Biomedical subjects

A Kuhröber

Publications and source records attributed to A Kuhröber.

14 recordsLinked to original sources

PubMed2000

HLA-DRB1,3,4,5 and -DQB1 allele frequencies and HLA-DR/DQ linkage disequilibrium of 231 German caucasoid patients and their corresponding 821 potential unrelated stem cell transplants.

Allelic matching within the HLA-DRB1 and -DQB1 loci significantly improves the clinical outcome of hematopoietic stem cell transplantation. Consequently, allelic typing of these loci is strongly recommended for the unrelated stem cell donor selection. In this study, the HLA-DRB1,3,4,5 and -DQB1 alleles of 231 patients and their corresponding 821 nonrandom potential stem cell donors were determined to define compatible donor/recipient pairs. Highly accurate HLA typing data were achieved by PCR-SSOP and a combination of group specific PCR-SSP and subsequent sequencing-based typing of nearly the whole second exon of each locus. The alleles DRB1*07, *09, and *10 were analyzed by PCR-reverse dot blot hybridization instead of sequencing. Additionally, DRB1 homozygosity was verified by temperature gradient gel electrophoresis. The identified 2104 HLA-DRB1 and HLA-DQB1 alleles as well as data on HLA-DRB3, -DRB4, and -DRB5 alleles were applied to a statistical program and absolute and relative delta values of DR/DQ linkages were calculated. The achieved data on the HLA-DRB1 allele distribution and on DR/DQ associations in terms of subtypes significantly ensure the typing reliability, since rare allele combinations will result in further investigations. Furthermore, detailed data on the DR/DQ allele associations allow estimations of the number of HLA-A, -B, and -DR matched unrelated stem cell donors necessary for the identification of DRB and DQB subtype identical donors.

Alleles↗

PubMed1998

Rapid method for successful HLA class I and II typing from cadaveric blood for direct matching in cornea transplantation.

BACKGROUND: The aim of the study was to establish fast methods for postmortem HLA class I and II typing of cornea donors using cadaveric blood. METHODS: The commercially available reagents Lymphokwik MN and Dynabeads were evaluated here to provide an enriched living mononuclear cell (MNC) population and B-cell population for HLA class I and II typing of cadaveric blood by serology. Cadaveric blood was obtained 1-80 h post mortem. After isolation of living B-cells and B-cell-depleted living MNC's, cells were serologically typed by double-fluorescence cytotoxicity assay for HLA class I and II antigens. RESULTS: In 373 (81%) of 461 cadaveric blood samples HLA class I typing, and in 36 (62%) of 56 cadaveric blood samples HLA-class II typing, by serology was successful and accomplished within 5 h. Results from the serological HLA class I typing were confirmed by the results of HLA class I typing by RNA-based sequencing in seven cases. To improve the HLA class II typing, DNA typing using PCR with sequence-specific primers was performed in 148 samples and reverse hybridization of PCR-amplified DNA to immobilized HLA class II specific primers in 270 samples. These data were confirmed by DNA-based sequencing in five cases and by sequence-specific oligonucleotide hybridization in all cases. CONCLUSIONS: These results lead to the following typing strategy: HLA class I typing should be performed by serology. HLA class II typing should be performed by DNA technology because of its relative independence of the quality of the blood sample. The strategy we have developed is very successful and fast for tissue typing post mortem, thus expanding the time available for ideal HLA matching, increasing the number of available HLA-matched corneas and therefore reducing the number of graft rejections.

Blood Grouping and Crossmatching↗

PubMed1998

Polyvalent vaccination against hepatitis B surface and core antigen using a dicistronic expression plasmid.

