Diversity of fliC gene in commensal Escherichia coli derived from various mammals.

Relations between the diversity of the fliC gene conditioning flagellum protein in E. coli and the source of the strain origin are presented. The fliC genes have been identified and characterized in commensal E. coli derived from 10 healthy animal species living in Zoo Safari Park (Poland). The fliC gene was found in 150 strains by the PCR method. The amplifiedfliC products revealed single bands within the range 1.26-2.16 kbp. Forty restriction patterns (classed by restriction analysis with the use of RsaI (PCR-RFLP RsaI; R-types) were determined. The neighbor-joining method was employed to illustrate the distribution of the kinds of R-types. There are 3-8 various R-types of a diversified frequency of occurrence in strains. Application of PCR-RFLP RsaI permitted the identification of alleles of fliC genes characteristic for E. coli and the estimation of their diversity among the animal species. The transmission ways of E. coli fliC+ between organisms of different species were determined and confirmed the role of transmission and horizontal gene transfer in the generation of the allelic diversity of fliC gene in natural E. coli populations.

Heterogeneity of Escherichia coli derived from artiodactyla animals analyzed with the use of rep-PCR fingerprinting.

Genetic polymorphism of 83 isolates of E. coli, derived from 4 species of artiodactyla animals living in a relatively close contact on the grounds of a theme park ZOO Safarii Swierkocin (Poland) was determined using the rep-PCR fingerprinting method, which utilizes oligonucleotide primers matching interspersed repetitive DNA sequences in PCR reaction to yield DNA fingerprints of individual bacterial isolates based on repetitive extragenic palindrome (REP) primers. The fingerprint patterns demonstrated the essential polymorphism of distribution of REP sequences in genomes of the examined isolates. The arithmetic averages clustering algorithm (UPGMA) statistical analysis of fingerprints with the use of the Jaccard similarity coefficient differentiated E. coli isolates into three similarity groups containing various numbers of isolates. The groups comprised isolates derived from two, three and four species of the source animals. The isolates derived from each source segregated in the dendrogram in a different way, both within the similarity groups and among them, indicating an individual repertoire of E. coli in the examined species of animals. The similarity relations among E. coli derived from the same source, illustrated in a dendrogram with a number of subclusters of a low mutual similarity (< or = 20%), indicated an essential interstrain differentiation in terms of the distribution of REP sequences. Our results confirmed the hypothesis of the oligoclonal characters of populations obtained from particular sources. The rep-PCR fingerprinting method with REP primers is simple and highly differentiating and can be recommended for use in explorations of large groups of animals and monitoring the variability of strains.

Rep-PCR--a variant to RAPD or an independent technique of bacteria genotyping? A comparison of the typing properties of rep-PCR with other recognised methods of genotyping of microorganisms.

The paper presents technical aspects of rep-PCR fingerprinting technique and compares its typing abilities, differentiation power and reproducibility with other recognised and recommended genotyping methods. Although rep-PCR fingerprinting is similar to MAAP techniques, it demonstrates some essentially different elements. The data presented in this review, indicate a rep-PCR genomic fingerprinting technique as a highly discriminating, independent screening method for determining the taxonomical diversity of bacterial population.

Application of rep-PCR fingerprinting for genotyping of Escherichia coli strains in Wojnowskie Wschodnie and Wojnowskie Zachodnie lake.

The paper presents usefulness of application of the PCR-based fingerprinting method, which uses enterobacterial repetitive intergenic consensus primers ERIC (rep-PCR (ERIC)) in analysing and characterising Escherichia coli population in the water environment. 46 E. coli isolates of homogenous biochemical properties were analysed. The received results prove considerable genomic diversity among the analysed isolates. The used technique has turned to be a reproducible and rapid method with a considerable differentiation power. The introductory research has revealed that the technique may be successfully used in qualitative research, for intra-species differentiation of microorganisms occurring in water environment.

A new lymphoblastoid cell line defective in class II HLA expression.

A lymphoblastoid cell line, HAJ, was derived by in vitro transformation with Epstein-Barr virus of peripheral blood lymphocytes (PBL) from a patient with renal insufficiency awaiting kidney graft. Cell surface expression of class I and class II HLA molecules was determined by flow cytofluorimetry using monoclonal antibodies and compared with that of cell line PAJ similarly derived from a healthy donor. HAJ cells expressed class I antigens at levels comparable with PAJ cells. In contrast, class II antigens were absent from the cell surface of HAJ cells while they were abundant on PAJ cells. Permeabilization and fixation of cells with acetone/formaldehyde solution revealed intracellular Ki-67 antigen but not class II HLA molecules. The genes for HLA-DR beta, DQ alpha and DP alpha were present in the HAJ genome as detected with polymerase chain reaction (PCR) using locus-specific primers amplifying a second exon. In RT (reverse transcriptase)-PCR, transcripts of DQA1 and DPA1 genes were easily detectable in PAJ (positive control) but not in HAJ cells. These results suggest a defect in HAJ cells of transcription of genes for all class II antigens. The cell line HAJ may prove to be an interesting model for in vitro studies of molecular mechanisms of the regulation of class II expression.

