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.

Major histocompatibility complex (MHC) class III genetics in two Amerindian tribes from southern Brazil: the Kaingang and the Guarani.

Population genetic studies of the major histocompatibility complex (MHC) class III region, comprising C2, BF and C4 phenotypes, and molecular genetic data are rarely available for populations other than Caucasoids. We have investigated three Amerindian populations from Southern Brazil: 131 Kaingang from Ivaí (KIV), 111 Kaingang (KRC) and 100 Guarani (GRC) from Rio das Cobras. Extended MHC haplotypes were derived after standard C2, BF, C4 phenotyping and restriction fragment length polymorphism (RFLP) analysis with TaqI, together with HLA data published previously by segregation analysis. C2 and BF frequencies corresponded to other Amerindian populations. C4B*Q0 frequency was high in the GRC (0.429) but low in the Kaingang. Unusual C4 alleles were found, viz. C4A*58, A*55 and C4B*22 (presumably non-Amerindian) and aberrant C4A*3 of Amerindian origin occurring with a frequency of 0.223 in the GRC. C4A*3 bands of homo- and heterozygous individuals carrying this variant were Rodgers 1 positive and Chido 1,3 positive, showed a C4A specific lysis type and a C4A like alpha-chain. Polymerase chain reaction studies and sequencing showed that this is based on a C4A*3 duplication with a regular C4A*3 and a partially converted C4A*0304 carrying the C4B specific epitopes Ch 6 and Ch 1,3. Associations of class III haplotypes with particular RFLP patterns were similar to those reported for Caucasoids. The previously described association between combined C4A and CYP21P deletions and the 6.4 kb TaqI fragment was not seen in these Amerindians. This fragment occurred within a regular two locus gene structure in the Kaingang, representing a "short" gene at C4 locus I. C4 and CYP21 duplications were frequently observed. The distribution of extended MHC haplotypes provides evidence for a close relationship between the KIV and KRC and a larger genetic distance between the two Kaingang groups and the GRC.

Molecular biology in diagnosis and epidemiology of Helicobacter pylori: PCR for the detection and AP-PCR for characterization of patient isolates.

Infection with Helicobacter pylori causes chronic active gastritis and has been associated with gastric and duodenal ulcer disease. In biopsy samples of 110 patients with clinical symptoms of active gastritis, H. pylori was detected by means of the polymerase chain reaction (PCR), using species-specific primers defining a 858 bp DNA fragment of H. pylori urease beta-subunit. Sensitivity and specificity of the PCR was compared with culture, histology and Warthin-Starry stain (WSs), detection of H. pylori urease antibodies in serum and urease testing with the Campylobacter-like organism (CLO) test. PCR yielded specific amplification products in 53 cases, whereas culture of the organisms was positive in a subset of 50 cases. Only direct detection in histological sections of biopsy specimens had a higher sensitivity, with 65 positive samples. In contrast, the CLO test was negative in eleven culture-positive and PCR-positive cases. Significant urease antibody titres were found in 39 patients with histologically confirmed diagnosis. These results placed the sensitivity of PCR between tat of the Warthin-Starry stain (WSs) and that of culture. Therefore, PCR can be proposed as a useful rapid and time-saving technique for the detection of H.pylori in gastritis. For epidemiological purposes, fingerprinting with arbitrarily chosen primers by AP-PCR was evaluated. Strain-specific patterns with up to 13 fragments were achieved with 10-nucleotide or longer primers (21-nt) with a G + C content > or = 55%. Thirty-five of 40 strains investigated by this method were distinguishable with a single primer. These results suggest a high level of DNA sequence diversity within this species with the possibility of confirming the clonality in consecutive isolates from a single individual. Alternatively, an increased in-vivo mutation rate could be responsible for DNA divergence, resulting in specific strains for each individual patient.

Campylobacter and Salmonella contaminating fresh chicken meat.

1853 packages of fresh chicken breast meat of German, Dutch and French origin were investigated for their contamination with Campylobacter and/or Salmonella. Swabs were taken and cultured from dripwater, meat surface, meat interior and packet bowl. Campylobacter was isolated from 619 meat samples (= 33%), Salmonella from 377 meat packages (= 20%). In 111 of these contaminated chicken samples, both Salmonella and Campylobacter were present. The contamination rate and the species spectrum observed differed depending on the origin of the packages and the time of control.

