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Biomedical subjects

F Emmrich

Publications and source records attributed to F Emmrich.

At least 73 records · Page 4Linked to original sources
PubMed1994

Cytomegalovirus infection in transplant recipients. The role of tumor necrosis factor.

Human cytomegalovirus (CMV) infection is an important cause of morbidity and mortality in transplant recipients. CMV infection commonly results from the reactivation of a latent infection. Using a set of monoclonal anti-CMV antibodies, we found CMV antigen expression in peripheral blood mononuclear cells (PBMNC), particularly in monocytes, in 312 of 816 samples from 190 allograft recipients. The detection of CMV-IE antigens and CMV-IE DNA in PBMNC indicates that positive cells may represent truly infected cells. The relation between increased cytokine plasma levels (particularly following treatment by pan-T cell antibodies) and the appearance of CMV antigens in PBMNC suggests that cytokines may play an important role in the reversal of CMV latency. This hypothesis is supported by our finding that tumor necrosis factor-alpha (TNF) is able to stimulate the activity of the CMV-IE enhancer/promoter region in the human monocytic cell line, HL-60. The interleukins 1, 2, 3, 4, 6, 8 and 10; transforming growth factor-beta; interferongamma; and granulocyte/macrophage colony-stimulating factor did not show any enhancing effect on the CMV promoter activity. Thus, TNF-alpha seems to play a key role in regulating the balance between latency and reactivation of CMV infection. Inhibition of TNF-alpha release or action may be an alternative strategy for preventing CMV-associated morbidity in allograft recipients.

Adult↗
PubMed1994

Ca2+ release and Ca2+ entry induced by rapid cytosolic alkalinization in Jurkat T-lymphocytes.

4-Aminopyridine (4-AP), a compound usually known as a K(+)-channel inhibitor, induced rapid cytosolic alkalinization from pH 7.15 to pH 7.4, and subsequently Ca2+ mobilization in the T-lymphocyte cell line Jurkat. Other weak bases, such as NH4Cl or triethanolamine, induced a smaller and/or slower increase in cytosolic pH, resulting in a lower or no detectable Ca2+ signal. In the presence of extracellular Ca2+, 4-AP mediated a rapid and sustained increase in the free cytosolic Ca2+ concentration similar to that obtained by T-cell receptor-mediated stimulation. In the absence of extracellular Ca2+, 4-AP transiently released Ca2+ from an intracellular store that is most likely identical with the agonist- and Ins(1,4,5)P3-sensitive Ca2+ pool of Jurkat T-cells. As possible mechanisms for Ca2+ release from this particular pool as induced by 4-AP we examined (i) formation of Ins(1,4,5)P3 and (ii) sensitization of the Ins(1,4,5)P3-receptor/Ca(2+)release system by increasing intracellular pH. Although 4-AP did not induce formation of inositol polyphosphates, as demonstrated by h.p.l.c. analysis, in permeabilized cells the dose-response curve for Ins(1,4,5)P3 was shifted to the left by changing the intracellular pH from 7.2 to 7.4. This indicated that sensitization of the Ins(1,4,5)P3-receptor/Ca(2+)-release system was responsible for the effects of 4-AP seen in intact cells. In conclusion, 4-AP appears a novel tool for depletion of the agonist-sensitive Ca2+ pool of T-cells without simultaneous formation of Ins(1,4,5)P3, thereby inducing capacitative Ca2+ entry in these cells.

4-Aminopyridine↗
PubMed1994

Adriamycin inhibits inositol 1,4,5-trisphosphate 3-kinase activity in vitro and blocks formation of inositol 1,3,4,5-tetrakisphosphate in stimulated Jurkat T-lymphocytes. Does inositol 1,3,4,5-tetrakisphosphate play a role in Ca(2+)-entry?

