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.

H-2/HLA cross-reactions. Absorption analysis of cytotoxic antihuman activity in anti-H-2 mouse sera.

Absorption experiments and subsequent retesting on human peripheral lymphocytes were performed with the congenic anti-H2f mouse serum known to give strong cytotoxic reactions with human cells which correlate with the presence of HLA-A2 antigen (mouse serum ASP 223, donor strain B10.M (H-2f), recipients (B10 X A.SW)F1 (H-2b/H-2s) hybrids; antibodies present in the serum anti-H-2.9, 8,37). Depending on the dose of A2-negative cells used for absorption, the serum can be rendered operationally monospecific to HLA-A2. Absorption experiments with lymph node cells of different mouse strains have shown that H-2f.p.w7 haplotypes share the ability to absorb completely the antihuman activity with about 15 X 10(6) cells. Consequently, the antihuman activity is not attributable to the presence of the anti-H-2.9 private specificity in the serum. H-2d.k.r haplotypes have weak absorbing capacity, because 500 X 10(6) lymph node cells are needed to absorb 70% of the antihuman cytotoxic acitivity. All strains that were able to absorb antihuman cytotoxic activity from ASP 223 shared the specificity H-2.8. Thus, the H-2.37 specificity seems to be responsible for strong and the H-2.8 specificity for weak absorbing capacity. The absorbing capacity of the H-2d haplotype was localized in the H-2.K end. Mouse lymph node cells have a 10- to 20-fold higher absorbing capacity for the antihuman activity than thymus cells. It was shown that H-2.K end public specificities predominantly expressed on lymph node cells are responsible for the generation of antihuman (anti-HLA) cytotoxic activity in anti-H-2 sera.

Mouse lymph node activating factor released during contact of HL-A-different blood lymphocytes.

Ten-fold concentrated supernatants from the 4-hr mixed cultures of lymphocytes from two humans different at two haplotypes were inoculated s.c. into the hind footpad of mice. Twenty-four to forty-eight hours later, the cellularity and the synthesis of ribonucleic acid increased significantly in the popliteal lymph nodes draining the site of injection. The supernatants from cultures of HL-A-identical cells of cells different at one haplotype lacked this activity. The results suggest that human imcompatible lymphoid cells upon mutual contact liberate a soluble mediator capable of activating the draining lymph nodes; i.e., it is able to trap cells from the circulation and to stimulate them. The mediator is species-nonspecific; the activity produced by human lymphocytes can be tested on mice.

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.

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.

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.

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.

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.

HLA molecule--cytoskeleton associations induced by cross-linking with specific antibodies.

Sera of patients with antibodies against HLA-typed panel, control human sera, anti-beta 2-microglobulin monoclonal antibody recognizing HLA class I molecules, anti-HLA class II monoclonal antibodies and control antibodies were used in indirect immunofluorescence study of living human peripheral blood lymphocytes and their cytoskeletons. Lymphocytes immobilized on concanavalin A-precoated glass slides enabling extraction procedures and repeated washings were found useful for such a study. If the lymphocytes were treated with human polyclonal anti-HLA antibodies only, or with two antibodies (i.e. with a specific monoclonal anti-HLA antibody and an anti-mouse Ig polyclonal antibody labelled with fluorescein isothiocyanate (FITC), the cross-linking of the surface molecules caused their resistance to the detergent effect. Murine monoclonal anti-HLA antibodies alone, bound to the cell surface molecules, formed immune complexes easily extracted with Triton solution. In such cases, the second anti-mouse Ig antibody labelled with FITC did not visualize specific molecules in the remaining matrix. No significant changes in the microtubules of cells treated with anti-HLA antibodies were observed.

HLA-D antigens in a Czech population and their association with HLA-DR antigens.

HLA-D specificities Dwl through 11 and HLA-DR antigens DRwl through WIA8 were studied on a panel of 101 unrelated individuals of the Prague HLA panel. THe HLA-D specificities were defined by means of the HLA-D homozygous typing cells in MLC test, the HLA-DR specificities were tested on B cells using VIIth Histocompatibility Workshop antisera. In the panel tested, the antigen and gene frequencies of eleven HLA-D determinants, the associations between the HLA-B and HLA-D antigens as well as between the HLA-D and HLA-DR antigens were determined. Significant associations between some of the HLA-D and HLA-DR antigens were found.

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.

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.

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.

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.

[Recurrence of idiopathic membranous glomerulopathy]. / Relapsy u idiopatické membranózní glomerulopatie.

In 30 patients with idiopathic membranous glomerulopathy during prednisone treatment and prednisone and cyclophosphamide treatment resp. partial or complete clinical remission was recorded. In the course of the investigation (118 +/- 77 months) in 7 patients 1-4 relapses occurred. The relapses occurred more frequently in women. There was no difference between the two groups as regards clinical manifestations and morphological findings at the onset of the disease. All relapses were treated in the same way as the first attack. Treatment had in all patients the same result as during the first attack and in the course of the investigation in none of the patients deterioration of renal functions was recorded. In case of effective immunosuppressive treatment the prognosis of patients with relapses is not worse than in patients without relapses.

[HLA antigens in type II diabetics]. / HLA antigeny u diabetiku II. typu.

The study examined the relationship between class I HLA antigens and type II diabetes with regard to insulin secretion and hyperlipoproteinemia. Out of 28 HLA antigens of A, B and C loci, 46 type II diabetics had statistically more significant HLA Cw1 - 15.9% as compared with 6.3% of the control group, the relative risk being 2.81. These patients were younger than the other diabetics but showed no difference in insulin secretion or hyperlipoproteinemia. Diabetics with HLA Cw4, just as patients after myocardial infarction with concomitant hyperlipoproteinemia, were also found frequently to have hyperlipoproteinemia (89 percentage cases as compared to 63% of the other diabetics). The findings may be indicative of potential genetic heterogeneity of type II diabetes. The paper stresses the necessity of establishing suitable genetic characters for early diagnosis of diabetes and its development.

[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.

[A non-classic form of congenital adrenal hyperplasia]. / Neklasická forma vrozené adrenálni hyperplázie.

The authors examined five children with the non-classical form of adrenal hyperplasia. The clinical symptoms of the disease comprise: early congenital pubic hair, small stature, accelerated bone maturation, infertility, in girls hirsutism, acne, menstrual disorders, amenorrhoea, in boys oligospermia, acne. Laboratory examination reveals slightly elevated 17-OH progesterone values during the synactene loading test. It is an autosomal recessively hereditary disease. It is due to an allelic variant of the gene for 21 hydroxylase. The authors revealed a link with antigen B14. Even in the small group examined antigen B14 was present in 40%, although the prevalence in our population is 2.9%.