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Biomedical subjects

G Pluschke

Publications and source records attributed to G Pluschke.

At least 37 records · Page 2Linked to original sources
PubMed1998

Crucial role of tumor necrosis factor receptor 1 expression on nonhematopoietic cells for B cell localization within the splenic white pulp.

During immune responses the initial activation of B cells takes place in T cell zones of periarteriolar lymphoid sheaths (PALS) of the splenic white pulp. After initial activation, B cells migrate into the primary follicles and, in association with follicular dendritic cells (FDCs), undergo clonal expansion and differentiation giving rise to germinal centers (GCs). Peanut agglutinin binding (PNA+) cells of the GC differentiate further into memory or plasma cells. Here we report that in tumor necrosis factor receptor 1-deficient mice (TNFR1(-/-)), the location of B cells was altered and that plasma cells were abnormally distributed in the splenic PALS. In contrast to lymphotoxin alpha-deficient mice (LTalpha-/-), bone marrow or fetal liver transplantation did not correct the abnormal organization of the spleen, location of B cells, the lack of an FDC network, nor the antibody response in TNFR1(-/-) mice. These results argue for a crucial role of TNFR1 expression on nonhematopoietic cells for the maintenance of the splenic architecture and proper B cell location. In addition, the lack in development of an FDC network after adoptive transfer suggests that either FDCs are not of bone marrow origin or that they depend on signals from nonhematopoietic cells for maturation.

Animals↗
PubMed1998

Sequence and diversity of T-cell receptor alpha V, J, and C genes of the owl monkey Aotus nancymaae.

We cloned and sequenced TcR alpha chain cDNA of three healthy Aotus nancymaae monkeys. Fifteen different TRAJ segments and 9 different TRAV genes were identified in the 29 rearrangements analyzed. As expected from the greater phylogenetic distance, A. nancymaae TRA gene sequences diverged more from the human sequences than those of the chimpanzee or the rhesus macaque. However, no Aotus TRAJ segment or TRAV gene was found which lacked a human counterpart. These counterparts were AJ02, AJ05, AJ09, AJ15, AJ22, AJ23, AJ28, AJ30, AJ32, AJ34, AJ37, AJ40, AJ42, AJ45, AJ52 and AV2S1, AV2S3, AV3S1, AV8S1, AV12S1, AV15S1, ADV21S1/DV5, AV22S1S and AV23S1, respectively. In most cases the identity of amino acid sequences between corresponding Aotus and human genes was greater than 80%. This marked conservation of TRA gene sequences indicates a close structural relationship of Aotus and human TcR and demonstrates that the TcR repertoire in primates is remarkably stable. The results support the concept of using Aotus monkeys, which are susceptible to infection with the human malaria parasite Plasmodium falciparum, as an animal model for the evaluation of molecularly defined malaria vaccine candidates.

Animals↗
PubMed1997

Titration calorimetry study of an anti-idiotypic antibody cascade in a human melanoma-associated antigen system.

The thermodynamic parameters of interactions between six variants of the anti-idiotypic monoclonal antibody (mAb) CGP 60686 produced by the hybridoma MK2-23 with an idiotypic mAb and five different anti-anti-idiotypic mAb were studied with high sensitivity titration calorimetry. CGP 60686 recognizes an epitope in the antigen-combining region of the human high-molecular-weight-melanoma-associated antigen (HMW-MAA)-specific mouse mAb CGP 76873 produced by the hybridoma 763.74. The five HMW-MAA-specific anti-anti-idiotypic mAbs GH 464, GH 518, GH 149, GH 386 and GH 586 were generated from mice immunization with mAb CGP 60686. All interactions between the anti-idiotypic mAb and the idiotypic mAb or the anti-anti-idiotypic mAb showed large exothermic binding enthalpies between -15 and -23 kcal/mol and binding affinities larger than 6 x 10(9) M-1. Four of the five anti-anti-idiotypic mAbs tested exhibited significantly higher binding enthalpies for the interaction with the anti-idiotypic than the idiotypic mAbs. Replacement of either the heavy or the light chain variable region of the anti-idiotypic mAbs with an unrelated variable region abolished the idiotype to anti-idiotype interaction. Thus, both the heavy and the light chain variable region of the anti-idiotypic mAbs are required for binding to the idiotype. The values of the binding enthalpy showed only small variations when binding of the idiotypic mAb CGP 76873 to four variants of the anti-idiotypic mAb CGP 60686 with different immunoglobulin constant regions, but identical variable regions were compared. Furthermore, Fab fragments of the idiotypic mAbs showed almost the same binding enthalpy per binding site as the whole IgG molecules. Immunoglobulin constant regions thus had little influence on the idiotype to anti-idiotype interactions. Taken together, the observed thermodynamic parameters suggest that the idiotype to anti-idiotype interactions studied here are enthalpy-driven processes with only minor entropic contributions. High sensitivity titration calorimetry was used to monitor protein-protein interactions within an anti-idiotypic antibody cascade. It was found that the direct measurement of the interaction enthalpy allowed a quantitative characterization of the binding processes studied.

