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Biomedical subjects

R Würzner

Publications and source records attributed to R Würzner.

At least 19 recordsLinked to original sources
PubMed1996

Molecular bases of combined subtotal deficiencies of C6 and C7: their effects in combination with other C6 and C7 deficiencies.

Combined subtotal deficiency of C6 and C7, in which both proteins are expressed at very low levels, has been observed in homozygous form in two families. A defect at the 5' splice donor site of intron 15 of the C6 gene explains the low molecular weight of the C6 protein and is probably responsible for its low expressed concentration. The C7 defect is more enigmatic: the protein is of normal molecular weight, low circulating concentration, and altered isoelectric point. An Arg > Ser codon substitution in exon 11 is the only molecular alteration within the mature C7 protein. These defects are associated with a characteristic set of polymorphic DNA markers in the C6/C7 region, forming a distinct haplotype. The haplotype has been found in combination with a number of other haplotypes containing defective genes that lead either to C6 or C7 deficiency, but with different consequences. Where it is combined with a C6-deficient gene, the serum C7 levels can be surprisingly high, possibly because there is no C6 generating C56 to consume the C7. In contrast, where the C7 genes are both defective (but still partially functional), there may be a profound deficit of circulating C7 because there is ample C6 to produce C56 and consume the already small amount of C7. Each molecular defect has also been found in isolation and has the expected effect.

Amino Acid Sequence↗
PubMed1996

Organ-specific contribution to circulating C7 levels by the bone marrow and liver in humans.

Many cells types can produce complement component C7, although the major site of C7 synthesis is unknown. Conversion from recipient to donor allotype following organ transplantation has demonstrated the synthetic sites of several complement proteins, but in the case of C7 this was not possible until recently. A novel C7 polymorphism (C7 M/N) has been described based on the reactivity with the monoclonal antibody WU 4-15 which identifies in allotype of C7 (C7 M). Bone marrow and hepatic C7 production was quantified in bone marrow transplant and liver transplant recipients, respectively, where a mismatch for the C7 allotypes distinguished by the monoclonal antibody had occurred. In the bone marrow transplant group, one informative transplant was identified and donor-derived C7 was detected by enzyme-linked immunosorbent assay. It contributed to 18-27% of the total circulating C7 during the post-transplant phase and was increased during episodes of inflammation. In the liver transplant group, the hepatic contribution to the C7 levels were 30% and 52%, respectively, in two patients identified prospectively. A further three informative liver transplant patients were identified retrospectively and in these individuals, 56-62% of the circulating C7 was liver-derived. This study demonstrates that the majority of the circulating C7 is derived from the liver and bone marrow with a lesser contribution from other sources. These findings provide further support for the concept that locally secreted complement proteins have an important role in inflammation.

Base Sequence↗
PubMed1996

CR1 stump peptide and terminal complement complexes are found in the glomeruli of lupus nephritis patients.

The number of CR1 on podocytes is reduced in nephropathies with severe glomerular damage, especially in the diffuse proliferative glomerulonephritis (DPGN) of systemic lupus erythematosus (SLE). Reduction of CR1 number on erythrocytes is due to proteolysis of CR1 by macrophage proteases activated by the reaction of their complement receptors, which leaves a 'CR1 stump peptide' on the erythrocyte. In the present study, we demonstrated the presence of the terminal complement complex (TCC) and the CR1 stump in histological sections of biopsies from patients with SLE by the indirect immunoperoxidase technique. Less severe glomerular lesions presented TCC deposits mainly in the mesangium (mesangial pattern). In lupus nephritis, with more severe glomerular damage, TCC deposits were detected both in the mesangium and in the capillary loops with podocyte involvement (mixed pattern). Patients with highly active DPGN presented a marked reduction of intact podocyte CR1 receptors in association with increased reactivity to the anti-CR1 stump antibody and with glomerular TCC deposits of mixed histological pattern. These results suggest that the decrease in the number of podocyte CR1 receptors in severe glomerular lesions of SLE may be due to a local proteolytic activity associated with activation and deposition of TCC.

