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M Merkenschlager

Publications and source records attributed to M Merkenschlager.

At least 37 records · Page 2Linked to original sources
PubMed1994

MHC control of the naive TCR alpha-chain repertoire.

The naive T cell repertoire is shaped by interactions between developing thymocytes and thymic stroma. Both positive and negative selection involve the clonotypic TCR and MHC molecules carrying self-peptides. Except for the MHC-dependent effects of superantigens on TCR V beta usage; there has been little evidence that the TCR structure of naive T cell varies with the selecting MHC products. To examine this point from another angle, in particular the TCR alpha-chain, we have analyzed alpha-chain usage in a system in which the vast majority of T cells express a transgene-encoded TCR beta-chain, compatible with efficient T cell development on a wide range of MHC haplotypes. Endogenous TCR alpha-chains are thus selected without interference from the forces known to act on TCR-beta, permitting us to observe MHC influences on alpha-chain selection. We have used V alpha-specific Abs to quantitate alpha-chain usage in MHC congenic, MHC recombinant, and MHC transgenic mice and provide evidence that the naive TCR alpha-chain repertoire is under MHC control. The data demonstrate a direct impact of known MHC class II products but also reflect more complex influences, apparently involving other gene products within the MHC. Sequence analysis of differentially selected TCR suggests that selection acts on the entire alpha-chain, including V alpha, J alpha, and the junctional region.

Amino Acid Sequence↗
PubMed1994

In vitro construction of graded thymus chimeras.

A simple in vitro approach is described for constructing chimeric thymi in which the degree of chimeric contribution can be tightly controlled. This is achieved by protease-assisted dissociation of fresh fetal thymic tissue, mixing the resulting cell suspensions at the desired ratios, and subsequent reaggregation. Analysis of these graded chimeras shows that stromal elements become evenly interspersed and form a microenvironment able to support the maturation of endogenous T cell precursors. Chimeras between normal and MHC-deficient thymi have been used to demonstrate the rescue of mutant, allotype-marked thymocytes by wild-type stromal cells. Other potential applications of the method include quantitative studies on positive and negative thymic selection, the functional role of defined stromal cell types in these processes, and the analysis of mutants (either natural or engineered) by complementation.

Animals↗
PubMed1993

Antioxidant treatment of thymic organ cultures decreases NF-kappa B and TCF1(alpha) transcription factor activities and inhibits alpha beta T cell development.

Using electrophoretic mobility shift assays (EMSA), we have recently shown that nuclear extracts of 14-day mouse fetal thymocytes contain abundant NF-kappa B transcription factor activity. To determine the functional role of NF-kappa B in early thymocyte development, we have exposed fetal thymus organ cultures to inhibitors of NF-kappa B activation, namely the antioxidants N-acetyl-L-cysteine and butylated hydroxyanisole. Both compounds caused a dose-dependent arrest of thymocyte differentiation toward alpha beta, but not gamma delta, T cells. This was associated with a profound decrease in nuclear content of NF-kappa B and TCF1(alpha) transcription factor activity, as determined by EMSA. In contrast, NF-Y was affected less strongly, and cyclic AMP-response-element-binding protein levels remained essentially unchanged by antioxidants. To test the idea that alpha beta T cell development is correlated with NF-kappa B and TCF1(alpha) activity, we conducted additional experiments in a submersion culture system in which the generation of alpha beta T cells can be manipulated. Standard submersion culture supports gamma delta but alpha beta T cell development. Under these conditions, EMSA showed that transcription factor activities were similar to those seen in the presence of antioxidants. Importantly, when the generation of alpha beta T cells in submersion culture was restored by elevating oxygen concentrations, there was a dramatic increase in TCF1(alpha) activity, and both NF-kappa B and NF-Y returned to control levels. Taken together, these results strongly suggest that NF-kappa B and TCF1(alpha), presumably in concert with other transcription factors, play an important role in the development of alpha beta T cells.

Acetylcysteine↗
PubMed1993

T cell development: new approaches.

The differentiation of T lymphocyte precursors into functionally mature progeny proceeds in distinct stages. Since these are identified by characteristic constellations of phenotypic markers, the effects of experimental manipulations on T cell development can be readily monitored. In order to complete their developmental program, thymocytes must interact with stromal elements, which positively and negatively select the functional T cell receptor (TCR) repertoire. This process normally assures self tolerance and immunocompetence. Disturbances are of practical importance for clinical disorders including immunodeficiencies and autoimmune phenomena, raising a particular interest in human T cell development and repertoire formation. Here, we discuss results and possible applications of a culture system for human thymocytes. Further, we describe an in vitro approach addressing the requirements for a crucial step in T cell development; the transition from the immature CD4 CD8 double positive (DP) to the mature CD4 single positive (SP) stage.

