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W Holter

Publications and source records attributed to W Holter.

At least 55 records · Page 3Linked to original sources
PubMed1992

IgM and IgG but not cytokine secretion is restricted to the CD27+ B lymphocyte subset.

In a recent study we reported that CD27 is expressed on a subpopulation of human B lymphocytes and presented circumstantial phenotypic evidence that CD27 expression may be acquired late during B cell differentiation. Here we present functional data showing that, after in vitro stimulation, CD27+ but not CD27- B cells secrete large amounts of both IgM and IgG. Using double immunofluorescence staining of CD27 and IgD, three functionally different B cell subsets representing distinct stages of B cell differentiation can be isolated: 1) the CD27- IgD+ B cells, which do not secrete appreciable Ig; 2) the CD27+IgD+ B cells, which exclusively secrete IgM; and 3) the CD27+IgD- B cells, which comprise the IgG-producing cells. Furthermore, costimulation of CD27- B cells with low m.w. B cell growth factor, in the presence or in the absence of a CD40 mAb, does not induce these cells to become Ig-secreting cells. Although CD27- B cells hardly secrete Ig of any isotype in response to Staphylococcus aureus+IL-2, these cells proliferate vigorously and express the IL-2R alpha chain (CD25) under these stimulatory conditions. Furthermore, both CD27- and CD27+ B cells are capable of producing similar amounts of IL-6 and TNF-alpha. Taken together, these findings indicate that CD27 is a unique non-Ig surface marker discriminating naive from primed B lymphocytes. Furthermore, the capacity to proliferate and to secrete the B cell differentiation factors IL-6 and TNF-alpha already exists at an early B cell differentiation stage at which the cells lack CD27 expression and are not induced to produce Ig.

Antigens, CD↗
PubMed1992

Regulation of interleukin-4 production in human mononuclear cells.

Interleukin (IL)-4 is a cytokine with a broad range of effects on immune cells, however, little is known regarding the regulation of its production in freshly isolated human peripheral blood mononuclear cells (PBMC). Here we report the production of IL-4 in such cells following stimulation with monoclonal antibodies (mAb) directed against different cell surface antigens. We show that triggering via CD2 is more efficient for IL-4 production than triggering via the CD3 complex. The addition of a CD28 mAb enhances IL-4 production approximately threefold. Cell depletion experiments show that among CD2 plus CD28-stimulated PBMC the production of IL-4 is restricted to the CD8-CD45RA-T cell subpopulation. mAb interfering with the binding of IL-2 to its receptor can inhibit the production of IL-4 in CD2 plus CD28-stimulated PBMC. As IL-2 induces cell proliferation and production of interferon-gamma, but not production of IL-4, it follows that IL-2 is necessary but not sufficient for IL-4 production.

Antigens, CD↗
PubMed1992

Evidence for the presence of activated CD4 T cells with naive phenotype in the peripheral blood of patients with rheumatoid arthritis.

We have investigated whether T cell activation in rheumatoid arthritis (RA) preferentially engages distinct T cell subpopulations in the peripheral blood (PB) and in the synovial fluid. We found that CD25 expression was enhanced among PB CD4 T cells of RA patients as compared with CD4 cells of patients with reactive arthritis, degenerative joint disease or of healthy controls. Within the CD4 T lymphocytes subset we found that the CD45RO- (naive) cells selectively in RA displayed higher levels of CD25 protein and of interferon-gamma mRNA expression when compared with the respective subset of all other investigated groups. These results show that in the PB of RA, but not in the PB of the other arthropathies or healthy controls, CD45RO-CD4 T lymphocytes exist which display well-defined signs of activation.

Aged↗
PubMed1992

Regulation of interleukin 4 production and of interleukin 4-producing cells.

