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R Blasczyk

Publications and source records attributed to R Blasczyk.

At least 55 records · Page 3Linked to original sources
PubMed2000

HLA-B*4012: a new allele with unique serological features.

We have defined the new allele HLA-B*4012, which had been isolated from a black individual. It was initially recognized as a serologically unique allele when typing her father for renal transplantation. The HLA class I phenotype was A*0201,*6602; B*4001,*4012; Bw6; Cw*0304,*1505. Sequencing from exon 1 through intron 3 of B*4012 was performed. B*4012 is identical to B*4001 and B*4010 in exon 3, and in the 3' part of exon 2, but it is unique in that exon 1 and the 5' part of exon 2 are identical to B*1503, B*1509, B*1510, B*1518, B*1523, and B*1529. The generation of this allele is best explained by a recombination event in exon 2 (break point between nucleotides 205 and 222 from the beginning of the coding region) of B*4001 or B*4010 with one of these B*15 variants as a donor allele. Its unique serological feature (B48, B60, B70, and B72 reactivity) is consistent with the sequence data of its donor alleles.

Alleles↗
PubMed2000

Increased diversity within the HLA-B*07 group: identification of the two novel alleles B*0709 and B*0710.

The identification of the two novel alleles, HLA-B*0709 and B*0710, is described. B*0709 differs from B*07021 by a nucleotide exchange at position 419 (A>C) which is located in exon 3. At the protein level the nucleotide exchange results in an amino acid residue difference (Tyr116Ser). The other newly detected allele, B*0710, differs from B*07021 by a nucleotide exchange at position 272 (A>G) which is located in exon 2. This nucleotide exchange also leads to an amino acid substitution (Tyr67Cys). The allogeneic potential in case of mismatch with other alleles of the B*07 group at bone marrow transplantation was assessed. The peptide motifs between B*0709 and B*0711 may be very similar and thus the alloreactive potential in case of mismatch may be low. In contrast, mismatches of B*0709 and B*0710 with other B*07 alleles are likely to stimulate alloreactive T cells.

Alleles↗
PubMed2000

Characterization of the novel allele HLA-B*4417: implications for bone marrow transplantation.

The identification of the novel allele HLA-B*4417, which was found in a blood donor of Caucasian origin, is described. In the sequence analysis the new allele differs from B*4402 by three nucleotides in exon 3. At the protein level, the new allele has two residue differences compared to B*4402. Due to substantial differences with other B*44 variants a possible mismatch may impair clinical outcome of bone marrow transplantation.

Alleles↗
PubMed2000

Two novel DRB alleles detected by PCR-SSO and confirmed by sequencing: DRB1*0317 and DRB3*0210.

We here describe two new DRB alleles (DRB1*0317 and DRB3*0210) which were detected by polymerase chain reaction using sequence-specific oligonucleotides (PCR-SSO) and confirmed by direct sequencing. The exon 2 sequence of DRB1*0317 is the result of an intergenic recombination event yielding a hybrid allele between 5'DRB1 and 3' DRB3 sequences with the recombination breakpoint located between codons 39-51. The new DRB3*0210 allele is closest related to DRB3*02021 except for a single nucleotide position at codon 51. Here, the sequence AGG at codon 51 which was a group-specific motif for all DRB3*02 alleles described previously, is replaced by the DRB consensus sequence ACG.

Alleles↗
PubMed2000

Establishment and characterization of colon carcinoma and renal cell carcinoma primary cultures.

Patients with metastatic renal and colon carcinoma have a very poor prognosis. In many cases, the tumor recurs after surgical excision and chemotherapy. Therefore, it might be beneficial for cancer patients to induce an immune attack against the tumor by inserting a cytokine gene into the tumor cells. Here, two different techniques for isolation of single tumor cells were compared. An enzymatic solution was superior to an EDTA/DTT isolation solution for establishing tumor primary cultures. In total, 18 primary cell cultures could be established from 68 patients with colon and renal cell carcinoma. Cells were further characterized concerning fibroblast contamination, cell proliferation and HLA-typing. These primary tumor cells might be of value for cytokine gene transfer and in vaccination protocols for cancer patients.