Animals↗
PubMed1992

Selective manipulation of the human T-cell receptor repertoire expressed by thymocytes in organ culture.

A recently described organ culture system for human thymocytes is shown to support the generation of a diverse T-cell receptor repertoire in vitro: thymocytes of the alpha beta lineage, including representatives of the V beta families 5.2/5.3, 6.7, and 8, accounted for the majority of T-cell receptor-positive cells throughout a 3-week culture period. Thymocytes bearing gamma delta receptors were also identified, particularly among the CD4 CD8 double-negative subset. The T-cell receptor repertoire expressed in organ culture responded to experimental manipulation with staphylococcal enterotoxins. Staphylococcal enterotoxin D (a powerful activator of human peripheral T cells expressing V beta 5.2/5.3 receptors) caused a marked reduction of V beta 5.2/5.3 expression, as determined with the V beta-specific antibody 42/1C1. Evidence is presented that this loss of V beta 5.2/5.3 expression resulted from the selective deletion of activated thymocytes by apoptosis, in concert with T-cell receptor modulation. These effects of staphylococcal enterotoxin D were specific (since staphylococcal enterotoxin E did not influence V beta 5.2/5.3 expression) and V beta-selective (since expression of V beta 6.7 remained unaffected by staphylococcal enterotoxin D). On the basis of these observations, we suggest that thymic organ culture provides a powerful approach to study the generation of the human T-cell repertoire.

Animals↗
PubMed1992

Human postnatal thymocytes generate phenotypically immature CD3dim, CD5dim, CD1abright progeny in organ culture.

We previously described an organ culture system that supports the in vitro development of human fetal thymocytes in murine alymphoid thymic rudiments without addition of exogenous factors. This approach to study human T cell precursors is limited by the requirement for fetal tissue. We show that human thymocytes from pediatric sources can be expanded under similar conditions. Organ cultures generated predominantly CD4 CD8 double positive cells, most of which maintained the phenotype CD3dim, CD5dim, and CD1abright, characteristic for double positive thymocytes ex vivo. This culture system should facilitate in vitro studies on human thymocyte development and repertoire selection.

Animals↗
PubMed1992

[The ionized calcium in the blood of domestic chickens: dependence on age and sex].

In the present study concentration of ionized calcium and the correlation between total and ionized calcium in blood of domestic fowl and its dependence on age and sex were investigated. Furthermore a possible influence of blood sampling procedure on the measured values was assessed. Leghorn chickens of either sex, aged 3, 9, 25, and 80 weeks, respectively, were used. Blood sampling was performed by vein-puncture and indwelling catheters, respectively, of both ulnaris vein and artery of animals which had been fasting for 12 hours. Determinations of ionized calcium concentration was carried out by use of an ion-selective electrode (ICA1), and of total calcium concentration in plasma using atomic absorption, respectively. After attainment of sexual maturity total calcium in roosters rose from 2.73 to 2.85 mmol/l, whereas ionized calcium fell from 1.45 to 1.40 mmol/l and per cent fraction of ionized/total calcium (extent of ionization) from 53.1 to 49.1%, respectively. After attainment of sexual maturity in hens, the content of total calcium in venous blood rose from 2.78 to 5.78 mmol/l, and of ionized calcium from 1.45 to 1.59 mmol/l, respectively, whereas the extent of ionization was significantly reduced from 52.2 to 27.1%. On comparison of the vein-puncture and indwelling catheter technique a marked influence of the blood sampling procedure on calcium concentrations measured in blood of chicken became evident. On attainment of sexual maturity both the arterial and the venous blood (sampled from indwelling catheters) of hens showed levels of ionized calcium about 8% higher as compared to roosters. The extent of ionization amounted to 39% in sexually mature females as opposed to 51% in males. Concentrations of calcium ions in venous blood averaged 0.07 to 0.08 mmol/l higher compared to arterial blood in either sex. Concentration of ionized calcium did not exhibit any correlation to total calcium in adult animals. However, the concentration of ionized calcium was inversely correlated to blood pH.

Age Factors↗
PubMed1992

[The ionized calcium in the blood of domestic chickens: dependence on the laying cycle].