Autoantigens↗
PubMed1996

How partial C7 deficiency with chronic and recurrent bacterial infections can mimic total C7 deficiency: temporary restoration of host C7 levels following plasma transfusion.

An apparently completely complement C7-deficient patient with refractory otitis media and two episodes of meningococcal disease was given therapeutic plasma transfusions in 1992 and 1994. Following these transfusions unexpected changes were found in C7 levels. Immediately after transfusion the serum C7 levels failed to rise to the expected levels but then rose to 5-10% of the normal mean during the next 5 days and remained at that level for more than 2 weeks before eventually returning to zero. The patient's DNA genotyped C7 M, and therefore C7 N donor plasma was selected for the second transfusion to allow identification of the source of the C7 circulating post-transfusion. This C7 phenotyped C7 M, demonstrating it to be of recipient origin. Therefore, the apparently completely C7-deficient patient was able to secrete some C7. By a combination of DNA typing and isoelectric focusing of the C7 appearing after transfusion, it was demonstrated that the patient was heterozygous for combined subtotal C6/C7 deficiency (inherited from his father) and a different, so far uncharacterized, subtotal C7 deficiency (inherited from his mother). The low amount of C7 secreted appeared to be constantly consumed, probably by generation of C5b6 as a result of his chronic infection. He had been shown to have circulating C5b6 most of the time, and thus only when sufficient exogenous C7 was given to consume the free C5b6 did his own C7 appear in circulation.

Bacterial Infections↗
PubMed1996

A new intronic polymorphism in the C7 gene 36 bp from the common expressed C7 M/N polymorphism.

We report a new polymorphism in the complement C7 gene that results from an A-C transversion in intron 12, 27 bp upstream of exon 13 (C712.-27) and 36 bp upstream of the point mutation that underlies the C7 M/N antigenic polymorphism. The C7 12.-27 polymorphism subdivides C7 M haplotypes, but not C7 N. It also sheds light on the evolution of the various types of deficiency genes at the adjacent C6 locus.

Base Sequence↗
PubMed1996

The Cayapa Indians of Ecuador: a genetically isolated group with unexpected complement C7 M/N allele frequencies.

The Cayapa Indians live in north-western Ecuador in close proximity to a Black population of African ancestry. C7 M/N allotyping has proved to be a good technique for plasma genetic analysis in several populations. Investigation of 124 Cayapa plasma samples revealed the highest allele frequency of C7*N observed in any population examined so far (0.36 versus 0.225 or lower). The marked difference in frequency compared with several Oriental populations, which are believed to have been derived from the same Asian population as native Amerindians, may reflect the effect of a small founder population followed by a high degree of genetic isolation. The allele frequency of 0.12 for C7*N determined for the neighbouring Black population supports the conclusion that there has been a lack of genetic admixture of Cayapas with other populations, confirming the results of ethnohistorical investigations and other protein polymorphism studies.

Alleles↗
PubMed1995

Human polymorphonuclear leukocytes store large amounts of terminal complement components C7 and C6, which may be released on stimulation.

Secretion of the C factors C7, C6, and C3 by human polymorphonuclear leukocytes (PMNs) and PBMCs was studied by ELISA and immunoblot. The release of C7 and C6 by PMNs during 24 h of culture was 16-fold and 6-fold higher, respectively, than the C3 release, with median concentrations of 50.2 ng/ml, 18.3 ng/ml, and 3.1 ng/ml, respectively. In PBMC cultures, C release was considerably lower, and there was a different secretory pattern with a 6-fold higher release of C3 compared with C7 and C6. Stimulation with PMA led to a more rapid and complete secretion of the components to the culture media, whereas treatment with unopsonized Candida species did not affect the release. PMN release of C factors was not dependent on protein biosynthesis, and there was no indication of a selective uptake of C7 from serum as demonstrated by incubating PMNs from a subject with allotype C7 N in C7 M serum. Thus, the C components were probably produced by the PMNs or their bone marrow precursors before ex vivo culture. In cell lysates of freshly isolated cells, median C7, C6, and C3 contents of 1 x 10(7) PMNs were 149.7, 60.1, and 10.4 ng/ml, respectively, whereas the corresponding values for 1 x 10(7) PBMCs were 3.2, 2.6, and 14.6 ng/ml, respectively. The C6 and C7 were shown to incorporate into the terminal complement complex, and their molecular integrity was supported by identical m.w. to C6 and C7 present in normal serum. PMNs may represent a major source of C7 and C6 and may be more important than monocytes or macrophages in contributing terminal C components at a site of inflammation. This suggests a new role for the PMN as a C membrane attack modulator.