Genes encoding the small (S) surface antigen (HBsAg) or the core (C) antigen (HBcAg) of hepatitis B virus (HBV) were cloned into the monocistronic expression vectors pCMV-1 or pCMV-2 under HCMV-IE promoter control. Coding fragments of these vectors were fused to generate a dicistronic expression construct pCMV/C-S in which the antigens HBcAg and HBsAg are coexpressed. Transient in vitro transfection studies demonstrated that HBcAg and HBsAg are coexpressed from this construct. Vaccination of mice of different H-2 haplotypes with mono- or dicistronic expression plasmids induced humoral and cellular immune responses to HBsAg and the HBcAg. In particular, intramuscular injection of 'naked' dicistronic plasmid DNA into mice elicited polyvalent humoral and cytotoxic T lymphocyte responses to HBsAg and HBcAg. The studies demonstrate that dicistronic expression plasmids are a novel way to construct a polyvalent vaccine against HBV that comprises HBsAg and HBcAg as immunogens.

Animals↗

PubMed1997

DNA vaccination with plasmids encoding the intracellular (HBcAg) or secreted (HBeAg) form of the core protein of hepatitis B virus primes T cell responses to two overlapping Kb- and Kd-restricted epitopes.

Plasmid DNA encoding either the intracellular form HBcAg or the secreted form HBeAg of the core protein of hepatitis B virus (HBV) was injected into the muscle of H-2b, H-2d or F1b x d mice. Serum antibody responses and class I-restricted cytotoxic T lymphocyte (CTL) responses to HBcAg/ HBeAg were detected in all mice tested. Stable murine H-2b and H-2d transfectants that express either intracellular HBcAg were secreted HBeAg were constructed. With these cell lines we restimulated in vitro T cells primed in vivo and detected their specific cytolytic reactivity against naturally processed peptides. CD8+ CTL responses elicited by DNA vaccination with plasmids encoding HBcAg or HBeAg were specific for the (previously described) Kd-binding HBcAg93-100 peptide MGLKFRQL in H-2b mice or the (newly defined) Kd-binding HBcAg87-96 peptide SYVNTNMGL in H-2d mice. The overlapping epitopes span residues 87-100 of HBcAg, and are present on HBcAg and HBeAg. CTL responses were equally well elicited in vivo by injecting HBcAg- or HBeAg-expressing plasmid DNA, and CTL efficiently recognize in vitro HBcAg- and HBeAg-expressing transfectants. DNA vaccination of F1b x d mice with HBcAg- or HBeAg-expressing plasmid DNA primed CTL populations that recognized the Kb- or the Kd-restricted epitope. Both Kb- and Kd-binding peptides are thus generated from cytoplasmic/nuclear HBcAg and secreted HBeAg. These data make it unlikely that the appearance of HBeAg-negative variants during chronic HBV infection results from CTL-driven selection. DNA vaccination is an efficient technique to prime CTL responses against overlapping epitopes present on intracellular or secreted viral protein antigens.

Animals↗

PubMed1996

DNA vector constructs that prime hepatitis B surface antigen-specific cytotoxic T lymphocyte and antibody responses in mice after intramuscular injection.

We tested the efficiency of induction of immune responses to the small hepatitis B surface antigen (HBsAg) in mice by intramuscular DNA immunization using different vector constructs that allow high levels of HBsAg expression in mouse cells. The HBsAg-specific responses of class I-restricted cytotoxic T lymphocytes (CTL) and of B cells (serum antibody titers) were measured. Following the intramuscular inoculation of 'naked' DNA, five different vector constructs of 4-8 kb, that contained or did not contain an intron and/or the neo gene, in which HBsAg expression was driven by promoter sequences derived from the immediate early region of HCMV, the SV40 enhancer/promoter region, or a retroviral 3' LTR efficiently primed responses of class I-restricted CD8+ CTL precursors. In contrast, the constructs in which HBsAg expression was driven by HCMV-derived promoter sequences stimulated significantly higher levels of HBsAg-specific serum antibody titers after intramuscular DNA injection than the SV40 or MPSV vector constructs. Large (15 kb) episomal vector constructs did not stimulate CTL or antibody responses. The data demonstrate that: (i) intramuscular DNA immunization represents an efficient technique for priming CTL and antibody responses to HBsAg; (ii) many vectors can be constructed that express an immunogenic product after intramuscular inoculation of 'naked' DNA; (iii) the efficiency of the tested vector constructs to prime after DNA immunization, either a CTL response, or an antibody response, differs.