Adenocarcinoma↗
PubMed1999

Monomorphic HLA class I-(non-A, non-B) expression on Ewing's tumor cell lines, modulation by TNF-alpha and IFN-gamma.

In this study, the expression of polymorphic and non-polymorphic MHC antigens in Ewing's tumor (ET) cells was examined by surface staining, Western blots and transcriptional analysis. Cell lines derived from Ewing's tumors largely lack polymorphic HLA class Ia antigens of both the HLA-A and the HLA-B loci but binding of monomorphic HLA antibodies indicates significant expression of HLA-C locus antigens and/or HLA class Ib molecules. HLA Ib molecules encoded by the HLA-E, -F or -G loci with a molecular mass of less than 44 kDa were not detected in lysates of either constitutive or TNF-alpha plus IFN-gamma treated ET cells. Two representative ET cell lines with either detectable HLA-A, -B antigens (A673) or absolutely non-detectable HLA-A, -B antigens (SK-ES-1) were further subjected to transcriptional analysis. A673 mRNA hybridized with HLA-A, -B, -C and HLA-E-specific probes in Northern blots. By contrast, mRNA specific for HLA-A, -B, -C was negative in SK-ES-1 but TNF-alpha plus IFN-gamma reconstituted HLA-A, -B, -C transcription in this cell line. HLA-E was transcribed in A673 but not in SK-ES-1. Combining mRNA and surface expression of HLA class Ia molecules results in a highly variable pattern of defective HLA class I expression in this type of neuroectodermal tumor. The involvement of the ET-specific fusion transcript EWS/Fli-1 in modulating the HLA-A and -B locus antigens is likely to occur by the upregulation of c-myc in these tumors. The exceptionally constant expression of HLA-C or some other non-A, non-B antigens (reactive with defined monoclonal antibodies) implies important consequences on tumor-cell resistance against specific CTL and NK activity in vivo.

Gene Expression↗
PubMed1999

Fluorotyping of HLA-DRB by sequence-specific priming and fluorogenic probing.

Similar to our recently described HLA-A and -C fluorotyping strategies, the aim of this study was to develop a sequence-specific primed polymerase chain reaction (PCR-SSP)-based fluorotyping method for HLA-DRB. Applying the fluorogenic 5' nuclease assay, it is possible to increase the sample throughput rate by abolishing all labor-intensive post-amplification steps. Additionally, problems related to contamination are eliminated. The method relies on the 5'-3' exonuclease activity of the Taq-DNA Polymerase which cleaves a target-specific and individually labelled fluorogenic probe during successful PCR. Different labelled probes specific for different targets can be applied in a single PCR, allowing independent detection of the specific HLA and the internal control product. The probe used to detect the HLA-DRB specific amplicons was labeled at its 5' end with FAM as the reporter and further 3' with TAMRA as the quencher. The probe hybridized within the 2nd exon to a conserved region which was covered by all primer mixes. In case of amplification, the cleavage of the fluorogenic probe led to an interruption of the TAMRA-mediated quenching effect and generated a significant increase of the FAM fluorescence. The HLA-DRB fluorotyping information was based on the FAM fluorescence released by 24 individual primer mixes. A TET-TAMRA-labelled probe was used to indicate amplification of the internal control sequence in each PCR reaction. So far, 170 PCR typed clinical samples representing all serologically defined HLA-DRB specificities were analyzed using this fluorotyping method. The results were 100% concordant with those obtained by conventional agarose gel detection.

Alleles↗
PubMed1999

Sequencing of HLA class II genes based on the conserved diversity of the non-coding regions: sequencing based typing of HLA-DRB genes.