[Effect of recombinant human erythropoietin on kinetics of alloantibodies in patients awaiting renal allograft]. / Wplyw leczenia ludzka rekombinowana erytropoetyna na kinetyke alloprzeciwcial u chorych oczekujacych na przeszczep nerki.

Iterative transfusions are one of the most important mechanism of the induction as well as maintenance anti-HLA antibodies. We evaluated the evolution of PRA (panel reactive antibodies) in 46 sensitized patients during and after withdrawal of blood transfusion. Twenty nine of them (22 women and 7 men in age 38 +/- 7 years) were treated with EPO for 13 to 60 months. With EPO therapy anemia improved, allowing transfusions withdrawal in 19 patients. PRA level decreased from 62 +/- 23% to 42.5 +/- 25% after 1 year of therapy. At 2 year PRA reduced to 37.7 +/- 30% and remained on this level at 3 year. PRA levels became negative in 4 patients, decreased in 11 (by 35%) and remained high in 7 patients. The results of the patients were compared with 17 sensitized patients (9 women, 8 men in age of 40 +/- 10) who did not receive EPO and only part of them required sporadicly transfusions (mean 3 unit of blood) during last 2-3 years. PRA levels decreased from 59 +/- 20% to 45 +/- 22% at 3 year. PRA became negative in 6 patients. The reduction in PRA levels in this group of patients was lower than in EPO treated patients (24.3% vs 14%) as well as in fewer number of patients PRA droped (53% vs 63%). Analysis of risk factors for persistent high levels of alloantibodies showed, that female sex and DR2 phenotype were significant factors.

Absorption with K562 erythroblastoma cells as a means for discrimination between auto- and alloantibodies in sera of hypersensitized renal patients.

We applied K562 erythroblastoma cell line as a substitute for autologous lymphocytes in the absorption of autoantibodies from sera of 26 hypersensitized renal patients exhibiting high degree of cytotoxic reactivity with a panel of lymphocytes from unrelated individuals (panel reactivity, PRA). All of analysed sera contained allo-reactivity before absorption. After removal of autoantibodies, 85% of the sera still contained alloantibodies whereas in 15% of the sera no alloreactivity was detectable. Absorption of high PRA sera with K562 cells and with autologous lymphoblastoid cell lines derived by transformation with Epstein-Barr virus (EBV-LCL) gave substantially the same results. As autoantibodies have reportedly no effect on kidney graft survival, the autoabsorption of high PRA sera may increase the chance of patients in whom only autoantibodies were detected to receive the transplant. Application of K562 cells, instead of autologous lymphocytes/lymphoblasts, is recommended whenever high numbers of patient's cells are not available. It may be particularly suitable for routine clinical laboratories, not equipped and prepared for derivation and propagation of EBV-LCL.

Determination of immunoglobulin class of alloantibodies in sera from hypersensitized renal patients.

26 sera from hypersensitized renal patients were analysed for immunoglobulin class of alloantibodies. To assess the participation of IgM and IgG in alloreactivity, sera were treated with dithiothreitol (DTT) or Staphylococcus aureus protein A (SpA), respectively. Treatment with both DTT and SpA was also performed to test the possible contribution of other immunoglobulin classes. Immunological complexes which may mimic alloreactivity were excluded by polyethylene glycol (PEG) treatment. Immunochemically modified sera were tested against a panel of allogeneic B and T lymphocytes. IgG class alloantibodies reactive with B lymphocytes were found two times more frequently than those reactive with T lymphocytes. In 22% of sera only IgM alloantibodies were detected. No contribution of other immunoglobulin classes of immune complexes was seen. Our results show that DTT and SpA treatment are technically simple and reproducible methods of discriminating between IgG and IgM alloantibodies and can be recommended for routine use in clinical analysis of sera from hypersensitized renal patients awaiting kidney graft.

HLA-A, B, C, and DR gene and haplotype frequencies in 600 poles by a maximum likelihood method of gene counting.

A population sample of 600 healthy, unrelated persons was used for estimating HLA-A, B, C and DR antigen, gene and haplotype frequencies. The Polish population is characterized by a high value of HLA-A10, B13, B27, DR1 and DR5 gene frequencies. Strong gametic associations between genes A1 and B8, A3 and B7, as well as between B8 and DR3, and B7 and DR2, typical for many European populations, are also present in the Polish population.