In the present study the plasma concentration of both ionized and total calcium in arterial as well as in venous blood of Leghorn hens and its dependence on the ovulatory cycle were investigated. During the ovulatory cycle, the concentration of ionized calcium in blood of hens follows the course of a sine curve. Maximum calcium ion concentrations (1.58 mmol/l in venous blood and 1.48 mmol/l in arterial blood, respectively) were observed 3 to 6 hours after oviposition, and minimum concentrations (1.26 and 1.19 mmol/l, respectively) at 18 to 21 hours after oviposition. Changes in total and ionized calcium concentration went parallel. Six to 9 hours after oviposition maximum values (4.24 and 4.15 mmol/l respectively) and 21 to 24 hours after oviposition minimum values (3.25 and 2.99 mmol/l, respectively) were measured. The arterio-venous difference was -0.22 mmol/l on average.

Animals↗
PubMed1991

Allorecognition of HLA-DR and -DQ transfectants by human CD45RA and CD45R0 CD4 T cells: repertoire analysis and activation requirements.

We have investigated the requirements for allogeneic stimulation of human CD4 T cells using HLA class II products expressed on various cellular backgrounds. Human (class II-negative RJ2.2.5 mutant) B cell lines transfected with HLA-DR or -DQ cDNA clones were efficient stimulators for highly purified CD4 T cells. HLA-DR-transfected mouse L cells or IFN-gamma-induced human fibroblasts, although able to function as accessory cells for T cell responses to the mitogen PHA, failed to stimulate strong T cell alloresponses. On the basis of these observations, we have employed class II transfectants to address the following questions: (a) do CD45RA and CD45R0 subpopulations differ in their allogeneic activation requirements, (b) are these subpopulations skewed in their recognition of HLA-DQ vs. HLA-DR in a manner which might support the concept that CD45RA T cells are involved in HLA-DQ-restricted suppressor inducer functions and (c) by using transfectants expressing individual HLA-DR or -DQ heterodimers in combination with limiting dilution analysis, can one for the first time obtain estimates of precursor frequencies for allogeneic cells recognizing each of these class II isotypes? Our results show that CD45RA and CD45R0 T cells respond comparably to optimal numbers of stimulator cells. However, when CD45RA and CD45R0 T cell populations depleted of endogenous accessory cells were cultured with limiting numbers of stimulator cells, CD45R0 cells generally responded more strongly, consistent with the elevated levels of various adhesion molecules known to be expressed by this population. Further, we found a similar representation of responses to HLA-DR and -DQ antigens among populations expressing CD45RA and CD45R0 isoforms. Finally, the precursor frequencies of allogeneic CD4 T cells responding to particular HLA-DR alleles were higher than to -DQ, but only by a factor of about 1.6, indicating that HLA-DQ recognition may occur more frequently than implied from previous antibody blocking studies.

Antibodies, Monoclonal↗
PubMed1991

CD45 isoform switching precedes the activation-driven death of human thymocytes by apoptosis.

A recently described chimaeric organ culture system is employed to investigate the effects of antibodies to the CD3 and CD2 cell surface structures on the fate of developing human thymocyte populations. Engagement of CD3 as well as CD2 with reagents that stimulate peripheral human T cell results in the activation-induced death of human thymocytes in organ culture. Death is preceded by a characteristic phenotypic change; thymocytes that bear CD45RA (determinants associated with high-molecular-mass isoforms of the leukocyte common antigen family, CD45) are first induced to co-express CD45RO (the low-molecular-mass isoform of CD45), and subsequently lose CD45RA expression. This antigenic change is followed by cellular DNA fragmentation, characteristic of apoptosis. We conclude that engagement of CD3 as well as CD2 can recruit human CD45RA+ thymocytes into the CD45RO+ population, where cell death occurs. Our results suggest that this phenotype conversion is a marker for thymocytes destined for programmed cell death.

Animals↗
PubMed1991

[Pteridine pattern in the urine of swine of different breeds and halothane genotypes in relation to nutrition and stress].

Urinary pteridine patterns and creatinine concentrations were determined in pigs of the breeds Piétrain, Deutsche Landrasse and their crossbreds. Also the influence of the halothane genotypes, of different feeding and of treadmill exercise was examined. We conclude that creatinine concentration in pig urine is not a reliable reference parameter for the pteridine excretion. Creatinine values were significantly influenced by breed and feeding. The pteridine pattern of pigs' urine depends on the breed and correlates with other metabolic parameters. Stress is reflected by altered pteridine concentrations.