Animals↗
PubMed1996

Molecular cloning of a human melanoma-associated chondroitin sulfate proteoglycan.

A human melanoma-associated chondroitin sulfate proteoglycan (MCSP), recognized by mAb 9.2.27, plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. We report here the molecular cloning and nucleotide sequencing of cDNA encoding the entire core protein of human MCSP and provide its deduced amino acid sequence. This core protein contains an open reading frame of 2322 aa, encompassing a large extracellular domain, a hydrophobic transmembrane region, and a relatively short cytoplasmic tail. Northern blot analysis indicated that MCSP cDNA probes detect a single 8.0-kb RNA species expressed in human melanoma cell lines. In situ hybridization experiments with a segment of the MCSP coding sequence localized MCSP mRNA in biopsies prepared from melanoma skin metastases. Multiple human Northern blots with an MCSP-specific probe revealed a strong hybridization signal only with melanoma cells and not with other human cancer cells or a variety of human fetal and adult tissues. These data indicate that MCSP represents an integral membrane chondroitin sulfate proteoglycan expressed by human malignant melanoma cells. The availability of cDNAs encoding MCSP should facilitate studies designed to establish correlations between structure and function of this molecule and help to establish its role in the progression of human malignant melanoma.

Adult↗
PubMed1995

Variation in N-linked carbohydrate chains in different batches of two chimeric monoclonal IgG1 antibodies produced by different murine SP2/0 transfectoma cell subclones.

Two chimeric human/murine monoclonal antibodies were constructed by substitution of the murine constant regions with human gamma 1 and kappa constant regions for heavy and light chains, respectively. The chimeric human/murine molecules are anti-idiotypic antibodies, meaning that they were directed against the antigen binding site in the variable region of another antibody. Antibody batches were produced under identical production conditions, using two selected SP2/0 myeloma cell subclones, which produce chimeric antibodies with different variable regions, but identical constant regions. Several samples were collected during the production of the antibodies in hollow-fibre reactors. The heavy chain, but not the light chain, of the two different chimeric IgG1 antibodies is glycosylated. Structural analysis of the enzymically released N-linked carbohydrate chains by 1H-NMR spectroscopy, as well as by chromatographic profiling, demonstrated that the collection of N-glycans comprises a small amount of monoantennary, and for the greater part diantennary structures. The N-glycans are completely (alpha 1-->6)-fucosylated at the innermost GlcNAc residue. The antennae of the neutral diantennary N-glycans are built up from GlcNAc beta 1-->2, Gal beta 1-->4GlcNAc beta 1-->2 or Gal alpha 1-->3G alpha 1 beta 1-->4GlcNAc beta 1-->2 elements, whereas the antennae of the neutral monoantennary carbohydrate chains have only (beta 1-->2)-linked GlcNAc residues. Galactosylation of the GlcNAc beta 1-->2Man alpha 1-->6 branch occurs four times more frequently than that of the GlcNAc beta 1-->2Man alpha 1-->3 branch, independently of the production batch. A small amount of the diantennary N-glycans are mono- or disialylated, carrying N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), exclusively (alpha 2-->6)-linked to beta Gal. Analysis of the different production batches demonstrates that the structures of the N-linked carbohydrate chains are identical in the two chimeric antibodies, but that the relative amounts of the major oligosaccharide components, the degree of sialylation and the molar ratio of Neu5Ac to Neu5Gc varies with the SP2/0 cell subclone, and only slightly with cell age.

Animals↗
PubMed1994

Oligoclonality and skewed T cell receptor V beta gene segment expression in in vivo activated human intestinal intraepithelial T lymphocytes.