Animals↗

PubMed1996

Expression of the human alpha2 integrin subunit in mouse melanoma cells confers the ability to undergo collagen-directed adhesion, migration and matrix reorganization.

Previous studies have shown that cultured human and other mammalian cell require the alpha2 beta1 integrin receptor to reorganize and contract 3-dimensional extracellular matrix lattices containing collagen type I. This function is of prime importance for the later phases of wound healing, in which fibroblasts reorganize and contract extracellular matrix components newly deposited in the granulation tissue. It is also known that highly aggressive human melanoma cells have acquired this function, possibly enhancing their invasive potential. To further study alpha2 beta1-mediated functions, we expressed the human alpha2 and beta1 chains of integrins in mouse melanoma cells (BULT). The parental cell line was unable to exert known alpha2 beta1-mediated functions: The cells did not adhere, spread, or migrate on collagen type I, and they did not reorganize 3-dimensional collagen I lattices. Transfection of the human alpha2 chain was sufficient to confer not only specific adhesion to collagen type I in static adhesion assays, but also the ability to exert complex functions such as migration on collagen and efficient reorganization of collagen-I-containing matrices. Coexpression of the human beta1 chain did not further enhance these functions in BULT melanoma cells. This was underscored by the observation that alpha1-specific monoclonal antibodies were able to completely block the newly introduced functions, whereas beta1-specific antibodies had no such effect. Moreover, in transfectants expressing both the human alpha2 and beta1 chains, the human alpha2 chain did not preferentially associate with the human beta1 chain as compared with mouse beta-chains. This may indicate that the molecular structures guiding the physical association of the human alpha2 chain with beta chains are extremely highly conserved between the species.

Animals↗

PubMed1994

Immunization with soluble hepatitis B virus surface protein elicits murine H-2 class I-restricted CD8+ cytotoxic T lymphocyte responses in vivo.

Immunization with soluble proteins only rarely induces a specific response of CD8+ CTL. We describe experiments that demonstrate the efficient and specific in vivo priming of CTL in BALB/c mice immunized with soluble hepatitis B virus (HBV)-derived surface (S) protein. A single (s.c., i.p. or i.v.) injection of a low dose (30 ng to 3 micrograms per mouse) of recombinant S protein particles without adjuvants induced a CTL response. This specific cytotoxic response was read out against a panel of different S protein-expressing transfected mouse cell lines. Effector cells of this response were Ld-restricted, CD3+ CD4- CD8+ CTL. H-2d/Ld+ (BALB/c, C.B-17) mice were responders; H-2d/Ld- (dm2) mutant mice and H-2b (C57BL/6) mice were nonresponders. Injections of various dosages of a S protein-derived, immunogenic, synthetic peptide into BALB/c mice by various routes did not prime CTL. After incorporation of S protein particles into IFA or aluminum hydroxide, these protein Ag lost their ability to specifically stimulate CTL in vivo. After priming of mice with S protein emulsified in IFA or adsorbed to aluminum hydroxide boost injections with native S protein particles were inefficient in stimulating a specific CTL response. These findings are of relevance for the design of synthetic subunit vaccines for which specific stimulation of CD8+ T effector functions is desired.

Adjuvants, Immunologic↗

PubMed1994

Vaccination with T cell receptor peptides primes anti-receptor cytotoxic T lymphocytes (CTL) and anergizes T cells specifically recognized by these CTL.