Interleukin 4 (IL-4) is a cytokine with a broad range of functions on immune cells including the stimulation of IgE synthesis by B cells. IL-4 is synthesized by freshly isolated T cells following appropriate stimulation with either lectins or antibodies against selected surface antigens. IL-4 production has also been observed by freshly isolated FceR+ non-T, non-B cells. With T cell clones, IL-4 is produced by clones of the Th2 and Th0, but not of the Th1 type. The molecular basis for restricted cytokine production by such clones as well as the signals responsible for differentiation into either phenotype are only at the beginning to be understood.

Animals↗
PubMed1992

Phenotype of human T cells expressing CD31, a molecule of the immunoglobulin supergene family.

The CD31 molecule is a leucocyte-surface glycoprotein of 130 kDa with homology to the immunoglobulin gene superfamily. In this study we report on the expression of CD31 on human T cells and demonstrate that it subdivides peripheral blood T lymphocytes into two novel subsets. CD31 is expressed by 38% of CD3+ lymphocytes. About 85% of CD31+ T cells display the CD45RA phenotype, 35% the CD45RO phenotype, 24% the CD4 phenotype and 72% the CD8 phenotype. There is also a correlation between CD31 expression and CD45RA expression in cord blood T cells; 89% of CD3+ cord blood cells express CD31, and most of them have the CD45RA phenotype. A discrepancy was found with thymocytes, which are positive for CD31 but negative for CD45RA. Stimulation of human T cells leads to down-regulation of CD45RA, while CD31 continues to be expressed. In functional studies, CD31 antibody binding to T lymphocytes does not lead to mobilization of intracellular calcium, proliferation or modulation of T-cell proliferation.

Antibodies, Monoclonal↗
PubMed1991

A simple and rapid slide blot method to quantify cytokine-mRNA in small numbers of lymphocytes.

In this study we describe an in situ hybridization protocol suitable for the measurement of interleukin mRNAs in small numbers of cells in suspension. In this procedure defined numbers of such cells are coated onto defined areas of microscopic slides using LAB-TEK tissue culture chamber slides. 32P-labelled oligonucleotides are then hybridized to them in situ. Binding of the probes is measured by autoradiography using X ray film followed by densitometry (slide blot) or, as in standard in situ protocols, by exposure to photoemulsion. In experiments designed to demonstrate the specificity and feasability of this protocol, peripheral blood T cells were activated with mitogenic stimuli and oligonucleotide probes specific for IL-2, IFN-gamma, or a control probe (IL-2 random) were hybridized to them. Both lymphokine genes were found to be transcribed in two discrete phases with the first peak at 12 h and another peak 48 h after stimulation, as measured semi-quantitatively on a per cell basis using X ray film exposure. The proposed experimental protocol supports the advantages of X ray film autoradiography such as rapidity, simplicity and the objectivity of densitometric evaluation, but also permits microscopic evaluation of individual cells as performed in classical in situ hybridization protocols using photoemulsion.

Base Sequence↗
PubMed1991

In vitro and in vivo activated T cells display increased sensitivity to PGE2.

In this study we report that in vitro activation of T cells increased the cyclic AMP response to subsequent prostaglandin E2 (PGE2) stimulation severalfold per cell. This sensitization of T cells to PGE2-induced cyclic AMP generation was observed when the T cells had been stimulated in vitro for 5 days with either the CD3 monoclonal antibody OKT3, phytohemagglutinin, or the combination phytohemagglutinin plus the phorbol ester PMA. Enhanced cyclic AMP generation following mitogenic activation was seen in response to both PGE2 and forskolin, direct activator of the adenylate cyclase, indicating that the amount of adenylate cyclase had increased during the in vitro activation course. In order to investigate whether various T cell subsets in general and in vivo activated T cells in particular would differ in their susceptibility to PGE2, we isolated CD4+, CD8+, CD4-CD8-, CD4+CD45RO+ ("memory"), and CD4+CD45RA+ ("virgin") T cells and studied PGE2-mediated inhibition of CD3-induced proliferation, as well as cyclic AMP generation in response to PGE2, respectively. We found that CD8+ T cells are more susceptible to PGE2 inhibition and produce more cyclic AMP than CD4+ T cells. Double-negative T cells (enriched for gamma delta T cell receptor positive cells) were found to be sensitive to PGE2 as well. Within the CD4+ T cell population, CD45RO+ ("memory") T cells were significantly more sensitive to PGE2-mediated suppression than CD45RA+ ("virgin") T cells. CD45RO+ cells required a 10-fold lower dose of PGE2 for half-maximum suppression of proliferation. However, no difference in cyclic AMP production could be demonstrated between these two subsets. We propose that substantial heterogeneity exists among peripheral blood T lymphocyte subsets regarding their sensitivity to the immunosuppressive action of PGE2 and that the sensitivity of individual cells changes in the course of an immune response.