Effects of the cytostatic drug adriamycin on inositol polyphosphate metabolism were analyzed in a human T-cell line (Jurkat) using a recently developed anion-exchange high performance liquid chromatography/post-column complexometric dye system. Treatment of intact T-cells with adriamycin prior to stimulation with an anti-CD3 monoclonal antibody induced a dose- and time-dependent decrease in the intracellular level of inositol 1,3,4,5-tetrakisphosphate (complete inhibition after 2 h at 10 microM adriamycin) and an increase in the level of inositol 1,3,4-trisphosphate without significantly changing the levels of other inositol phosphates. A marked inhibition of the inositol 1,4,5-trisphosphate 3-kinase activity and a slight activation of the inositol 1,3,4,5-tetrakisphosphate 5-phosphatase activity were observed in cytosolic extracts in the presence of adriamycin, providing an explanation for the drug-induced metabolic effect. Adriamycin thus seems to be an extremely valuable tool for further dissecting inositol polyphosphate metabolism, as well as signaling pathways. Along these lines, we observed that adriamycin did not change the free cytosolic Ca2+ concentration of Jurkat T-lymphocytes and, in particular, did not modulate Ca2+ influx upon T-cell receptor stimulation. We conclude that (i) inositol phosphate signaling pathways constitute an as yet undescribed target for the action of adriamycin and that (ii) an increase of inositol 1,3,4,5-tetrakisphosphate is not necessary for sustained Ca(2+)-entry in stimulated T-cells.

Biological Transport↗
PubMed1994

Purification and analytical characterization of an anti-CD4 monoclonal antibody for human therapy.

A purification process for the monoclonal anti-CD4 antibody MAX.16H5 was developed on an analytical scale using (NH4)2SO4 precipitation, anion-exchange chromatography on MonoQ or Q-Sepharose, hydrophobic interaction chromatography on phenyl-Sepharose and gel filtration chromatography on Superdex 200. The purification schedule was scaled up and gram amounts of MAX.16H5 were produced on corresponding BioPilot columns. Studies of the identity, purity and possible contamination by a broad range of methods showed that the product was highly purified and free from contaminants such as mouse DNA, viruses, pyrogens and irritants. Overall, the analytical data confirm that the monoclonal antibody MAX.16H5 prepared by this protocol is suitable for human therapy.

Animals↗
PubMed1994

Karyotypic characterization of human T-cell lines immortalized by Herpesvirus saimiri.

Herpesvirus saimiri subgroup-C strains transform non-neoplastic human T lymphocytes in vitro to antigen-independent continuous growth. The karyotypes of 8 of these T-cell lines, derived from peripheral blood or bone marrow, are analyzed here. In general, these lines showed a normal diploid karyotype, although one of them had acquired a clonal chromosomal rearrangement after 21 months in culture. Chromosomally aberrant cell clones were found more frequently in cell lines derived from blood lymphocytes of leukemia patients than in cultures from healthy donors. However, the observed aberrations, some of which were defined in detail using chromosome in situ suppression hybridization (CISS) with chromosome-specific recombinant DNA libraries, did not show any relation between the various cell lines nor to the respective types of leukemia.

Cell Line↗
PubMed1994

Engagement of the CD4 receptor inhibits the interleukin-2-dependent proliferation of human T cells transformed by Herpesvirus saimiri.

Infection with Herpesvirus saimiri, a tumor virus of non-human primates, transformed human CD4+ T cell clones to permanent interleukin (IL)-2-dependent growth without need for restimulation with antigen and accessory cells. The IL-2-dependent proliferation of these cells was dramatically inhibited by soluble anti-CD4 whole antibodies, F(ab')2 and Fab fragments, and also by gp 120 of human immunodeficiency virus. The inhibition was not due to cell death and could be overcome by high concentrations of exogenous IL-2. Cell surface expression of CD4, and to a lesser degree the density of the IL-2 receptor alpha chain, were reduced upon anti-CD4 treatment. After long lasting (> 12 h) incubation with anti-CD4, abundance and activity of CD4-bound p56lck were diminished while the free fraction of p56lck remained unchanged. Since IL-2 binding to its receptor activated only the CD4-bound fraction of p56lck, the IL-2-induced p56lck activity was diminished after long-term CD4 ligation. Taken together, our results suggest a cross talk between CD4- and IL-2 receptor-mediated signaling via p56lck.