Intraepithelial intestinal T lymphocytes (IEL) bearing alpha beta or gamma delta T cell receptors (TCR) are positioned to serve as a first line of defense against enteric pathogens. To investigate whether intestinal IEL are subject to antigenic selective forces distinct from that influencing (xp T cells in the peripheral blood (PBL), we performed a comparative analysis of V beta gene segment usage in IEL and PBL of immunologically normal donors by quantitative PCR. Primers for 22 different human TCR V beta gene segments of V beta gene segments families were used to analyze the repertoire of TCR beta chain transcripts in colonic IEL (c-IEL), in corresponding colonic lamina propria lymphocytes (c-LPL), and in peripheral blood lymphocytes. In each of the three individuals examined, a limited number (1-4 out of 22) of TCR V beta families predominated and accounted for more than 50% of the total beta chain transcripts from c-IEL, whereas in PBL and c-LPL a more even distribution of V beta gene families was observed. The dominating V beta gene families were V beta 2, V beta 3, V beta 6, V beta 8 and V beta 14. In one individual, V beta 3 comprised more than 40% of the entire repertoire of c-IEL beta chain transcripts. Sequence analysis of the predominant V beta 3 family in this individual revealed identical sequences in 13 of 17 clones analyzed. Human alpha beta TCR+ c-IEL could not be driven to proliferate or exhibit cytotoxic function in vitro however, PCR analysis for detection of lymphokine mRNA revealed constitutive production of several lymphokines known to exert trophic effects on intestinal epithelial cells and pro-inflammatory activities. Taken together, the striking degree of oligoclonality may indicate that the intraepithelial intestinal immune system is targeted to a limited set of hitherto unknown self- or foreign antigens present in the intestine and orchestrates intramucosal inflammatory and regenerative processes.

Base Sequence↗
PubMed1993

Analysis of T cell receptor V beta regions expressed by rheumatoid synovial T lymphocytes.

The T cell receptor (TCR) V beta gene segment repertoire of T lymphocytes derived from peripheral blood of two healthy individuals and synovial tissue, synovial fluid and peripheral blood of three rheumatoid arthritis (RA) patients was analyzed. A sensitive assay based on the amplification of cDNA by the polymerase chain reaction (PCR) was used to analyze the levels of expression of 20 TCR V beta gene segment families. The relative expression of V beta gene segments in lymphocytes derived from peripheral blood, synovial tissue and synovial fluid was conserved over 155 days in one patient. V beta 9 transcripts were undetectable in the cells of this individual. In the two other patients the frequency of V beta 2 transcripts in synovial T cells of affected joints was significantly higher than in their peripheral blood lymphocytes. Dominance of distinct rearrangements among the V beta 2 transcripts from the synovial cells of these patients support the idea that the synovial T cell response is driven by antigen.

Amino Acid Sequence↗
PubMed1993

HLA-DRB1 gene sequences in HLA-DR4 positive and negative patients with rheumatoid arthritis.

The second exon of the DRB1 gene encoding for the first domain of the HLA-DR beta 1-chain was sequenced in 16 patients (10 DR4/DR1 positive, 6 DR4/DR1 negative) with seropositive rheumatoid arthritis (RA). We could confirm the strong association of susceptibility to RA with functionally equivalent conformations on otherwise distinct MHC molecules. At least one HLA-DR allele in all of the analysed DR4 or DR1 positive patients showed such an epitope with a minimal variability limited to residue 71. However, in HLA-DR4 and -DR1 negative patients such a similar epitope could not be detected.

Alleles↗
PubMed1992

Autoaggressive myocytotoxic T lymphocytes expressing an unusual gamma/delta T cell receptor.

Polymyositis mediated by gamma/delta T cells is a unique disease in which autoaggressive T lymphocytes surround, invade, and destroy muscle fibers. Histochemically, the vast majority of muscle-infiltrating T cells in a patient with polymyositis were reactive with a pan-gamma/delta T cell receptor (TCR)-specific monoclonal antibody (TCR-delta 1+), but unlike > 90% of peripheral blood gamma/delta T cells, these lymphocytes did not react with V delta 1- or V gamma 9-specific antibodies (A13- and Ti gamma A-, respectively). Differential reactivity with two different V delta 2-specific monoclonal antibodies (BB3-/TiV-delta 2+) indicated that the infiltrating T cells express a V delta 2-containing TCR with unusual additional structural features. Using conventional and anchored polymerase chain reaction for the analysis of TCR transcripts, we found a striking predominance of one unusual V delta 2-J delta 3 recombination and one V gamma 3-J gamma 1 recombination. Both the unusual phenotype (TCR-delta 1+/A13-/Ti gamma A-/BB3-/TiV-delta 2+) and the dominance of distinct TCR transcripts are compatible with the assumption that one T cell clone, which expresses a V gamma 3-J gamma 1-C gamma 2/V delta 2-J delta 3-C delta disulfide-linked TCR, dominates among the infiltrating T cells of the polymyositis muscle specimen analyzed.