Antibodies, Monoclonal↗
PubMed1991

Expression of GTP-binding proteins and prostaglandin E2 receptors during human T cell activation.

GTP-binding proteins (G-proteins) are a family of closely related, yet structurally distinct signal transducing proteins. In this study the presence and relative abundance of several G-proteins and of their corresponding mRNAs were measured in resting and activated human T lymphocytes. We found that T lymphocytes contain RNA coding for Gs, Gi2, and Gi3. No Gi1- and Go-specific RNA could be detected. Membrane fractions of resting and activated lymphocytes were studied in immunoblot experiments. Again, Gs, Gi2, and Gi3, but not Gi1 and Go, were detected. Upon mitogenic activation, a relative increase in mRNA for Gs and Gi3, but not for Gi2 could be demonstrated in Northern blot experiments. Immunoblotting indicated an increase in Gs and Gi3 density in membrane fractions of T cells as well. Paralleling the increase in Gs, we found that activated T cells produce five to seven times more cAMP per cell in response to prostaglandin E2 (PGE2) than resting lymphocytes. Finally, PGE2 binding studies showed that the number of receptors for this hormone increased from 435 +/- 322 to 1035 +/- 357 per cell following in vitro stimulation. We propose that in vitro T cell activation is paralleled by an increase in sensitivity to PGE2-induced cAMP generation. This sensitization is accompanied by both an increase in cell surface PGE2 receptor numbers as well as by increased expression of the signal transducing protein Gs and may physiologically be important for limiting an immune response.

Cyclic AMP↗
PubMed1991

Sequence-specific toxicity of transfected retroviral DNA.

Experimental gene transfer and viral infections can result in the accumulation of unintegrated DNA in target cells. The effects of such accumulation on target cell metabolism have not been directly studied. The experiments reported in this paper show that transfection of cloned retroviral long-terminal-repeat (LTR) DNA, or of a variety of eukaryotic promoters, into proliferating HeLa cells results in rapid, sequence-specific, and dose-dependent cell death. Plasmids containing the Rous sarcoma virus LTR or the human immunodeficiency virus LTR cloned in pUC-related plasmids are 5 to 10 times more toxic than pUC19. The demonstrated sensitivity of eukaryotic cells to exogenously introduced DNA has important implications for the interpretation of gene transfer experiments and may be relevant to the pathogenic mechanisms in the course of retroviral infections such as AIDS.

Avian Sarcoma Viruses↗
PubMed1991

[Phenotypical and functional characterization of leukocytes--the CD-system].

In several workshops held over the past few years 89 different cluster molecules have been identified on the surface of human leukocytes by the use of monoclonal antibodies. In this review we describe the principal of the CD clustering system and present a short survey of its application in clinic and basic research along with a complete listing of the CD molecules.

Antibodies, Monoclonal↗
PubMed1990

CD27 expression by a distinct subpopulation of human B lymphocytes.