Antibodies, Monoclonal↗
PubMed1994

Imaging rheumatoid arthritis joints with technetium-99m labelled specific anti-CD4- and non-specific monoclonal antibodies.

A direct comparison of the joint-imaging properties of inflammation-specific- and non-specific monoclonal antibodies (Mabs) was possible in a patient suffering from long-standing, severe rheumatoid arthritis (RA). This patient received an anti-CD4- and an anticarcinoembryonic antigen (anti-CEA) Mab, both labelled with technetium-99m, 9 days apart from each other. The anti-CD4 Mab was superior to the isotype-matched anti-CEA Mab in imaging inflamed joints. In the knee joint, the target-to-background ratio of the synovial membrane (SM) activity in comparison to that of adjacent large vessels was 1.22 (SM/muscle 1.55) for the anti-CD4 Mab and 0.53 (SM/muscle 0.92) for the anti-CEA Mab, in both cases 4 h after injection of the immunoglobulin. Since the CD4 antigen is present on the surface of T-helper lymphocytes and macrophages infiltrating the inflamed synovial membrane, imaging with the anti-CD4 Mab may allow more specific detection of inflammatory infiltrates in RA.

Aged↗
PubMed1994

The effect of anti-CD4 on helper function of CD4,45RA+ versus CD4,45RO+ T cells.

Here we have investigated and compared the effects of anti-CD4 on helper function of CD4,45RA+ versus CD4,45RO+ T cells. Only CD4,45RO+ cells, but not CD4,45RA+ cells were able to promote B cell differentiation resulting in immunoglobulin production in vitro (IgM as well as IgG) which could be inhibited by anti-CD4 MoAbs (MAX.16H5 and T151). In pokeweed mitogen (PWM)-induced B cell proliferation a similar pattern of responsiveness was obtained. When we studied the anti-CD4 effects on cytokine production in T cells stimulated in mixed lymphocyte reaction (MLR) or by mitogens, we found that neither IL-2 nor IL-4 production was dramatically influenced by anti-CD4 in CD4,45RO+ cells. This led us to the conclusion that the inhibitory effect of anti-CD4 on B cell proliferation and immunoglobulin secretion was not due to inhibition of cytokine production. To clarify this point, we investigated the ability of anti-CD4 to inhibit conjugate formation between B and T cells. It was found that CD4,45RO+ T cells formed more conjugates than CD4,45RA+ cells, and that only the conjugate formation by CD4,45RO+ T cells was inhibited by anti-CD4. These results suggest that (i) anti-CD4 inhibits T helper functions primarily by affecting CD4,45RO+ cells, and (ii) this effect is probably mediated by contact inhibition in the early phase of T-B collaboration.

Antibodies, Monoclonal↗
PubMed1994

Synovial tissue implants from patients with rheumatoid arthritis cause cartilage destruction in knee joints of SCID.bg mice.

OBJECTIVE: To establish in SCID.bg mice a model in which joint destruction is initiated by human inflammatory cells from patients with rheumatoid arthritis (RA). METHODS: Development of a surgical technique and immunohistologic analysis. RESULTS: Initial experiments with single cell suspensions failed because more than 70% of the cells injected intraarticularly left the mouse knee joint within 16 h without causing destruction. This was observed with peripheral blood mononuclear cells, T cell lines reactive to mouse or rat collagen type II, and synovial mononuclear cells. Cell immigration was reduced but not prevented by preactivation with mitogens. In contrast, small tissue implants from human synovial membrane which were transferred by surgical intervention into mouse knee joints remained at the site of injection and could be easily localized within the mouse joint (observation period up to 8 weeks). The human synovial membrane implants induced pannus formation and erosion of cartilage and bone while only a mild and transient synovitis was observed with normal synovial membrane and control tissues like human thymus. The predominant cells at the site of destruction were human (CD68+) and murine (Mac-2+) monocytes/macrophages. CONCLUSION: The human/murine SCID arthritis is a useful model for studying pathogenetic aspects of joint destruction as well as effects of new drugs or novel treatment strategies.

Animals↗
PubMed1993

Immortalization of human T cell clones by Herpesvirus saimiri. Signal transduction analysis reveals functional CD3, CD4, and IL-2 receptors.