Serum antibody reactivity to recombinant mig and whole cell antigens in Mycobacterium avium infection.

Mycobacterium avium is a significant opportunistic pathogen in immunocompromised patients. Moreover, the prevalence of infections in patients without known predisposing conditions has also been increasing in recent years. Patients would greatly benefit from early diagnosis of disseminated infection. Serodiagnostic tests have already been promising in tuberculosis and immunocompetent patients but studies in HIV-infected patients and humoral response to M. avium antigens resulted in conflicting data. We have evaluated the use of the phagocytosis-induced MIG protein of M. avium as a diagnostic antigen. Serum antibody levels of M. avium-infected, HIV-negative patients were significantly elevated for the recombinant MIG (p < 0.001) and also for M. avium whole-cell antigens (p < 0.025) as compared to controls. In contrast, HIV-infected patients with disseminated M. avium infection demonstrated also elevated levels of antibody for the whole-cell antigen (p < 0.00001) but a decreased reactivity for the MIG antigen (p < 0.007). The recombinant antigen proved to have no cross-reactivity with M. tuberculosis antigens as antibody levels were decreased in tuberculosis patients (p < 0.001). Therefore, a simultaneous serological test using recombinant MIG and the whole cell antigens might be helpful in the sometimes problematic diagnosis of M. avium infections in patients without predisposing conditions.

Detection of 4-quinolone resistance mutation in gyrA gene of Shigella dysenteriae type 1 by PCR.

To study 4-quinolone resistance, the N-terminal coding region of gyrA from nalidixic acid-susceptible and -resistant isolates of Shigella dysenteriae type 1 was amplified by PCR, cloned, and sequenced. DNA sequence analysis of gyrA from nalidixic acid-resistant isolates revealed a C-to-T transition at nucleotide position 248 leading to a Ser-83-to-Leu substitution which was absent in susceptible clinical isolates. Direct HinfI digestion of PCR-amplified DNA detected similar mutations. Thus, DNA gyrase A subunit mutation Ser-83 to Leu is implicated in 4-quinolone resistance in S. dysenteriae type 1.

An estimate on the frequency of duplicated haplotypes and silent alleles of human C4 protein polymorphism. II. Investigations in healthy Negro families.

The first investigation of complete MHC marker data in South African Negroes by segregation analysis in 11 families with up to three generations is presented, including quantitative evaluation of C4 allotype patterns and C4 beta chain determinations according to Steuer et al. (1). The frequency of homo- and heteroduplicated, hybrid, and non-expressed C4 alleles was determined from C4 protein phenotyping, including C4 alpha and beta chains, quantitative estimates of the relative electrophoretic C4 banding patterns by scanning densitometry, and from the other classical MHC markers by submitting all results to the family analysis program (FAP). From unrelated non-diseased individuals (n = 105) in these families with 62 haplotypes, the following frequencies were observed for non-expressed alleles: C4A*Q0 0.1189, C4B*Q0 0.2552, and for the total of heteroduplicated alleles: C4A 0.0645, C4B 0.0608. Applying additionally quantitative determinations of C4 banding patterns, homoduplications such as C4A*3 A*3, C4B*1 B*1, C4B*3 B*3, and the heteroduplication C4A*3 A*2 were assumed. In the investigated individuals the heteroduplications of C4A*12 and C4A*3 with the A*91 allele and of C4B*2 with C4B*92 were observed. It was concluded that not only allele frequencies but also the frequency of heteroduplications seems to be of specific ethnic character. Furthermore, the prior hypothesis that deletion or non-expression at one C4 locus is accompanied by duplication at the other was only confirmed for non-expressed B-alleles with C4A*3 A*91 or C4A*12 A*91.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic variability of the MHC class III complement proteins C2, BF, C4A and C4B in southern Brazil.

We describe here the genetic variability of C2, C4 and BF in 225 healthy adult individuals from southern Brazil, including 172 Caucasoids, 47 Mulattos, 3 Blacks and 1 Amerindian. C2 gene frequencies were in accordance with those described for other populations, and two rare variants were observed. The BF allotype frequencies were slightly different between the Caucasoids and Mulattos, the latter having a higher BF*F frequency. A new BF*S variant, identified as S05 was observed in a Caucasoid individual. The frequencies of C4A and C4B in the Caucasoids were similar to other reported Caucasoid populations; a decrease of the silent allele B*Q0, and several rare variants were observed. A higher C4A*3 frequency and a remarkable decrease of C4A*Q0 were observed in the Mulattos. In addition, several C4 heteroduplications and aberrant allotypes were observed. Considering the high genetic variability found in a limited number of individuals, one may conclude that due to genetic admixture much heterogeneity might be expected for the MHC class III region in different Brazilian populations.