In this paper, we present a novel sequencing based typing strategy for the HLA-DRB1, 3, 4 and 5 loci. The new approach is based on a group-specific amplification from intron 1 to intron 2 according to the serologically-defined antigens. For this purpose, we have determined the 3' 500 bp-fragment of intron 1 and the 5' 340 bp-fragment of intron 2 of all serological antigens and their most frequent subtypes. We discovered a remarkably conserved diversity characterized by lineage-specific sequence motifs. This lineage-specificity of non-coding motifs in the 1st and 2nd intron offered the possibility to establish a clear serology-related amplification strategy. The method allows the complete analysis of the 2nd exon and the definition of the cis/trans linkage of sequence motifs by intron-mediated polymerase chain reaction (PCR)-based separation of the haplotypes in nearly all serologically heterozygous samples. In particular, the non-coding variabilities between the DR52-associated DRB1 groups made their independent amplification possible. Thus, compared to the standard procedures using exon-based amplification primers, the groups DR3, DR12, some DR13 alleles (1301, 1302) and the DR14 group could be amplified by specific primer mixes. The DR8 could be amplified with an individual primer mix not co-amplifying the DR12. The DR11 and DR13 did not show any individual motif in intron 1 or intron 2. In order to achieve a separate amplification, they had to be amplified by multispecific primer mixes (DR3/11/13/14; DR3/11/13 or DR11/13/14) excluding the other haplotype. Thus, exclusively the alleles in rare DR11,13 heterozygosities without a DRB1*1301 or 1302 could not be amplified separately. Fourteen primer mixes are used to amplify the specificities DR1-14, and 6 primer mixes for the specificities DR51-53. The sequence homology of the 3' end of intron 1 facilitated the application of only three different sequencing primers for all DRB alleles.

Alleles↗
PubMed1999

Unusual association of the DRB4 null allele, DRB4*0103102N, with HLA DRB1*0402 in a sample of Austrian patients.

Tissue typing for HLA class II antigens is routinely performed by serological, and/or DNA-based methods. Accuracy, absence of cross-reactivity and controllable level of resolution are striking advantages of molecular methods. However, a disadvantage of molecular typing, compared to serological methods, is the identification of unexpressed alleles. Whereas serology allows a more or less direct insight into antigen presence, molecular biology is an indirect method and results must also be interpreted by considering the biological pathways of protein expression. We believe that identification of nonexpressed MHC alleles is of importance for transplantation, since nonexpressed MHC allele positive individuals could give rise to antibody formation against the respective expressed MHC product in donor tissue. Usually, the nonexpressed DRB4*0103102N is encountered in association with DRB1*0701-DQB1*03032, which facilitates correct DNA typing. Here we describe the unusual association of this unexpressed DRB4*0103102N, with DRB1*0402-DQB1*0302 in a sample of Austrian patients.

Adult↗
PubMed1999

Antibodies to private and public HLA class I epitopes in platelet recipients.

BACKGROUND: Transfusions or pregnancies can cause immunization against private HLA determinants and public epitopes shared by more than one private HLA antigen. HLA antibodies are correlated with febrile transfusion reactions, lower platelet response following platelet transfusion, and an increased rate of renal transplant rejection. Until now, antibody specificities in alloantisera from platelet recipients have been poorly characterized. STUDY DESIGN AND METHODS: Consecutive serum screens from platelet recipients were analyzed for antibodies against private HLA class I antigens and public HLA epitopes using a serum analysis program based on the 2 x 2 table analysis of correlations. Serum screens of highly immunized patients and of patients with new alloimmunization events were reviewed separately. RESULTS: Of the serum screens from 566 platelet recipients, 1577 indicated alloimmunization (panel-reactive antibodies >5%). The program assigned a specificity in 1024 of these screens (64.9%) and at least once in 522 of 566 patients (92.2%). In 267 patients, antibodies detecting public epitopes in the combined A- or B-locus cross-reacting groups were found; other public markers were detected in 39 patients. Patterns of reactivity were remarkably less stable than in patient groups previously studied. In many patients, antibodies with apparent private epitope specificity preceded the identification of antibodies against a shared marker of the same cross-reactive group. However, the disappearance of antibodies (whether or not this was followed by a new antibody against a private or public marker belonging to another cross-reacting group) was also observed. CONCLUSION: The computerized analysis of microlymphocytotoxicity tests enhances the rate of antibody specification in sera from platelet recipients with lymphocytotoxic antibodies. The identified antibodies should be taken into account in the selection of platelet donors. The data confirm and extend previous observations on HLA class I antibodies and elucidate new alloimmunization events.

Antibodies↗
PubMed1998

Human herpesvirus type 6 variants identified by single-strand conformation polymorphism analysis.