Investigation of human activated T cells has been complicated by the need for periodic restimulation with Ag/mitogen and accessory cells and by the limited life span of most human T cell clones. To overcome these problems, we have transformed established human T cell clones to permanent growth with Herpesvirus saimiri, a lymphoma-inducing virus of nonhuman primates. Three human CD4+ T cell clones were investigated in detail. They have been growing in the presence of exogenous IL-2 but without restimulation with mitogen or feeder cells for more than 11 mo with doubling times between 2 and 4 days. In contrast, their nontransformed parent clones needed to be restimulated with PHA and feeder cells every 14 to 21 days. To compare responses of H. saimiri-transformed clones with those of their parent clones, we stimulated the cells with IL-2 or with anti-CD3 and/or anti-CD4 mAb with and without cross-linking on the cell surface. Transformed and nontransformed T cell clones were strikingly similar in parameters of early signal transduction, namely, tyrosine phosphorylation and mobilization of calcium. Ligation of their TcR/CD3 complexes by mAb or by Ag in the presence of autologous accessory cells increased the proliferation and the secretion of IFN-gamma. Taken together, we have shown that human T cell clones immortalized with H. saimiri express functional CD3, CD4, and IL-2R. They constitute a simple, stable, reproducible and accessory cell-free model system for the investigation of signal transduction events in activated human T cells.

Antigens, Bacterial↗
PubMed1993

Intracellular Ca2+ pools in Jurkat T-lymphocytes.

Jurkat T-lymphocytes comprise at least four intracellular Ca2+ pools. Pool I was agonist-sensitive and contained 23 +/- 8% (n = 18) of the total Ca(2+)-storage capacity, as shown in intact cells in the presence of EGTA. The time courses of the agonist-induced formation of Ins(1,4,5)P3 and of the Ca2+ release from pool I were nearly superimposable, indicating that the agonist-sensitive pool I is emptied by Ins(1,4,5)P3. Likewise, in permeabilized cells, the size of the Ins(1,4,5)P3-sensitive Ca2+ pool I was 27 +/- 11% (n = 14). Pool II contained 26 +/- 5% (n = 9) of intracellularly stored Ca2+ and was liberated by thapsigargin, an inhibitor of the endoplasmic-reticulum (ER) Ca(2+)-ATPase. Addition of thapsigargin before addition of agonist abolished the agonist-induced Ca2+ release in both intact and permeabilized cells, indicating that pool I is a subcompartment of the ER Ca2+ pool. The content of this ER Ca2+ pool (pools I and II) amounted to 51 +/- 15% (n = 9) in intact cells and 49 +/- 16% (n = 16) in permeabilized cells. Caffeine released Ca2+ even when the ER pool (pools I and II) was emptied by previous addition of thapsigargin, indicating the presence of a third pool independent of pools I and II. Pool III contained 23 +/- 6% (n = 8) in intact cells, but 41 +/- 8% (n = 5) in permeabilized cells. The remaining intracellularly stored Ca2+ was released by addition of the Ca2+ ionophore ionomycin. This fourth pool contained 27 +/- 8% (n = 9) in intact cells, but less than 10% in permeabilized cells. The size of pool III was increased when pools I and II were emptied before addition of caffeine, whereas the size of pool IV was decreased under such conditions. In conclusion, this first comprehensive description of intracellular Ca2+ pools in Jurkat T-lymphocytes demonstrates the presence of four different Ca2+ pools, provides estimates of their sizes and describes relationships between each other. Release of Ca2+ from pool I [Ins(1,4,5)P3-sensitive] has previously been shown to play a major role in T-cell activation, whereas the physiological role of pools II-IV remains to be established.

Antibodies, Monoclonal↗
PubMed1993

Mass changes of inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate during cell cycle progression in rat thymocytes.