Humoral and cellular immunity in HIV positive and HIV negative Helicobacter pylori infected patients.

The prevalence of H. pylori associated gastritis seems to be different in HIV positive and HIV negative patients. Therefore a correlation to immunodeficiency can be postulated. The histology of gastritis, status of H. pylori infection and parameters of humoral and cellular immune response were investigated in 41 HIV positive and 47 HIV negative patients, who were subjected to upper endoscopy for the evaluation of gastrointestinal symptoms. In HIV positive patients 37% had active chronic gastritis against 62% of the HIV negative patients. In 73% of HIV positive cases of active chronic gastritis H. pylori was detected by bacteriological culture and/or Warthin-Starry stain. In HIV negative patients active chronic gastritis was always associated to H. pylori infection. Production of antibodies as measured by two commercially available ELISA tests was significant in HIV positive and HIV negative patients; both tests correlated well with H. pylori detection by culture or direct microscopy. Immunoglobulin class specific immunoblots corresponded to the ELISA results in HIV negative patients but to a lesser extent in the HIV positive group which was assumed to be related to unspecific polyclonal activation in these patients. Systemic cellular immunity was investigated by proliferation assays of peripheral blood mononuclear cells (PBMC). Proliferative response to the unspecific mitogen PHA was reduced in HIV positive patients. A sonicated H. pylori antigen failed to induce lymphocyte proliferation. The antimitogenic effect was also seen in case of coincubation with PHA. This observation was independent of H. pylori and HIV infection status. We conclude that in HIV positive as in HIV negative patients active chronic gastritis is predominantly related to H. pylori infection. The prevalence of H. pylori associated gastritis in HIV positive patients is significantly reduced (p < 0.025) compared to HIV negative controls. Decreased susceptibility to H. pylori infection in HIV positive patients may not be explained by the abnormal reactivity of their humoral or cellular immune response.

Helicobacter pylori antibodies in sera of children suffering from chronic abdominal pain.

107 pediatric patients aged 9 to 18 with persistent gastric complaints were examined serologically and bacteriologically for Helicobacter pylori. Helicobacter was identified in 48 (45%) of individuals. 51 (48%) of children were found to be seropositive when H. pylori antibodies were detected by the ELISA; 56 (52%) when the passive haemagglutination test was used, and 41 (38%) in the latex agglutination test. 25% of culture-negative patients were found to be seropositive. The percentage of raised H. pylori antibody titres in the control (healthy subjects) varied from 20 to 27%, depending on the method applied.

Association of C4B deficiency (C4B*Q0) with erythema nodosum in leprosy.

A considerable number of studies have postulated significant associations between susceptibility to the different clinical manifestations of leprosy and the MHC. In this investigation, the association between the MHC class III complement proteins C2, BF, C4A and C4B and leprosy in a patient population of Southern Brazil was studied. A total of 109 non-related leprosy patients was investigated; 73 presented with lepromatous leprosy (LL), 46 of them had the immunopathological reaction of erythema nodosum (ENL), the remaining 36 were tuberculoid, borderline and indeterminate leprosy (TIBL) patients. The control group included 172 healthy individuals matched with the patients according to their ethnic and geographical origin. C2, BF, C4A and C4B allotypes were determined by standard technologies including Western blots for C2 and C4 variant alleles with monoclonal and polyclonal antibodies. Non-expressed ('silent') C4 alleles in hemizygously deficient individuals were estimated semiquantitatively on the basis of the C4A and C4B isotype ratio and by the MASC ('minimal chi-square') method. The results showed a significantly elevated presence of the non-expressed C4B allele (C4B*Q0) in the LL and ENL patient groups in comparison with the controls. The most significant difference was observed in the ENL group when compared with the controls. In addition, all patients who were homozygously C4B-deficient had ENL, and most of them had the BF*F1 allele. The comparison between LL patients with and without ENL also showed a statistically significant difference in the presence of C4B*Q0, indicating that C4B deficiency itself is associated with ENL. The relative risk of LL patients with the C4B*Q0 allele suffering from ENL was 5.3 compared with LL patients without C4B*Q0. Since immune complexes (IC) are considered to be the pathogenic cause of ENL, our findings indicate that C4B deficiency may play an important role in the abnormal immune response against Mycobacterium leprae and in the lack of IC clearance, leading to ENL reactions. Individuals with this allele seem to be at a higher risk of developing pathological immune reactivity in lepromatous leprosy.