Candida↗
PubMed1995

Molecular basis of the complement C7 M/N polymorphism. A neutral amino acid substitution outside the epitope of the allospecific monoclonal antibody WU 4-15.

The allotypes of the C7 M/N polymorphism are determined by comparing the ELISA reaction pattern of the allospecific mAb WU 4-15 with polyclonal anti-C7 IgG. To characterize the molecular basis of this polymorphism, the WU 4-15 epitope was mapped by expression of cDNA fragments. It was found to be located within the boundary region of the two short consensus repeats of C7 (amino acid residues 595-612). Coincidentally an A or C substitution was found in the course of investigation of the gene structure at nucleotide 1759. This leads to an electrically neutral Pro/Thr substitution at residue 565 and alternative Sau961 or RsaI restriction sites, respectively. Enzymatic digestion or sequencing of PCR-amplified genomic DNA from C7 M/N-typed individuals showed a perfect correspondence of the genotype with the phenotype of the C7 M/N polymorphism. In contrast, no association was found with the genotypes of a second polymorphic site at residue 367. Thus, a threonine at amino acid residue 565 allows access of WU 4-15 to its epitope but its substitution with proline almost completely inhibits access in the native molecule, although the epitope is about 40 amino acid residues distant from the polymorphic site in the primary structure. Furthermore, the different conformational structure is probably responsible for the hypomorphic appearance of C7*N.

Alleles↗
PubMed1995

Decay-accelerating factor (CD55) protects human immunodeficiency virus type 1 from inactivation by human complement.

HIV-1, in contrast to animal retroviruses, is not lysed by human complement, but is readily inactivated by the sera from different animal species. To identify a possible species-specific protection mechanism. HIV-1 was expressed in cells of non-human origin. Recombinant HIV-1 virions that could encode the chloramphenicol acetyltransferase (CAT) protein were produced in African green monkey COS-1 cells, mink cells and, as a control, in human HEp-2 cells and were then used to infect CD4-positive target cells. Analysis of the CAT activity of the target cells revealed that fresh HIV-1-negative human serum reduced the infectivity of HIV-1 derived from monkey and mink cells five- to tenfold, but had no effect on HIV-1 produced in human cells. In addition, human serum efficiently lysed HIV-1 produced in non-human cells in contrast to HIV-1 originating from human cells, suggesting lysis as an important mechanism of virus inactivation. Mammalian cells are protected against lysis by homologous complement by membrane-bound regulatory molecules. Two of these complement inhibitors, namely decay-accelerating factor (DAF) and, to a lesser extent, CD59 were found on the surface of HIV-1 virions by means of a virus capture assay. Antibodies against DAF, but not against other host cell molecules found on the viral surface, efficiently blocked the resistance of HIV-1 produced in human cells to human complement. These results suggest that the acquisition of DAF during the budding process from human cells protects HIV-1 in a species-specific way against the attack of human complement.

Animals↗
PubMed1995

Study of the in vitro activation of the complement alternative pathway by Echinococcus granulosus hydatid cyst fluid.

In the present study we have investigated the fluid phase activation of the complement (C) alternative pathway by Echinococcus granulosus sheep hydatid cyst fluid (SHCF) and its higher molecular weight fraction (SHCF-I) by quantitating the formation of both the terminal C intermediary C5b6 complex and the terminal C complex (TCC). Our results show that in vitro C activation progresses beyond the C5 step suggesting that potentially lytic complexes may be generated in vivo. In addition, SHCF and SHCF-I glucidic moieties are probably involved in C activation since 80% and 86% of SHCF and SHCF-I activity respectively was destroyed by periodate oxidation. Furthermore, partial deglycosylation with Peptide N-Glycosidase F of SHCF-I which had been digested with Pronase E, released an active fraction (MW < 14 KDa) which bound to Soybean agglutinin, suggesting that N-linked oligosaccharides containing alpha- or beta-linked N-acetyl galactosamine play a role in C activation by SHCF.