CD27 is present on the surface of a major subset of peripheral blood T lymphocytes. In this report we show that CD27 is also expressed on a subpopulation of the normal human B cell lineage which is absent from cord blood but present in tonsils and in the peripheral blood of adult individuals. CD27+ B lymphocytes are characterized by the following criteria: (a) in terms of physical properties, the CD27+ B cells form a population with an increased cell size combined with a decreased cell density; (b) the CD27 expression of tonsillar B lymphocytes is postively correlated with mIgA but negatively correlated with membrane IgM/membrane IgD positivity; (c) CD27 on B cells can be induced selectively by the combination of Staphylococcus aureus plus interleukin 2, but not by either treatment alone, and (d) CD27+ B lymphocytes express high levels of the adhesion structures LFA-1 (CD11a), ICAM-1 (CD54), LFA-3 (CD58) and of the lymphocyte homing receptor CD44. These latter findings suggest that CD27+ B cells are predispose to form cell-cell interactions. Accordingly, within 3 h of cell culture CD27+, but not CD27-, B lymphocytes were found to form LFA-1-mediated homotypic B cell clusters.

Antibodies, Monoclonal↗
PubMed1990

Transient analysis for antiproliferative gene activity.

A subset of tumor suppressor genes presumably functions by the inhibition of cellular proliferation; however, antiproliferative activity after transfection with putative suppressor genes has been difficult to demonstrate and often requires lengthy selection either in nude mice or in vitro. A rapid alternative is presented here that utilizes a gene encoding a surface marker protein to identify transfectants in a transient expression assay. In this assay the labeling index, rate of DNA synthesis, cell-cycle distribution, and surface receptor display are measured by flow cytometry. Human beta-interferon, a gene with proven antiproliferative activity, was studied using the transient analysis system. The beta-interferon gene was introduced into human tumor cells along with the marker gene encoding the 55-kDa subunit of the human interleukin 2 receptor. Within a few days of transfection, analysis of transfectants by flow cytometry revealed a decrease in the fraction of cells in G2/M and an increase in the fraction of cells in G1/G0 and S phases. The distortion of the cell cycle was accompanied by as much as a 69% reduction in the rate of DNA synthesis and, in some experiments, an unanticipated increase in the labeling index. Therefore, cells accumulating in S phase were not blocked but continued to synthesize DNA although at a reduced rate. These studies on DNA synthetic rates revealed the caveat that screening for antiproliferative candidate genes with a labeling index alone could, in certain circumstances, exclude potentially interesting sequences from further consideration. Although this transient analysis system was developed for studies on cellular proliferation, it may prove suitable for phenotypic assays on other genes as well.

Bromodeoxyuridine↗
PubMed1989

Efficient gene transfer by sequential treatment of mammalian cells with DEAE-dextran and deoxyribonucleic acid.

A variation of the classical DEAE-dextran method of gene transfer was developed for efficient transfection of HeLa cells with plasmid DNA. A brief exposure of the cells to medium containing DEAE-dextran was found to be sufficient for subsequent uptake of pRSVcat and to be superior to cocultivation of the cells with DEAE-dextran plus DNA. This sequential method of gene transfer is nontoxic and yielded up to 60% of HeLa cells positive for a surface protein encoded by the transfected sequence. The implications of this sequential transfection technique regarding the mechanisms of gene transfer are discussed.

Animals↗
PubMed1988

T cell stimulation via CD2 molecules is regularly accompanied by an increase in cytoplasmic pH. Different effects of lectins and CD3 antibodies.

The stimulation of different cell types with growth factors is often accompanied by a rapid intracellular alkalinization. By using mitogenic lectins, cluster of differentiation (CD)2 and CD3 mAb, as stimuli, we studied early changes of the intracellular pH in the activation process of resting human PBL. We found increases in free cytoplasmic Ca2+ levels and DNA synthesis but no intracellular alkalinization in the early activation phase upon stimulation with the mitogenic lectins, Con A, and PHA. Similarly stimulation with CD3 mAb led in most instances to no detectable pH shifts. Only in 7 out of 30 experiments was CD3 mAb-induced alkalinization observed. In contrast, stimulation with mitogenic combinations of anti-CD2 mAb led in all instances to rapid and clear-cut intracellular pH shifts very similar to those observed upon stimulation with PMA. In medium lacking sodium bicarbonate the intracellular alkalinization via the CD2 structure could be blocked by the amiloride analogue 5-(N-methyl-N-isobutyl)amiloride (MIA), which indicates that this increase in pH is mediated by the amiloride-sensitive Na+/H+ antiporter. Blockade of this antiporter had no negative effect, however, on T cell proliferation as measured by thymidine incorporation. In contrast, significantly enhanced proliferation rates were observed after stimulation with mitogenic combinations of anti-CD2 antibodies in the presence of MIA. No such effect of MIA could be observed in lectin induced T cell stimulation. These findings indicate that stimulation of the Na+/H+ antiporter via the CD2 structure is neither a prerequisite for T cell proliferation nor does it promote T cell growth. It rather seems to function in a regulatory role. In its absence, superinduction of proliferation can be achieved.

