Glomerular C4d deposition in proliferative glomerular diseases.

INTRODUCTION: The aim of this study was to evaluate the immunohistochemical expression of C4d in native renal biopsies of proliferative glomerular diseases, complement pathways in these diseases, and assess the relationship of C4d with histological and clinicopathological parameters, other complement proteins, and immunoglobulin markers. METHODS: This cross-sectional study was conducted during the year 2018-19 involving 107 native renal biopsies with histologically diagnosed cases of proliferative glomerular diseases. C4d immunohistochemical evaluation of renal tissue sections was performed using polyclonal antihuman C4d as the primary antibody. Patients were classified as positive and negative groups based on their glomerular C4d deposition. RESULTS: The overall prevalence of C4d positivity was 80.4% in proliferative glomerular diseases ranging between 60.0% in C3 glomerulonephritis to 92.9% in membranoproliferative glomerulonephritis. Mixed capillary and mesangial deposition were noted in all cases of proliferative glomerulonephritis. Classical pathway was dominantly involved in all glomerular diseases except C3 glomerulonephritis and IgA nephropathy. Multivariate logistic regression analysis revealed that glomerular IgG staining (aOR: 5.86, 95% CI: 1.26-27.14) and IgM staining (aOR: 3.90, 95%CI: 1.07-14.18) were significantly associated with C4d positivity. CONCLUSION: C4d staining along with immunoglobulin markers such as IgG and IgM and complement proteins can be useful in delineating different complement activation pathways in glomerular diseases and understanding the disease pathogenesis.

The follicular dendritic cell restricted epitope, FDC-M2, is complement C4; localization of immune complexes in mouse tissues.

We have identified the murine follicular dendritic cell (FDC) marker, FDC-M2, recognized by monoclonal antibody mAb209, as complement component C4. Consistent with this, FDC-M2 was detectable at sites of immune complex-mediated inflammatory disease. Analysis of FDC-M2 distribution in complement-deficient mice highlighted the differences in immune complex clearance between these mice, andshowed that a population of uncharacterized FDC-M2+ reticular and perivascular cells in the spleen, distinct from FDC, are also involved in immune complex capture and possibly retention. These results demonstrate that mAb209, in addition to its role as an FDC marker, is a valuable reagent for the analysis of complement deposition in vivo.

Reference typing report for complement component C4.

During the 7th Complement Genetics Workshop, Mainz, Germany, May 1998, a complement component C4 typing exercise took place with the aim of applying present technologies to the definition of reference C4 alleles/phenotypes and the recognition of nonexpressed (Q0) C4 alleles within expressed haplotypes. Eleven samples were submitted from 3 laboratories and tested by 14 participating laboratories with basic protein-typing technologies; in addition, each laboratory contributed data from local expertise. The samples were introduced to the reference typing for one or more characteristic allotype or for partial or total nonexpression of one isotype. The blinded samples were centrally evaluated and the results discussed among the participants at a plenum meeting. From the results, the samples could be classified into a group of common, easy to diagnose pheno-/allotypes, less common but still unanimously recognised variants, and a third group with difficult pheno-/allotypes. Within the latter group, the allotypes were either new (C4A '92'; C4B '93') and/or showed partial or total reversed antigenicity and unusual Rodgers/Chido (Rg/Ch) PCR subtypes (C4A '92'; C4A 12; C4B '35'; C4B '13'). Semiquantitative C4-alpha-chain estimates of relative isotype levels correlated well with the number of alleles seen at each locus by agarose gel electrophoresis, and were superior to other isotype quantitation methods. From the evaluation of the reference typing it was concluded that the recognition of rare, aberrant or hybrid C4 alleles with partial or total reversed Rg/Ch antigenicity or monoclonal reactivity is still difficult in most instances; besides isotype-dependent lysis, relative migration values, immunoblots with Rg- and Ch-specific monoclonal antibodies, Rg/Ch PCR typing, side-by-side comparison with already described allotypes will ultimately be required. The recognition of nonexpressed alleles within C4A and C4B expressed phenotypes remains the major obstacle in C4 genetic typing. Finally, a conclusive interpretation of DNA typing results will be achieved only in the context of complete allotyping results at the protein level, and at present cannot replace conventional protein allotyping.

Inheritance of serum autoantibody, reduced serum IgA and autoimmune disease in a canine breeding colony.