Six human herpesvirus 6 (HHV-6) variants were analyzed for heterogeneity using the polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP). Two independent DNA regions were selected: a fragment of the gene U11 (position 18966-21578) coding for a basic phosphoprotein, the major antigenic structural protein pp100; and a fragment from an open reading frame (ORF) area of the gene U67, previously referred to as 13R (position 102458-103519), coding for a product of unknown function. The two PCR systems based on the above DNA sequences yielded products of 187 bp and 223 bp, respectively. DNA obtained from three laboratory reference strains (U1102, R104 and St.W.) and from HHV-6 infected peripheral white blood cells of bone marrow transplant patients and blood donors was used to test the applicability of two different SSCP analysis systems for the identification of HHV-6 variants using amplicons derived by PCR from the two genomic regions described above (U11 [pp100], U67 [13R-ORF]). The generation of characteristic SSCP patterns enables the rapid differentiation of HHV-6 A and B strains for the classification of variants derived from clinical samples, reducing the need for expensive and time-consuming direct sequencing analyses.

Base Sequence↗
PubMed1998

Treatment of relapse after allogeneic bone marrow transplantation with unmanipulated G-CSF-mobilized peripheral blood stem cell preparation.

Donor lymphocyte infusions (DLI) are an effective treatment of leukemia relapse after allogeneic bone marrow transplantation. Undesired side-effects are the development of graft-versus-host disease (GVHD) and the occurrence of pancytopenia in some patients. In a pilot study, we investigated if unmanipulated G-CSF-mobilized peripheral blood stem cells which naturally contain large numbers of T lymphocytes (D-PBSC/LI) would be equally effective or even superior than DLI in generating a graft-versus-leukemia reaction (GVL) but could mitigate or prevent the development of pancytopenia. We treated 12 patients with CML chronic phase (n = 5), CML blast crisis (n = 2), AML (n = 2), ALL (n = 1), CLL (n = 1) and multiple myeloma (n = 1). In five patients with acute leukemia or CML blast crisis D-PBSC/LI followed intensive chemotherapy (group A), in seven patients D-PBSC/LI were given without any prior chemotherapy (group B). In group A two patients were evaluable for hematologic toxicity. Leukopenia <1000/microl lasted for 10 and 19 days, and thrombocytopenia <20,000/microl for 11 and 13 days, respectively. In group B leukopenia <1000/microl and thrombocytopenia <20,000/microl was observed in only one patient. Moderate cytopenia developed in four of five evaluable patients. A complete remission could be achieved in all seven patients with CML who all developed acute and/or chronic GVHD. None of the remaining five patients achieved a complete remission despite acute and/or chronic GVHD in two of them. Four patients died from disease progression, one patient from a secondary lymphoma, and one patient as a result of uncontrolled GVHD. In conclusion, D-PBSC/LI is effective in inducing GVL reaction but it does not prevent pancytopenia in each case. It remains unclear if it mitigates the incidence and severity of pancytopenia.

Adult↗
PubMed1998

The nature of polymorphism of the HLA-DRB intron sequences is lineage specific.

The sequence database of HLA-DRB genes is mainly derived from mRNA analysis or has focused exclusively on the polymorphism of the 2nd exon. Little is known about the non-coding sequences of the different DRB alleles which represent about 94% of the genes. In this study we have determined the sequence of the 3' 500 bp intron 1 fragment adjacent to exon 2 in all serologically defined HLA-DRB genes and their most frequent allelic subtypes. The intron sequences turned out to be highly polymorphic. Similar to the class I introns, this variability was not characterized by random point mutations but by a highly systematic diversity reflecting the lineage-specific relationship of the HLA-DR alleles. With a few exceptions in DRBI*15, 13 and 08 as well as DRB4 and 5, the variability mirrors the serological diversity. As well as delivering insight into the genetic relationship between the different DRB alleles, these sequences will provide an extremely valuable basis for developing advanced DRB sequencing strategies for clinical purposes.

B-Lymphocytes↗
PubMed1998

Fluorotyping of HLA-A by sequence-specific priming and fluorogenic probing.

