A complement receptor-1 polymorphism with high frequency in malaria endemic regions of Asia but not Africa.

Complement receptor-1 (CR1) is a ligand for rosette formation, a phenomenon associated with cerebral malaria (CM). Binding is dependent on erythrocyte CR1 copy number. In Caucasians, low CR1 expressors have two linked mutations. We determined the Q981H and HindIII RFLP distribution in differing population groups to ascertain a possible role in adaptive evolution. We examined 194 Caucasians, 180 Choctaw Indians, 93 Chinese-Taiwanese, 304 Cambodians, 89 Papua New Guineans (PNG) and 366 Africans. PCR/RFLP used HindIII for CR1 expression and BstNI for the Q981H mutation. DNA sequencing and pyrosequencing were performed to resolve inconclusive results. Gene frequencies for the L allele were 0.15 in Africans, 0.16 in Choctaws, 0.18 in Caucasians, 0.29 in Chinese-Taiwanese, 0.47 in Cambodians and 0.58 in PNG. Allelic frequency for 981H were 0.07 in Africans, 0.15 in Caucasians, 0.18 in Choctaws, 0.29 in Chinese-Taiwanese, 0.47 in Cambodians and 0.54 in PNG. The Q981H polymorphism correlates with the HindIII RFLP in most groups except West Africans and appears to be part of a low CR1 expression haplotype. The gene frequency for the haplotype is highest in the malaria-endemic areas of Asia, suggesting that this haplotype may have evolved because it protects from rosetting and CM.

A simple method for accurate quantification of complement receptor 1 on erythrocytes preserved by fixing or freezing.

The mean number of complement receptor 1 (CR1) molecules on erythrocytes differs between normal individuals within the range of 100-1000 molecules per cell. In some disease states such as systemic lupus erythematosus (SLE), acquired immune deficiency syndrome (AIDS), insulin-dependent diabetes mellitus and malaria, erythrocyte CR1 levels are reduced and CR1 function may be impaired. Current methods for determining erythrocyte CR1 levels by flow cytometry require the use of freshly drawn blood samples because CR1 is lost from erythrocytes during storage. In order to facilitate field studies of associations between erythrocyte CR1 levels and disease, we have developed and validated an assay to quantify CR1 on both healthy and diseased erythrocytes that have been fixed in 5% formaldehyde or frozen in glycerol. These methods enable blood samples to be collected in areas lacking the facilities for flow cytometry and stored for later accurate quantification of CR1. Such procedures will be of particular benefit for future investigations of erythrocyte CR1 expression level and malaria susceptibility.

Section 1B: Rh flow cytometry. Coordinator's report. Rhesus index and antigen density: an analysis of the reproducibility of flow cytometric determination.

Fifty-seven IgG monoclonal anti-D antibodies were evaluated in the Rh flow cytometry section, in which 12 laboratories participated. Staining protocols and a fluorescein (FITC)-conjugated Fab fragment goat anti-human IgG (H + L) as a secondary antibody were recommended but not mandatory. A CcDEe red blood cell (RBC) sample that was shown to be homozygous for RHD by molecular methods was supplied and used as internal 'standard RBC' throughout all experiments. An RBC panel comprising two partial D and four weak D types was supplied as well. The use of standard RBC reduced the variability of the data among the laboratories and allowed the conversion of fluorescence data into epitope densities, which were compounded in an antigen density (antigen D per RBC). The highest antigen density was determined for DVI type III, followed by DVII and weak D type 3; the lowest antigen density were determined for weak D type 1 and type 2. Nine of the 12 participating laboratories discriminated three groups of aberrant RhD that had similar Rhesus indices (RI): D category VI with RI = 0; weak D type 2 and type 3 with an high RI; and D category VII and weak D type 1 with an intermediate RI. The antigen densities and the Rhesus indices obtained correlated well among the laboratories of this Workshop section despite different staining protocols, secondary antibodies and instrumentation.

New HLA-DRB1*07 allele identified from microlymphocytotoxicity/SSP discrepancies.

A HLA-DRB1*07 variant allele has been identified in a cadaver kidney donor. Serological typing using monoclonal antibodies detected HLA-DR4 and HLA-DR7. HLA class II DNA typing using sequence-specific primer (PCR-SSP) polymerase chain reaction only detected DRB1*04, while sequence-specific oligonucleotide (PCR-SSO) polymerase chain reaction confirmed the presence of both DRB1*04 and DRB1*07 alleles, although two extra reactions were also found. Exon 2 of the HLA-DRB1*07 was isolated using allele-specific PCR, then cloned and sequenced. Four mutations, at positions 170 (T --> C), 171 (C --> T), 174 (C --> G), and 179 (C --> A), were observed. These mutations changed codons 57 and 60 (V --> A; S --> Y, respectively). This amino acid sequence at position 56-61 is only found in DRB1*0811.

Distribution of the synergistic haemolysin genes hld and slush with respect to agr in human staphylococci.

Several staphylococcal species express a synergistic activity which potentiates hemolysis by beta-hemolysin. In Staphylococcus aureus, this activity is mediated by the delta-hemolysin, coded by the hld gene within the agr locus. In S. lugdunensis, the equivalent activity results from the production of 3 small peptides coded by an operon named slush, distinct from hld and located outside the agr region. We examined 15 clinically relevant staphylococcal species for the presence of hld, slush and agr by specific hybridisation. All species contained a recognizable agr-related locus. Three species never produced synergistic haemolysis and contained neither hld nor slush. Of the 12 producer strains, 5 contained hld, apparently within the agr region, 4 contained slush and one (S. caprae) contained both. Two other producer species (S. hominis and S. simulans) hybridised to neither probe.

Synergistic hemolytic activity of Staphylococcus lugdunensis is mediated by three peptides encoded by a non-agr genetic locus.

Some strains of the coagulase-negative Staphylococcus lugdunensis produce a synergistic hemolytic activity (SLUSH), phenotypically similar to the delta-hemolysin of S. aureus. Reverse-phase high-pressure liquid chromatography of supernatants from S. lugdunensis 307 yielded three late-eluting peaks of 3.5 kDa with synergistic hemolytic activity. A degenerate oligonucleotide probe was designed from partial amino acid sequences of the 23-amino-acid (aa) tryptic fragments from one of the three peaks and hybridized to a single 2.8-kb HindIII chromosomal fragment. The relevant portion of this fragment was cloned by PCR, and sequencing showed the presence of three related open reading frames (ORFs), SLUSH-A, SLUSH-B, and SLUSH-C, preceded by an unrelated short potentially coding sequence (ORF-X), cotranscribed on a polycistronic 838-nucleotide mRNA. The amino acid sequences of the peptides from the three peaks align perfectly with the predicted sequences from the three SLUSH ORFs (peak I = SLUSH-B; peak II = SLUSH-C; peak III = SLUSH-A). These three peptides are closely related (amino acid homology, >76%) and do not show significant homology to S. aureus delta-hemolysin but do resemble a Salmonella typhimurium invasin and the "gonococcal growth inhibitor," a bacteriocin secreted by Staphylococcus haemolyticus. The predicted ORF-X gene product is a 24-aa peptide with no homology to the SLUSH peptides.