Description of novel class I alleles encountered during routine registry typing in 2007.

Twenty-six novel human leukocyte antigen (HLA) class I alleles are described: 3 HLA-A alleles, 19 HLA-B alleles and 4 HLA-C alleles. Only one of the novel alleles (HLA-B*0753) was found in multiple individuals and likely is not uncommon in the population. Nineteen (approximately 70%) of the 26 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Four of these single nucleotide variants are silent substitutions, and one creates a null allele. The remaining novel alleles differ from their most similar allele by two to six nucleotide substitutions. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-B lows (codons 30, 67 and 72), while HLA-Cw*0347 encodes an amino acid change at a position not previously reported to be polymorphic for this locus.

Prognosis of pediatric patients transplanted for Ph+ chronic myeloid leukemia in the period from 1989 to 2006 in the Czech Republic.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by clonal proliferation of primitive hematopoietic stem cell. The median age at diagnosis is 55 to 60 years with less than 10% of patients younger 20 years. Incidence of CML in children in the Czech Republic is 0.106 cases/100 thousands per year. Here we report outcome of 38 pediatric patients (median age 12.5 years; range 1.8 - 17.3) with Ph-positive CML diagnosed between years 1989 to 2006. Primarily chronic phase of the disease was diagnosed in 32 (84%) patients. 32 (84.2%) patients underwent hematopoietic stem cell transplantation (HSCT) with the median age at transplantation of 14.9 years (range 6.9 - 20.5 years). Out of transplanted patients 16 (50%) obtained graft from unrelated donor, 13 (41%) from matched sibling donor, 2 from haploidentical family donor and autologous transplantation has been performed in one case. 6 patients were not transplanted, 4 of them died (median 1.2 years from diagnosis), 2 are alive 0.6 and 17.8 years from the diagnosis. Overall survival (OS) in 25 patients after HSCT at our department during the whole period is 66.7% with 15/16 being in stable continuous molecular-genetic remission (94%). During the period of time results of transplantations have been significantly improved (p=0.0071). OS after HSCT until year 1997 is 25% while from year 1998 until now is 87.5%. All centers OS of patients after HSCT is 71%. Results of HSCT in children with CML obtained from the year 1998 at our center are fully comparable with results achieved in large and experienced centers. HSCT remains the only proven and effective method for the treatment of CML. Clinical studies assessing the role of tyrosine kinase inhibitors in children instead of early HSCT should be planned carefully in order to avoid sub-optimal outcomes.

Identification of HLA alleles with low or no cell surface expression in the Czech population.

The presence of the A*24020102L allele is implicated in one donor from the CBMD who serologically was typed as A2; B44, B55; Cwl, Cw7. The DRB4*01030102N allele was identified in one healthy donor and in one patient with MDS during routine HLA class II DNA typing. The DRB4*01030102N allele was identified in the patient's father, who had CML, and was associated with the HLA-A3-B7-Cw7-DRB1*0701-DQB1*0303 haplotype, which is common for European populations. In order to avoid mistyping, both techniques, serology and molecular biology must be used for HLA typing, especially for cases where just one antigen appeared to be present using serological methods.

Alloresponses of cord blood cells in primary mixed lymphocyte cultures.

The aim of this study was to compare the alloreactive responses against HLA antigens of cord blood cells with those of adult peripheral blood cells. In primary mixed lymphocyte cultures and bulk cell-mediated lympholysis experiments cord blood cells demonstrated significantly decreased proliferation and cytotoxicity. Experiments analyzing the specificity of anti-HLA cytotoxic T lymphocytes (CTL) revealed that cord blood (CB) CTL reacted only partially with third-party cells expressing the stimulating HLA antigens. Lower frequencies of IL-2 producing helper, cytotoxic T-cell precursors and IL-4 producing CB cells were found, whereas the frequencies of IFN-gamma producing cells, as determined by ELISpot experiments, were equivalent to the frequencies of adult IFN-gamma producing cells. Our results imply that, although CB cells have significantly decreased proliferative and cytotoxic alloresponses in bulk mixed lymphocyte cultures, their IFN-gamma production is comparable with that of adult mononuclear cells. Preserved production of IFN-gamma may be a risk factor for the development of graft-versus-host disease and should be taken into consideration when evaluating the possibility for stem cell transplantation with HLA-mismatched CB.

The epitope recognized by pan-HLA class I-reactive monoclonal antibody W6/32 and its relationship to unusual stability of the HLA-B27/beta2-microglobulin complex.