Animal Nutritional Physiological Phenomena↗
PubMed1990

T cell alloresponses against HLA-DQ and -DR products involve multiple epitopes on the CD4 molecule. Distinct mechanisms contribute to the inhibition of HLA class II-dependent and -independent T cell responses by antibodies to CD4.

The involvement of the human CD4 molecule in T cell alloresponses to transfected HLA-DQ and -DR Ag was investigated with antibodies to defined CD4 epitopes. Anti-CD4 reagents inhibited T cell proliferation in a dose-dependent manner, and affected responses to HLA-DQ and -DR products equally well. As previously observed for conventional alloresponses, saturating concentrations of CD4 antibodies were required (and sufficient) for substantial blocking of T cell responses to transfected HLA-DQ and -DR products, and antibodies to a wide range of CD4 epitopes were inhibitory. In contrast to these results on class II-dependent T cell proliferation, MHC-independent T cell activation (via CD3 antibodies) was largely resistant to inhibition with the same dose range of CD4 mAb (provided that CD3 and CD4 reagents could not compete for the same class of FcR). This observation validates our conclusion that despite the substantial sequence differences between HLA-DQ and -DR heterodimers, the participation of CD4 epitopes in T cell responses to these molecules is conserved.

Antibodies, Monoclonal↗
PubMed1990

Functional epitope analysis of the human CD4 molecule. The MHC class II-dependent activation of resting T cells is inhibited by monoclonal antibodies to CD4 regardless whether or not they recognize epitopes involved in the binding of MHC class II or HIV gp120.

This study was designed to define regions on the human CD4 molecule important for the class II-dependent activation of resting, polyclonal CD4 T cells. With the use of mAb to known epitopes on CD4, we assayed the degree of CD4 saturation and functional effects on T cell activation over a range of antibody concentrations in parallel titration experiments. This approach allows a quantitative comparison of different reagents, regardless of parameters such as affinity for CD4. In sharp contrast to results reported for preactivated T cells and CD4 transfected T cell hybridomas, all 22 CD4 mAb tested did inhibit proliferative responses of freshly isolated CD4 T cells to MHC class II Ag. At the lowest saturating concentration of each antibody, T cell proliferation was reduced by 45 to 82%. Inhibition did not depend on antibody-induced modulation of CD4 expression. Strikingly, no correlation was found between the functional effects and the specificity of the mAb for different epitopes on CD4, such as the putative binding sites for MHC class II or HIV glycoprotein gp120.

Antibodies, Monoclonal↗
PubMed1990

The influence of nutritional factors on biopterin excretion in laboratory animals.

Nutritional influences on urinary total biopterin levels in rats and pigs were investigated. During the first nights in metabolic cages with food deprivation a significant increase in biopterin values was found in rats. This could be diminished either by feeding, adaptation to food deprivation or by oral glucose application. With food deprivation under normal housing conditions, this increase could not be found. Rats that were fed a cellulose preparation without metabolizable energy had no increase in biopterin excretion. The circadian rhythm of biopterin excretion was influenced by food deprivation as well as by cellulose. Alterations in water intake and urinary output had no effect on biopterin levels related to creatinine. Remarkable changes in biopterin excretion are more likely due to hormonal functions and regulations related to stress than to nutritional factors. More investigations into these problems are being performed.

Animal Nutritional Physiological Phenomena↗
PubMed1990

Human thymocyte development in mouse organ cultures.

A novel system to study human thymocyte development is described in which embryonic mouse thymic rudiments are seeded with human precursor cells in vitro. In these cultures human thymocytes proliferate extensively (greater than 20-fold increase in cell number) and mature, as evidenced by the accumulation of double and single positive (CD4+ and/or CD8+) cells. Data presented here suggest that the survival and ordered development of the mature human thymocytes in chimeric thymuses is dependent on human stromal elements. Immature CD4-CD8- human thymocytes failed to colonize or minimally recolonized mouse thymic lobes unless provided with high density (greater than 1.077 g/ml) human thymic cell fractions. These fractions contain multicellular complexes of epithelial/nurse cells, thymocytes, and dendritic cells/macrophages which dramatically enhanced the recolonizing capacity of purified CD4-CD8- thymocytes. The chimeric organ culture system described here provides not only a new approach for studying human T cell ontogeny but also a direct means for the future dissection of stromal interactions necessary for successful transition of precursor cells (CD4-CD8-) to immature double positive (CD4+CD8+) and mature single positive cells (CD4+ or CD8+) in the thymus.

Animals↗
PubMed1989

Memory T cells.

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Antigens, CD↗