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Rainer Blasczyk

Publications and source records attributed to Rainer Blasczyk.

At least 19 recordsLinked to original sources
PubMed2007

Regulating MHC expression for cellular therapeutics.

BACKGROUND: In the past decade, regenerative medicine and cell-based therapies have emerged as new science and technology, with the main goal of repairing, replacing, or regenerating new tissues. A critical issue in this field is the high polymorphism of HLA, which compromises immune acceptance. The lentivirus-mediated delivery of short-hairpin RNAs (shRNAs) has proved to be an efficient method to inhibit the translation of a specific gene. STUDY DESIGN AND METHODS: A lentiviral-based vector system was used for drug-inducible expression of shRNA sequences that target either beta2-microglobulin (beta2m) or HLA heavy-chain transcripts. RESULTS: The transduction of inducible RNA interference cassettes containing the sequences for shRNAs targeting beta2m or HLA heavy chain suppressed HLA class I expression by up to 90 percent in HeLa and B-lymphocyte cell lines as well as in peripheral blood monocytes. The expression of HLA class I antigens was fully restored in these cells after the drug had been discontinued. It was demonstrated that HLA class I knockdown was effective in preventing antibody-mediated cell lysis and CD8+ T-cell response. The residual HLA expression in HLA-silenced cells may provide sufficient protection against natural killer cell-mediated lysis. CONCLUSIONS: These data demonstrate the feasibility of controlling HLA expression by genetically modifying cell-based therapeutics to overcome the limitations of immune rejection, bringing cellular therapies closer to reality.

CD8-Positive T-Lymphocytes↗
PubMed2007

The molecular diversity of Sema7A, the semaphorin that carries the JMH blood group antigens.

BACKGROUND: Semaphorin 7A (Sema7A), the protein that carries the JMH blood group antigen, is involved in immune responses and plays an important role in axon growth and guidance. Because previous serologic studies on red blood cells (RBCs) suggested a considerable diversity of Sema7A, the present study was designed to elucidate the complex picture of the molecular diversity of this protein. STUDY DESIGN AND METHODS: The JMH antigen status was determined by serology, flow cytometry, and Western blot. Genomic and transcript analysis of SEMA7A was performed by nucleotide sequencing. Recombinant Sema7A proteins were used for genotype-phenotype correlation. A three-dimensional model of Sema7A was generated for topologic analyses. RESULTS: Our studies on 44 individuals with unusual JMH phenotypes and their family members revealed that aberrant Sema7A expression can be an inherited or an acquired phenomenon and is based on reduced surface expression or qualitative changes in Sema7A. These different phenotypes are caused by variations of the SEMA7A gene or seem to be generated by autoimmune-related or RBC lineage-specific mechanisms. The variant JMH phenotypes were related to the presence of missense mutations in SEMA7A, predicting amino acid changes in the semaphorin domain of Sema7A. Sequence analysis of the variant SEMA7A alleles revealed mutations affecting codons 207 and 460/461. Topologic analyses showed that Sema7A polymorphisms were prominently located on the top and bottom of the semaphorin domain, suggesting a functional relevance of these sites. CONCLUSION: These findings provide a basis with which to delineate the various ligand-binding surfaces of Sema7A.

Antigens, CD↗
PubMed2006

A modular concept of HLA for comprehensive peptide binding prediction.

A variety of algorithms have been successful in predicting human leukocyte antigen (HLA)-peptide binding for HLA variants for which plentiful experimental binding data exist. Although predicting binding for only the most common HLA variants may provide sufficient population coverage for vaccine design, successful prediction for as many HLA variants as possible is necessary to understand the immune response in transplantation and immunotherapy. However, the high cost of obtaining peptide binding data limits the acquisition of binding data. Therefore, a prediction algorithm, which applies the binding information from well-studied HLA variants to HLA variants, for which no peptide data exist, is necessary. To this end, a modular concept of class I HLA-peptide binding prediction was developed. Accurate predictions were made for several alleles without using experimental peptide binding data specific to those alleles. We include a comparison of module-based prediction and supertype-based prediction. The modular concept increased the number of predictable alleles from 15 to 75 of HLA-A and 12 to 36 of HLA-B proteins. Under the modular concept, binding data of certain HLA alleles can make prediction possible for numerous additional alleles. We report here a ranking of HLA alleles, which have been identified to be the most informative. Modular peptide binding prediction is freely available to researchers on the web at http://www.peptidecheck.org .