A broadly used pan-HLA class I-reactive monoclonal antibody W6/32 is believed to recognize a conformational epitope dependent on association between heavy chains and beta2-microglobulin (beta2m). However, in the present study we report that W6/32 does recognize at least some free HLA class I heavy chains under the partially denaturating conditions of nonreducing Western blotting, namely nearly all HLA-B allelic products. Furthermore, we confirm and largely extend our previous observation that complexes of beta2m with heavy chains of a few HLA class I allelic forms (most notably HLA-B27) exhibit unusual resistance to dissociation by SDS, which is reminiscent of MHC class II molecules. In addition, our data indicate the existence of covalent (disulfide-linked) heterodimers of certain HLA class I heavy chains (namely Cw1 and Cw4) and beta2m.

[Prediction of insulin-dependent diabetes mellitus in children of first-degree relatives of diabetic patients]. / Predikce inzulin dependentního diabetes mellitus u detských prvostupnových príbuzných diabetických pacientu.

BACKGROUND: Individuals at risk for insulin dependent diabetes mellitus (IDDM) can be identified using a combination of genetic, immunological and metabolic markers. Our study was aimed at prediction of IDDM in a cohort of children having a first-degree relative with IDDM. METHODS AND RESULTS: In the period of three years, we investigated 208 non-diabetic children and adolescents, aged 10.0 +/- 5.3 (mean +/- SD), mostly siblings of diabetic children. The genetic risk was determined by the HLA-DQB1, -DQA1 genotyping and subtyping of the DRB1*04 alleles carried on the DQB1*0302 haplotypes. Insulitis was detected using a combination of autoantibody tests against three molecular-defined antigens (insulin, GAD65, IA-2). Prevalence of insulitis (defined as confirmed positivity of at least one autoantibody) was 9/208 (4.3%). In children carrying the IDDM highest-risk genotype (HLA-DQB1*0201-DQA1*05/DQB1*0302-DQA1*03), insulitis was almost 10 times more frequent (5/24, 21%) than in children with other genotypes (4/184, 2.2%, P = 0.003). In all subjects with insulitis, the first phase insulin response (FPIR) was determined by the intravenous glucose tolerance test. Three of the nine children had decreased FPIR, of whom two were later diagnosed with IDDM. None of the remaining children developed IDDM. CONCLUSIONS: We present the first IDDM prediction study in the Czech population, emphasising the utility of genetic risk investigation in the prediction scheme.

Analysis of paternal alleles in nucleated red blood cells enriched from maternal blood.

The purpose of our study was to identify paternal alleles in NRBC enriched from maternal peripheral blood for detection of the presence of foetal cells in the maternal circulation and to establish a reliable non-invasive method which should allow following genetic testing. For enrichment of foetal cells from peripheral maternal blood we combined Ficoll-Paque density gradient centrifugation and MACS. Maternal leukocytes were firstly depleted using anti-CD14 and anti-CD45 microbeads. NRBC were sorted from the CD14-/CD45- fraction by positive selection using CD71 microbeads. Paternal alleles in the CD14-/CD45-/CD71+ fraction were indicated by the PCR method using HLA (DRB1, DQB1, DQA1) and Polymarker System (LDLR, GYPA, HBGG, D7S8, GC) as genetic markers. Different paternal alleles of studied 8 loci were detected in 13 out of 19 samples of cells enriched from maternal peripheral blood between the 13th and 36th week of gestation. Our results demonstrate that foetal cells enriched from maternal peripheral blood may be used as a source of foetal DNA for prenatal diagnosis, paternity testing and other application.

Characterization of patient antibodies after kidney transplantation.

OBJECTIVE: The aim of this study was to characterize patient antibodies before and after cadaver and/or living-donor kidney transplantation and to correlate these data with the clinical course after transplantation. METHODS: Sera from 69 cadaver, 9 living-related and 2 patients waiting for living-donor kidney transplantation were analyzed by the complement dependent cytotoxicity (CDC) test, flow cytometry (FCXM) and ELISA. RESULTS: FCXM revealed that 15.0% of patients before transplantation and 16.7% after transplantation had antibodies to donor cells. 10.3% patients were positive before and after transplantation (+/+), while 6.8% developed antibodies early after transplantation (-/+). Analysis of the specificity of those antibodies by ELISA showed that it was directed to: 1) mismatched donor HLA antigens 2) antigens belonging to the same cross-reacting group (CREG) as the mismatched donor antigens 3) HLA antigens not expressed by donor cells or, probably, to non-HLA antigens. CONCLUSION: Anti-HLA antibodies were detected in patients before and after transplantation and in most cases their anti-HLA specificity could be determined. Fast and precise characterization of antibodies in patients before and after transplantation can be performed by both sensitive methods--FCXM and ELISA--which may help predict the onset of immunological complications and, consequently, improve the prognosis after organ transplantation.

