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M Merkenschlager

Publications and source records attributed to M Merkenschlager.

At least 19 recordsLinked to original sources
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↗
PubMed1989

Evidence for differential expression of CD45 isoforms by precursors for memory-dependent and independent cytotoxic responses: human CD8 memory CTLp selectively express CD45RO (UCHL1).

On the basis of differential CD45 expression, human T cells can be separated into approximately reciprocal populations. The mAb UCHL1 detects a 180 kd molecular mass isoform, CD45R0. The CD45RA cluster of mAbs reacts with CD45 isoforms of 205 and 220 kd molecular mass. These reagents subdivide both CD4 and CD8 T cell populations. CD4 T cells that proliferate in response to memory-dependent (recall) antigens have been shown to selectively express CD45R0. We extend these observations to a model for cytotoxic responses that allows the functional analysis of CD8 T cells with differential CD45 expression. Precursors for allo-specific CTL responses are readily detectable in CD45R0 as well as CD45RA populations. In contrast, we find memory CTLp greatly enriched among CD45R0 cells. In combination with earlier work, these results suggest that differential expression of CD45 isoforms is associated with memory formation for different classes of immune responses in both major T cell lineages.

Antigens, Differentiation↗
PubMed1988

Characterization and functional properties of a novel monoclonal antibody which identifies a T cell receptor in chickens.

A monoclonal antibody, mAb6, was produced that specifically bound to chicken T lymphocytes. Immunofluorescence analysis using a fluorescence-activated cell sorter revealed that the antibody reacted with approximately 50% of blood lymphocytes and with approximately 40% of splenocytes and thymocytes. It did not react with bursal cells and erythrocytes. Among different types of hemopoietic cell lines, only a T cell line was reactive with mAb6. When coupled to Sepharose 4B beads, mAb6 was found to be highly mitogenic for chicken T cells. In soluble form, mAb6 inhibited concanavalin A-induced T cell proliferation and the cytotoxic activity of allosensitized T cells, the inhibition occurring in a dose-dependent manner for both assays. Thus, the tissue distribution and the effects of this antibody on T cell responses suggest that mAb6 recognizes a T cell receptor present on a large proportion of chicken T cells.

Animals↗
PubMed1988

Limiting dilution analysis of proliferative responses in human lymphocyte populations defined by the monoclonal antibody UCHL1: implications for differential CD45 expression in T cell memory formation.

Using limiting dilution analysis, we investigated proliferative responses of UCHL1+ and UCHL1- T cell populations to compare the precursor cell frequencies following recall and alloantigen stimulation, and the complexity of cellular interactions within UCHL1+ and UCHL1- populations. We find high frequencies of recall antigen responses among UCHL1+, but not UCHL1- T cells. In contrast, both populations contain similar frequencies of alloantigen responsive cells. Our results are consistent with single-hit kinetics in recall as well as alloantigen responses and show no complex cellular interactions within the responding populations. In conclusion, the difference between the recall antigen response of UCHL1+ and UCHL1- cells observed in conventional proliferation assays is most probably due to a high frequency of recall antigen-responsive UCHL1+ cells and not to suppressive phenomena in the UCHL1- population. These data suggest that memory T cells are largely of the UCHL1+ phenotype. The relation of post-thymic T cell maturation and differential CD45 expression is briefly discussed.

Antibodies, Monoclonal↗
PubMed1988

Phenotypic diversity of the CD45 antigen and its relationship to function.

Monoclonal antibodies (mAbs) have identified further heterogeneity within the major CD4 and CD8 T-lymphocyte subsets. Antibodies to different polypeptides of the CD45 complex separate two largely non-overlapping subsets that may represent stages in the maturation of T cells or separate functionally distinct subsets.

Animals↗
PubMed1988

Rate of incorporation of radiolabelled nucleosides does not necessarily reflect the metabolic state of cells in culture: effects of latent mycoplasma contamination.

In response to cell-free conditioned medium derived from the human bladder carcinoma line T24 (T24 SN), we found greatly reduced incorporation of tritiated thymidine and uridine ([3H]TdR, [3H]UR) by the human carcinoma lines UCHNCu (small-cell lung carcinoma) and LS174T (colon carcinoma). The effect was not due to an excess of nucleosides or cytokines known to be present in T24 SN. Cell-cycle distribution, increase in cell numbers, and de novo nucleoside synthesis in the indicator cells were only slightly altered. This was in contrast to the gross reduction in [3H]TdR/[3H]UR incorporation and seemed to indicate selective downregulation of pyrimidine-salvage pathways, despite ongoing polynucleotide synthesis. Spontaneous [3H]TdR uptake remained low for several passages in vitro but was readily restored by pharmacological inhibition of de novo pathways with 5-fluoro-deoxy-uridine (5-FUdR). This suggested a stable but reversible regulatory effect of T24 SN on the pyrimidine metabolism of the indicator cells. Further investigation showed degradation of [3H]TdR by a particle-bound activity in T24 SN. Mycoplasma contamination of T24 had not been detectable using standard cultural and staining methods, but became apparent when T24-cell lysates were hybridized with a recently described DNA probe (Goebel & Stanbridge, 1984). We conclude that latent mycoplasma contamination can stimulate changes in cellular pyrimidine metabolism. Our results provide an example for latent mycoplasma infection mimicking metabolic changes in cultured cells by direct interference of a microbial enzyme with the assay system. We describe a rapid and simple bioassay to detect and distinguish particle-associated and soluble phosphorylase activity by [3H]TdR degradation. It may be a useful screening assay for mycoplasma contamination in tissue culture.

Cell Line↗