Acetylgalactosamine↗
PubMed1995

DNA polymorphisms of the complement C6 and C7 genes.

The linked C6 and C7 loci are rich in genetic markers, both at the protein and DNA levels. There are now seven common DNA polymorphisms distributed over about 300 kbp of chromosome 5p12-14. We report a new TaqI RFLP for C7 and a method for typing a C7 variant (T368S) hitherto known only from cDNA clones. We have re-investigated the published RFLPs to provide information on their frequency in North European Caucasian (predominantly British and Irish) subjects and have revised some of the published parameters, especially the sizes of polymorphic restriction fragments. Their precise locations within the genes are also reported: the three markers for C6 are in exon 3, intron 3 and adjacent to exon 17 and the four markers for C7 are in introns 15 and 13 and in exons 13 and 9. The gene frequencies of the second commonest allele of all seven markers lie in the range 0.2 to 0.37, except C6 A/B in the Japanese, where the frequencies of both common alleles are about 0.45. We have estimated the gene frequencies for the DNA polymorphisms which correlate with C7 M/N phenotype and for the C6 A/B phenotype and find them to be the same as the phenotypic estimates in Caucasians and in the Japanese respectively. The markers provide the possibility of defining 128 haplotypes, many (28) of which have been observed. Allelic associations in these genes are generally surprisingly weak.

Alleles↗
PubMed1995

Complement component C6 and C7 haplotypes associated with deficiencies of C6.

Both complete C6-deficiency (C6*Q0) and subtotal C6-deficiency (C6*SD) have been described as simple recessive traits and C6*SD has been described in combination with subtotal deficiency of the C7 coded at an adjacent locus. The trace of C6 protein found in both C6*SD traits is phenotypically indistinguishable, being smaller than normal C6 and having different isoelectric properties. A defect has been found in the C6 gene which plausibly explains the C6*SD phenotype, and this defect is also common to both C6*SD traits. We present data from seven DNA markers of the C6 and C7 genes which show that although at least four haplotypes are associated with C6*Q0, most South African C6*Q0 patients carry a common defective haplotype. The most common haplotype associated with C6*Q0 has been observed only once among unaffected haplotypes of relatives. In one family, the cases of C6*SD share a complete haplotype with both cases of combined deficiency and are probably heterozygous for this condition and complete deficiency of C6. In another family, the C6*SD is on a slightly different haplotype and C7 is normally expressed. Thus, the C6 defect is not sufficient on its own to explain the C7 deficiency in the combined deficient haplotype. The haplotype associated with the combined deficiency is found not only in normal control subjects, but also in one case of complete C6 deficiency. In this case the molecular defect seen in combined or C6*SD cases is absent.

Alleles↗
PubMed1995

Molecular basis of subtotal complement C6 deficiency. A carboxy-terminally truncated but functionally active C6.

Individuals with subtotal complement C6 deficiency possess a C6 molecule that is 14% shorter than normal C6 and present in low but detectable concentrations (1-2% of the normal mean). We now show that this dysmorphic C6 is bactericidally active and lacks an epitope that was mapped to the most carboxy-terminal part of C6 using C6 cDNA fragments expressed as fusion proteins in the pUEX expression system. We thus predicted that the abnormal C6 molecule might be carboxy-terminally truncated and sought a mutation in an area approximately 14% from the carboxy-terminal end of the coding sequence. By sequencing PCR-amplified products from this region, we found, in three individuals from two families, a mutation that might plausibly be responsible for the defect. All three have an abnormal 5' splice donor site of intron 15, which would probably prevent splicing. An in-frame stop codon is found 17 codons downstream from the intron boundary, which would lead to a truncated polypeptide 13.5% smaller than normal C6. This result was unexpected, as earlier studies mapped the C5b binding site, or a putative enzymatic region, to this part of C6. Interestingly, all three subjects were probably heterozygous for both subtotal C6 and complete C6 deficiency.