The Czech Bone Marrow Donor Registry.

OBJECTIVE: The Czech Bone Marrow Donor Registry (CBMD)--established 9 years ago, operates within the National HLA Centre, a constituent part of the Department of Immunology at the Institute for Clinical and Experimental Medicine. The Czech Cord Blood Register (CSCB) was recently established (in 1996) and started its activities. METHODS: CBMD is responsible for maintaining a database of HLA typed volunteer donors, for performing national and international searches in the file of bone marrow transplantation as well as for coordinating the communication between participating centres. RESULTS: The operation of the CBMD registry requires the modern communication technology for the exchange of data with local organisations (donor and transplant centres) and with international BM organisations and networks abroad (Bone Marrow Donors Worldwide--BMDW, National Marrow Donor Program--NMDP, European Donor Secretariat E.D.S., European Marrow Donor Information System--EMDIS). CONCLUSIONS: The CBMD is fully integrated into international cooperation. The HLA typed unrelated stem cells from Prague can be selected for patients in the whole world.

HLA-association in patients with intolerance to mercury and other metals in dental materials.

A group of selected 25 patients with serious intolerance to heavy metals used for dental restoration were examined for HLA antigens. A significant increase for HLA -- B37, B47 and DR4 was found. The value of the relative risk is not significant after correction for the number of antigens tested and therefore further studies of more patients are needed.

Strong association of HLA-B27 heavy chain with beta 2-microglobulin.

Monoclonal antibody TG1 recognizes specifically antigens HLA-B27, B7, B22 and B17 on cell surface in cytotoxicity and cytofluorometry tests. When cell detergent extracts were subjected to SDS PAGE under mild conditions (no heating and no reduction of the sample) followed by Western blotting, TG1 detected exclusively a complex of B27 heavy chains with beta(2)-microglobulin (as a 50 kDa band) whereas the other B-locus antigens (B7, B22, B17) were detected as free 43 kDa heavy chains under the same conditions. When the samples were boiled prior to SDS PAGE, TG1 detected again the 43 kDa free heavy chains of B7, B22 and B17 but no zone corresponding to B27 could be detected indicating that the epitope in free B27 chains is more sensitive to denaturation by SDS. Thus, our main finding is that the interaction of HLA-B27 heavy chain with beta(2)-microglobulin appears to be stronger than that of the other HLA-B chains. The resistance of the HLA-B27/beta(2)-microglobulin complex to the SDS dissociation is strikingly similar to the behavior of MHC class II molecules under similar conditions. Thus, it may be speculated that HLA-B27 complexes can be also more stable than other MHC class I molecules under more physiological dissociative conditions (e.g. in endosomal compartments). This feature might potentially influence antigen presentation by HLA-B27 and contribute to the well known disease linkage of HLA-B27.

An overview of the Czech Bone Marrow Donor Registry.

The Czech Bone Marrow Donor registry (CBMD) was founded in 1991 in the National HLA centre at Prague's Institute for Clinical and Experimental Medicine. In the same year, the CBMD submitted its data to the Bone Marrow Donors Worldwide (BMDW). Another line of CBMD's international cooperation is accomplished through computer linkup with the European Donor Secretariat (E.D.S) network. Donors are being recruited constantly through blood transfusion units and other volunteers are enrolled through the mass media. All the methodology used is developed in compliance with the standards of the European Federation for Immunogenetics (EFI). CBMD closely cooperates with clinical centres for transplantation of bone marrow, stem cells (PBSC) and cord blood from unrelated donors. More than 7,000 potential bone marrow typed in HLA-A, B locus have been registered. Besides potential bone marrow donors, frozen cells of cord blood are kept by CBMD. Search requests from registries all over word come via E.D.S. daily except for weekends. Since its foundation in 1991, nearly 20,000 international requests have been handled. During the last two years, 5 CBMD donors provided their bone marrow to Czech patients, one donor provided stem cells (PBSC) and one donor provided bone marrow + stem cells (PBSC). To date, more than 20 transplantations from unrelated donors have been performed in Prague's transplant centres.

Flow cytometry cross-match before kidney transplantation in relation to early postransplant rejection.

