside use of complement protein (C3 and C4)... (Comparative Study)
Comparative Study Meta-Analysis
The lack of credible reference materials and satisfactory methods for quantifying serum levels has limited the bedside use of complement protein (C3 and C4) measurements. However, great technological strides have been made in the last few years. The remaining barrier to a more relevant and cost-effective use of serum protein data for diagnosis and prognosis is the availability of reliable reference intervals from birth to old age for both males and females. Fifty-one publications reporting reference intervals were identified that meet the criteria used in our prior four studies, and these were analyzed statistically. Previous small studies with constrained age ranges agree, on average, with our larger series of life-long reference ranges. This meta-analysis provides support for our reference ranges and places them in the context of previous publications.
Topics: Chemistry, Clinical; Cohort Studies; Complement C3; Complement C4; Humans; Reference Values; White People
PubMed: 14730551
DOI: 10.1002/jcla.10095 -
Frontiers in Immunology 2019Antiphospholipid syndrome (APS) is a chronic and disabling condition characterized by recurrent thrombosis and miscarriages mediated by antibodies against...
Antiphospholipid syndrome (APS) is a chronic and disabling condition characterized by recurrent thrombosis and miscarriages mediated by antibodies against phospholipid-binding proteins (aPL), such as betaglycoprotein I (βGPI). Complement is involved in APS animal models and complement deposits have been documented in placenta and thrombotic vessels despite normal serum levels. Analysis of circulating blood cells coated with C4d displays higher sensitivity than the conventional assays that measure soluble native complement components and their unstable activation products in systemic lupus erythematosus (SLE). As C4d-coated blood cell count has been reported to be more sensitive than serum levels of complement components and their activation products in systemic lupus erythematosus (SLE) patients, we decided to evaluate the percentage of C4d positive B lymphocytes (BC4d), erythrocytes (EC4d), and platelets (PC4d) in primary APS patients and asymptomatic aPL positive carriers as marker of complement activation in APS. We assessed by flow cytometry the percentages of BC4d, EC4d, and PC4d in primary APS (PAPS; n. 23), 8 asymptomatic aPL positive carriers, 11 APS-associated SLE (SAPS), 17 aPL positive SLE, 16 aPL negative SLE, 8 aPL negative patients with previous thrombosis, 11 immune thrombocytopenia (ITP) patients, and 26 healthy subjects. In addition, we used an model to evaluate the ability of a monoclonal anti-βGPI antibody (MBB2) to bind to normal resting or activated platelets and fix complement. EC4d and PC4d percentages were significantly higher in PAPS and aPL carriers as well as aPL positive SLE and SAPS than in aPL negative controls. The highest values were found in PAPS and in SAPS. The EC4d and PC4d percentages were significantly correlated with serum C3/C4 and anti-βGPI/anti-cardiolipin IgG. studies showed that MBB2 bound to activated platelets only and induced C4d deposition. The detection of the activation product C4d on circulating erythrocytes and platelets supports the role of complement activation in APS. Complement may represent a new therapeutic target for better treatment and prevention of disability of APS patients.
Topics: Antiphospholipid Syndrome; Biomarkers; Blood Cells; Blood Platelets; Case-Control Studies; Complement Activation; Complement C3; Complement C4; Erythrocytes; Female; Humans; Male
PubMed: 31031764
DOI: 10.3389/fimmu.2019.00773