Alleles↗
PubMed2006

Aberrant expression of HLA-B*3565Q is associated with a disrupted disulfide bond.

The identification of expression variants is a challenge in HLA diagnostics. We here describe the identification of the novel allele HLA-B*3565Q. The serological HLA class I type, as determined by a lymphocytotoxicity test, was A11,24; B38; Bw4; Cw-; whereas PCR-sequence-specific primers resulted in A*11,*24, B*35,*38; Cw*12, thus suggesting the presence of a nonexpressed B*35 allele. To clarify the lack of serological HLA-B35 reactivity, exons 2 and 3 were sequenced following haplotype-specific amplification. At position 564 from the beginning of the coding region (exon 3), a transversion (C-->G) was observed, which, at the amino acid level, results in a substitution from cysteine to tryptophane at position 164 of the mature polypeptide. Because this position is essential for the formation of a disulfide bond linking the cysteine residues at positions 101 and 164, which is strongly conserved in functional class I molecules of vertebrates, the disruption of this bond is very likely to be the reason for the lack of serological detectability. We later found the same novel allele in a second unrelated individual, of whom we were able to establish a lymphoblastoid cell line (B-LCL). Serological testing of this B-LCL indicated a very low aberrant expression of HLA-B*3565Q, which cannot be expected to be detected by standard serology techniques.

Alleles↗
PubMed2006

The replacement mutation in HLA-DRB1*1211 affects a likely keystone position.

Currently, 10 different amino acid variants of the HLA-DRB1*12 family are known. We here report the identification of a new HLA-DRB1*12 allele in a healthy Caucasian male individual. The allele was detected by sequencing-based typing during confirmatory high-resolution typing of an unrelated, male, potential donor from the Czech National Marrow Donors Registry. Compared with DRB1*120101, to which it is closest, the new variant is characterized by a new replacement mutation (T-->C) at nucleotide position 126 of exon 2, resulting in the amino acid substitution Phe-->Leu at position 47. Computational analysis reveals that position 47 functions as a keystone in the beta(1) domain, joining both segments of the alpha helix with the beta sheet, and plays a major role in the structural conformation of the binding groove. Additionally, position 47 is part of pocket E of the peptide binding groove and is directly involved in peptide binding. The new allele, DRB1*1211, is therefore likely to differ substantially from other DRB1*12 alleles in its peptide binding repertoire and alloreactive potential.

Amino Acid Substitution↗
PubMed2006

Class-, gene-, and group-specific HLA silencing by lentiviral shRNA delivery.

HLA incompatibility is the most relevant immunologic barrier to cell-based therapies. Improvement of histocompatibility is essential to achieving better survival of allogeneic cells in the foreign organism. RNA interference technology can be used to selectively and stably reduce cellular HLA class I expression. In the present study, we designed small interfering RNA (siRNA) molecules that target either beta2-microglobulin (beta2m) or HLA-A heavy chain transcripts and identified sensitive sites on the target RNAs using an in vitro transcription/translation (IVTT) system. Transfection of siRNA into B-lymphocyte cell lines (B-LCLs) resulted in specific reduction of HLA class I or HLA-A antigen expression by 79% at the mRNA and protein levels. An allele-specific HLA silencing rate of 65% was achieved in a B-LCL heterozygous for HLA-A*24,*68 allospecificities using HLA-A*68-specific siRNA. Lentiviral delivery of short hairpin RNA into HeLa and B-LCL cells resulted in selective and permanent silencing of HLA class I or HLA-A by up to 90% even under inflammatory conditions. In cytotoxicity and proliferation assays, it was demonstrated that HLA class I knockdown was effective in preventing antibody-mediated cell lysis and CD8+ T cell response, while the residual HLA expression in HLA-silenced cells was protective against NK-cell-mediated lysis. The present data strongly suggest that silencing of HLA expression in a class-, gene-, and group-specific manner is an effective approach that may provide a new basis for developing new immunotherapies in the field of regenerative medicine.