Immunological parameters were examined in 113 English cocker spaniel dogs from two breeding kennels. Dogs from kennel 1 (n = 86) were grouped as having idiopathic cardiomyopathy (n = 19), autoimmune or other disease (n = 7) or being clinically normal (n = 60). Dogs from kennel 2 (n = 27) were all clinically normal and used for comparative purposes. There was a high incidence of serum antinuclear antibody (ANA) amongst all groups from kennel 1 (39/82 dogs tested), with anti-thyroglobulin and anti-erythrocyte antibodies also recorded in a dog with systemic lupus erythematosus. Thirty percent of dogs with idiopathic cardiomyopathy had anti-mitochondrial antibody. Thirteen dogs from kennel 1 had reduced serum IgA (< or = 0.3 mg/ml), but there was no consistent abnormality in the concentration of serum IgG, IgM, complement C3 or C4 in these thirteen dogs, or other dogs from this kennel. No immunological abnormality was recorded in dogs from kennel 2. Pedigree analysis of dogs from kennel 1 revealed inheritance of autoimmune disease, serum ANA and low serum IgA within several breeding lines. Inheritance of idiopathic cardiomyopathy was recorded through three generations and a strong association demonstrated between the presence of this disorder and a particular complement C4 phenotype (C4: 4).

Genetic polymorphism of the fourth component of human complement: population study and proposal for a revised nomenclature based on genomic PCR typing of Rodgers and Chido determinants.

The fourth component of human complement (C4) is coded for by two homologous genes, C4A and C4B, located in the class III region of the major histocompatibility complex (MHC). Genetic typing of C4A and B alleles is routinely carried out by high-voltage agarose gel electrophoresis. The electrophoretic C4 polymorphism can be further subdivided by the Rodgers (Rg) and Chido (Ch) blood groups, which are antigenic determinants of the C4A and B alpha-chains, respectively. We have used a recently described direct PCR typing method using sequence-specific primers (PCR-SSP) in combination with electrophoretic C4 typing as well as genomic RFLP analysis to determine the frequency of C4 allotypes, Rg/Ch subtypes and C4A-B haplotypes in a family study of the German population. As the current C4 allele designation does not provide any information about the presence or absence of Rodgers and Chido antigens, we have developed an extension to the existing C4 nomenclature. This revised allele designation combines the existing numerical allotypes defined by electrophoretic mobility with eight subtypes (01-08) based on Rg/Ch PCR genotyping results. Using this approach, most electrophoretic allotypes could be subdivided. Among the C4A allotypes, the most common allele was A*0301 (59.9%), and the most common subtype among all electrophoretic allotypes was 01 (85.1%; = Rg1,2-positive, Ch-negative). For C4B, the most common allele was B*0101 (64.3%), and the most common subtype was 01 (79.6%; = Ch1,2,3,4,5,6-positive, Rg-negative). The subtypes 03, 04, 07 and 08 of the C4A allotypes, and the subtypes 03, 07 and 08 of the C4B allotypes, were not detected in this study. The analysis of duplicated C4 alleles revealed considerable heterogeneity of their subtypes. The results demonstrate that all known C4 allotypes can now be assigned unambiguously, which facilitates the identification of MHC haplotypes relevant for transplantation and disease association studies.

Association of complement C4 and HLA-DR alleles with systemic lupus erythematosus in Koreans.

OBJECTIVE: To examine the association of complement C4 and HLA-DR to systemic lupus erythematosus (SLE) susceptibility in Korea. METHODS: Complement C4 protein typing was carried out by immunofixation and immunoblotting methods using EDTA-plasma from 60 patients with SLE and 72 healthy controls. Restriction fragment length polymorphism analysis of C4 genes was also carried out using TaqI or HindIII for restriction enzymes. HLA-DR was determined by polymerase chain reaction amplification with sequence specific primers using genomic DNA from 67 patients with SLE and 72 healthy controls. RESULTS: The frequency of the C4AQ0 allele was significantly higher in the patients with SLE than in controls (41.7 vs 25.0%, p < 0.05). The deletion of the C4A gene commonly found in Caucasian patients with SLE was not observed in any patients. For HLA-DR, a significant increase of the haplotype DRB1*1501 was observed in the patients (26.9 vs 12.5%, p < 0.05) and DR9 was also significantly increased (23.9 vs 11.1%, p < 0.05). An increase in each DR2 and DR9 was independent of an increase in C4AQ0. The frequencies of DR2 and DR9 were significantly decreased in patients with renal involvement and alopecia, respectively. CONCLUSION: Our data suggested that the presence of C4AQ0 allele, DRB1*1501-DRB5*0101 haplotype and DR9 contributed to susceptibility to SLE in Koreans and that Korean SLE is based on a different genetic background from Caucasian patients.

Revised nomenclature for human complement component C4. WHO-IUIS Nomenclature Sub-Committee.

This note describes the recommended designations for allotypes of human complement component C4, which were approved by the Nomenclature Committee of the International Union of Immunological Societies (IUIS).

C4 nomenclature statement (1990).