The aim of our study was to develop a fluorotyping strategy for the HLA-A locus. In contrast to conventional sequence-specific primed PCR (PCR-SSP), which is based on an agarose gel electrophoresis, fluorotyping eliminates the drawback of low sample throughput, low potential for automation and problems related to contamination. Additionally, fluorotyping is capable of delivering quantitative results depending on the system set-up. The fluorescence-based PCR-SSP assay relies on target-specific and individually labeled fluorogenic probes allowing a simultaneous and differential detection of the specific HLA and the internal control product. The probe used to detect the HLA-A specific amplicons was labeled at its 5' end with 6-carboxyfluorescein (FAM) as the reporter and at its 3' end with 6-carboxy-tetramethylrhodamine (TAMRA) as the quencher. The probe hybridized within the 2nd intron to a conserved region which was found to be identical in all HLA-A alleles and was covered by all primer mixes. During successful PCR the cleavage of the FAM-labeled probe through the 5'-3' exonuclease activity of the Taq DNA-polymerase led to an interruption of the TAMRA-mediated quenching effect and generated a significant increase of the FAM fluorescence. The specific HLA-A typing information was based on the FAM fluorescence released by 24 individual PCR primer mixes. The internal control amplicon was detected with a tetrachloro-6-carboxyfluorescein-TAMRA-labeled fluorogenic probe. Since the HLA-A amplicons had to include the 2nd intron as the target for the fluorogenic probe, the sequence motifs which could be used as priming sites were limited. Therefore, some primer mixes covered more than one specificity resulting in ambiguous amplification patterns in 31 of 231 possible allele or group combinations of HLA-A1-A80. These ambiguities, which all involved the inability to discriminate a particular heterozygous genotype from a homozygous genotype, may be resolved by the quantitative data revealed by fluorotyping. This feature is also helpful to detect new alleles which are not amplified by the current primer mixes.

DNA Primers↗
PubMed1997

Preparation of autologous bone marrow grafts for cryopreservation using the AS104 cell separator.

We evaluated the AS104 cell separator (Fresenius AG, Bad Homburg, Germany) for ex vivo processing of bone marrow (BM) grafts of 43 patients suffering from germ cell cancer (GCC, n = 22), acute lymphocytic leukemia (ALL, n = 13) and malignant lymphoma (ML, n = 8). Recoveries of total nucleated cells (TNC), mononuclear cells (MNC) and colony-forming units granulocyte-macrophage (CFU-GM) were determined in the BM concentrates prepared for cryopreservation. Hematopoietic reconstitution was analyzed in patients who underwent autologous transplantation following high-dose radio-/chemotherapy (HDRCT). Processing of the BM suspension with a median volume of 1,013 ml (range: 422-1,574) resulted in 156 ml (80-186) within 50-120 min (median: 90). In the BM concentrates, medians of 28.6% TNC (10.6-69.6), 37.9% MNC (22.3-86.4), and 52.4% CFU-GM (20.8-96.4) were recovered. Twenty-six patients underwent HDRCT with reinfusion of autologous BM and were evaluable for engraftment. They received a median of 0.8 x 10(8) MNC/kg (0.3-1.6 x 10(8)) and 2.2 x 10(4) CFU-GM/kg (0.6-12.8 x 10(4) for hematopoietic rescue. Engraftment with neutrophils > 500/microliter occurred in a median time of 12 days (8-33) in all patients. We conclude that ex vivo processing of autologous BM with median recovery rates of 37.9% for MNC, and 52.4% for CFU-GM, results in a cell population that can rescue patients from HDRCT. The described technique is convenient, time-efficient, and provides reliable results in preparing BM autografts for cryopreservation.

Adolescent↗
PubMed1997

German consensus on immunogenetic donor search for transplantation of allogeneic bone marrow and peripheral blood stem cells.

In Germany allotransplantation of bone marrow or peripheral blood stem cells is presently performed by 34 different teams operating more or less independently. Thus, strategies of immunogenetic donor search, use of the various tissue typing techniques and policy on acceptable HLA mismatches in related and unrelated settings may vary considerably from one transplant centre to another. This paper summarises the results of the first German consensus meeting on immunogenetic donor search for bone marrow/peripheral blood stem cell grafting. The main goal of the participating transplant physicians and immunogeneticists was to define national standards for the above issues.

Bone Marrow Transplantation↗