B-Lymphocytes↗
PubMed2006

A weak blood group A phenotype caused by a translation-initiator mutation in the ABO gene.

BACKGROUND: Weak blood group A and B phenotypes are correlated with ABO glycosyltransferases exhibiting single-amino-acid changes and/or C-terminal modifications. STUDY DESIGN AND METHODS: A healthy donor diagnosed as having weak A antigen expression and his two children were subjected to extensive ABO typing. HeLa cells were used to transfect ABO expression plasmids. RESULTS: The donor's red blood cells were type A(weak)B and his serum sample contained weakly reactive anti-A(1) antibodies. A single T>C transition identified at the +2 position of the start codon of an ABO*A101 allele predicted the disruption of this methionine codon. In the transfection studies, a significant reduction of A activity was observed on HeLa cells transfected with a plasmid containing the variant ABO*A allele. Coexpression of the respective antithetical ABO*B101 wild-type construct further reduced cell surface A antigen expression. Similar expression results were obtained with ABO constructs in which the Met(1) start codon and five alternative start sites at codons 20, 26, 43, 53, and 69 had successively been interrupted. CONCLUSION: The donor's weak blood group A phenotype most likely resulted from expression of an N-truncated A transferase triggered by alternative translation start sites in the transmembrane domain or stem region.

ABO Blood-Group System↗
PubMed2006

Tetanus toxoid provides efficient T-cell help for the induction of HA-1(H) cytotoxic T cells.

BACKGROUND: In vitro generation and expansion of leukemia-reactive T cells may improve the efficacy and specificity of cellular immunotherapy against hematologic malignancies in the context of allogeneic stem cell transplantation. Since the expression of minor histocompatibility antigen HA-1(H) is limited to hematopoietic cells, ex vivo generated HA-1(H)-specific CD8+ cytotoxic T lymphocytes (CTLs) can be used for adoptive immunotherapy. STUDY DESIGN AND METHODS: Numerous studies have shown that primary CTL induction from naïve precursors requires professional antigen-presenting cells. Here, the feasibility of ex vivo induction of HA-1(H)-specific CD8+ CTLs is demonstrated from unfractionated peripheral blood mononuclear cells (PBMNCs) from healthy blood donors when CD4+ T-cell help is provided during primary stimulation. As a stimulus for the induction of T-cell help, tetanus toxoid (TT) was used. RESULTS: After the second restimulation cycle, approximately 1 percent of CD8+ T cells stained positively with the HLA-A*0201/HA-1(H) pentamer. Positive T cells were further expanded more than 1000-fold by antigen-independent stimulation with anti-CD3/CD28 monoclonal antibodies. HA-1(H)-induced T cells showed the classical phenotype for CD8+ memory effector cells: the phenotype changed from a mixed CD45RA/RO phenotype to an activated phenotype characterized by high expression of CD45RO and no expression of CCR7. The generated T cells revealed a very potent CTL response, even at low E:T ratios. CONCLUSION: This study demonstrates that TT provides a very potent and cost-effective tool for the in vitro induction of antigen-specific CTLs from precursor PBMNCs that can easily be adapted to GMP conditions for translational purposes.

Antigen Presentation↗
PubMed2005

A dose-response analysis of lenograstim plus dexamethasone for neutrophil mobilization and collection.