Base Sequence↗
PubMed1991

Biased T cell receptor V alpha region repertoire in the synovial fluid of rheumatoid arthritis patients.

Synovial T lymphocytes seem to contribute to the pathogenesis of rheumatoid arthritis (RA). Since very little is known about the structural heterogeneity of their T cell antigen receptors (TcR), we analyzed TcR alpha chain mRNA of synovial fluid T cells from two RA patients. TcR alpha chain cDNA was amplified by the polymerase chain reaction with single-sided specificity for the alpha chain constant (C alpha) gene segment, and the nucleotide sequences of 51 functionally rearranged cDNA clones were determined. Twenty different V alpha genes and 26 different J alpha gene segments were utilized in these cDNA clones. Three of the V alpha gene segments which are frequently (8%-17% total) expressed in synovial fluid T cells have rarely been found in the TcR repertoire of peripheral blood T cells from healthy individuals. The T cell responses in the rheumatic synovia analyzed here are not oligoclonal, but the usage of TcR V alpha genes is biased.

Antibody Diversity↗
PubMed1991

Humoral and cellular defense against intestinal murine infection with Yersinia enterocolitica.

The role of phagocytes and the complement system as potential host defense mechanisms against bacterial infection were studied in mice with two isogenic strains of Yersinia enterocolitica serotype O8 differing in pathogenicity because of differences in plasmid content. Complement depletion in mice by intraperitoneal injection of cobra venom factor did not affect the course of colonization of the intestinal tissue by each strain, indicating that in mice complement is not essential for the elimination of these bacteria. This conclusion is supported by the fact that fresh murine serum had no bactericidal effect in vitro either on the pathogenic or on the nonpathogenic strain. However, in the intestinal tissue as well as in the peritoneal cavity, only the pathogenic, plasmid-bearing Y. enterocolitica strain survived, while the nonpathogenic, plasmidless strain was rapidly eliminated. Since elimination from the peritoneal cavity is due to phagocytosis by polymorphonuclear leukocytes and macrophages, resistance to phagocytosis in vivo seems to be the decisive factor determining the virulence of pathogenic Y. enterocolitica strains.

Animals↗
PubMed1991

[Applications of PCR in molecular immunology].

Since a wide panel of monoclonal antibodies specific for antigen receptor variable (V) regions is unavailable, PCR has emerged as the primary analytical method for determining the expressed repertoire of T- and B-lymphocyte populations. Furthermore, PCR amplification of immunoglobulin V-region genes is exploited for the production of monoclonal antibodies by repertoire cloning as a replacement of the B-cell-hybridoma technique and for the humanization of murine monoclonal antibodies. The heavy and light chains of immunoglobulins as well as the transmembrane-protein chains which build the heterodimeric T-cell receptors consist of a constant and of an antigen-binding variable segment. Conventional PCR makes use of two specific oligonucleotide primers that flank a sequence of interest to prime replication of the bracketed DNA. In the case of antigen receptor encoding DNA one flanking sequence of the variable region is unknown or heterogeneous. Therefore, modifications of the conventional PCR had to be developed for the cloning and analysis of antigen-receptor DNA: (1) the usage of known V-gene segments by a lymphocyte population can be analyzed by quantitative PCR with panels of V-specific primers; (2) since PCR primers do not have to be completely complementary to the target DNA, it is possible to amplify a variety of different V regions with degenerate primers for relatively conserved sequences of the V genes; (3) enzymatic addition of a homopolymeric dG tail to the variable end of the antigen receptor DNA makes the amplification completely independent of the structure of the variable region because the dG-tailed DNA can be amplified with a nonspecific poly-dC oligonucleotide combined with a specific primer for the constant gene segment of interest.

Allergy and Immunology↗
PubMed1989

Isolation of rat IgM to IgG hybridoma isotype switch variants and analysis of the efficiency of rat Ig in complement activation.