We selected three peptides from the germ-line sequence of the V beta 8.2 and J beta 2.3 gene segments of the murine T cell receptor for antigen (TCR) which contained putative Kd- and Ld-restricted epitopes. Immunization of BALB/c (H-2d) mice with the V beta 8.2(67-90) 23-mer peptide 1 as well as the 15-mer V beta 8.2(95-108)-peptide 2 efficiently primed specific CD8+ cytotoxic T lymphocyte (CTL) responses in vivo against natural TCR-V beta 8.2 epitopes. V beta 8.2+ T cells were not deleted in TCR peptide-immunized mice because the fractions of V beta 8.2+ CD4+ and V beta 8.2+ CD8+ T cells in spleen and lymph nodes were not altered. The proliferative response of V beta 8.2+ T cells to stimulation by monoclonal antibody F23.2 was selectively suppressed (by 60-80%) in peptide-immunized BALB/c mice, indicating partial anergy of this T subset. Immunization of BALB/c mice with the J beta 2.3-derived peptide 3 stimulated a CD8+ CTL response against a class I-restricted epitope within this J beta segment that was also generated during natural "endogenous" processing of this self antigen. These data confirm the predictive value of major histocompatibility complex class I allele-specific motifs. The described experiments indicate that TCR peptide-primed CD8+ CTL recognize class I-restricted, natural V beta/J beta-TCR epitopes. Such anti-TCR CTL may, thus, operate in V beta-specific immunoregulation of the T cell system suppressing their functional reactivity without deleting them.

Amino Acid Sequence↗

PubMed1994

A self-reactive class I-restricted T-cell response of H-2b mice to determinants of the V beta 8.2 domain of the T-cell receptor for antigen.

We studied the induction of a self-reactive cytotoxic T lymphocyte (CTL) response to determinants of the variable V beta 8.2 region of the beta-chain of the T-cell receptor (TCR) for antigen in C57BL/6 (H-2b) mice. A CTL response was elicited in vivo by TCR peptide vaccination, and detected in vitro using syngeneic transfectants expressing a rearranged V beta 8.2+ TCR beta-chain. The first series of experiments used a 15-mer peptide representing residues 68-82 of the V beta 8.2 domain and containing Kb and Db allele-specific motifs. Immunization with this peptide stimulated an autoreactive CTL response that cross-reacted with V beta 8.2 epitopes presented by transfectants endogenously processing a V beta 8.2+ TCR beta-chain. These transfectants expressed a construct derived from a murine, rearranged V beta 8.2/D beta 2/J beta 2.3/C beta 2 TCR beta-chain cDNA. The V beta 8.2+ T-cell subset of peptide-primed mice was not deleted but its proliferative response to stimulation by the V beta 8.2-specific monoclonal antibody (mAb) F23.2 was suppressed. In a second series of experiments we immunized mice with a 23-mer peptide representing residues 41-63 of the V beta 8.2 domain that does not contain putative, allele-specific H-2b class I-restricted motifs. This TCR peptide vaccination stimulated a CD8+ CTL response reacting against syngeneic, peptide-pulsed targets but not cross-reacting against transfectants processing/presenting epitopes of the beta-chain. V beta 8.2+ T cells of these peptide-primed mice were not anergized. These data demonstrate that vaccination with an immunogenic peptide representing a naturally processed epitope of the V beta 8.2 domain of the TCR beta-chain induces a self-reactive CD8+ CTL specific for this V beta 8.2 epitope; and anergizes (but does not delete) V beta 8.2+ T cells.

Amino Acid Sequence↗

PubMed1993

Immunization of mice with the N-terminal (1-272) fragment of simian virus 40 large T antigen (without adjuvants) specifically primes cytotoxic T lymphocytes.