Amino Acid Sequence↗
PubMed1995

Complement C7 M/N allotyping in infectious diseases.

The allotypes of the C7 M/N polymorphism are determined by ELISA by comparing the reaction pattern of the allospecific monoclonal antibody WU 4-15 with that of polyclonal anti-C7 IgG. In order to find disease associations of the two alleles C7*M and C7*N we tested 528 hospitalised patients, most of them suffering from infectious diseases. No significant association of either of the two C7 M/N alleles to a particular disease was found, in particular refuting the hypothesis that Lyme borreliosis may be more frequent in homozygous carriers of the hypomorphic allele C7*N.

Alleles↗
PubMed1995

Complement factor H mRNA in Epstein-Barr virus-transformed B lymphocytes of a factor H-deficient patient: detection by polymerase chain reaction.

A Spanish family with a hereditary deficiency of factor H was identified in previous studies. The deficiency was subtotal as low amounts of a dysmorphic molecule with partial identity to factor H were detected in her serum. The aim of this study was to obtain further characterisation of her deficiency employing her immortalised lymphocytes. After purification of mRNA from her Epstein-Barr virus (EBV)-transformed lymphocytes and from Raji cells, and cDNA synthesis we succeeded in amplifying cDNA which codes for factor H domains 13-15 from both cell lines, using specific oligonucleotides. The amplified fragments were indistinguishable from the one which was amplified from the control cloned cDNA template. The failure to amplify the 3' end of the patient's factor H mRNA suggests that the mRNA is truncated, which is consistent with the characteristics of the dysmorphic protein. Thus, factor H mRNA was detectable in Raji cells and the patient's EBV-transformed lymphocytes. We suggest that immortalised B lymphocytes can be used for the study of a range of inherited complement deficiencies.

B-Lymphocytes↗
PubMed1995

Importance of the third thrombospondin repeat of C6 for terminal complement complex assembly.

The anti-C6 monoclonal antibody WU 6-4 was shown to be unequivocally native restricted since it neither binds to the terminal complement complex (TCC) nor to C5b6. In addition, it was shown to inhibit TCC formation by interfering with C5b6 generation. Using the pUEX expression system and C6 cDNA the WU 6-4 epitope was mapped to the third thrombospondin repeat of C6. This protein domain may therefore contribute to the C5 binding site of C6 and be involved in terminal complement complex assembly. The presence of the epitope in rabbit C6 indicates a useful model for studying inhibition of TCC formation in vivo.

Animals↗
PubMed1994

Complement component C7. Assessment of in vivo synthesis after liver transplantation reveals that hepatocytes do not synthesize the majority of human C7.

C7 M/N typing, the determination of the allotypes of the recently described C7 M/N protein polymorphism, was conducted on serum samples from donors and recipients of 100 liver transplantations to determine whether the liver is the predominant site of in vivo synthesis of human complement protein C7. Twenty-one cases were informative as the recipient was transplanted with the liver obtained from a donor with a different C7 M/N allotype. The determination of the C7 levels and phenotypes of up to 10 post-transplantation (p.t.) samples revealed that there was at most only a 50% contribution of the transplanted liver toward the C7 M/N allotype at 2 to 3 wk p.t.; that influence decreased with time and was approximately 10% in the samples obtained later than 6 wk after transplantation. C7 is thus the only terminal complement component not predominantly synthesized by hepatocytes, which is compatible with the observation that C7 is not an acute phase reactant. The transient contribution of the donor phenotype appears to be attributable to cells of the mononuclear phagocyte lineage, Kupffer cells in particular that are replaced by cells of recipient origin. Various cells of that lineage that are known to synthesize C7 in vitro, therefore, contribute more toward the C7 concentration than previously anticipated. Enhanced local C7 synthesis at the site of inflammation might add further to the basic C7 level especially because C7 is often the limiting factor for terminal complement complex generation.

Complement C7↗