Lymphadenopathy in patients with multiple sclerosis undergoing treatment with glatiramer acetate.
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Glatiramer Acetate↗
Biomedical subjects
R Blasczyk
Publications and source records attributed to R Blasczyk.
The noncoding regions of HLA-DRB uncover interlineage recombinations as a mechanism of HLA diversification.
The mechanisms generating new alleles at the MHC loci are still unknown in detail, and several proposals have been made to explain the extent of polymorphism. The patchwork pattern of polymorphism in the 2nd exon of HLA-DRB1 recommends this locus as a model for the study of the potential of interallelic gene conversion. In general, the inference of gene conversion-like events based exclusively on exon sequence comparisons may be misleading because the identity of the putative donor allele remains unknown. In this study, we describe five alleles of the HLA-DRB1 gene, which intron regions give evidence for interlineage recombination events either strictly located at the 2nd exon or involving the adjacent introns. Furthermore, we show that the noncoding regions provide important clues to the mechanisms of the generation of new alleles, and our results indicate that interlineage recombinations may be hidden and are perhaps more frequent than currently expected.
Alleles↗
Identification of the novel allele HLA-B*0809 in a Caucasian individual: estimation of allogeneic potential between B*08 variants.
The identification of the new allele HLA-B*0809, which was found in a Caucasian individual, is described. In the sequence analysis the new allele differs from B*0801 by six nucleotides located in exon 3. As the structure is identical to a variety of other HLA-B alleles, it is likely that the new allele originated by gene conversion. At the protein level, the new allele has two amino acid differences compared to B*0801. Comparing the residues of the alpha1 and alpha2 domains of the B*08 variants to each other, a high degree of polymorphism was found. Three alleles differ from B*0801 by only one amino acid residue whereas the other comparisons revealed at least two disparities. B*0809 differs from the other B*08 variants by at least two amino acid residues, suggesting that mismatching may provoke alloreactivity and thus impair clinical outcome of bone marrow transplantation. Among the B*08 alleles with a single amino acid difference, the mismatch combinations B*0801 vs. B*0805 and B*0801 vs. B*0807 appear to be the most compatible based on structural data.
Alleles↗
Identification of the novel allele HLA-B*1545: assessment of alloreactivity in case of mismatch with other B*15 alleles.
The novel allele HLA-B*1545, which has been serologically typed as B62, was identified in a male Caucasian. In the sequence analysis the new allele differs from B*1507 by nucleotide 419 which is located in exon 3. Its structure suggests that it may have originated by a point mutation or by gene conversion with a variety of HLA-B alleles. At the protein level, the nucleotide substitution results in an amino add exchange compared to B*1507 (Tyr116Ser). Due to probably substantial differences to B*1507 and the other B*15 variants with regard to peptide binding, a mismatch is likely to impair clinical outcome of bone marrow transplantation.
Alleles↗
Identification of the novel allele HLA-B*1546 which belongs to the serological B72 type: implications for bone marrow transplantation.
The identification of the novel allele HLA-B*1546, which has serological B50 and B72 reactivity, was found in two members of a family of Turkish origin. In the sequence analysis the new allele differs from B*1501 by four nucleotides in exon 2. Its structure suggests that it may have originated by gene conversion with B*40, B*41, B*44, B*4501, B*47, B*4901 or B*50. At the protein level, the new allele has three amino acid differences compared to B*1501. Sequence alignment demonstrates that amino acid residue 46 is crucial for serological B72 reactivity. Due to substantial differences with other B*15 variants a possible mismatch may impair clinical outcome of bone marrow transplantation.
Alleles↗
HLA-A*2416: a new allele of the HLA-A19 lineage.
We here report on a new HLA-A19 allele (A*2416) found in a German kidney recipient. Serological class I typing revealed HLA-A11,19 without clear definition of the A19 split antigen. As with serology, polymerase chain reaction (PCR)-based typing also revealed inconclusive results. We therefore sequenced the gene from the 5' flanking region through the 3'-end of exon 4 of this allele after haplotype-specific PCR amplification. The sequence analysis revealed a new HLA-A allele which is identical to A*3101 with the exception of the 3' half of exon 2 which is identical to the common A9 alleles. The phylogenetic analysis constructed with the nearest-neighbor algorithm and based on exons 1-4 or introns 1-3 clearly indicated, that A*2416 belongs unequivocally to the A19 lineage.
Alleles↗
Identification of the novel allele HLA-A*6813 in two members of a family of Syrian origin: implications for bone marrow transplantation.
The identification of the new allele HLA-A*6813, which was found in a woman of Syrian origin and her son, is described. In the sequence analysis the new allele differs from A*68011 by positions 259 (A>G) and 261 (C>G) in exon 2. As the structure is thus identical to the HLA-A consensus sequence it is likely that the new allele originated by gene conversion. At the protein level, the new allele has one amino acid difference from A*6801 (Asn63Glu), which results in a distinct banding pattern in one dimensional-isoelectric focusing. Amino acid residue 63 contributes to the formation of pocket A and B and is thus important for peptide binding. A*6813 was serologically detectable only by two of six polyclonal, but by three monoclonal antisera. The restricted serological A68 activity may be explained by altered peptide binding as presented peptides can affect the serological recognition of major histocompatibility complex (MHC) class I molecules. Moreover, our findings suggest that a possible mismatch with the other known A*68 variants may impair clinical outcome of bone marrow transplantation.
Alleles↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