Immunization of C57BL/6 (B6) mice (H-2b) with the "large tumor antigen" (T-Ag) of simian virus 40 (SV40) in its soluble form without adjuvants primed CD8+ cytotoxic T lymphocytes (CTL) in vivo. CD8+ CTL primed in vivo by this non-structural 708-amino acid (aa) viral protein, and specifically restimulated in vitro, lysed H-2b target cells, either transfected with an SV40 T-Ag-encoding vector, or transformed by SV40 infection. H-2b RMA-S transfectants expressing the complete 708 aa T-Ag (which fail to transport peptides through the endoplasmic reticulum membranes) were not lysed. CTL were also efficiently primed in vivo by injection of the N-terminal 272 aa fragment of the T-Ag. Hence, this fragment contains the structure(s) required for a soluble protein to enter the "endogenous" class I-restricted antigen processing and presentation pathway for CD8+ CTL activation. In soluble form, the complete T-Ag or the N-terminal T-Ag fragment sensitized in vitro RBL5 cells for lysis by T-Ag-specific CTL lines and clones. This in vitro sensitization was blocked by brefeldin A. In contrast, specific recognition of RBL5 cells pulsed in vitro with synthetic, immunogenic nonapeptides (derived from N-terminal T-Ag epitopes) by CTL lines was insensitive to brefeldin A. Hence, T-Ag and its 272-aa N-terminal fragment can enter the "endogenous" processing pathway and prime CD8+ CTL in vivo and in vitro.

Adjuvants, Immunologic↗

PubMed1993

Diversity of functional T-cell receptor delta-chain transcripts from bone marrow cells of athymic nude mice.

CD3+ cells are detectable in bone marrow of athymic mice homozygous for the nude mutation. As previously shown, cells expressing the gamma delta T-cell receptor (TcR) represent 30-40% of this T-cell population. Using V delta-specific, V alpha 4-specific, and C delta-specific primers, TcR delta-chain transcripts were reverse transcribed and polymerase chain reaction (PCR)-amplified from total RNA prepared from bone marrow cells (BMC) of 6-month-old NMRI nu/nu mice. Amplified TcR delta-chain cDNA was cloned, and 49 randomly selected clones derived from seven amplification reactions were sequenced. Sequence analyses showed: (1) more than 80% of the sequenced clones represented in-frame transcripts of the TcR delta-chain; (2) in-frame transcripts containing V delta 1-, V delta 2-, V delta 3-, V delta 4-, V delta 5-, V delta 6- and V alpha 4-gene segments were detectable in nude BMC; (3) V delta 2-, V delta 4- and V delta 5-containing transcripts were more abundant and more diverse than V delta 1- and V delta 3-containing transcripts; (4) extensive N-region diversity was present in the V delta-D delta 2 (N1), D delta 2-D delta 1 (N2) and D delta 1-J delta 1 (N3) junctional regions; (5) P nucleotide additions were present in many transcripts; and (6) unusual truncated, in-frame transcripts with deleted D- and J-region genes were detected. A large potential TcR delta-chain repertoire is thus present in nude BMC.

Amino Acid Sequence↗

PubMed1992

Immunization with soluble simian virus 40 large T antigen induces a specific response of CD3+ CD4- CD8+ cytotoxic T lymphocytes in mice.

C57BL/6 (B6) mice (H-2b) were immunized with the large tumor antigen (T Ag) of simian virus 40 (SV40). Intraperitoneal or subcutaneous sensitization with soluble T Ag specifically primed cytotoxic lymphocyte precursors (CTLp). T Ag-specific cytotoxic T lymphocytes (CTL) were detected in a cytotoxicity assay after specific in vitro restimulation of effector cell populations from mice immunized with 2-10 micrograms purified, soluble T Ag and boosted with an injection of 2 micrograms T Ag 2-4 weeks after priming. Cells used for in vitro restimulation and as targets in cytotoxicity assays were syngeneic (B6-derived) RBL5 lymphoma cells expressing SV40 T Ag after transfection with a T Ag-encoding expression vector. Effector cells of this response were H-2 class I-restricted CD3+ CD4-CD8+ CTL. The magnitude of the anti-T Ag CTL response of B6 mice stimulated by soluble virus protein was comparable to the anti-T Ag CTL response of SV40-infected B6 mice. Injections of denatured or native T Ag protein primed CTLp equally well, but immunization with an equal dose of antigen emulsified in incomplete Freund's adjuvants inefficiently stimulated CTLp.