Improvement of BF typing and of BF F subtyping after neuraminidase treatment in the unconverted and converted factor B.

When neuraminidase-treated sera are analyzed by agarose gel isoelectric focusing, the factor B (BF) banding pattern is reduced to predominantly one major band without cathodically positioned bands. This not only makes unequivocal typing of BF allotypes possible but also the reliable distinction of all BF F subtype phenotypes with delimitation of "BF F subtype variants". With this new method, serum aging affects the BF determination to a lesser extent than when applying methods that separate native sera. We show that sialylation is not responsible for the BF F subtype polymorphism. All of the investigated BF allotype bands, including those characteristic of the subtypes, show functional hemolytic activity. The banding pattern after removal of neuraminic acid residues ranges from pH 6.8 to 7.3 for factor B, from pH 5.3 to 5.9 for the Ba fragment, and from pH 8.2 to 8.7 for the Bb fragment. The protein structure of factor B is also discussed. Eliminating the superimposition of bands in different BF allotypes, as demonstrated by these methods, proved to be necessary for the detection of hypomorphic BF gene products (BF QL), which are expressed by assumed BF*Q0 alleles in heterozygous genotypes. This allows investigation of BF*Q0 alleles on a protein level, which complements molecular genetic approaches.

Apparently non-expressed alleles of factor B (BF) code for hypomorphic proteins.

In three families with an apparent non-expressed factor B (BF) allele (BF*Q0), advanced methods of isoelectric focusing for the determination of BF F subtypes revealed different hypomorphic BF products (BF QL) with functional hemolytic activity expressed by the assumed BF*Q0 allele. A Taq I and a Msp I restriction fragment length polymorphism as well as the Ba fragment of the expression products showed banding patterns for the BF*QL alleles corresponding to BF S types, whereas an altered Bb fragment was seen in two BF QL products. In one family an intragenic recombination site within the Bb part of the BF gene was assumed. Investigations of factor B and its conversion fragments, as demonstrated by the used methods, allow to complement molecular genetic investigations of BF*Q0 alleles in heterozygous genotypes on a protein level. We conclude that apparently non-expressed alleles of factor B code for hypomorphic but functionally active proteins.

Association of major histocompatibility complex class III complement components C2, BF, and C4 with Brazilian paracoccidioidomycosis.

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.

Restriction fragment length polymorphism for the identification of Campylobacter jejuni-isolates.

Companion animals ("pets") are occasionally carriers of organisms pathogenic for man. In the present, study fecal samples of clinically inapparent animals with direct contact to 204 patients, suffering from campylobacter enteritis, were investigated for C. jejuni or C. coli (CJC). CJC positive animals were seen in the environment of only five patients (= 2.4%). By comparison of biotypes and serotypes of thermostable and thermolabile antigens from human and animal isolates no clear epidemiological relationship could be deduced. Using chromosomal DNA of the strains, genetic identity of the isolates was studied for HaeIII-restriction fragment length polymorphism (RFLP), applying a biotinylated commercial CJ probe. The probe was found to be specific for most CJ strains and revealed a pattern of one to four bands. In contrast to biotyping no identity of patient strains and animal isolates was seen in three cases; one case with different biotypes had identical RFLP patterns; one patient CJ strain did not show any pattern with the CJ probe. Serotypes were identical for a larger number of animal strains but differed in HaeIII RFLP and vice versa. Comparing the results from the different technological approaches it seems impossible to give a clear statement on the epidemiology of campylobacter infections or carrier state by biotyping alone. It is concluded that DNA RFLP patterns are a useful additional tool, but for epidemiological analysis a set of different methods should be used.

Early onset sensorineural hearing loss: association studies with major histocompatibility class III (complement) markers.