BACKGROUND: The objective was to evaluate the dose-response relationship of lenograstim plus dexamethasone for neutrophil mobilization and collection. STUDY DESIGN AND METHODS: In a prospective study, 260 healthy volunteers received oral dexamethasone (8 mg) plus a single subcutaneous injection of glycosylated granulocyte-colony-stimulating factor (G-CSF; lenograstim) at medians of 1.5 (1.0-2.3) microg per kg (n = 43), 3 (2.4-4.1) microg per kg (n = 73), 6 (4.3-7.9) microg per kg (n = 123), and 12 (8.2-17.2) microg per kg (n = 21) and underwent neutrophil collections with a polymorphonucleated neutrophil (PMN) program. White blood cell (WBC) counts and PMN mobilization and collection results were compared, and the severity and clinical significance of donor adverse reactions were evaluated. Fifty-two neutropenic patients (29 children, 23 adults) underwent 271 neutrophil transfusions (GTXs) every other day to maintain WBC levels continuously above 0.5 x 10(9) per L. RESULTS: Within the dose range 1.5, 3, and 6 microg per kg, each doubling step was associated with a 10 to 15 percent PMN increase in peripheral blood up to 32.8 (19.1-49.2) x 10(9) per L (6 microg/kg; p </= 0.00032) as well as in the neutrophil concentrate up to 79 (34-150) x 10(9) per U (6 microg/kg; p </= 0.00042). A further doubling to 12 microg per kg achieved neither better mobilization nor better apheresis results. The rate of clinically important adverse reactions increased already with the 6 microg per kg mobilization step. The GTX resulted in median peak WBC increments to 3.8 (0.4-18.2) x 10(9) per L (children) and 1.6 (0.3-9.4) x 10(9) per L (adults), but in adults the WBC threshold of 0.5 x 10(9) per L was not continuously exceeded. CONCLUSIONS: The most effective dose-response ratio for PMN mobilization was demonstrated in the 6 microg per kg lenograstim group. In neutropenic adults, GTX treatment on an every-other-day schedule may be ineffective.

Adjuvants, Immunologic↗
PubMed2005

Nondeletional ABO*O alleles frequently cause blood donor typing problems.

BACKGROUND: Difficulties in the demonstration of expected isoagglutinins is a common problem in ABO reverse typing. Some nondeletional ABO*O alleles have been shown to encode for the expression of minimal amounts of A antigen, resulting in very weak anti-A activity in some cases. It is unknown whether minor problems with ABO reverse typing are related to specific ABO*O alleles. STUDY DESIGN AND METHODS: Among 2196 blood group O red cell (RBC) donations, the ABO alleles of those donations in which the isoagglutinins were incorrectly identified were analyzed with an autoanalyzer. The presence of nondeletional ABO alleles was determined by sequence-specific priming and sequencing. RESULTS: Fifty (2.3%) of the group O RBC donations tested had to be typed manually because of isoagglutinin detection problems in automated typing: reduced anti-A activity was observed in 45 cases, reduced anti-B activity in 4 cases, and variably reduced isoagglutinin activity in 1 case. The nondeletional ABO*O alleles ABO*O03 and ABO*Aw08 were implicated in 38 of these 50 cases (1.7% of all blood group O donors). The remaining samples, including those with reduced anti-B activities, were homozygous for deletional ABO*O alleles. CONCLUSION: Nondeletional ABO*O alleles are the most frequent cause of isoagglutinin detection problems in blood group O donors.

ABO Blood-Group System↗
PubMed2005

Missense mutations outside the catalytic domain of the ABO glycosyltransferase can cause weak blood group A and B phenotypes.

BACKGROUND: Only little is known about the impact of amino acid substitutions outside an enzyme's active site on A and B transferase activity. STUDY DESIGN AND METHODS: A panel of blood group A- and B-specific plasmids containing the six known missense mutations of the coding sequence upstream of exon 6 of the ABO gene were constructed. HeLa cells were used to transfect ABO expression plasmids. RESULTS: Expression of ABO variants containing single or multiple missense mutations in HeLa cells resulted in a significant decrease in the percentage of antigen-expressing cells (up to 29%) and in mean fluorescence intensity (MFI; up to 50%) compared to transfection with ABO*A101 or ABO*B101. Coexpression of the respective antithetical wild-type construct (ABO*A101 and ABO*B101, respectively) further reduced cell surface expression of variant ABO constructs in regard to the percentage of expressing cells (up to 53% decrease) and MFI (up to 59% decrease). CONCLUSION: Weak A and B subgroups can arise from transferases with amino acid changes in the N-terminal domain, particularly in AB phenotypes, where normal A1 or B1 glycosyltransferases compete for the same substrates.

ABO Blood-Group System↗
PubMed2005

Nondeletional ABO*O alleles express weak blood group A phenotypes.