Amiloride↗
PubMed1988

Purification of transiently transfected cells by magnetic affinity cell sorting.

A method was developed to purify transiently transfected HeLa cells or African green monkey kidney CV-1 cells by magnetic affinity cell sorting. Monolayer cultures were transfected with mammalian expression vectors coding for either of two novel cell surface antigens, the Tac subunit of the human IL-2 receptor or vesicular stomatitis virus G protein. During the transient expression phase, cell populations were placed in suspension and mixed with monoclonal-antibody-coated magnetic particles in the presence of a sorting solution designed to minimize nonspecific cell/cell and cell/particle interactions. Transfected cells expressing the vector-encoded cell surface antigen were then isolated by application of a magnetic field. Reconstruction experiments indicated that IL-2 receptor-positive cells were bound about 100-fold more efficiently than receptor-negative cells. In transient transfection experiments, populations of greater than 90% antigen-positive cells were reproducibly obtained.

Animals↗
PubMed1988

Regulation of the CD2 alternate pathway of T cell activation by CD3. Evidence for heterologous desensitization.

Normal resting T cells were stimulated through the alternate CD2 pathway. A CD3 mAb VIT3 completely blocked their proliferative response. The time interval for 50% inhibition lasted for 24 h after the onset of CD2 stimulation. Mitogen-activated cloned long term cultured T cells could also be stimulated via CD2. This proliferative response was again inhibitable by VIT3, indicating that CD3 regulates the CD2 pathway not only in resting cells, but also in lymphocytes actively involved in an Ir. T cells were further loaded with Quin2 and their free cytoplasmic Ca2+ levels were monitored in response to CD3 and CD2 stimulation. Antibodies directed against both surface R triggered a rapid elevation of Ca2+ levels. Both responses were abrogated when the cells had been treated overnight with VIT3. The free cytoplasmic Ca2+ levels of VIT3-pretreated cells, however, were not higher than those of control cells. These results point to a functional interaction between CD3 and CD2 possibly at the level of signal transducing proteins. Finally, cholera toxin was found to inhibit the Ca2+ response in Jurkat T cells. Both the CD3 and CD2 stimulation were sensitive to cholera toxin, indicating that a GTP-binding protein may be involved in signal transduction for both surface structures.

Antibodies, Monoclonal↗
PubMed1988

A new HTC, a PLT and a PLT clone define a split of HLA-DR2 in the Austrian population.

An LD defined split of HLA-DR2, different from HLA-Dw2, Dw12 and DB9, is observed in the Austrian population. The narrow HLA-D specificity, defined by a new Austrian Homozygous Typing Cell (HTC), a primed lymphocyte (PLT) cell line and a PLT clone, is tentatively termed HLA-D"WH". Investigation of three informative families shows that HLA-D"WH" segregated together with HLA-DR2. In one family, even an individual heterozygous for HLA-DR2/Dw2 and HLA-DR2/D"WH" can be identified. A panel study including 90 non-related Austrians shows that HLA-D"WH" occurring in 6.7% of the whole panel, that is in 20% of the HLA-DR2 positive panel members, is completely included in HLA-DR2. HLA-D"WH" might be comparable to other LD specificities related to HLA-DR2, "MN2", "FJO", "AZH" or "GEN".

Austria↗