The aim of this study was to compare flow cytometry cross match (FCXM) results in patients before first kidney transplantation with the incidence of rejection episodes and kidney graft survival after transplantation. Sera of 51 patients obtained immediately before transplantation were tested on spleen cells of respective kidney donors. We found no correlation between a positive FCXM result before transplantation and the occurrence of immunological complications after transplantation.

Herpes viruses in multicase families with rheumatoid arthritis.

OBJECTIVE: To investigate the rate and extent of infection by Epstein-Barr virus (EBV), cytomegalovirus, and herpesvirus 6 in families (affected and nonaffected members) with multiple cases of rheumatoid arthritis (RA). METHODS: Viral DNA was detected by polymerase chain amplification in cells from saliva and peripheral blood. Human leukocyte antigen pedigrees were characterized. RESULTS: Viral DNA, particularly EBV, was detected in increased frequency (p = 0.029) in the patients with RA compare to their nonaffected relatives. CONCLUSION: We suggest that in RA multicase families, increased frequency of viral infection is likely a consequence of the disease state and/or due to gene(s) as yet unidentified.

Role of hydrophobic amino acids at position 74 of DRB1 chain in rheumatoid arthritis.

We have analyzed HLA class II alleles in a group of 153 Czech children with rheumatoid arthritis by PCR and hybridization with oligonucleotide probes. When we try to find a common sequence for all DRB1 alleles involved in juvenile and adult arthritis, we can notice hydrophobic amino acid at position 74, which is present in all these alleles, but not in nonsusceptible alleles, where is the hydrophilic amino acid at position 74. In our model, we speculate that the hydrophilic amino acid at position 74 creates a such kind of epitope which is not suitable for rheumatoid-associated peptides or T cells, and only hydrophobic amino acid can permit binding of these peptides or recognition by certain T cells. Analyses of the DPB1 sequences have shown that alleles which have a negatively charged amino acid at position 69, are more frequent in pauciarticular patients while those with a positively charged amino acid are more frequent in polyarticular patients. A positively charged amino acid at position 69 might present the same rheumatoid associated peptide as susceptible DRB1 alleles. The presence of more rheumatoid-associated peptide on the cell surface may cause conversion to more severe polyarticular forms. A negatively charged amino acid at position 69 could not present this peptide and a low concentration of the peptide on the cell surface presented just by DRB1 molecules keeps disease in a relatively benign condition of pauciarticular forms.

Class II alleles in juvenile arthritis in Czech children.

OBJECTIVE: Patients with pauciarticular and polyarticular onset rheumatoid factor (RF) negative juvenile arthritis (JA) have been reported to have a variety of HLA associations. The reason for the differences found in several recent studies is not known. We compare a new series of patients investigated in Prague, Czechoslovakia with those we reported from Dallas. METHODS: Czech patients with JA (N = 153) were classified clinically using the same criteria as in our studies in Dallas. The RF negative group included 56 patients that had persistent pauciarticular disease, 42 pauciarticular with polyarticular course and 39 with polyarticular onset. RF was present in 13 additional patients. HLA class II alleles were determined by oligotyping as previously described from our laboratory. RESULTS: DRB1*0801 was increased and DRB1*0701 was decreased in all the RF negative groups. The persistent pauciarticular group was associated with DRB1*11 and DPB1*0201 and lacked the association with DRB1*1301 seen in Dallas. Also found in Prague and not in Dallas were an increase in the frequency of DR2 in pauciarticular patients with early conversion and of DRB1*1201 in patients with iritis. Certain HLA associations (DRB1*0801, DPB1*0201) appear to be present in patients with JA in most studies; others (DRB1*1301, DPB1*0301) are more variable. CONCLUSION: The reason for differences in the HLA risk factors observed in our 2 populations is not known. Clinical heterogeneity not detected by our method seems the most likely explanation. Genetic and environmental factors may also play a role.

Sequences of HLA alleles associated with arthritis in adults and children.

Adult rheumatoid factor (RF) positive rheumatoid arthritis (RA) and RF positive arthritis of childhood are associated with DRB1*0401 in Caucasians, and DRB1*0405 in Orientals, and in Ashkenazi and nonAshkenazi Jews. Certain other DRB1 alleles (DRB1*0101,1001) which have a similar sequence in the 3rd hypervariable region of the 1st domain are also associated with RA, but they appear to function as weaker risk factors. The difference in the relative strength of the associations is likely to be due to structural differences in the 1st and 2nd variable regions of the first domain of these alleles. Similarities and differences in the HLA associations between North American Caucasoid patients with juvenile arthritis in Dallas, TX, USA, and in Prague, Czechoslovakia, are discussed.