A common and revised nomenclature of the allotypes of the fourth component (C4) of human complement has been proposed. It is based on the results of the C4 Reference Typing of the VIth Complement Genetics Workshop and Conference, Mainz, FRG, 1989, the previous C4 nomenclature and the guidelines for human gene nomenclature (ISGN). The designation of allotypes derives from their relative electrophoretic mobility, the distinction between C4A and C4B proteins from their relative hemolytic activity. Common alleles retain their single digit numeric designation, intermediate variants their two- or three-digit designations; newly discovered alleles should not interfere with already described variants. At least 13 C4A alleles, 16 C4B alleles as well as non-expressed genes at each C4 locus are presently known. There are also duplicated loci of each C4 gene; they should be designated by repetition of the locus symbol at the haplotype or genotype level. As a phenotype they will be placed in parenthesis without repetition of the locus symbol. Aberrant allotypes or hybrid genes should be explained by a special suffix. No special nomenclature is recommended for restriction fragment length polymorphisms. Their designation should follow the general rules of the ISGN.

Relative electrophoretic migration distances for the classification of C4 allotypes.

For the definition of common C4 allotypes relative electrophoretic migration (RM) values were determined. A set of standard C4 variants were investigated by prolonged agarose gel electrophoresis and subsequent immunofixation with specific antiserum. RM distances were measured by laser densitometry. Using an arbitrary standard of 100 units for the migration distance between the C4B 1 and C4A 3 bands a total deviation of +/- 6.45% in more than 108 single determinations was found. The common C4 alleles used for standardization were C4A*6, C4A*4, C4A*3, C4A*2, C4B*5, C4B*3, C4B*2, C4B*1. In addition, side by side comparison and admixture of known variants will be necessary for the differentiation of some of the closely migrating allotypes. RM values are now available for eight alleles frequently found in all populations for future comparison and designation of newly discovered C4 allotypes.

Purification and characterisation of ovine C4: evidence for two molecular forms in ovine plasma.

A relatively rapid procedure is described for the isolation of the fourth component of complement (C4) from ovine plasma. The method, which recovers approximately 30% C4, is based upon DEAE Sephacel anion exchange chromatography of PEG precipitated plasminogen depleted plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration. SDS-PAGE of purified ovine C4 under reducing conditions revealed a complex pattern of bands which was interpreted on the basis of a three polypeptide chain structure for each of two distinct species, or isotypes, of C4 molecule herein termed C4A and C4B. Each isotype differs in the mol. wt of the alpha chain--108 and 95 K respectively. Nucleophilic substitution of immunoprecipitated ovine C4 with radiolabelled methylamine revealed that both C4 species contained a reactive thiol ester site and that each could be cleaved into an activated form (presumably C4b) characterised by a truncated alpha' chain some 8 K lower in mol. wt. A comparison of the isotype composition of purified C4 with that of immunoprecipitated C4 from the same animal indicated that the purification procedure favoured isolation of the C4B isotype. The mol. wts of both the alpha and beta chains were lowered following digestion of ovine C4 with neuraminidase.

High resolution electrophoresis for the allotyping of human C4. Proposal of a relational nomenclature.

Among the major histocompatibility complex (MHC)-linked complement genes, the loci for C4A and C4B exhibit the most extensive structural polymorphisms. Therefore the differentiation of variant and complex C4 phenotypes often proves difficult in conventional immunofixation electrophoresis. To improve the available technique of C4 typing a closed horizontal electrophoresis system was combined with poly- and monoclonal alkaline phosphatase immunoprobing on contact diffusion blots. The high resolution and sensitivity of this method not only facilitated C4 allotyping but also revealed additional polymorphic variation. Relative electrophoretic mobilities specific for each C4 allotype were established by computerized remission densitometry and provided the basis for a quantitative denomination of C4 variants. Typing by high resolution electrophoresis and the proposed relational C4 nomenclature could be valuable for further immunogenetical studies of the C4 protein polymorphism.

Serum C4 concentration in the monitoring of systemic lupus erythematosus: requirement for C4 allotyping.

We have examined the influence of genetic and other factors on the serum concentration of the fourth component of complement (C4) in four patients with systemic lupus erythematosus (SLE) studied over 3 to 8 years. Complement allotyping was performed to determine the number of C4 null genes in each patient. Two patients with C4 null genes had relatively low serum C4 concentrations with normal serum anti-DNA binding and no evidence of active disease. By contrast two patients without null alleles appeared to be consuming C4 when the serum C4 concentrations were within the conventional reference range. We therefore propose the use of appropriate reference ranges adjusted for the number of null alleles. Such adjusted reference ranges may improve the utility of serum C4 concentration in monitoring disease activity.