Sequential sublining was used in combination with enzyme-linked immunosorbent assays to isolate mu----gamma isotype switch variants of the rat IgM secreting mouse-rat B cell hybridoma line BA1.8. Switch variants to all four subclasses of IgG were obtained. The variant antibodies retained the antigen specificity of the parental IgM for the O18 (lipopolysaccharide) antigen of Escherichia coli. In sodium dodecyl sulfate-polyacrylamide gels the apparent molecular mass of the gamma heavy chains decreased in the order gamma 2b greater than gamma 1 greater than gamma 2c greater than gamma 2a. IgM, IgG1, IgG2a, IgG2b and IgG2c of the BA1.8 variant family and IgG2b, IgE and IgA of the previously described BA1.2 family were used for a comparative analysis of the capacity of rat Ig to activate complement. Efficient lysis of sheep erythrocytes coated with the O18 antigen was observed with IgM and all IgG subclasses, but no lysis was triggered by IgE or IgA. One hundred to 1000 IgG molecules were required to mediate the same hemolytic activity as one IgM molecule. The four IgG subclasses were equally efficient at mediating lysis by rat or human complement, while IgG2a was less efficient with guinea pig complement than the other three IgG subclasses. Antibody-triggered binding of C3 to pathogenic O18:K1 E. coli bacteria was measured using serum containing 125I-labeled C3. K1-encapsulated strains did not fix C3 efficiently in the absence of specific antibodies while acapsular mutants fixed C3 via the alternative pathway. IgM and all IgG subclasses triggered C3 binding to the K1 encapsulated bacteria. The capacity of IgM to mediate C3 fixation was not greater than that observed with IgG.

Animals↗
PubMed1989

Roles of spleen and liver in the clearance of Escherichia coli K1 bacteraemia in infant rats.

The age-dependent increase in resistance of infant rats to K1 E. coli infection was studied by analysing the clearance of intravenously injected radiolabelled O18:K1 E. coli bacteria. In susceptible 7-day-old rats, the rate of reticuloendothelial clearance could not compete with bacterial multiplication, while the resistance of 18-day-old rats was attributed to the increased sequestration of bacteria in the spleen. After passive immunization with rat monoclonal anti-O18 IgG2b, the O18:K1 E. coli were rapidly cleared by the liver in both age groups. O1:K1, O18:K5 and O18:K- E. coli, which activate complement in an antibody-independent manner and are apathogenic for 7-day-old rats, were also rapidly sequestered in the liver. 7-day-old rats developed a fulminant bacteraemia after receiving 100 O18:K1 E. coli intravenously. After several hours a transient decrease in the level of bacteraemia was observed. However, the clearance remained incomplete, resulting in persistent bacteraemia and death. Endotoxin-responsive infant mice are able to clear the bacteraemia completely after a similar initial phase of bacterial multiplication while no clearance is observed in endotoxin-hypo-responsive mice. The marked host specificity of K1 E. coli infection thus appears to be related to differences in a clearance mechanism which may be sustained by an endotoxin-induced inflammatory response.

Age Factors↗
PubMed1989

Recombinant interleukin-1 stimulates clearance of Escherichia coli K1 bacteraemia.

Enhancement of non-specific resistance to neonatal Escherichia coli K1 infection by interleukin-1 (IL-1) was analysed. Recombinant human IL-1 administered prophylactically to newborn LPS-non-responsive C3H/HeJ mice induced rapid clearance of E. coli 018:K1 bacteraemia. The effect was dose-dependent and was observed with mice treated immediately to 1 day before bacterial challenge, whereas treatment 2 days before challenge was ineffective. was ineffective. Clearance of intravenously injected radiolabelled 018:K1 E. coli suggested that IL-1 triggered defence mechanisms that contribute to bacterial sequestration and killing in the spleen and liver. Comparable increase in bacterial clearance occurred in naturally resistant LPS-responsive mice that had been subjected to transient E. coli K1 bacteraemia and showed increased resistance to reinfection. In the course of E. coli K1 bacteraemia a strong synthesis of acute phase reactants was observed in both susceptible and resistant mouse strains, which indicated that these proteins alone cannot confer natural resistance to E. coli K1. IL-1 induced a very rapid synthesis of acute phase proteins. The clearance of K1 E. coli when still viable in IL-1-treated animals suggested that acute phase proteins are not likely to be major mediators of the IL-1-enhanced non-specific resistance.

Acute-Phase Proteins↗