Changes in the cellular mass and the cellular concentration of the highly phosphorylated inositol derivatives inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) and inositol hexakisphosphate (InsP6) were observed during a complete cell cycle of proliferating rat thymocytes. Inositol polyphosphates were determined by a recently developed anion-exchange HPLC/post-column complexometric dye system (Mayr, G. W. (1988) Biochem. J. 254, 585-591), and data were expressed as mass amounts per cell number or as absolute intracellular concentrations based on cell volume determinations by the cell analyzer system CASY 1. After a very early transient rise in both Ins(1,3,4,5,6)P5 and InsP6 at the beginning of the cell cycle, a decrease of the intracellular concentration of both compounds occurred which then remained on a low level between 24 and 48 h. Between 48 and 72 h the cells divided resulting in a decrease of the cell volume by a factor of approximately 2. During this phase a pronounced increase of Ins(1,3,4,5,6)P5 and InsP6 was observed. These increases were already seen when the data were expressed as mass per cell number, but were even enhanced when expressed as absolute intracellular concentrations. In conclusion, we present as a novel finding long term changes in the intracellular concentrations of Ins(1,3,4,5,6)P5 and InsP6 in proliferating thymocytes. This may indicate a role for these compounds during cell cycle progression.

Animals↗
PubMed1993

Cholera toxin inhibits T cell receptor signaling by covalent modification of the CD3-zeta subunit.

In the Jurkat T cell line, triggering of the TCR leads to activation of phospholipase C, resulting in an increase in inositol trisphosphate (IP3) release followed by a rise in intracellular Ca2+. This signaling pathway is interrupted by cholera toxin (CTX) treatment. To possibly explain this inhibition, we demonstrate that CTX can affect the TCR/CD3 complex itself by causing a covalent modification of the CD3-zeta subunit. After exposure of Jurkat cells to CTX, CD3-zeta increases its apparent m.w. and becomes more acidic in isoelectric focusing. The time course of the modification correlates well with the reduction in IP3 generation and Ca2+ release after CTX treatment, suggesting that the modification of zeta might be the cause of the impaired TCR/CD3 signaling. As is true for the CTX-mediated decrease in TCR signaling, the change in CD3-zeta was cAMP-independent and cannot be evoked by the enzymatically inactive CTX-B subunit alone.

CD3 Complex↗
PubMed1993

Cloning of a functional cDNA for human cytidine deaminase (CDD) and its use as a marker of monocyte/macrophage differentiation.

We have identified a cDNA clone for human cytidine deaminase (EC 3.5.4.5) during an investigation which aimed at cloning novel gene expression products related to monocyte/macrophage differentiation. The derived amino acid sequence of the clone comprises 145 residues yielding a molecular mass for the polypeptide of 16.1 kDa and exhibits a nearly 50% homology to cytidine deaminase from Bacillus subtilis. Cytidine deaminase activity of the cloned sequence could be demonstrated in a prokaryotic expression system. The mRNA is highly expressed in granulocytes while expression is very low in fibroblasts, chondrocytes, monocytes, and T- as well as B-cell lines. The mRNA can be induced in monocytes, the monocytoid cell line U937 and the myeloblastic line HL 60 by the differentiation inducer calcitriol.

Amino Acid Sequence↗
PubMed1993

Biased T cell receptor V gene usage in rheumatoid arthritis. Oligoclonal expansion of T cells expressing V alpha 2 genes in synovial fluid but not in peripheral blood.

OBJECTIVE: To analyze the T cell receptor (TCR) variable (V) region gene usage in the rheumatoid joint. METHODS: Monoclonal antibodies (MAb) were used to determine the prevalence of selected V elements on T cells in synovial fluid (SF) from rheumatoid arthritis (RA) patients and in peripheral blood (PB) from RA patients and normal controls. V alpha 2-positive PB and SF T cells from 1 patient were cloned by immediate limiting-dilution and analyzed by restriction mapping. RESULTS: In 9 of 14 RA patients, SF was enriched in at least 1 of the selected V elements, compared with PB. TCR genes of the V alpha 2 family were the most frequently overrepresented in the SF (4 patients). The expanded V alpha 2-positive cells were oligoclonal in SF but heterogeneic in PB. CONCLUSION: Our data showing biased and clonally restricted TCR elements in the rheumatoid joint indicate major histocompatibility complex-restricted antigen recognition, rather than a "superantigen," in the pathogenesis of RA.

Adult↗