BACKGROUND: Owing to a single-base deletion, the vast majority of ABO*O alleles encode for a truncated and catalytically inactive ABO glycosyltransferase, leading to the generation of a premature stop codon. Less frequent nondeletional ABO*O alleles such as ABO*O03, in contrast, have nonsynonymous mutations that may abolish the protein's enzyme activity by altering its sugar-binding site. STUDY DESIGN AND METHODS: Extensive ABO phenotyping and genotyping were performed in healthy blood group O donors with weak anti-A isoagglutinins and their relatives as well as in blood group O donors selected for the presence of ABO*O03. HeLa cells were used to transfect ABO expression plasmids. RESULTS: Donors or relatives carrying ABO*O03 and/or its rare variant ABO*Aw08 in homozygous (n = 2) or heterozygous (n = 14) form showed weak A antigen expression detectable only by adsorption-elution (n = 15) or by monoclonal anti-A typing (n = 1). The serum samples of most donors (n = 13) contained weak anti-A; in the remaining donors, anti-A isoagglutinin reactivity was in the normal range. In the transfection studies, weak A antigen expression on HeLa cells transfected with plasmids containing ABO*O03 or ABO*Aw08 expression constructs was detectable only by adsorption-elution. CONCLUSION: The data provide evidence that nondeletional ABO*O03-like alleles produce detectable amounts of A antigens.

ABO Blood-Group System↗
PubMed2005

ABO glycosyltransferases as potential source of minor histocompatibility antigens in allogeneic peripheral blood progenitor cell transplantation.

BACKGROUND: Most studies indicate that the incidence of graft-versus-host disease (GVHD) is not increased in ABO-mismatched allogeneic peripheral blood progenitor cell transplantation. These studies exclusively looked at ABO phenotypes without considering the fact that different genotypes hide behind identical phenotypes that encode for different sets of glycosyltransferases, thus providing a source for minor histocompatibility antigens (mHags). STUDY DESIGN AND METHODS: Therefore, whether peptides derived from ABO glycosyltransferases are capable of stimulating peptide-specific T cells was investigated. T-cell responses were identified by measuring intracellular interleukin-2 expression. RESULTS: Individuals with ABO genotypes encoding glycosyltransferases lacking the peptide sequences used for stimulation showed T-cell responses, whereas those expressing glycosyltransferases containing the respective peptide sequences proved to be tolerant, indicating that ABO peptides are allogeneic and may act as mHags. Interestingly, even ABO*O individuals were tolerant to O glycosyltransferase-derived peptides, which strongly suggests that truncated O transferases are expressed. CONCLUSION: Considering allelic ABO sequences, at least 15 percent of all phenotypically ABO-matched transplant pairs can be expected to have genotype constellations relevant to GVHD. Therefore, the genotype behind the ABO blood group phenotype should be considered to answer the question of whether ABO mismatch is a risk factor of GVHD.

ABO Blood-Group System↗
PubMed2004

Peptide-binding motif of HLA-A*6603.

The peptide motif of HLA-A*6603 was determined and compared with the available data on the peptide motifs of A*6601 and A*6602. A*6601 differs from A*6602 by two amino acids at positions 90 (Asp90Ala; outer loop) and 163 (Arg163Glu; pocket A). A*6603 differs from A*6601 and A*6602 by a single amino-acid exchange at position 70 (His70Gln; pockets A, B and C). No significant differences were found between the A*6602 and A*6603 peptide motifs suggesting that the Gln70His variation is of minor importance. However, the auxiliary anchors at position P1 of peptides bound by A*6601 (polar/acidic: Asp, Glu) and A*6602/6603 (polar/neutral: Ser) had striking differences. This finding may be best explained by the Arg163Glu substitution that results in a shift towards higher acidity in pocket A of A*6602/6603, apparently leading to the loss of preference for acidic auxiliary anchors. The similarity of A*6602 and A*6603 peptide motifs suggests low allogenicity when mismatched in stem cell transplantation. Inversely, the differences in A*6601 versus A*6602/6603 peptide motifs suggest that mismatches will have a higher allogenicity. These data will contribute to both assessing permissive mismatches in the A*66 group and weighting the impact of this individual amino-acid variation for matching and peptide binding algorithms.

Amino Acid Motifs↗
PubMed2004

A single amino-acid polymorphism in pocket A of HLA-A*6602 alters the auxiliary anchors compared with HLA-A*6601 ligands.