Animals↗

PubMed1992

Bone marrow cells of athymic nude mice express functional T cell receptor alpha chain transcripts rearranged to V delta 2, 3, 4, 5, 6 genes.

From bone marrow cells (BMC) of athymic nude mice, T cell receptor (TcR) alpha chain transcripts were selectively amplified by polymerase chain reaction (PCR) using V delta 2-, V delta 3-, V delta 4-, V delta 5-, V delta 6- and C alpha-specific primers. Amplified DNA fragments were cloned, and 32 randomly selected clones from 5 PCR were sequenced. Twenty-three distinct rearrangement events were detected, of which 87% (20/23) were in-frame. All five tested V delta genes (V delta 2, 3, 4, 5, 6) rearranged in-frame to J alpha-C alpha. N-region diversity in V delta-J alpha junctions present in most clones was limited to two to five nucleotides. P-nucleotide additions in this region were also detected. The V delta 5 gene located 3' of C delta in reversed transcriptional orientation was rearranged to J alpha by inversion. The J alpha usage pattern of the sequenced clones was strongly biased towards rearrangement of the most 5' genes (located nearest to C delta) of the J alpha cluster: the most 5' J alpha (J alpha TA1) was used by 30% of all clones, and 78% of all J alpha rearranged to V delta were located in the 5' 12 kb of the 60-kb J alpha cluster. As distinct V delta/C delta and V alpha/C alpha TcR usage patterns are prevalent in peripheral T cell populations, our data suggest that these TcR usage patterns results from repertoire selections operating in alpha beta and gamma delta T cell lineages, but not from preferential V delta-C delta and V alpha-C alpha rearrangement patterns.

Animals↗

PubMedDate not supplied

Donor selection process for allogeneic hematopoietic stem cell transplantation at the university hospital of Düsseldorf (1997-1998).

BACKGROUND: Transplantation of hematopoietic stem cells (HSC) is an effective treatment for a number of patients with life-threatening hematologic diseases. HSC donors can be found in the family of the patient or in registries of unrelated donors. In the present study, the search procedure within the last two years for an allogeneic HSC donor at the University of Düsseldorf is analyzed. PATIENTS AND METHODS: During 1997 and 1998, an early search for a related HSC donor in the family was performed for 70 high risk pediatric patients. During the same period, the search for an unrelated HSC donor for 116 adult and pediatric patients was performed. Low resolution HLA-A and -B typing was performed by serology in combination with DNA-typing. High resolution typing of HLA-A, -B and -C was carried out by DNA-sequencing. Low resolution HLA-DRB- und HLA-DQB1-typing was done solely by DNA-typing and high resolution typing of these genes was performed by DNA-sequencing. MAIN RESULTS: For 51 of 70 high risk pediatric patients (73%), no family donor could be defined, 16 of 70 patients (23%) had a genotypically identical sibling and for three of 70 patients (4%) an HLA-acceptable donor in the extended family could be identified. The search for an unrelated HSC donor was successful in 74% of the adult and pediatric patients lacking such a family donor. Most noteworthy, nearly all of the HLA-acceptable donors were identified from that group of donors in the registries, which were not only HLA-A and HLA-B, but also HLA-DR pretyped. CONCLUSION: These data show, that a growing number of pediatric patients with high risk leukemia need an unrelated HSC donor and that HLA-ABDR-pretyped registries present the optimal prerequisite to identify an HSC donor for most of the patients. In addition, 25% of the patients with no family or unrelated HSC donor require HSC transplants from alternative donors like unrelated Cord Blood (CB) from high quality cord blood banks.

Adult↗