In 39 families with at least one child suffering from moderate or severe bilateral sensorineural hearing loss (SNHL), major histocompatibility complex (MHC) class III complement phenotypes were retrospectively determined by standard methods; MHC class I segregation data was also available. The families were treated in the Hospital for Communication Disorders and selected for the HLA-B16 and B18 specificities, respectively. Haplotype and allele frequencies were derived from segregation analysis in the families. From 31 unrelated children with random and familiar forms of SNHL significant deviations in the distribution were seen for the following MHC class III alleles using as a control population 60 German healthy individuals: duplicated C4A alleles (C4"DA") p = 0.009, silent C4A alleles (C4A*Q0) p = 0.006, duplicated heavy C4 beta-chain alleles (C4 beta"DHH") p = 0.0003, and silent C4 beta-chain alleles (C4 beta*Q0) p = 0.0075. In serum samples from patients with an assumed genetic disposition according to clinical criteria indications for an association were found for C4"DA" (p = 0.03), C4A*Q0 (p = 0.003), C4B*3 (p = 0.046), C4 beta"DHH" (p = 0.004), and C4 beta*Q0 (p = 0.02). The underrepresentation of C4A*Q0 may be an indicator for aberrant or duplicated C4 alleles on the same haplotype or exhibit a protection mechanism for acquiring the inheritable forms of early onset SNHL.

Major histocompatibility complex class I to III allotypes in patients with AIDS-related complex/Walter-Reed 5, disseminated Kaposi's sarcoma and in normal controls. The ARC-IVIG Study Group.

In HIV-infected patients major histocompatibility complex (MHC) class I and II (= HLA-A, B, C, DR) association has been controversial. Of the MHC class III coded complement components C2, BF, C4A/C4B especially C4 allotypes appear of major immunogenetic relevance for their potential differences in virus neutralizing potency and immune complex formation. In the present study 29 patients with AIDS-related complex and Walter-Reed 5 ARC/WR5), 35 patients with disseminated Kaposi's sarcoma (KS), and 160 HIV-negative control individuals were compared for MHC class I to III allotypes. Diagnosis of ARC and KS (WR criteria) was done by clinical and laboratory parameters, MHC testing, by standard procedures. An increase in frequency (p less than or equal to 0.05) was observed between ARC/WR5 patients and controls for HLA-B35/CW4, DRW14, a decrease for B16, CW6/DR7. However, values were not significant if corrected for the number of tested antigens. No significant differences were seen between KS and ARC patients or controls for class III allotypes, nor for previously reported associations, e.g. for B8, DR2, DR3, and especially DR5, including the DR5 splits DRW11, 12. The results indicate the lack of a strong MHC association with the investigated antigens in West German Caucasoids, and support the hypothesis of ethnic dependence of HIV-related diseases. The HLA-B35/CW4 increase, also associated with the duplicated C4 A*3 A*2 and the silent C4B*Q0, was more pronounced in ARC patients with progression to AIDS-OI. The increased frequency of C4B*Q0 alleles in these patients was thought to be secondary to a hypothetical increase in 'converted' and dysregulated C4 genes not seen to be associated in this study.

Complement levels and circulating immune complexes in a controlled, longitudinal, multicentre study on effects of intravenous immunoglobulin in adults with AIDS-related complex/Walter-Reed 5. The ARC-IVIG Study Group.

The complement system, as the effector mechanism of the antigen-antibody reaction, and the levels of circulating immune complexes in a 1-year, double-blind, randomized, placebo-controlled study served as laboratory parameters to assess the effect of long-term high-dose intravenous immunoglobulin (IVIG) therapy in 30 adult patients (2 x 15) with AIDS-related complex/Walter-Reed 5 (ARC/WR5). We obtained no evidence of an adverse effect in such patients of high-dose IVIG administered over a 6-month period: none of the parameters studied showed a significant difference between the two groups of patients. In both groups, using data before the first infusion or using data of the whole study, a correlation between circulating immune complexes and classical complement pathway activation was found. The most striking increase was seen in the two groups of patients for functional serum factor D. The accumulation of serum factor D was not paralleled by an increase in serum creatinine. In patients with disease progressing from ARC to AIDS within the study period, an accumulation of serum factor D was not more or less pronounced than in those who remained in the ARC stage. Accumulation of factor D was not related to the clinical score as assessed in this study.