In this study we have sequenced peptides eluted from a truncated recombinant HLA-A*6602 molecule, and compared their features with data reported for peptides presented in the A*6601 molecule. A striking change in the amino-acid binding preferences was observed at peptide position P1, which interacts with pocket A of the HLA peptide-binding region. For A*6601, aspartic acid and glutamic acid, both of which possess polar acidic side-chains, have been described as auxiliary anchors. This is in marked contrast to A*6602, where we observed serine, which has a neutral polar side-chain, as auxiliary anchor at P1. Accordingly, this shift in the physico-chemical properties of the auxiliary anchor may be best explained by the HLA amino-acid polymorphism at position 163, where arginine (hydrophilic, alkaline) in A*6601 has been replaced by glutamic acid in A*6602. This amino-acid exchange results in a shift towards higher acidity in pocket A, apparently resulting in the loss of preference for acidic auxiliary anchors, and leading to the preference for the neutral amino acid serine. The change of the auxiliary anchor residue at P1 is likely to alter the spectrum of peptides presented by A*6602 compared with A*6601, which may result in allogenicity in the case of a mismatch in allogeneic stem cell transplantation.

Arginine↗
PubMed2003

The nature of diversity and diversification at the ABO locus.

In this study we analyzed the complete genomic sequences, except intron 1, and 2 regulatory regions of 6 common (ABO*A101, ABO*A201, ABO*B101, ABO*O01, ABO*O02, and ABO*O03) and 18 rare ABO alleles, 3 of which were new. This was done by phylogenetic analysis and correlating sequence data with the ABO phenotypes. The study revealed multiple polymorphisms in noncoding regions. The intron-based phylogenetic analysis revealed 5 main lineages: ABO*A, ABO*B, ABO*O01, ABO*O02, and ABO*O03. The genomic sequences of most rare ABO alleles differed slightly from those of the common alleles. Singular mutations or hybrid alleles were most common, but a few exhibited mosaic sequence pattern containing multiple exon and/or intron motifs from other ABO lineages. Thus, both an accumulation of mutations as well as an assortment of the mutations by recombination seems to be responsible for the ABO gene diversity. The prevalence of replacement mutations indicates positive selection for allelic diversity. Phenotype-genotype correlation showed that sequence variations within the complete coding sequence can affect A- and B-antigen expression. All variant ABO*A/B alleles and one new ABO*O03-like allele were associated with weak ABO phenotypes. These findings are suggestive of the requirement of a comprehensive coding sequence database for sequence-based phenotype prediction.

ABO Blood-Group System↗
PubMed2003

Systematic analysis of the ABO gene diversity within exons 6 and 7 by PCR screening reveals new ABO alleles.

BACKGROUND: Mutations critical for ABO blood group phenotypes have predominantly been found in exons 6 and 7 of the ABO gene, both of which encode the catalytic domain of ABO glycosyltransferase. To design rapid and reliable ABO genotyping assays, a profound knowledge of the prevalent alleles is required and a reliable sequence database needs to be established. STUDY DESIGN AND METHODS: A PCR screening system was established consisting of 102 different PCRs, each specific for a single nucleotide (nt) variation. The primer mixes were developed to walk from the 5' to the 3' end of exons 6 and 7 of the ABO gene to screen for nt mutations at 50 known polymorphic sites. A total of 109 unrelated individuals with common and rare ABO characteristics were screened. All blood samples in which the PCR results were inconclusive or inconsistent with the ABO phenotypes were subjected to sequence analysis of exons 6 and 7. RESULTS: The results of PCR screening were conclusive and consistent with the ABO phenotypes in 90 cases. In the remaining 19 cases, PCR screening revealed unusual allele combinations or amplification results that were incompatible with known ABO allele combinations or subgroups predicted by serologic analysis. In these 19 cases, sequencing revealed new ABO alleles (one ABO*Ael allele, one ABO*B(A) allele and two ABO*O alleles) in two individuals with common and seven individuals with variant ABO phenotypes. CONCLUSION: This PCR screening strategy is an effective tool for obtaining deeper insight into the ABO gene diversity and diversification and may be useful to increase the quality of the ABO sequence database.

ABO Blood-Group System↗